Practical Guide To Antimicrobial PDF

Practical Guide to Antimicrobial
Active Packaging
Rafael Gavara
Practical Guide to
Antimicrobial Active
Packaging
Rafael Gavara, Gracia López-Carballo,

Pilar Hernández-Muñoz, Ramón Catalá
Virginia Muriel-Galet,
Josep P. Cerisuelo and Irene Domínguez
A Smithers Group Company
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P
reface
Active packaging technologies are being developed to control the various problems
associated with food deterioration or spoilage, such as the control of oxygen,
carbon dioxide, ethylene or humidity inside the packaging, addition of chemical
preservatives, elimination of off-odours and undesirable substances, and the control
of microbiological contamination. Among these technologies, antimicrobial active
packaging systems, defined as those that beneficially interact with food or the
surrounding environment to inhibit or reduce microbial growth, have undoubtedly
become a fully accepted alternative to the direct addition of preservatives to foods
and have excellent future prospects for improving the quality and extending the
shelf life of industrially produced foods. Accordingly, active packaging has become
one of the so-called emerging non-thermal food technologies, which are being
implemented to provide the consumer with natural, fresh, quality food without
compromising safety.
The aim of this book is to develop a working knowledge and understanding of

antimicrobial packaging. After a brief introduction on the antecedents and basics
of active packaging, the various issues to be considered in order to manufacture
successful, efficient active antimicrobial packaging are covered. In Chapters 1–5,
the antimicrobial agents most commonly used and their mechanisms of action are
described, the manufacturing methods available to manufacture the active system are
detailed, the parameters that are critical to make an effective product are discussed
and tools to optimise them are provided, in addition the various in vitro and in vivo
methods for measuring the effectiveness of the antimicrobial system are described
in detail. Chapter 6 provides a collection of references detailing the most interesting
developments and commercially available antimicrobial packaging systems. Finally,
Chapter 7 presents a case study, from conception to manufacture, validation and
optimisation.
This book also aims to develop an understanding of why a specific agent is selected
for a particular food product, or why a specific polymeric material and manufacturing
iii
Practical Guide to Antimicrobial Active Packaging
technology are chosen. The reader will become familiar with the different procedures
for improving the activity of the developed packaging solution and ways of testing its
efficacy. This will accelerate the formulation of the active packaging concept, reducing
development time with respect to the trial-and-error processes common in many of
the reports for which references are given. Finally, it will help to identify the best and
most cost-effective solutions.
iv
A
cknowledgements
The authors acknowledge the financial support of the Spanish Ministry of

Economy and Competitiveness for Project AGL2012-39920-C03-01. Irene
Domínguez thanks the CSIC for the provision of a postdoctoral contract (JAE-
DOC), which was cofunded by ESF. The authors are also grateful to Karel
Clapshaw for language services.
v
C
ontents
Preface.......................................................................................................... iii
Acknowledgements.........................................................................................v
Contents .......................................................................................................vii
About the Authors – Affiliation .................................................................... xi
1 Introduction to Active Packaging .................................................................. 1

1.1 Packaging and Active Packaging .......................................................... 1
1.2 Plastic Materials for Packaging ............................................................ 3
1.2.1 General Characteristics of Plastics ........................................... 3
1.2.2 Important Properties of Plastics for Packaging ........................ 5
1.2.2.1 Mechanical Properties ............................................. 5
1.2.2.2 Optical Properties ................................................... 5
1.2.2.3 Thermal Properties .................................................. 5
1.2.2.4 Mass Transfer Properties ......................................... 6
1.2.3 Principal Plastics used for Packaging ....................................... 7
1.2.3.1 Polyolefins ............................................................... 7
1.2.3.2 Polystyrenes ............................................................ 9
1.2.3.3 Vinyl Polymers ...................................................... 10
1.2.3.4 Polyesters .............................................................. 11
1.2.3.5 Polyamides ........................................................... 12
1.2.3.6 High-barrier Polymers ........................................... 12
1.2.3.7 Bioplastics ............................................................. 13
1.2.4 Packaging Production Technologies ...................................... 13
1.2.4.1 Flexible Packaging................................................. 13
1.2.4.2 Rigid Packaging .................................................... 14
1.2.5 Food/Plastic Packaging/Environment Interactions ................. 15
1.2.5.1 Permeability .......................................................... 16
1.2.5.2 Sorption ............................................................... 17
1.2.5.3 Migration ............................................................. 17
vii
1.3 Active Packaging: Basic Characteristics ............................................. 18

1.3.1 Antimicrobial Active Packaging ............................................ 21
References ................................................................................................... 22
2 Antimicrobial Agents................................................................................... 25
2.1 Minimum Inhibitory Concentration and Minimum
Lethal Concentration ......................................................................... 25
2.2 Antimicrobial Agents used in Active Packaging ................................. 27
2.2.1 Enzymes ................................................................................ 28
2.2.1.1 Bacteriolytic Enzymes ............................................ 28
2.2.1.2 Antimicrobial Oxidoreductase Systems ................. 29
2.2.2 Bacteriocins ........................................................................... 30
2.2.3 Bacteriophages ...................................................................... 33
2.2.4 Surfactants ............................................................................ 34
2.2.5 Plant Extracts ........................................................................ 36
2.2.6 Polysaccharides ..................................................................... 39
2.2.7 Organic Acids ....................................................................... 40
2.2.8 Metals ................................................................................... 41
2.3 Factors which affect the Properties and Stability of Antimicrobial
Agents during the Processing of Packaging Films ............................... 43
References ................................................................................................... 45
3 Active Packaging Systems ............................................................................ 53

3.1 Active Component Preparation .......................................................... 53
3.1.1 Use of an Inorganic Substrate ................................................ 54
3.1.1.1 Clay Nanocomposites ........................................... 56
3.1.1.2 Metal Nanoparticles .............................................. 58
3.1.1.3 Mesoporous Silica ................................................. 60
3.1.2 Encapsulation in an Organic Substrate .................................. 61
3.1.2.1 Criteria for Antimicrobial
Agent Encapsulation ............................................. 62
3.1.2.2 Matrices for Encapsulation ................................... 63
3.1.2.3 Encapsulation Methods ......................................... 65
3.2 Independent Devices .......................................................................... 69
3.3 Incorporation into Packaging Structures ............................................ 72
3.3.1 Incorporation into Packaging Structures by
Thermomechanical Methods ................................................. 72
viii
Contents
3.3.1.1 Preparation of Active Materials by

Compounding ....................................................... 74
3.3.1.2 Preparation of Active Materials by
Extrusion .............................................................. 75
3.3.1.3 Extrusion Coating ................................................. 77
3.3.2 Wet coating ........................................................................... 78
3.3.2.1 Coating ................................................................. 79
3.3.2.2 Spraying ................................................................ 84
3.3.3 Surface Anchorage ................................................................ 85
3.3.3.1 Sample Preparation ............................................... 86
3.3.3.2 Surface Modification ............................................. 87
3.3.3.3 Attachment of Biomolecules
to the Surface ........................................................ 88
3.3.3.4 Surface Analysis and Determination
of Film Activity ..................................................... 90
References ................................................................................................... 90
4 Modelling and Optimisation of Active Packaging:

Parameters Related to Antimicrobial Efficiency ........................................... 97
4.1 Mass Transport.................................................................................. 98
4.2 Determination of Mass Transport Parameters.................................. 105
4.3 Release Control ............................................................................... 110
4.4 Modelling Release of the Agent ....................................................... 115
4.4.1 Modelling and Description using the
Finite Difference Method .................................................... 117
4.4.2 Modelling and Description using the
Finite Element Method ........................................................ 120
References ................................................................................................. 122
5 Methods for the Analysis of Antimicrobial Packaging Efficiency ............... 125

5.1 In Vitro Methods ............................................................................. 127
5.1.1 Disc Diffusion Method ........................................................ 128
5.1.2 Dilution Method ................................................................. 131
5.1.3 Japanese Industrial Standard Method ................................. 137
5.1.4 Surface Growth Method ..................................................... 139
5.1.5 Electron Microscopy ........................................................... 140
5.1.6 Atomic Force Microscopy ................................................... 142
5.1.7 Flow Cytometry .................................................................. 144
ix
5.2 In Vivo Methods.............................................................................. 147

5.2.1 Total Microbial Load .......................................................... 149
5.2.2 Deliberate Contamination ................................................... 151
5.2.2.1 Inoculum Preparation .......................................... 151
5.2.2.2 Method of Inoculation ........................................ 155
5.2.2.3 Storage Conditions .............................................. 156
5.2.2.4 Data Interpretation ............................................. 157
References ................................................................................................. 158
6 Review of Antimicrobial Packaging Systems.............................................. 161
References ................................................................................................. 187
7 Case Study: Active Packaging of Minimally Processed Vegetables ............. 201
7.1 Description of the Product ............................................................... 201

7.2 Decontamination Technologies and Antimicrobial Agents
Applied to Fresh Vegetables ............................................................. 204
7.2.1 Chlorine .............................................................................. 204
7.2.2 Chlorine Dioxide ................................................................ 205
7.2.3 Ozone ................................................................................. 205
7.2.4 Irradiation........................................................................... 206
7.2.5 Modified Atmosphere Packaging ......................................... 206
7.2.6 Hurdle Technology.............................................................. 211
7.2.7 Novel Technologies ............................................................. 212
7.2.7.1 Active Packaging ................................................. 212
7.3 Antimicrobial Active Packaging ....................................................... 214
7.3.1 Active Material Manufacture: Laboratory Development ..... 216
7.3.2 Full Characterisation of Properties Prior to Film Scale-up ... 221
7.3.3 Industrial Production of the Active Film ............................. 225
7.3.4 Modelling and Optimisation of the Active System .............. 229
7.3.4.1 Modelling an Antimicrobial Package ................... 230
7.3.4.2 Solving the Package Model .................................. 233
References ................................................................................................. 236
Abbreviations .................................................................................................... 243
Index ................................................................................................................. 247
x
A
bout the Authors – Affiliation
The Packaging Group of the Instituto de Agroquímica y Tecnología de Alimentos

(IATA), which belongs to The Spanish National Research Council (CSIC), has been
conducting research in the field of food packaging technology since 1991. The group’s
main line of research involves analysing the interaction between plastic packaging
and food by characterising the barrier properties with regards to water vapour,
oxygen, carbon dioxide and organic volatiles, while also investigating the migration
problems present in polymeric materials used in packaging production, as well as
developing specific packaging technologies. In recent years, the group’s research areas
have essentially focused on developing new materials and packaging technologies,
placing particular emphasis on the use of nanotechnology to improve conventional
packaging materials, obtaining biodegradable materials from biomass and integrating
these factors into new packaging technologies. The latter area focuses on studies
involving packaging in a modified atmosphere and active packaging. Over these last
few decades, in the vanguard of food packaging science and technology, the group
has received financial support from Spanish ministries, regional authorities, European
research programmes and industrial projects, allowing the establishment of a suitable
laboratory infrastructure and an excellent multidisciplinary working group.
In relation to the aim of this book, the first studies featuring active packaging started
in 1999 and endeavoured to offer alternatives for fruit preservation by using minimal
processing, with interesting results in connection with certain natural compounds for
the avoidance of strawberry mould, as well as removing oxygen from the packaging
headspace. Since then the group has investigated the antimicrobial capacity of
natural substances obtained from plants in addition to procedures for incorporating
these antimicrobial agents into food packaging systems, initially by manufacturing
independent devices to be placed with the food in a conventional container, and
progressively by trying to incorporate them into the packaging walls. In this area,
thermomechanical, solution-casting and surface-anchorage techniques have been
explored by the group, with successful applications for various food products. In
addition, microbiological techniques for measuring the efficacy of the antimicrobial
materials have been set up and used for active materials with various mechanisms of
xi
action. The expertise and knowledge obtained over many years of research has led
to the writing of this guide.
Gracia López-Carballo, Virginia Muriel-Galet, Josep P. Cerisuelo, Irene Domínguez,

Pilar Hernández-Muñoz, Ramón Catalá and Rafael Gavara
Packaging Group, Instituto de Agroquímica y Tecnología de Alimentos (IATA, CSIC),

Av. Agustín Escardino 7, 46980 Paterna, Spain
xii
1
Introduction to Active Packaging
1.1 Packaging and Active Packaging
Food deteriorates with time due to the activity of living organisms (moulds, bacteria,
insects, rodents and so on), the physico-chemical activity of the environment
[temperature, relative humidity, oxygen (O2), radiation and so on] and the biological
activity of the food itself. The technological measures taken to avoid or minimise the
adverse effects of the aforementioned factors have led to the development of food
preservation techniques which involve a wide range of operations, very varied in
nature and complexity, and include packaging.
For some foods packaging is simply a form of presentation, a means of commercial

distribution and/or a way of providing the consumer with information, e.g., fresh
food intended for immediate consumption or food items that can be considered as
shelf-stable because of their physico-chemical characteristics. However, the marketing
of food products requires strict control of the conditions in which they are processed,
distributed and stored. The function of packaging, which is the barrier between food
and the environment, is to reduce the impact of external factors, protect the integrity
of the product and avoid or delay loss or deterioration of the nutritional, sensory
and safe characteristics that determine its quality and acceptance for consumption.
Thus packaging is a fundamental part of the food preservation system. In general,
whatever form of protection is applied, packaging is essential, and for many foods
the form of packaging determines the preservation technology.
The packaging used for the preservation and marketing of foodstuffs has changed
over the years with the gradual evolution of technology to its current state; it is
now characterised by a wide and varied range of materials and designs, with new
application processes and new packaging technologies. The packaging of the present
is required to be considerably more than a mere container or receptacle, whereas
packaging of the future will have to comply with increasing requirements. In a
society with easy access to an array of high-quality products, the consumer demands
constant innovation and improved safety and convenience of packaged foods. In this
1
context, the new paradigm of packaging is smart behaviour, understood as active

participation in maintaining and even improving the quality of the packaged food.
This has led to the concepts of ‘active packaging’ and ‘intelligent packaging’, terms
that refer to ways in which packaging functionality goes beyond the conventional
functions of containing, protecting, presenting and providing information about
food [1]. Although the terms active packaging and intelligent packaging are often
used as synonyms, they really refer to concepts that are different but very close, and
that may be complementary in many cases.
Traditionally, one of the most highly valued characteristics of packaging was its inertia
with regard to the food it contained. The packaging had to act as a mere container
and a passive barrier that provided insulation from the external environment, with
a minimal effect on the packaged product. Active/intelligent packaging, on the other
hand, aims to use or enhance food system/packaging/environment interactions so that
they act in a coordinated way to improve the healthiness and quality of the packaged
food product and extend its shelf life. In other words, the packaging ceases to be a
mere container and starts to play an active role in maintaining or even improving the
quality of the packaged food by correcting deficiencies in the preservation system.
Thus packaging with active containers or ‘active packaging’ can be considered as an
emerging food preservation technology [2].
With this redefinition of the concept of active packaging and its acceptance in
international health legislation [3], it is now possible to think about designing packaging
and packaging technologies in terms of the requirements of different products and
consumer markets, providing new ways of preserving and marketing foodstuffs,
but mindful of the fact that every food has a specific mechanism of deterioration,
which must be studied and understood in order to develop and apply the technology
most suitable for optimum control of product quality. There is no doubt that active
packaging responds to society’s increasing demand for food quality and safety.
Active packaging has subsidiary components that are deliberately added to it, or to
the material of which it is composed, in order to improve the functionality of the
system in various ways, i.e., either by acting on the composition of the atmosphere
inside the packaging by means of permselective materials or substances that emit or
retain gases and vapours, or by modifying the composition and/or characteristics of
the food, releasing substances that act positively on the food or which absorb/retain
undesirable components. Thus the function of active packaging is the preservation of
the packaged product. Intelligent packaging, on the other hand, contains an internal
or external indicator that provides information about aspects of the packaging or the
packaged product. Intelligent packaging always involves the complete food/packaging/
environment system; it analyses the system, processes the information and presents
it, generally without taking any action, whereas active packaging does take action.
Thus these two functions may be complementary and are not exclusive.
2
Active packaging and intelligent packaging have been gradually introduced

commercially and a wide variety of uses have been proposed for them [4]. Diverse
active packaging technologies have been developed to control various problems of
food deterioration or spoilage, such as control of O2, carbon dioxide (CO2), ethylene
or humidity inside the packaging, addition of chemical preservatives, elimination of
off-odours and undesirable substances, and control of microbiological contamination.
Active packaging has undoubtedly become a fully accepted alternative to traditional
packaging, with excellent future prospects for improving the quality and extending
the shelf life of foods produced on an industrial scale [5, 6].
This practical guide presents and discusses theoretical and practical aspects of the
technology of antimicrobial active packaging. After a short introduction regarding the
basis of active packaging and the materials used for its preparation, there is a review
of active systems that have been developed, some of them already used in practical
applications, together with a presentation and discussion of the various techniques
for preparing these packaging materials, methods for evaluating their effectiveness
and their possible applications.
1.2 Plastic Materials for Packaging
All kinds of packaging materials have been used for active packaging, although plastics
provide the best possibilities for this technology. The use of plastic materials for food
packaging began in the 1950s and became increasingly important, gradually taking
over from other traditional materials for some applications. The versatility of these
materials enabled the development of a wide range of packaging to satisfy the various
requirements of food packaging processes and new types of packaging.
The basic characteristics of plastic packaging materials are: lightweight, good chemical
inertia, versatility of shape, permitting the use of flexible packaging and processes
in which manufacturing and packaging are integrated, easy printability, suitable
mechanical resistance and heat sealability. On the other hand, they present certain
disadvantages, such as permeability to gases and aromas, the migration of some minor
components of plastics into the packaged product and thermostability issues, so that
the use of these materials is sometimes limited for the packaging of certain foods [7, 8].
1.2.1 General Characteristics of Plastics
Plastic materials can be defined in simple terms as easily identifiable high molecular
weight (MW) organic polymers formed by chemically bonded chemical units (the
constituent unit or segment) which are repeated. The basic components are carbon,
hydrogen and O2, and occasionally include other elements such as nitrogen, chlorine
3
or sulfur. The polymer matrix also generally contains various additives, fillers and/
or pigments, which are added to protect the polymer from degradation during
synthesis, facilitate its transformation or provide it with properties that it would
not otherwise possess. The considerable length of the chains means that they have
many intermolecular interactions, so they are usually solid. In this state, polymer
chains are usually entangled and arranged in a disorganised way, forming a material
which has an amorphous matrix. Some polymers manage to partially arrange their
chains in a regular structure, forming crystals and producing materials described
as semicrystalline. Whether they are amorphous or semicrystalline, most polymers
have a temperature range in which they soften or melt, which means that they can
be moulded when in a viscous state.
The raw materials for making conventional plastics are derived from petroleum,
although new biopolymers obtained from renewable sources are being developed
and can satisfactorily replace conventional materials for many purposes [9]. They
are generally obtained by polymerisation of one or more monomers, either by
polyaddition or polycondensation, giving rise to homopolymers or copolymers with
a great variety of properties and possibilities for practical applications. Various
classifications of plastics have been established, based on the synthesis reaction
mechanism, chain structure, groups, chemical structure of the repeating unit and so
on. One of the most common classifications is based upon their mechanical behaviour,
dividing them into three main groups: elastomers, thermoplastics and thermosetting
plastics or thermosets. Elastomers (rubbers) are very elastic polymers that respond
to any stress by deforming but then return to their original state when the stress is
removed. These materials are used for making seals, but it is difficult to use them for
the fabrication of packages. Thermoplastics soften when they are heated and harden
when they cool down, so they can be moulded and transformed into packaging
structures. Their softening temperature varies with the polymer type and MW. They
generally offer high impact resistance and ease of processing. Thermoplastics are the
plastic materials most widely used at present, especially for manufacturing packaging
[10, 11]. Thermosets undergo chemical transformations during processing resulting
in a permanent structure which is insoluble and infusible. They cannot generally be
used on their own, but are employed as fillers and reinforcements for other polymers.
Some thermosets, such as phenolic resins or urea-formaldehyde resins, were used in
the past for making bottles and stoppers, but nowadays their use in the packaging
industry has dwindled because of their high cost and the difficulty of transforming
them in comparison with thermoplastics.
There are a large number of polymers, although almost all the plastics used for
packaging belong to about 20 groups, the most commercially important of which are
the ones known as commodity or standard plastics: polyethylene(s) (PE), polypropylene
(PP), polystyrene (PS), polyvinyl chloride (PVC), polyethylene terephthalate (PET)
4
and polyamide(s) (PA). For many uses, the pure single materials on their own are not
always able to satisfy the specifications of the final product. In practice, therefore,
there is wide use of complex or multilayer structures, which are the result of bonding
laminas of different plastic materials.
1.2.2 Important Properties of Plastics for Packaging
The properties of plastic materials are a consequence of their chemical nature,

morphology, formulation, manufacturing technology and conditions of use. There are
significant properties common to all plastics in packaging applications, irrespective
of the specific uses for which they are intended.
1.2.2.1 Mechanical Properties
The behaviour of plastics in response to mechanical stress of any kind, which is of

great importance in their practical use for packaging, can be described by means of
various parameters that relate to resisting the effect of various physical actions to
which packaging may be subjected to in its transformation and use, such as abrasion,
traction, tearing, bursting, flexion, impact, perforation and so on. The packaging must
remain intact and the integrity of the product it contains must also be maintained
against these factors.
1.2.2.2 Optical Properties
Transparency, gloss and turbidity are the properties of greatest practical interest.
In their natural state, most plastics are opaque or translucent to a greater or lesser
degree, depending on their degree of crystallinity and, of course, the wall thickness
of the final product. Control of the optical properties of the final product can be
achieved by altering the polymerisation conditions, modification of the polymer
composition by blending with additives or nucleating agents, thermal treatment of
the product and adjusting the conditions for processing the material. By optimising
these factors it is possible to obtain films and packages that are transparent or have
selective transparency and a specific colour.
1.2.2.3 Thermal Properties
Thermoplastics are characterised by a mechanical behaviour which is affected by

temperature, especially with regard to their glass transition temperature (Tg). The Tg
5
is the temperature at which the material ceases to be relatively rigid and begins to
soften [12]. A polymer exhibits the characteristics of a ‘rubbery material’ (flexible
and tough) at temperatures above the Tg, whereas at temperatures below the Tg it
exhibits the characteristics of a ‘glassy material’ (rigid and fragile). Polymers with
a low Tg (below room temperature or the temperature at which they are used) are
generally flexible at low temperatures, while those that have a high Tg become brittle
at typical refrigeration temperatures but have a better capacity for withstanding
high temperatures. Semicrystalline thermoplastics also have a melting temperature
(Tm), at which the crystalline regions of the polymer are transformed into a molten
liquid state. Some potentially semicrystalline thermoplastics can be solidified into an
amorphous state by suitable thermal and mechanical transformation. When these
materials are heated they may exhibit a cold crystallisation temperature, at which the
solid amorphous polymer is reorganised and becomes semicrystalline. The crystallinity
of semicrystalline polymers ranges between 20 and 80% and is affected not only by
the inherent chemical nature of the material but also by other factors such as the
presence of other substances, e.g., nucleating agents, pigments and fillers, the rate
of cooling from the molten state, the presence or absence of mechanical stresses
during solidification, MW and so on. As the crystallinity increases the polymer
becomes denser and generally more rigid and resistant at high temperatures, which
means that polymers with high crystallinity are more suitable for applications at
high temperatures.
The temperature range over which polymers can be used is large, ranging from
-80 to over 200 °C. However, none of the materials that are generally used can
withstand this entire range, or the range normally used for food packaging, without
some modification of their functional properties. Therefore the packaging selection
and design must consider the temperature conditions and time of exposure that
are required. Also of great practical interest is the heat-sealing temperature. This
temperature, or rather temperature range, is the one at which the polymer is partially
melted by the application of heat and at which two polymer surfaces are joined
together by the application of pressure, producing a hermetic seal that is very useful
for shaping and sealing packages.
The behaviour of plastics in response to temperature can be described by the following

parameters: Tg, Tm, softening temperature, maximum/minimum temperatures of use
and heat-sealing temperature, cold seal strength and hot tack strength.
1.2.2.4 Mass Transfer Properties
Plastics are not a complete barrier to mass transfer, i.e., they are not impermeable like
glass or metal, and there is always the possibility that some degree of mass transfer
6
may take place in the environment/polymer/food system. The use of plastics for food
packaging is determined by their ability to prevent an undesirable transfer of the
packaging components to the product and vice versa, and also their ability to limit
the entry of atmospheric and environmental components that might cause undesirable
alterations to the product and/or affect its period of preservation.
The various mechanisms of mass transfer correspond to permeability phenomena, i.e.,

the transfer of low MW molecules (O2, CO2, H2O, aromas and so on), through the
packaging, the sorption and retention of components of the product in the packaging,
and migration, i.e., transfer of low MW molecules (additives, monomers and so
on) from inside the polymer to the environment or the product. These properties
are the object of more detailed study in the section on packaging/food/environment
interactions.
1.2.3 Principal Plastics used for Packaging
Of the commercially available polymer materials, the principal families of plastics

used in the manufacture of packaging are polyolefin(s) (PO), styrene polymers, vinyl
polymers, polyesters and PA.
1.2.3.1 Polyolefins
• Polyethylene and ethylene copolymers
PE are non-polar semicrystalline thermoplastics with various degrees of branching

and, consequently, the crystallinity ranges from about 40 to 80%. They are all
characterised by a good barrier to moisture and a low barrier to O2 and gases in
general. PE are generally classified according to their density, which ranges from
0.910 to over 0.950 g/cm2 [13]. The increase in density is accompanied by an increase
in resistance to traction, gas and water vapour barrier properties and thermal stability.
On the other hand, there is a reduction in transparency, impact resistance, stretching
percentage and suitability for heat sealing.
• Low-density polyethylene
Low-density polyethylene (LDPE) was the first plastic to be developed as a packaging

material and is the one which is most commonly used in practice. It is a semicrystalline,
semitransparent branched polymer. It is characterised by its easy processability and
can be used to make tough, flexible products with easy heat-sealing and good water
vapour barrier properties but very low gas barrier properties. It can be transformed
7
by practically all current technologies and is most commonly applied as a flexible

film, obtained by blown film extrusion, because of its low cost and its effectiveness.
The minimum temperature of use ranges from -50 to approximately 60 °C, although
it can be much greater for short times.
• High-density polyethylene
High-density polyethylene (HDPE) is semitransparent, exhibits a low degree of

branching and is the most rigid member of the homopolymer PE family. Its higher
MW and its high crystallinity (60–80%) give it the best gas and water vapour
barrier properties in the PE range. It can be used for injection moulding, extrusion
blow moulding and coextrusion. Its Tm is higher, ranging between 125 and 140 °C,
which makes heat sealing during flexible packaging more difficult. The maximum
temperature of use is about 95 °C. If the MW is high the embrittlement temperature
increases although it is well below 0 ºC.
• Ionomers
Ionomers are obtained by the copolymerisation of olefins with a comonomer that contains
an acid group and the subsequent reaction with metal hydroxide. The monomers most
commonly used for synthesising ionomers are ethylene and methacrylic acid, although
in principle any system capable of providing an acid group would be possible. The metal
ion takes the place of a hydrogen atom in the carboxylic group. Although ionomers are
much more expensive than LDPE or ethylene-vinyl acetate copolymer (EVA), they are
widely used in packaging because of their advantageous properties in many applications,
especially as a sealing layer in multilayers. The greatest advantage of ionomers is their
excellent behaviour during heat sealing; their excellent hot tack enables them to maintain
the integrity of the packaging, even though small particles of the product may be trapped
in the sealing area as a result of the filling process. They also have a greater ability to
absorb water than the aforementioned copolymers and homopolymers.
• Ethylene-vinyl acetate copolymers
Vinyl acetate polymerises with ethylene in any proportion, and the properties and
applications of the copolymer differ according to the relative quantity of the two
monomers. As the proportion of vinyl acetate in the copolymer increases, its properties
approach those of an elastomer in terms of flexibility and therefore impact resistance.
There is also an increase in transparency and gas permeability, and a decrease in
resistance to traction, melting point and resistance to heat. The proportions used in
packaging range between 10 and 30%. With high percentages of vinyl acetate, EVA
is mainly used to promote adhesion. EVA copolymers with lower proportions of
vinyl acetate are used to make films which exhibit high gas permeability. EVA mixed
with LDPE is used to provide intermediate properties. A family of materials has been
8
developed, known as ethylene-vinyl alcohol (EVOH) copolymers, which are derived

from the hydrolysis of EVA copolymers; due to its special properties it is described
later, in the section on high-barrier polymers.
• Polypropylene
It is similar to PE in many aspects but has a more complex structure. The presence
of a substituent, –CH3, provides the possibility of various chain architectures, which
are reflected in the functional properties of the material obtained [14]. When it is
manufactured using a Ziegler−Natta catalysis and under the right conditions, it is
possible to obtain PP with a very regular isotactic crystalline structure, which results
in a thermoplastic material that is very useful in packaging design (isotactic PP). If
the spatial arrangement of the chain segments is not controlled, i.e., it has an atactic
structure, the resulting polymer is soft and sticky and is only useful as an adhesive.
With a density of approximately 0.90 g/cm2, PP is the lightest of the plastics used
for packaging and one of the most widely used in rigid food packaging. It can be
injection moulded and extrusion blow moulded or thermoformed from sheets. It is
also used in flexible packaging as a film, obtained by extrusion, and is laminated in
complex structures and polymeric multilayers. In many of these applications, random
copolymers with ethylene in concentrations of 1 to 5% are used because they exhibit
excellent clarity and a lower melting point than homopolymer PP.
The properties that have made it possible to extend the use of PP are its low water
vapour permeability, toughness and low cost. As an O2 barrier, PP has a high
permeability but it is lower than PE. It is possible to obtain oriented films (oriented-PP),
which improve its optical properties and reduce the gas permeability with respect to
non-oriented PP. Its Tm is somewhat higher than that of HDPE, 158–168 °C, and the
temperature of use is 110 °C or a little higher, depending on the additives used. The
embrittlement temperature is around 0 °C.
1.2.3.2 Polystyrenes
Polymers and copolymers of styrene are high MW thermoplastics that are characterised
by their rigidity, ease of fabrication by extrusion and injection moulding and their low
density. There are a large number of commercial plastics based on the chemistry of styrene,
ranging from homopolymers to various types modified for impact resistance [high-impact
polystyrene (HIPS) or styrene-butadiene] or expandable polystyrene (EPS) [15].
The PS commonly known as crystal PS is the homopolymer of unmodified styrene.

Despite its common name, it is a totally amorphous thermoplastic, transparent
(like glass) and brittle, with low heat resistance (below 70 °C), and it has low gas
9
and water vapour barrier properties. It can be thermoformed from laminas obtained
by extrusion and it can be injection moulded. Orientation can increase resistance to
traction in the direction of alignment of the chains and biaxial orientation increases
resistance to traction in both directions.
HIPS are graft copolymers which contain butadiene and were developed for high-
impact resistance. HIPS is translucent white in colour and has less water vapour
permeability, much greater resistance to stress cracking and greater stability with
regard to heat deformation in comparison to PS.
• Expanded polystyrene
EPS contains a swelling agent that causes the granules of PS to swell during extrusion,
when laminas are fabricated for thermoforming, or during moulding, when containers
are made, giving rise to products known as EPS. In the first case, trays can be made,
and in the second, all kinds of containers adapted to the shape of the product. Using
extrusion it is also possible to obtain cushioning material for packing.
Products made with EPS provide excellent thermal insulation and lightweight. The
specific properties depend on the quantity of swelling agent and the transformation
conditions which are determined by the density of the final product; if a high density
is achieved the packaging is light and rigid, whereas a low density product will tend to
be soft and flexible. The water vapour and gas barrier properties of EPS are very low.
1.2.3.3 Vinyl Polymers
• Polyvinyl chloride
PVC is a material widely used for packaging [16]. Like PS, it is an amorphous polymer
with high resistance to tension and good transparency. It can be presented as a rigid or
flexible material and in the latter case it contains high concentrations of plasticisers. It
can be extruded to make films and it is also possible to make hollow bodies, especially
bottles, by extrusion moulding. In typical packaging applications it is presented as a
highly transparent, chemically inert material with high mechanical resistance. Its gas
and water vapour barrier properties are moderate. Its typical resistance to temperature,
about 80 °C, can be improved with formulations that make it possible to increase its
use to temperatures close to 90 °C. Its resistance to low temperatures can reach at
least -20 °C, depending on the formulation.
• Polyvinylidene chloride
Polyvinylidene chloride (PVDC) is an inert, semicrystalline polymer that forms very

transparent films which exhibit low gas and vapour permeability [17]. It is generally
10
presented as a copolymer with vinyl chloride, acrylates or nitriles to adapt its properties
to a variety of uses. Due to its high tendency to degrade or decompose as a result
of heat, it is usually not processed thermally via extrusion or injection. In the field
of packaging, PVDC is applied as a coating onto a flexible film imparting high gas,
water vapour and aroma barrier properties.
1.2.3.4 Polyesters
Polyesters are a large family of plastics widely used for food packaging. They are
materials that can be presented in amorphous or semicrystalline form, depending
on the thermal treatment to which they are subjected and the addition of nucleating
agents which facilitate crystallisation.
• Polyethylene terephthalate
PET is a linear thermoplastic obtained from terephthalic acid and ethylene glycol. The
polymer has an unusual combination of properties, being simultaneously resistant,
rigid and tough while in a semicrystalline state, at temperatures below the Tg (45 °C),
when the mobility of the chains is restricted. It can be oriented by stretching the
chains during moulding and extrusion, which can increase its resistance to traction
and rigidity. It has moderate water vapour and O2 barrier properties and can be
extruded to make films and extrusion blow moulded or injection blow moulded to
make bottles. New forms of PET have been developed which contain nucleating agents
and are added to increase the crystallinity by between 50 and 75%. This crystalline
PET is opaque, withstands much higher temperatures of up to 200 °C and can be
used as oven trays. The tendency of PET to crystallise during transformation can
be reduced by the addition of a second glycol in the polymerisation reaction. The
resulting material, which is completely amorphous and known generically as PET
glycol, is easily blow moulded and is exceptionally transparent, but exhibits a decline
in barrier properties.
• Polycarbonates
Polycarbonate(s) (PC) are technical plastics in which the structural units are essentially
of the carbon type. The most important commercial forms of PC are based on
bisphenol A. They are characterised by high resistance and rigidity and considerable
extensibility without becoming brittle. They are highly transparent and have
intermediate gas and water vapour barrier properties. Their high Tm, approximately
172 °C, enables them to be used in applications that require high temperature. They
can be transformed by coextrusion to make multilayers and they can be injection
blow moulded to make bottles.
11
1.2.3.5 Polyamides
Under the generic name of Nylon, PA are a large family of polymers whose main
identifying characteristics are their high heat tolerance, puncture resistance and
ductility. They are obtained by the condensation of monomers that contain amine
groups and acid groups.
The most common PA, PA 6, is the linear polymer that is obtained via the ring
opening polymerisation of caprolactam [18]. As PA 6 plastics tend to absorb water
other materials are added in order to avoid this effect. They can be used in multilayer
structures, e.g., as film and in extrusion blow moulding. Their greatest attribute is
their high mechanical resistance, especially puncture resistance, and they also provide
a high gas barrier and exhibit excellent resistance to fats.
1.2.3.6 High-barrier Polymers
High-barrier polymers are polymers that have a high resistance to the transmission of
gases. There is no standardised criterion for establishing a classification with regard
to permeability, although commercial practice takes O2 permeability as the basis for
this classification.
Copolymers of vinylidene chloride with vinyl chloride or acrylonitrile were the first
high-barrier plastics to be developed and are still widely employed, although they have
been replaced for most uses by EVOH copolymers, produced via the hydrolysis of
EVA copolymers [17]. The presence of ethylene constitutes 27−48% of the copolymer
composition which provides various formulations; owing to the presence of the –OH
group all the copolymers are characterised by very low O2 permeability, high sensitivity
to moisture and a tendency to absorb water. Therefore EVOH must be protected
against moisture in multilayer structures, e.g., using PO. It can be coextruded to
make films or hollow bodies via coextrusion blow moulding.
A well-established alternative to multilayer structures containing EVOH is the

application of transparent coatings of silicon oxide or aluminium on conventional
materials, i.e., PET and PP, which have applications as heat-sealed lids or tops for
pots and trays.
Other new high-barrier materials that have been developed are aliphatic polyketones,
aminopolyethers, liquid crystal polymers and, in particular, certain aromatic PA whose
unique characteristic is the improvement of their barrier properties as the humidity
increases. They are transparent and for short periods resist high temperatures (120 °C),
with good mechanical properties.
12
1.2.3.7 Bioplastics
The environmental impact attributed to conventional plastic materials, because they

are obtained from petroleum and their slow degradation in the environment generates
a great accumulation of packaging waste, has prompted the development of bioplastics
consisting of biopolymers obtained from renewable sources. These biomaterials must
exhibit characteristics of biodegradability and suitable mechanical, thermal, optical
and barrier properties for use as packaging materials that provide an alternative to
materials made with synthetic polymers or petroleum derivatives.
Biopolymers can be obtained from a wide variety of sources [9, 19]:
• From agricultural, livestock and marine products, e.g., polysaccharides such as

cellulose, starch and chitosan(s) (CS), and proteins such as casein, gluten and
collagen.
• By chemical synthesis from biomass monomers such as polylactic acid (PLA).
• Produced by natural or genetically modified microorganisms, such as

polyhydroxyalkanoates.
At present these are still materials with limited applications and high prices, but
they are in a period of considerable expansion. Among the biomaterials currently on
the market as replacements for PO, starch derivatives are the most widely used and
cheapest, but PLA is also an option.
1.2.4 Packaging Production Technologies
With the assistance of a variety of techniques, it is possible to transform plastic

materials into a wide variety of packages, of different designs and formats, which
are suitable to meet food marketing requirements.
1.2.4.1 Flexible Packaging
A thin film or lamina is the simplest form of presentation of a flexible plastic material.
The basic method of fabrication is blown film extrusion or cast film extrusion of
the polymer, obtained in the form of chips or pellets, together with the additives
required to process it and give it the properties desired for its use. A hopper unloads
the chips into a heated cylinder inside which a helical screw transports them while at
the same time gradually melting them, producing a homogeneous mass. At the end of
13
the extruder barrel the film is obtained from a tubular nozzle or a flat slit, either in
the form of a tube (blown film extrusion) or as a flat film (cast film extrusion) [10].
There are various procedures for combining two or more separate films to produce
a complex or laminated material, i.e., a multilayer structure. To obtain complex
films consisting of only thermoplastic polymeric materials, increasing use is made of
coextrusion processes, i.e., combined extrusion of the polymeric materials in the same
installation. Lamination using adhesives is the most widely used technique because of
its suitability for combining all kinds of substrates, including non-polymeric films such
as paper sheet or aluminium sheet, especially when a high bonding force is required.
A wide range of adhesives are used, specific for each type of substrate, although the
most common ones are vinyl, acrylic and polyurethane. Coating a substrate with a
molten polymeric material or a polymer dispersion is another lamination technique
that is widely used due its cost-effectiveness and versatility for all kinds of laminates.
Once the film of single or complex polymeric material has been obtained, various
techniques are used for packaging fabrication, depending on the type of packaging
and the product to be packaged. The packaging can be shaped prior to use, leaving an
opening for filling and final closing, although there is a general preference for integrated
processes in which the packaging fabrication and its use take place in succession in a
continuous automated process, so that it all constitutes a single operation.
The fabrication of bags, pouches, sacks and so on starts with a film in the form of
a tube or with one or two flat films, with bonding performed by heat sealing (the
application of heat and pressure) or by means of adhesives when the materials cannot
be heat welded. Vertical and horizontal feed installations have been developed for
fabrication and filling; vertical feeding is suitable for the packaging of products that
can flow freely, such as powder, granules and liquids, whereas a horizontal feed can
be used for the packaging of products of any shape and nature.
1.2.4.2 Rigid Packaging
Rigid packaging is considered to comprise of bottles, pots, trays, casks, tanks, jerrycans
and other kinds of hollow-body packaging with a three-dimensional shelf-stable
presentation and some degree of consistency [11].
The various kinds of packaging are generally manufactured by moulding processes,

working directly with the polymeric material in the form of granules or powder, or by
thermoforming from films or sheets obtained previously by means of the techniques
mentioned earlier. Like flexible packaging, rigid packaging can also be preformed or
obtained by means of integrated processes.
14
Various procedures can be used to manufacture packaging directly from the material,
with the various forms of extrusion moulding and injection moulding being widely
used for thermoplastic materials. In all cases the process begins with the softening
of the material, when the plastic granules are agitated in a hot extrusion barrel and
extruded by a helical transporter.
During extrusion blow moulding the material emerges from the nozzle of the heating
barrel in the form of a tube and in a much more viscous state than during injection
moulding. The machine cuts a suitable length of tube, still in a plastic state, and air
is blown into it under pressure. Blown extrusion/coextrusion techniques are used;
during both techniques the plastic is forced into a cold mould by means of compressed
air. The air pressure causes the material to expand and adapt to the shape of the
mould, and when the material cools the mould is opened for removal of the finished
container. The blown extrusion technique is performed in a single step and can be
used to obtain large-capacity containers.
During injection moulding, the molten plastic is injected at high pressure by a piston
through a narrow nozzle into a mould, where it takes on the desired shape. This
procedure can be used to make all kinds of open containers, such as cups, pots, trays
and so on. However, it is not possible to make bottles and other hollow narrow-necked
containers; to produce these items, after the injection process the piece or preform
is transferred to a second mould, where it undergoes a blow moulding process. The
packaging obtained via the blow injection moulding process can be used to obtain
containers with a better finish than those obtained by extrusion, especially with
regards to the closure mechanism, and of small capacity.
Many rigid packaging designs can also be produced by thermoforming or heat

moulding the thermoplastic material obtained previously, via extrusion, coextrusion
or lamination, into the form of a flat sheet or thin film, by applying heat and vacuum/
pressure in a suitable mould. The thermoforming technique is very simple and
economical and is particularly suitable for integrated packaging processes, in which
there are two reels of film, one to make the container (tray, pot and so on) and the
other for the lid.
1.2.5 Food/Plastic Packaging/Environment Interactions
Together with their many advantages, plastic materials also present limitations and
specific problems, partly resulting from interactions with the environment and the
packaged product. The packaging, together with the packaged food and environment,
constitutes a ternary system in which mass transfer phenomena of low MW molecules,
such as gases, water vapour or residues and additives of the material itself, take
15
place [20]. These phenomena, known as permeability, sorption and migration, can
produce chemical, nutritional and sensory changes in the packaged food and may have a
negative impact on the preservation of the food, even leading to its loss. Figure 1.1 gives
a schematic description of the mass transfer processes and their practical consequences.
1.2.5.1 Permeability
Permeability, or permeation, is defined as the passage of gases and vapours through the
packaging. Whatever their nature, polymers are, to a greater or lesser degree, permeable
to the molecular diffusion of fluids inside them, with the penetration taking place in the
direction of the gradient of concentration, i.e., high to low, through the empty spaces
that are left between the networks of macromolecular chains that constitute the polymer
matrices. The molecules of the permeant (O2, humidity, aromas and so on) are initially
sorbed into the interphase of the packaging in contact with the external environment,
and once in the material they diffuse throughout the thickness and are finally desorbed
into the interphase in contact with the food or interior environment.
OD ER T
FO YM EN CONSEQUENCES
POL ON
M
IR
ENV
• Sensory food degradation
O2, CO2, ... PERMEABILITY
moisture, • Container deterioration
aromas (loss of quality and possible
rejection)
Fats,
dyes, SORPTION
others • Sensory and nutritional food
degradation (loss of texture,
browning, rancidity of fats,
O2, moisture,
aroma degradation, etc.)
aromas
PERMEABILITY
Radiation • Sensory food degradation
and possible toxic effects.
Monomers, Container deteroration
MIGRATION
additives,
MIGRATION solvents
Figure 1.1 Food/packaging/environment interactions in plastic packaging materials
Generally speaking, the barrier capacity (impermeability) is determined by the nature of

the polymer (structure, crystallinity, orientation and so on) and that of the permeant fluid
(size, configuration, polarity, concentration and so on) and also by the environmental
conditions of the system. In practice, the barrier properties define the capacity of the
16
packaging to resist the absorption/desorption of vapours, the permeation of gases,

vapours and aromas, and the passage of light through the films. The study of the barrier
properties of different materials for a particular gas or vapour is therefore necessary
in order to be able to select the appropriate materials that are most suitable for the
packaging of a specific product.
The form of expression of the barrier characteristics of films of polymeric materials is

their permeability coefficient (P) or quantity of a substance (q) that diffuses through
the material of thickness (L), under specific pressure and temperature conditions,
per unit of time (t) and surface (A) when it separates two environments that have
different partial gas pressures (∆p):
q×L
P = D× S = (1.1)
A × t × ∆p
The permeability coefficient is an intensive magnitude value that is very useful in

packaging design as a basic parameter for objective evaluation of barrier properties
and therefore for defining the suitability of plastic packaging materials. With a good
knowledge of the effect of the packaging atmosphere on the sensory characteristics
that define the quality of a particular food, measurements of permeability even enable
us to estimate the shelf life that can be expected for the packaged product.
1.2.5.2 Sorption
Sorption is the retention of food components in the polymeric material structure of

the packaging. The components potentially adsorbed in the packaging are principal
constituents, such as water, oil or fat, and minor constituents, such as sugars, alcohols,
aroma components, colourants and so on. Sorption phenomena are generally not
involved in food loss processes, given that the retention of components is not related
to microbiological or toxicological effects. For this reason, sorption is the mass
transport process that has been least studied. Nevertheless, the retention of some
components may lead to consumer rejection, e.g., a product which has undergone
the partial loss of an organoleptically very active component. The sorption of other
important compounds (vitamins or sugars) is not perceptible when the product is
consumed. Sorption processes can also be used beneficially when the sorbate is an
undesired component from a sensory or nutritional viewpoint.
1.2.5.3 Migration
Migration is the transfer or desorption of low MW molecules initially present in the

packaging to the packaged product or environment. Plastics contain numerous low
17
MW substances that can be transferred. Some migrants are residues from the synthesis
of the polymer, such as monomers, oligomers, catalysts, detergents, solvents and so on.
Others are added to the polymers to improve their properties, such as photostabilisers,
antioxidants, lubricants, plasticisers, antifogging agents, printing pigments and so
on. These substances (once they are present in the packaging) diffuse through the
structure until they reach the interphases and are partially transferred to the headspace
and/or are dissolved in the food. These processes cause loss of product quality and
therefore reduce its shelf life. Moreover, some of the transferrable substances may
be toxic if swallowed and consequently may induce toxicity via their consumption.
For this reason migration is regulated by national and international laws. These laws
define the terms global migration (total mass released in the food irrespective of its
composition) and specific migration [mass of a specific compound of special (toxic
or sensory) importance] and they also establish the maximum limits of admissible
migration and the analytical procedures for evaluation that the materials must pass
to allow them to be used in food packaging.
As has been mentioned, knowledge of mass transfer properties is critical for

establishing the degree of material suitability for the requirements of a specific product.
These properties, which differentiate polymeric materials from traditional packaging
materials (glass and metal) have made it possible to develop packaging technologies
for foods in which a total barrier is not necessary or is even counterproductive. As we
shall see later in this book, these properties, to a large extent, have led to advances
in the development of active packaging systems.
1.3 Active Packaging: Basic Characteristics
As defined in Article 3 of European Regulation (EC) No.450/2009, active materials

are materials that are designed to deliberately incorporate components that will release
or absorb substances into or from the packaged food or the environment surrounding
the food. Thus packaging ceases to be a mere container for food and takes on an
active role in the preservation or improvement of its quality and shelf life.
Active packaging can be obtained by various means, but there are basically two ways
in which it functions, either with the active agent inside the packaging with the food
but separate from it, in an independent device, or with the active agent forming part
of the packaging material itself.
Since the beginning of the development of these technologies, the active element has
been introduced via a small bag, sachet or label containing the agent (e.g., iron to
remove residual O2 in the packaging or silica gel to eliminate humidity) which was
placed inside the packaging together with the packaged product [4]. The bag is made
18
of polymeric material which is sufficiently permeable to allow the release and/or

activity of the active substance, but generally without coming into contact with the
product. It is, of course, necessary to use active materials that do not endanger the
safety of the packaged product. This is how most of the systems of the first generation
of active packaging functioned and it is still a technique that is widely used. However,
this form of active system functionality has been questioned because it means the
presence inside the packaging of elements that are foreign to the natural composition
of the food, which may lead to rejection by the consumer and also complicates the
packaging process because it involves an extra operation to insert the bag into the
packaging. Moreover, this technique is generally not applicable to liquid products.
The alternative, which is now extensively used for many technologies and will
undoubtedly become more widespread in the future, is to incorporate the active
substance in the packaging material itself. This is a more attractive method for
consumers, who will not find any strange matter inside the packaging that might
attract their attention and cause them to doubt the safety of the food that they are
about to consume; in addition, it simplifies the packaging technology by eliminating
the operation of inserting the active system in the packaging and is applicable to all
kinds of products [21].
Various forms of active packaging have been proposed to control most of the problems
concerning the deterioration or spoilage of food quality, such as control of gases in the
packaging headspace (O2, CO2, ethylene and so on), regulation of humidity, addition
of antioxidants and chemical preservatives, incorporation of aromas or functional
compounds to improve food characteristics, elimination of off-odours and undesirable
substances, and control of microbiological contamination.
The basic materials that have been used to develop active packaging are paper and
cardboard, plastics and metals or combinations of them, but the developments of active
packaging technologies have generally employed plastic materials, with which they
find their greatest possibilities. As mentioned earlier, plastics are not totally inert with
regard to food, as they allow the exchange of gases and vapours between the headspace
inside the packaging and the exterior, and sorption of low MW compounds from the
food to the wall of the packaging or migration of some components from the packaging
to the food. Undesirable interactions of this kind may negatively impact on the quality
and safety of the packaged food. However, these interactions can also be used for the
benefit of the product, by taking advantage of these mechanisms when designing active
packaging, e.g., instead of releasing undesirable substances to the food they release
substances with a beneficial effect which have previously been incorporated into the
packaging (antioxidants, antimicrobials, preservatives, aromas and so on), or they
remove undesirable components of the food by sorption or permeation (cholesterol,
lactose, amines, odours and so on). The active components can be present in the
19
packaging material, either inside it, forming part of its composition, or deposited on
its surface, as illustrated in Figure 1.2.
Figure 1.2 Ways in which an active material functions
In the design of active plastics, various factors must be taken into account to
make their industrial application viable. The polymeric material must be easily
processable using conventional technologies, either by mechanical/thermal processing
(extrusion, injection) or applied as a coating onto the packaging material without
appreciably affecting the processing. Of course, the properties of the polymer must
not be compromised by the incorporation of the active agent, especially if they are
fundamental to the packaging design. Moreover, the active agent must retain its
activity once it has been incorporated into the polymer and it must be released into
the medium with suitable kinetics for it to exert optimal activity. Another aspect of
great importance is that the material should not begin its activity until the product is
packaged, thus avoiding loss of the material before it is used, so there is a need for
a mechanism to trigger its activity the moment it comes into contact with the food
in the packaging.
Among the polymeric materials used in food packaging, PO are the most employed
for applications in which there are no particular gas and vapour barrier requirements.
When greater impermeability of the packaging is required, the alternatives are
polyesters or PA and especially multilayer structures that include high-barrier
materials, such as copolymers of EVOH or metallised copolymers. As an alternative to
20
the current conventional polymers obtained from petroleum, attention is increasingly

turning to biopolymers derived from renewable sources. During the development of
active materials, all polymeric materials can be used as vehicles for active agents,
because the mass transfer processes mentioned earlier take place in all of them. The
choice depends on the method of fabrication, the requirements of the food and the
requirements of the agent and the agent/food/packaging system, which are covered
in Chapters 3 and 4 of this book.
The nature of the active agents of interest are varied, e.g., organic acids, enzymes,
nutrients, natural plant extracts, aromatic components, bactericides, fungicides,
antioxidants and so on, all of which are being used to develop active packaging to control
the various problems that are posed by the need to preserve and improve food quality.
1.3.1 Antimicrobial Active Packaging
One of the forms of active packaging that arouses greatest interest and is increasingly
finding practical application is controlling the microbiological contamination of food.
Several reviews have described some relevant developments on this issue [6, 22–25].
The growth of microorganisms is one of the main causes of food spoilage. Yeasts,
moulds and bacteria can cause food deterioration by acting in specific ways on
different foods according to pH, water activity, partial pressure of O2 and CO2, and
temperature. Microbiological contamination takes place mainly on the surface of the
food, as a consequence of the operations of obtainment, preparation and handling
to which it is subjected until the moment of consumption. Various antimicrobial
substances are customarily used to control contamination in products that are
consumed fresh, or with minimal preservation treatments, and are applied directly
onto the product, as a complement or alternative to physico-chemical preservation
techniques. Direct application of antimicrobial substances is not always sufficiently
effective, given that the activity of many of these substances may be reduced by
their neutralisation by the food or rapid diffusion within the food. Incorporation of
antimicrobial substances into the packaging can certainly provide an alternative way to
maintain their effective activity, thus constituting antimicrobial active packaging that
allows the slow release and incorporation into the food of bactericidal or fungicidal
agents to control contamination of the packaged food and prolong its shelf life, whilst
also ensuring a greater guarantee of quality and safety. For a product to be used as
an antimicrobial active agent incorporated in packaging, an essential requirement is
that it should be accepted as being of food grade.
Antimicrobial active packaging technology provides a response to consumer demand

for products which are subjected to less aggressive treatments than conventional
21
ones, treatments that do not alter the quality of the product and preserve its freshness
and original nutritional quality. The potential applications of antimicrobial active
packaging have made it an object of attention for many research groups, and various
materials have already been developed for the preservation of foods such as fruits,
vegetables, chicken, cheese, meat and so on.
The antimicrobial activity in active packaging can be based on the release of volatile
substances in the packaging headspace or on migration of the active component
incorporated in the packaging material to the packaged food; antimicrobial polymers
allow the slow release of bactericidal or fungicidal substances or antimicrobial
additives which are compatible with food. Another option is the chemical or physical
immobilisation of the active agent in the packaging material, so that it exerts its activity
by direct contact of the product with the packaging surface. There are also polymers
that exhibit antimicrobial activity, as in the case of CS, or an antimicrobial activity
can be created by modification of the surface, as happens with some PA treated using
irradiation. Chapter 2 describes some of the antimicrobial agents that are best known
and most widely used in the development of active packaging.
The materials developed generally use conventional synthetic polymers as a basis, mostly
PO, although currently there is growing interest in the use of biopolymers obtained
from renewable sources. Biopolymers based on polysaccharides such as cellulose and
its derivatives, starch, alginates, carrageenates and CS, and also protein derivatives
such as corn zein, wheat gluten, casein, soy isolates, or collagen and gelatin, among
others, have been used as a basis for developing antimicrobial active biopolymers.
References
1. R. Catala and R. Gavara, Arbor-Ciencia Pensamiento Y Cultura, 2001, 168,

109. [In Spanish]
2. R. Catala, G. Lopez-Carballo, P. Hernandez-Munoz and R. Gavara in

Poly(lactic acid) Science and Technology: Processing, Properties, Additives
and Applications, The Royal Society of Chemistry, London, UK, 2015, p.243.
3. D. Dainelli, N. Gontard, D. Spyropoulos, E. Zondervan van den Beuken and

P. Tobback, Trends in Food Science & Technology, 2008, 19, S99.
4. M.L. Rooney in Active Food Packaging, Ed., B.A. Professional, Chapman &
Hall, London, UK, 1995.
5. J. Gomez-Estaca, C. Lopez-de-Dicastillo, P. Hernandez-Munoz, R. Catala and

R. Gavara, Trends in Food Science & Technology, 2014, 35, 42.
22
6. G. López-Carballo, J. Gómez-Estaca, R. Catalá, P. Hernández-Muñoz and

R. Gavara in Emerging Food Packaging Technologies: Principles and Practice,
Eds., K.L. Yam and D.S. Lee, Woodhead Publishing, Cambridge, UK, 2012, p.27.
7. R.J. Hernandez in Handbook of Food Engineering Practice, Eds.,

K.J. Valentas, E. Rotstein and R.P. Singh, CRC Press, Boca Raton, FL,
USA, 1997, p.291.
8. W.E. Brown in Plastics in Food Packaging: Properties, Design and

Fabrication, Marcel Dekker, Inc., New York, NY, USA, 1992.
9. G. Robertson in Environmentally Compatible Food Packaging, Ed.,

E. Chiellini, Woodhead Publishing, Cambridge, UK, 2008, p.3.
10. T.E. Rolando in Flexible Packaging − Adhesives, Coatings and Processes,

Rapra Technology Ltd, Shawbury, UK, 2000.
11. F. Hannay in Rigid Plastics Packaging: Materials, Processes and Applications,

Smithers Rapra Ltd, Shawbury, UK, 2002.
12. B. Wunderlich in Thermal Analysis of Polymeric Materials, Springer-Verlag

Berlin Heidelberg, Berlin, Germany, 2005.
13. C. Vasile and M. Pascu in Practical Guide to Polyethylene, Rapra Technology

Ltd, Shawbury, UK, 2005.
14. D. Tripathi in Practical Guide to Polypropylene, Rapra Technology Ltd,

Shawbury, UK, 2002.
15. J.R. Wünsch in Polystyrene: Synthesis, Production and Applications,

16. S. Patrick in Practical Guide to Polyvinyl Chloride, Rapra Technology Ltd,

Shawbury, UK, 2005.
17. W.J. Koros in Barrier Polymers and Structures, Ed., W.J. Koros,
American Chemical Society, Washington, DC, USA, 1990.
18. I.B. Page in Polyamides as Engineering Thermoplastic Materials,

19. S. Imam, G. Glenn, B.S. Chiou, J. Shey, R. Narayan and W. Orts

in Environmentally Compatible Food Packaging, Ed., E. Chiellini,
Woodhead Publishing, Cambridge, UK, 2008, p.29.
23
20. O.G. Piringer and A.L. Baner in Plastic Packaging Materials for Food: Barrier
Function, Mass Transport, Quality Assurance, and Legislation, John Wiley &
Sons, New York, NY, USA, 2008.
21. A.L. Brody, E.P. Strupinsky and L.R. Kline in Active Packaging for Food
Applications, CRC Press, Baco Raton, FL, USA, 2010.
22. J.H. Han, Food Technology, 2000, 54, 56.
23. K. Cooksey, Additives for Polymers, 2001, 2001, 6.
24. P. Appendini and J.H. Hotchkiss, Innovative Food Science & Emerging
Technologies, 2002, 3, 113.
25. P. Suppakul, J. Miltz, K. Sonneveld and S.W. Bigger, Journal of Food Science,
2003, 68, 408.
24
2
Antimicrobial Agents
2.1 Minimum Inhibitory Concentration and Minimum

Lethal Concentration
Many compounds and mixtures have received attention because of their ability to
inhibit microbial growth. However, as Paracelsus said, ‘Everything is poison, there is
poison in everything. Only the dose makes a thing not a poison’, which means that the
antimicrobial activity of a compound depends on its concentration. In microbiology,
the lowest concentration of an active agent which inhibits the visible growth of a
specific microorganism under in vitro conditions, within a defined period of time, is
known as the minimum inhibitory concentration (MIC) of that substance for that
particular microorganism [1].
At the MIC, a substance produces a bacteriostatic or fungistatic effect against a specific

bacterium or fungus, respectively. Although the MIC can be determined on agar or in
a liquid medium, the traditional method of determining the MIC is using the broth
dilution technique, where serial dilutions of the antimicrobial agent are added to a
broth medium in microtitre plates (Microdilution Broth Method) or in culture tubes
(Macrodilution Broth Method). Each well or tube contains increasing concentrations
of the antimicrobial agent and is inoculated with a known and fixed amount of the
microorganism under investigation. Standardised protocols are required for intra-
and interlaboratory reproducibility, because results may be significantly influenced
by the method employed.
To determine the MIC of a specific bacterium or fungus in a liquid medium, a cell

suspension of the microorganism (100 µL) is inoculated into 10 mL of culture medium
(Macrodilution Method) containing different amounts of active agent and incubated
for 24–48 h (usually overnight) at the optimum growth temperature. Turbidity is
determined after 24 and 72 h using a spectrophotometer. The MIC is the lowest
concentration of the active agent which inhibits microbial growth.
25
The minimum lethal concentration (MLC) is defined as the lowest concentration

of an antimicrobial agent able to kill the majority (99.9%) of a microbiological
inoculum [2]. The MLC produces a bactericidal or fungicidal effect against bacteria
or fungi, respectively. The MLC may be considered as the lowest concentration at
which no growth is observed after subculturing into a fresh medium [3]. All tubes
showing no growth may be subcultured onto an agar medium to ensure the non-
viability of the culture.
As mentioned throughout this book, antimicrobial agents incorporated in polymer

films can effectively inhibit the growth of microorganisms present in food products.
The effectiveness of a given preservative against a specific microorganism depends on
how it is applied, its concentration, the food type and a long list of other factors; it is
therefore difficult to compare the effectiveness of the in vitro test described above with
the effectiveness of the agent in a food product as discussed in Chapter 5. Similarly,
it is difficult to compare the activity observed after direct addition of the preservative
to food with the effectiveness of antimicrobial packaging containing the same
preservative. When direct addition is used, the whole amount of preservative is added
to the food surface; however, via dispersion in liquid food or diffusion in solid food the
additive tends to be homogenously distributed throughout the food. As explained in
Chapter 5, the results obtained using in vitro conditions with standard growth media
and optimum microbial growth temperature cannot usually be confirmed using real
food due to interactions with the food matrix or food components such as proteins,
lipids, cations and so on. Thus, the MIC of the whole product (surface and bulk) has
to be determined. A large amount of active agent is required to reach the MIC of the
whole food product, which can result in unacceptable changes in food organoleptic
properties. When the active agent is attached to a polymer film, the release of the
antimicrobial agent occurs slowly and under control, and is more concentrated in the
food which is closer to the packaging, i.e., the food surface where it is needed more,
thus reducing the amount of agent actually present in the food; however sometimes,
when diffusion is too slow, the surface concentration is below the MIC or MLC and
the active packaging system lacks efficiency.
In general, the antimicrobial effect increases upon increasing the concentration of the
active agent. However, a limit might exist beyond which the effect does not increase,
thus losing efficiency owing to the interaction with other compounds. As mentioned
throughout this book and with particular emphasis in Chapter 4, the antimicrobial
activity of active agents incorporated into films often depends upon the rate of agent
release from the packaging material to the food surface, which is governed by the
diffusion of the antimicrobial compounds incorporated into the packaging material.
Very rapid release causes loss of the agent within a short period of time, and therefore
the antimicrobial effect against microbial growth is not maintained during the shelf
life of the product. On the other hand, release that is too slow might maintain a
26
constant dose during a long period of time but the dose may not be effective if it
does not reach the MIC. The rate of antimicrobial agent release depends on several
factors including the carrier material and its interactions with the active substance,
swelling of the polymer matrix by food constituents, chemical affinity between the
food and polymer, solubility of antimicrobial agents in the food product and storage
temperature. A more detailed description of these factors is given in Chapter 4.
As mentioned earlier, the mechanism of action of most antimicrobial films occurs

via release of the agent into the food. Therefore the aim of these releasing systems
is to serve as vehicles for the agent, to transfer it from the polymer matrix to the
food, and preferably, to keep a desired concentration of the active compound in
the packaged food during the shelf life of the product. However, there are also
antimicrobial packaging systems where the agent is immobilised in the packaging
and exerts its action via the packaging surface in contact with the food, i.e., without
releasing the active agent. Non-migratory active packaging offers the potential for
improving food safety and quality while minimising the release of the active agent
into the food.
Numerous studies have investigated food applications for antimicrobial packaging

via the incorporation of diverse agents. In this chapter some of the most important
antimicrobials applied in food are described.
2.2 Antimicrobial Agents used in Active Packaging
There are numerous antimicrobial substances and mixtures; therefore they are
commonly classified into antimicrobial groups according to their chemistry or origin,
as Table 2.1 shows.
Table 2.1 Examples of antimicrobial agents in active packaging

Classification Antimicrobial agent
Enzymes Lysozyme, glucose oxidase, lactoperoxidase
Bacteriocins Nisin, pediocin
Surfactants Ethyl lauroyl arginate
Bacteriophages EcoShieldTM, ListShieldTM
Plant extracts Essential oils, tea extract, grape extract
Polysaccharides Chitosan
Organic acids Acetic, lactic, propionic, sorbic and benzoic acids
Metals Silver, gold, titanium dioxide and zinc NP
NP: Nanoparticle(s)
27
2.2.1 Enzymes
Antimicrobial enzymes are ubiquitous in nature. Some enzymes which play a significant
role in an organism’s defence mechanisms against microbial infection are being used
in food preservation, e.g., antimicrobial oxidoreductases exert their effects by in situ
generation of reactive molecules and hydrolytic antimicrobial enzymes degrade the
key components of bacterial and fungal cells walls [4].
2.2.1.1 Bacteriolytic Enzymes
The mechanism of action of the antimicrobial agent determines the type and extent
of microbial damage. Some active agents are able to inhibit synthesis of the cell
wall, a structure that is critical for the survival of bacterial species. Lysozyme is a
bacteriolytic enzyme obtained from hen egg-white, hence it is considered a natural
antimicrobial agent, and it has recently been incorporated into packaging materials.
The mechanism of action of this enzyme involves hydrolysation of the beta 1–4
glycosidic bonds between N-acetylmuramic acid and N-acetylglucosamine. These
bonds are present in peptidoglycans, which form 80 to 90% of the cell wall of
Gram-positive bacteria. Consequently, Gram-positive bacteria are very susceptible
to the action of lysozyme. As Figure 2.1 shows, lysozyme acts on the peptidoglycans
present in the cell wall, increasing cell porosity and permeability and finally resulting
in cell lysis. In order to further improve the antimicrobial effectiveness of the enzyme,
coadjuvant agents such as detergents and chelators [ethylenediaminetetraacetic acid
(EDTA)] are usually added.
Peptidoglycan Lysozyme Permeability increase Cell lysis
Cell membrane
Cytoplasm
Figure 2.1 Scheme of the mechanism of action of lysozyme against

Gram-positive bacteria
Like any other enzyme involved in an enzymatic process, lysozyme has a mechanism
of action which is highly dependent on its three-dimensional (3D) structure. Thus,
when lysozyme is employed as an antimicrobial agent in a packaging system, the
incorporation process should consider that the macromolecule is only active if the
required target structure is reached, i.e., the method of incorporation should allow the
28
polypeptide to reach the target structure after partial or full release of the molecule
from the material. Consequently, severe thermal processes or occlusion in a very
dense polymer matrix are not the best incorporation procedures for the production
of efficient active packaging.
Lysozyme has been immobilised on ethylene-vinyl alcohol (EVOH) films with

antimicrobial packaging applications [5]. In this study a polymeric surface was treated
to generate the suitable chemical environment to react with lysozyme, creating covalent
bonds between the packaging polymer chains and the enzyme, while allowing freedom
of movement and action in order to inhibit the growth of Listeria monocytogenes.
Immobilisation of the antimicrobial agent enabled the agent to be effective on the
food surface without migration into the product.
Other authors developed gliadin films crosslinked with cinnamaldehyde using a casting
procedure and incorporated lysozyme as an antimicrobial agent. This hydrophilic
polymer swells in the presence of water, releasing lysozyme molecules into the food
medium, thus showing effectiveness against Listeria innocua [6].
In another approach, metal-chelating active packaging films were prepared by grafting

metal-chelating polyacrylic acid onto the surface of polypropylene (PP) films. These
grafts were reported to create an environmental chemistry that could reduce or enhance
the antimicrobial activity of lysozyme, which was added to the liquid food simulant,
against Listeria monocytogenes, depending on the ionic strength of the food [7]. This
research demonstrates that metal-chelating substances, such as these grafts or EDTA
scavenging iron or magnesium/metal cations, are needed to stabilise cell membranes.
Lysozyme may be used in combination with other preservation techniques. The use
of sorbate, ethanol, temperature and low pH or other antimicrobial enzymes, such
as glucose oxidase and lactoperoxidase (LPS), may increase the microbial safety of
food products.
2.2.1.2 Antimicrobial Oxidoreductase Systems
Oxidoreductase enzymes do not possess inherent antimicrobial activity; these enzymes

generate cytotoxic products which exhibit antimicrobial activity. LPS and glucose
oxidase are the most widely used oxidoreductase enzymes.
LPS is an enzyme found in milk, saliva and tears secreted by the mammary,
salivary and lachrymal glands of mammals, respectively. The LPS system consists
of 3 components: LPS, thiocyanate and hydrogen peroxide (H2O2). The enzyme
catalyses the oxidation of the thiocyanate anion (SCN−) via H2O2 and generates
29
intermediate antimicrobial products such as the hypothiocyanite anion (OSCN−) and

hypothiocyanous acid [8]. These products have a broad spectrum of antimicrobial
activity against bacteria, fungi and viruses. The oxidation of cell components such
as sulfhydryl groups or NADH results in damage to the cytoplasmic membrane and
transport system and the denaturation of proteins, resulting in growth inhibition
or cell death.
LPS was incorporated at 0.7% in whey protein films and these films, when deposited
on agar media, inhibited the growth of Listeria monocytogenes [4.2 log colony forming
unit(s) (CFU)/cm2 compared with control films]. In addition, when used as coatings
on contaminated smoked salmon, they significantly reduced the count of Listeria
monocytogenes (>3 log CFU/g) and total aerobic microorganisms (1 log CFU/g) [9].
In alginate films they inhibited the growth of Escherichia coli, Listeria innocua and
Pseudomonas fluorescens. The extent of the inhibitory action depended on enzyme
activity and the initial concentrations of H2O2 and potassium thiocyanate (KSCN)
[8]. LPS has been incorporated into a chitosan (CS) edible film for mango packaging
and coating, and exhibited antimicrobial effects against pathogenic microorganisms.
This technology can help to prevent the spoilage of mango fruit by fungi and bacteria
during storage and transport, thus extending the shelf life [10].
All the above-mentioned examples are active films manufactured from biopolymers
using casting procedures. LPS is thermosensitive and its thermal denaturation has
been used as an indicator of overtreated food products [11].
2.2.2 Bacteriocins
Bacteriocins are proteins or complex proteins produced by microorganisms that are

biologically active and have antimicrobial action against other bacteria, principally
closely related species [12], hence they can be considered as natural preservatives.
Bacteriocins can be synthesised by yeast and Gram-positive and Gram-negative
bacteria, and their synthesis generally takes place when these microorganisms are
exposed to stress conditions. The most frequently employed bacteriocins used in food
preservation are produced by lactic acid bacteria. Bacteriocins produced by lactic acid
bacteria are active over a nanomolar range and have no reported toxicity [12]. The
main advantage of bacteriocins is that after consumption (as an additive in a food
product) they are rapidly digested by proteases in the human digestive tract [12].
These compounds are ribosomally synthesised peptides, so their characteristics can
be changed to enhance their activity and spectra of action [13].
Since there are a large number of bacteriocins, with more than 227 collected and
described on a web database (http://bactibase.pfba-lab-tun.org/about.php), they
30
need to be grouped and classified. Among the various classifications available, the
following is often used:
I) Lantibiotics: small heat-stable peptides containing lanthionine and beta-methyl-

lanthionine which have a molecular mass <5 kDa. The lantibiotic bacteriocins
were initially divided into two subclasses:
• Subclass Ia includes relatively elongated, flexible, positively charged peptides

which act by forming pores in the cytoplasmic membrane. Nisin is the best-
known bacteriocin in this group.
• Subclass Ib are characteristically globular peptides, more rigid in structure

and either negatively charged or with no net charge; they act by interfering
with essential enzymatic reactions of sensitive bacteria.
II) A non-lantibiotic group of small heat-resistant peptides with a molecular mass

<10 kDa. These bacteriocins are formed exclusively by unmodified amino
acids, ribosomally synthesised as inactive prepeptides that are activated by
posttranslational cleavage of the N-terminal leader peptide. They contain the
sequence -Tyr-Gly-Asn-Gly-Val-Xaa-Cys.
• Subclass IIa includes peptides which are active against Listeria, e.g., pediocin.
• Subclass IIb are bacteriocins formed by two peptides, such as lactococcin G.
• Subclass IIc are small peptides which are transported by leader peptides.
III) These are large bacteriocins with a molecular mass >30 kDa and are heat-labile
proteins.
IV) These are complex bacteriocins with lipid or carbohydrate moieties.
V) These are circular bacteriocins and not modified post-translationally.
Lantibiotics are the group of bacteriocins which are most studied and applied
industrially. Nisin produced by Lactococcus lactis subspecies lactis is used as an
additive in foods. Although bacteriocins generally inhibit only closely related
species, nisin presents a broad antimicrobial spectrum and is effective against many
Gram-positive organisms, such as Listeria monocytogenes, Bacillus cereus and
Clostridium botulinum. Nisin is a peptide formed by 34 amino acids with a MW
below 5 kDa. This relatively small length facilitates its release from antimicrobial
active packaging after contact with solid or liquid food, as observed in several
packaging developments.
31
The antimicrobial activity of bacteriocins is based on an interaction with the bacterial

membrane, forming pores and dissipating the proton motive force. These peptides
are usually effective against Gram-positive microorganisms. Bacteriocins of lactic
acid bacteria may be unable to inhibit Gram-negative organisms because the outer
membrane blocks the site for bacteriocin action. Deegan and co-workers [14] showed
the effectiveness of nisin against Gram-negative organisms such as Salmonella sp.,
Escherichia coli and other members of the Enterobacteriaceae family by combining
nisin with EDTA, a chelating agent. EDTA acts by binding to Mg 2+ ions in
lipopolysaccharides resulting in disruption of the outer membrane of Gram-negative
bacteria.
Consumer demand for natural food preservatives has been increasing over recent
decades. Since bacteriocins are considered natural products, they might have good
acceptance from customers who demand healthier, natural food additives. Nisin
has often been used in ‘hurdles’ technology in combination with other preservation
methods, such as low pH, high salt concentrations or modified atmosphere packaging.
Nisin, E234, is authorised for food preservation in the European Union (EU) by
Directive 95/2/EC which details food additives other than colours and sweeteners.
Nisin is permitted in ripened cheese and processed cheese, certain puddings, clotted
cream and mascarpone. In the USA, nisin is labelled as a natural preservative, defined
as ‘generally recognised as safe (GRAS)’, and the maximum level varies according to
the product and member state. Nisin has been applied in meat and meat products,
and vegetarian and dairy foods.
Nisin has been incorporated in edible films made with tapioca starch and has been
tested against Listeria innocua [15]. This bacteriocin has also been included in whey
protein, zein and gelatin films, and all these films were successfully used to inhibit the
growth of Listeria monocytogenes [16–18]. Nisin combined with organic acids in soy
protein showed an antibacterial effect against Listeria monocytogenes, Escherichia coli
and Salmonella Gaminara [19]. In all these examples, hydrocolloids were dissolved
in water or an aqueous mixture, nisin was added to the solution and the films were
obtained via casting. Nisin, alone or combined with natamycin, directly applied onto
Galotyri cheese efficiently suppressed fungal growth and reduced the population of
lactobacilli and lactococci [20].
Nisin has been incorporated into a cellulose-based packaging material, which inhibited
Staphylococcus aureus growth on cheese slices for 3 months under refrigerated
storage [21]. Pediocin and nisin incorporated into cellulose films showed an
antimicrobial effect against Listeria monocytogenes in meat and poultry products for
12 weeks at 4 °C [22]. Cast films of wheat gluten and zein containing nisin showed
significant activity against Lactobacillus plantarum in aqueous media at different
temperatures, although its release and activity were incomplete [23].
32
2.2.3 Bacteriophages
Bacteriophages, or phages, are a bacterial virus discovered by Ernest Hankin in 1896

and in 1915 Frederick Twort described their antibacterial activity. However, Felix
d’Herelle (1919) was probably the first scientist who used phages as a therapy to treat
severe dysentery. Bacteriophages are obligate parasites able to lyse the bacterial host.
Bacteriophages have an average size of 20−200 nm and a simple structure comprising
of genetic material, in the form of deoxyribonucleic acid (DNA) or ribonucleic acid
(RNA), with a surrounding proteinaceous capsid. The function of the protein capsid
is to protect the phage genome and to facilitate the specific adsorption of the phage
into the host cell to infect it. Bacteriophages interact with bacterial cells via receptors
present in the tail fibres (in myoviridae, siphoviridae and podoviridae bacteriophages)
or on the capsid (leviviridae bacteriophages) and inject the phage contents into
the bacteria creating pores or channels in the cell wall. In the case of lytic phages,
these viruses invade bacterial cells, disrupt bacterial metabolism, produce multiple
phage replicates and finally lyse the bacteria. Alternatively, the presence of an excess
of phage particles, with respect to the number of bacterial cells, increases the number
of pores and channels formed in the bacterial cell wall leading to loss of cell integrity
and cell death. In this case cell lysis occurs without phage replication.
Phages may be used as natural antimicrobials to control the presence of bacteria

at various stages of food production. In addition, bacteriophages may be used as a
biopreservative in animal feed to reduce the risk of subsequent contamination, to
decontaminate surfaces and equipment, and to extend the shelf life of perishable
foods. Furthermore, phages may be used in ‘hurdles’ technology in combination
with other preservation methods. The antimicrobial activity of phages has been
tested against Gram-positive and Gram-negative bacteria in meat, poultry and dairy
products [24, 25].
Bacteriophages are considered to be safe and several commercial phage-based

products, for direct food applications, have been declared as GRAS by the US Food
and Drug Administration (FDA).
EcoShieldTM is a commercially available preparation composed of three lytic

bacteriophages specific for Escherichia coli O157:H7. This product reduced the
microbial growth of Escherichia coli O157:H7 in tomatoes, spinach, broccoli and
ground beef [25]. LMP-102TM, later renamed ListShieldTM, is a commercially available
preparation composed of six lytic bacteriophages specific for Listeria monocytogenes.
ListShieldTM exhibited antilisterial activity and reduced growth by 2.0–4.6 log units
on melons and 0.4 log units on apples. Combined treatment with nisin significantly
reduced the bacterial counts, by 5.7 and 2.3 log units, on cut melon and apples,
respectively [26]. Phages have been reported to be effective against Salmonella sp.;
33
the phage mixture was shown to reduce Salmonella sp. populations by approximately
3.5 logs on honeydew melon slices stored at 5 and 10 °C, and by approximately 2.5
logs on slices stored at 20 °C [27].
Commercial phage products are usually applied by spray or direct addition to food
surfaces; however, this method could reduce phage effectiveness due to dilution in
the food matrix. Spraying phages in sufficiently high numbers could eliminate all the
target bacterial cells from the food surface, however, it can be very expensive and
allow the development of niches where phage counts are low and phage resistance
may develop. In this sense, the immobilisation of phages onto food packaging could
be a better alternative. Anany and co-workers [28] immobilised phages on modified
cellulose membranes (based on charge difference) and evaluated the antibacterial
activity against Listeria monocytogenes and Escherichia coli O157:H7 in raw and
ready-to-eat meats. Other authors have encapsulated phages, e.g., Salmonella sp.
phages have been encapsulated in CS-alginate microspheres [28].
Bacteriophages are abundant in the environment and resistant to adverse conditions

such as desert sand. However, they are usually present in environments with pH 5−9
and at temperatures below 60 ºC. Phages are present in fresh and non-processed foods
such as fresh fruits and vegetables, fresh ground beef, milk and cheese. Thus, the
direct application of phages to the food surface could eliminate or significantly reduce
bacterial contamination, making the food safe to consume and without changes to the
organoleptic quality. However, consumers may not accept the use of bacteriophages
applied to food as a biocontrol agent.
2.2.4 Surfactants
Ethyl lauroyl arginate (LAE) is a novel food preservative produced by Laboratorios

Miret, S.A. (LAMIRSA, Spain). The preparation and application of this product is
described in ES Patent Applications 512643 A1 [29] and WO 03/034842 A1 [30].
It is one of the most potent antimicrobial agents among novel food additives, with
a broad spectrum of antimicrobial activity against Gram-negative and Gram-
positive bacteria, fungi and yeasts due to its chemical properties as a cationic
surfactant. LAE is electrostatically absorbed onto the microbial cell surface causing
disruption and instability of the cell membrane as shown in Figure 2.2. LAE acts
in various ways and the antimicrobial effect depends upon the structure of the
microorganisms. LAE exhibits slightly more activity against Gram-positive bacteria
than Gram-negative bacteria. Gram-negative organisms are less susceptible to the
action of this antimicrobial agent because they possess an outer membrane which
surrounds the cell wall and restricts the diffusion of hydrophobic compounds
through its lipopolysaccharide layer [31]. LAE disrupts the cytoplasmic membrane
34
and outer membrane of Gram-negative bacteria, whereas, in Gram-positive bacteria

changes were observed in the cell membrane and cytoplasm. These changes produce
disturbances in the membrane potential which consequently inhibits cell growth and
leads to a loss of viability without cell lysis [32, 33].
LAE
Bilayer lipid memebrane
Figure 2.2 Mechanism of action of LAE. Scheme showing the disruption of the
bilayer lipid membrane by electrostatic interactions with the cationic surfactant
LAE is rapidly metabolised in the human body into natural endogenous compounds
which are present in the human diet, mainly arginine and ornithine. As a result, LAE
has been classified as GRAS and approved as a food additive to improve food safety
and quality; the US Department of Agriculture has approved its use in meat and
poultry products, but it is currently not approved in dairy products [34].
Martin and co-workers [35] applied LAE to control the growth of Listeria
monocytogenes on the surface of frankfurters. The results demonstrated a reduction
of at least 1 log CFU/cm2 upon the application of 22 µg/mL of LAE. Porto-Fett and
co-workers [36] also studied the antimicrobial effect of LAE on commercially produced
frankfurters, in combination with potassium lactate and sodium diacetate, against
Listeria monocytogenes. The study demonstrated that the number of pathogens
decreased by 2 log CFU/package within 2 h and remained relatively unchanged over
the 120-day storage period.
LAE was shown to exert a bactericidal effect against Campylobacter jejuni tested in
vitro. The antimicrobial efficacy of LAE was also tested against Campylobacter jejuni
on chicken breast fillets and the results demonstrated the antimicrobial effect of LAE
with 200 and 400 mg/kg treatments. Lauric arginate at 400 mg/L gave a maximum
reduction of 1.5 log CFU/g of Campylobacter jejuni during 7 days of storage at 4 °C
without any change in the meat pH. LAE caused a reduction of 2.3 log CFU/g in
psychrotrophs compared with the control on day 0 of storage but this antimicrobial
effect was not sustained after 3 days [37].
35
Several studies have incorporated LAE into films with the objective of obtaining
antimicrobial polymers, e.g., EVOH films containing LAE were applied on chicken
stock and ready-to-eat surimi sticks and were found to inhibit the total microbial
load for 10 days under storage at 4 °C whilst exerting an antibacterial effect against
Listeria monocytogenes and Escherichia coli [38, 39]. CS films incorporating LAE
reduced the total microbial load in fresh poultry products [40]. Edible antimicrobial
coating solutions with CS, LAE and nisin were applied to reduce foodborne pathogen
contamination on ready-to-eat turkey [41]. In that report, foods were directly coated
with the CS solutions or wrapped with polylactic acid films coated with the active
solution. Packaging with polyethylene terephthalate coated with oregano essential
oil(s) (OEO) and LAE has been applied to sheep cheese, producing an antimicrobial
effect against Escherichia coli O157:H7 [42].
2.2.5 Plant Extracts
Many plant extracts and essential oils have been observed to be potential natural food
preservatives. Essential oils are aromatic oily liquids usually obtained by expression,
fermentation, extraction and, more frequently, via steam distillation from plants
(flowers, buds, seeds, fruits, roots and so on). The composition of essential oils may
differ with several factors: seasons, geographical sources, parts of the plants, methods
of extraction and so on. The composition of essential oils obtained from different parts
of the same plant can vary widely, e.g., essential oils obtained from coriander seeds or
leaves have a different composition. Similarly, essential oil extracted from cinnamon
bark is rich in cinnamaldehyde while that from cinnamon leaves is rich in eugenol.
The antimicrobial activities of essential oils and plant extracts have been studied for a
long time, and considerable research has been published on the antimicrobial activities
of plant essential oils against foodborne pathogens: Escherichia coli, Salmonella
typhimurium, Bacillus cereus, Staphylococcus aureus, Listeria monocytogenes,
Shigella dysenteria and so on. Essential oils of oregano, thyme, bay and clove are the
most active extracts against Escherichia coli. Chemical analysis has shown that the
principal components of these essentials oils are carvacrol, thymol, citral and eugenol.
About 3,000 essential oils are known and more than 300 are commercialised as
flavours and fragrances. Besides their antibacterial properties, essential oils and their
components exhibit antiviral, antitoxigenic, antimycotic, antiparasitic and insecticidal
activities. However, the mechanism of action is not yet clear because of the variable
composition of essential oils, the large number of chemical groups and the various
targets present in the cell. Since essential oils are rich in volatile terpenoids and phenolic
compounds, these compounds are commonly associated with the inhibitory activity of
essential oils. Generally, the active components of essential oils inhibit microorganisms
36
by disturbing and damaging the cytoplasmic membrane and protein membrane. This
structure is present in eukaryotic and prokaryotic cells, hence the action of this type
of antimicrobial is usually less selective and can produce a toxic effect in humans.
Essential oils exhibit activity against a wide spectrum of microorganisms, which is

improved by synergic factors such as low water activity (aw), low pH, chelators or
mild heat. The combined effect of essential oils or their components with some food
additives (sodium chloride, sodium nitrite or nisin) may increase the antimicrobial
activity, although antagonistic interactions have also been reported. A synergy between
sodium chloride and mint oil has been observed in their antimicrobial activity against
Salmonella Enteritidis and Listeria monocytogenes [43]. However, sodium chloride
exhibited an antagonistic interaction with carvacrol and p-cymene when tested against
Bacillus cereus on rice [44]. Carvacrol and p-cymene showed synergy but this effect
is reduced when sodium chloride is added.
The presence of several active components in an essential oil can produce a synergistic
effect, e.g., the major compounds of OEO, carvacrol and thymol, had an additive
effect when tested against Staphylococcus aureus and Pseudomonas aeruginosa [45].
Mixtures containing different combinations of cilantro, coriander, dill and eucalyptus
essential oils produced synergistic or antagonistic effects [46]. Carvacrol, the main
component of OEO, disrupts the outer membrane of Gram-negative bacteria,
releasing lipopolysaccharides and increasing cytoplasmic permeability [47]. Thymol,
present in OEO, has a structure very similar to carvacrol, with the hydroxyl group
at a different location on the phenolic ring. Essential oils can produce other effects,
such as depletion of the proton motive force, electron flow and active transport,
and inhibition of protein synthesis; the latter is essential for the multiplication and
survival of bacterial cells [47].
Essentials oils have been widely applied as antimicrobials in food. Since their
principal components are hydrophobic, the antimicrobial activity of essentials oils
could decrease when applied to aqueous food products. An alternative is the use
of stabilisers, such as lecithin, which would avoid the coalescence of hydrophobic
droplets by increasing the viscosity of the aqueous medium.
Another relevant issue is the strong organoleptic impact that they cause in the product
and what level is acceptable to the consumer. The flavour, odour and colour produced
by the incorporation of essential oils in food is usually accepted by consumers when
the chosen foods are associated with herbs or spices such as meats, fishes, cheeses,
salads, sauces or soups.
Several publications have reported the development of food packaging employing

essential oils [48]. These compounds can also be incorporated into food packaging
to achieve a more effective, rational use, although this step is especially problematic
37
when working with volatile or thermolabile molecules, as a large amount of the

compound is lost or inactivated during processing and the remaining amount in the
polymer may not be enough to exert its antimicrobial effects on the food, an issue
which is discussed in Chapter 3. In addition, the antimicrobial compound must be
chemically compatible with the polymer matrix to allow good dispersion in the film
but not inhibit its release. Despite these issues, there are several studies regarding
the incorporation of essential oils, or their components, into biopolymer films
obtained by casting and applied to food packaging: oregano, rosemary and garlic
essential oils were incorporated in whey protein films [49], thyme and OEO in soy
protein [50], oregano and rosemary extracts in gelatin [51], essential oils of clove
(Syzygium aromaticum L), fennel (Foeniculum vulgare Miller), cypress (Cupressus
sempervirens L), lavender (Lavandula angustifolia), thyme (Thymus vulgaris L), herb-
of-the-cross (Verbena officinalis L), pine (Pinus sylvestris) and rosemary (Rosmarinus
officinalis) were incorporated in gelatin and CS blends [52], clove in sunflower protein
concentrate [53], oregano in cassava starch-CS blends [54], lemongrass in alginate
[55], eugenol in zein [56], and Zataria multiflora in carboxymethyl cellulose films
[57]. Salad packaging was improved by combining a modified atmosphere with an
active bag consisting of PP/EVOH film containing OEO or citral [58]. These essential
oils exhibited antimicrobial activity against natural microflora present in salad, and
against the pathogenic microorganisms Escherichia coli, Salmonella enterica and
Listeria monocytogenes in vitro and also on food.
Essential oils have also been incorporated into conventional polymers via thermal
processing. Basil, oregano and thyme essential oils or their components have been
added to low-density polyethylene (LDPE), although significant losses due to
evaporation were reported [59–61]. To reduce these losses the incorporation of clays
has been attempted [62, 63].
Natural polyphenols have also been extracted from fruits, vegetables, cereals, medical
plants, microalgae and edible wild flowers. High concentrations of these compounds
have been found in the plant extracts of grapes, olives, blueberries, sweetsops, mangoes
and citrus fruits [64]. Green teas are rich in flavan-3-ols (epicatechin, catechin and
galloylated derivatives), which are responsible for the majority of their biological
activities, i.e., anti-inflammatory, anti-tumour, antioxidative and antimicrobial [65].
The mix of catechins is responsible for the high antioxidant effect of green tea and
other reports have shown that different types of catechins are responsible for the
overall antimicrobial activity of green tea [66].
Plant extracts, such as green tea or grape seed extracts, have been classified as food
additives by the EU and have been incorporated into packaging films applied to
food in order to extend product shelf life by means of their inherent antioxidant
and antimicrobial properties [67]. In fact, packaging can have two functions: as
38
active packaging (improving shelf life and the quality of food by reducing counts of
Escherichia coli and Listeria monocytogenes) and as a vehicle to deliver functional
substances to the food [68]. Green tea extract and OEO were incorporated into EVOH
and showed antimicrobial properties against Listeria monocytogenes, Escherichia coli
and Penicillium expansum [69]. Films with green tea and probiotic bacteria were able
to extend the shelf life of hake for at least a week, decreasing the count of hydrogen
sulfide (H2S)-producing bacteria and total viable count, and increasing the beneficial
lactic acid bacteria in fish [70]. Green tea has been incorporated into CS film, enhancing
the antioxidant and antimicrobial properties of the film and prolonging the shelf life
of sausages by reducing counts of lactic acid bacteria, moulds and yeasts [71].
Grapefruit and grape extracts have shown a broad range of bioactive properties,
including an antimicrobial effect. LDPE coextruded film containing grapefruit
seed extract extended the shelf life of beef stored at 3 °C, inhibiting the growth of
Listeria monocytogenes, Escherichia coli, Staphylococcus aureus and Bacillus subtilis
[72]. Grape seed extract was incorporated into soy protein edible film, showing a
positive result against Listeria monocytogenes, Escherichia coli and Salmonella
typhimurium [73].
2.2.6 Polysaccharides
CS is a cationic linear aminopolysaccharide composed of randomly distributed

(1→4)-linked D-glucosamine and N-acetyl-D-glucosamine units obtained by the
partial N-deacetylation of chitin, which is primarily obtained from seafood processing
waste. CS is a natural, biodegradable, biocompatible polymer with applications in a
variety of technological areas, such as agrochemistry, pharmacy, biomedicine, textiles
and food packaging [74].
Applications of CS as a biocide are only effective in an acidic medium because its

antimicrobial action is based on its cationic nature, i.e., activity only occurs when the
amine groups are positively charged. The interaction between positively charged CS
and microorganisms leads to cellular membrane depolarisation and microbial death.
The activity of CS has been reported to depend upon molecular weight (MW), degree
of deacetylation and derivatisation, if any, as well as on the target organisms [75]. In
this regard, CS has shown antimicrobial activity against a wide range of foodborne
filamentous fungi, yeast and especially bacteria.
No and co-workers [76] studied the antibacterial activities of six CS samples

and six CS oligomers with different MW against different strains of Gram-
negative bacteria (Escherichia coli, Pseudomonas fluorescens, Salmonella
typhimurium and Vibrio parahaemolyticus) and Gram-positive bacteria (Listeria
39
monocytogenes, Bacillus megaterium, Bacillus cereus, Staphylococcus aureus,

Lactobacillus plantarum, Lactobacillus brevis and Lactobacillus bulgaricus).
The results showed that CS inhibited the growth of most of the bacteria tested;
however, the inhibitory effect depended on the MW of the CS and the type of
bacterium. The CS oligomer reduced bacterial growth by 1–5 log cycles at a
1.0% concentration, but the effects were more limited than those of CS alone.
CS generally showed stronger bactericidal effects against Gram-positive bacteria
than Gram-negative bacteria.
The antimicrobial effectiveness of CS has been shown in fruits and vegetables against
Bacillus cereus, Listeria monocytogenes, Enterobacter aeromonas, Brochothrix
thermosphacta, Lactobacillus curvatus, Lactobacillus plantarum, Lactobacillus
sakei, Pediococcus acidilactici and Pseudomonas fluorescens [77]. Strawberries are
among the most perishable fruits and are susceptible to physical damage and fungal
infection caused by Botrytis cinerea and Rhizopus spp. El Ghaouth and co-workers
[78, 79] investigated the effect of a CS coating applied to strawberries. Strawberry
fruits were inoculated with a spore suspension of Botrytis cinerea or Rhizopus
stolonifer and subsequently dipped in CS solutions (1.0 and 1.5% in 0.25 N HCl).
The results showed that the CS coating reduced the decay of strawberries compared
with the control. Edible coatings can be used as a protective barrier to reduce fruit
respiration and transpiration, retard microbial growth and improve the texture
quality of fruits. CS has been widely studied as a hydrophilic polymer in the design of
reservoir delivery systems for the slow release of active compounds over an extended
period of time. CS has also been reported to possess a film-forming property for
use in edible films or coatings [80–82]. A CS coating can improve the storability
of perishable foods by modifying the internal atmosphere as well as decreasing
transpiration loss.
2.2.7 Organic Acids
Organic acids and their salts are widely used as food additives to prevent food
deterioration and extend its shelf life. The most effective organic compounds are acetic,
lactic, propionic, sorbic, ascorbic, citric, propionic, succinic, tartaric and benzoic
acids. These additives increase food acidity, producing an unfavourable environment
for microbial growth. The antibacterial effect is produced by disruption of the cell
membrane and interfering with nutrient transport or metabolism [83]. Given the
weak acid nature of most of these compounds, food pH is considered to be a decisive
factor for effectiveness because it affects the concentration of undissociated acids.
The undissociated forms of organic acids can easily penetrate the lipid membrane
of the bacterial cell and, once internalised into the neutral pH of the cell cytoplasm,
dissociate into anions and protons [84].
40
Organic acids and their salts have been incorporated in several biopolymer matrices
via casting procedures, and their antimicrobial characteristics have been tested against
various microorganisms, e.g., the release of sorbic acid from wheat gluten films
was studied under different conditions [85], gallic acid in CS inhibited the growth
of Escherichia coli, Salmonella typhimurium, Listeria innocua and Bacillus subtilis
[86], the inhibition of grey mould, caused by potassium sorbate incorporated into
starch, was enhanced by the presence of citric acid [87], stearic acid, ascorbic acid
and citric acid in methylcellulose also improved the oxidative stability of foods [88].
Organic acids and salts are fairly stable when exposed to thermal treatments and this
characteristic has been used to develop active films via extrusion, e.g., sorbic anhydride,
the acid anhydride form of sorbic acid, was incorporated into polyethylene (PE) films
and these films showed a higher activity against slow-growing fungi (Penicillium)
than against fast-growing fungi (Aspergillus) [89], the antifungal activity against
Aspergillus niger and Penicillium spp. of benzoic and sorbic acid incorporated in
PE-co-methacrylic acid was tested as a function of pH [90], potassium sorbate in
ethylene-vinyl acetate/linear LDPE did not show any relevant activity against Candida
spp., Pichia spp., Trichosporon spp. or Penicillium spp. owing to insufficient release
into aqueous food [91], and benzoic anhydride in PE was used to wrap a tilapia fillet
and effectively inhibited the growth of the habitual microflora [92].
2.2.8 Metals
Metals are mostly used as additives in various applications such as wood preservation,
cosmetics, medical devices, antifouling paints, antibacterial textiles and so on.
For decades metals have been used as antimicrobials; this property, like many others,
is enhanced as the size of the metal particle decreases and the surface-to-volume ratio
increases. Thus the antimicrobial activity of metal NP, with a very high surface area
in contact with the microorganism, can be very potent. Recently, NP from various
metals (including silver, gold and zinc) were studied and their antimicrobial effect
was reported [93]; metal NP were shown to exhibit bactericidal, antifungal and
antiviral activity.
At present, NP or nanotechnological devices are frequently incorporated into

packaging materials. NP can be employed directly in functionalised food (e.g.,
regulatory peptides from plants, nanodroplets/-clusters and nanowater) as food
additives or supplements, which can increase their bioavailability in comparison
with conventional packaging formulations [94]. However, owing to this increased
bioavailability, nanomaterials themselves constitute a new generation of unknown
toxic chemicals with insufficient information available regarding the possible health
41
or safety risk they may pose. The mechanism of action of these materials depends
on the size of the NP [95].
There are a great variety of chemical and physical processes for the synthesis of metal
NP, most of which involving the formation of colloidal NP or their incorporation into
other materials to constitute nanocomposites, e.g., silver NP have been incorporated
into polymer matrices for the development of antimicrobial films and coatings [40].
Silver NP, less than 10 nm in diameter, create pores in the bacterial cell walls;
consequently, the cytoplasmic content is released to the exterior of the cell, resulting
in cell death without affecting the intracellular and extracellular proteins and nucleic
acids of the bacterium. Silver NP have been tested for their antimicrobial properties
against a wide range of foodborne pathogens. NP with a size of less than 25 nm showed
an antimicrobial effect against multi-resistant bacteria such as methicillin-resistant
Staphylococcus aureus, methicillin-resistant coagulase-negative Staphylococcus
epidermidis, vancomycin-resistant Enterococcus faecium and extended-spectrum
beta-lactamase (ESBL)-positive Klebsiella pneumoniae [96].
Silver NP present in a polymer matrix acted as nanoreservoirs for the delivery of silver
ions, ensuring their availability in the substrate over time [97]. Silver NP have been
generated in situ in a CS matrix and the final nanocomposite exhibited antimicrobial
properties against Escherichia coli and Staphylococcus aureus. Similar activity was
observed for absorbent pads in which silver NP had been deposited on the surface
of the fibres [98].
Gold NP are widely used because of their biocompatibility. Gold NP create holes in
the cell wall, resulting in the leakage of cell contents and cell death. They can also bind
to bacterial DNA which alters DNA transcription [99]. Gold NP have been used as a
coating for surfaces such as implants, fabrics for wound treatment and glass surfaces
to maintain sterilised conditions [100]. In the report just cited, gold NP inhibited the
growth of several Gram-negative and Gram-positive pathogenic bacteria as well as
the yeasts Saccharomyces cerevisiae and Candida albicans.
Titanium dioxide NP are used to prevent biofilm formation [101]. The antimicrobial
activity is due to the photocatalytic generation of free hydroxyl radicals when titanium
dioxide NP are illuminated with ultraviolet (UV) light at wavelengths below 385 nm.
The antimicrobial activity of titanium dioxide NP has been demonstrated against
Escherichia coli in water [102]. In another study Escherichia coli cells were damaged
and exhibited membrane disorganisation, which increased the membrane permeability,
leading to the accumulation of zinc oxide (ZnO) NP in the bacterial membrane and
the cellular internalisation of ZnO NP.
ZnO NP exert a minimal effect on human cells but they have selective toxicity against
microorganisms such as pathogenic bacteria. These NP exhibit a potent antimicrobial
42
activity, hence they are suitable for application as antimicrobials in the agricultural
and food industries. ZnO NP are able to completely lyse foodborne bacteria by
damaging the cell membrane and increasing cell permeability. The mechanism of
action of zinc NP is due to two factors: the generation of peroxides from the NP
surface, and as the activity is produced at the metal surface, the smaller the particle,
the greater the generation of H2O2 and secondly, the release of ions that can damage
the cell membrane and interact with intracellular compounds [102].
ZnO NP have been studied in diverse food-related bacteria, e.g., Bacillus subtilis,
Escherichia coli, Pseudomonas fluorescens and so on. They exhibited antibacterial
activity against Listeria monocytogenes, Salmonella Enteritidis and Escherichia coli
O157:H7 in culture medium [103].
Metals are not degraded under the standard processing conditions used for
thermoplastic polymers (~200 °C). This property can be further extended to
antimicrobial metal NP as it seems that, as in other NP applications, their addition
to polymer matrices could be the fastest route to take commercial advantage of their
enhanced properties [104].
2.3 Factors which affect the Properties and Stability of Antimicrobial

Agents during the Processing of Packaging Films
In the literature there is a long list of methods for manufacturing antimicrobial

packaging systems which are based on the incorporation of agents into polymeric
materials and can be classified into three groups: a) application of an antimicrobial
coating onto film, paper or food surfaces from a solution containing the agent and the
polymer; b) melt blending of the antimicrobial compounds with plastic resins during
thermomechanical procedures such as extrusion, coextrusion or injection processes;
and c) immobilisation of antimicrobials onto film surfaces via chemical modification
of the polymer surface and covalent bonding between the film and agent [105].
The addition of antimicrobial agent(s) to a film may result in changes to the properties
of the film, such as the optical and mechanical properties, tensile strength, diffusion
coefficients, O2 and water barriers, and so on. The concentration of active agent
incorporated and the possible effects of the additive on the final packaging film,
especially on colour and transparency, are important factors to be considered because
these alterations can render materials invalid for use in packaging applications.
Furthermore, heat, pressure, friction or other processing conditions in the manufacture
of active films could cause loss, degradation or inactivation of the antimicrobial agents.
Therefore processing conditions and active agent compatibility must be considered in
order to ensure efficient product development. For instance, some natural antimicrobial
43
agents such as enzymes exhibit low resistance to high temperatures, active agents such
as extracts with a high polyphenol content (e.g., grape extract) may be degraded by
high temperatures and the thermal degradation of catechins has been reported to be
a limiting factor in the manufacture of active packaging applications [106].
As mentioned in Chapter 3, processes based on dissolution of the polymer and the

agent in a common solvent or solvent mixture are, in general, not detrimental to the
agent. The resulting film should contain a homogeneously distributed agent; however,
during the solvent evaporation process volatile agents are converted into vapour and
released prior to film formation.
This type of processing is usually employed to develop coatings on film surfaces.

Thus, coating technologies are non-aggressive thermal methods, appropriate for
thermolabile non-volatile agents such as enzymes, polypeptides or surfactants like
LAE. This is also the procedure commonly employed for CS because it is a non-
plastic biopolymer. Another method for incorporating polypeptides, such as enzymes
or bacteriocins, into packaging films is by direct coating or absorption onto the
polymer surface. The labile bonding of the agent to the surface will result in a fast
release into the food.
On the other hand, extrusion processes expose the antimicrobial agent to high
temperatures, pressures and friction. A high specific mechanical energy value, a
measurement of the severity of process conditions, might result in degradation of the
agent. The solubility and reactivity of antimicrobial agents within the polymer at the
extrusion temperature are also critical factors. Solubility involves the homogeneous
distribution of the antimicrobial agent in the polymer matrix, and reactivity is related
to the potential degradation, via chemical reactions, of the antimicrobial agents by
themselves or with other components of the extrudate.
Organic acids and their salts exhibit excellent resistance to thermal treatment, hence
several antimicrobial films have been developed using the extrusion method, as
already mentioned in Section 2.2.6. Similarly, metal NP and metal oxides can be
homogeneously distributed via extrusion processes.
Essential oils are difficult to incorporate into a polymer matrix. The volatile nature
of these components, especially active ones, results in inevitable loss via volatilisation
during extrusion and solvent evaporation during coating procedures. Indeed, EVOH
coatings applied on PP films by gravure printing containing OEO or citral lost between
35 to 50% of the initially added agent during the drying process [107].
Another technique for obtaining antimicrobial films involves the immobilisation

of non-volatile antimicrobials onto a polymeric surface, where they exert their
antimicrobial activity by being in direct contact with the food. In this case the active
44
agent is not released, so its activity is limited to the contact surface only. These films
are applied in vacuum packaging and as slice separators. Bacteriocins and enzymes
were selected as the antimicrobial agents to be immobilised onto film surfaces.
Immobilisation is achieved via wet chemical treatment with strong acids, bases, ozone,
corona, UV irradiation, ion and electron beam irradiation, and plasma [108]. These
and other techniques are explained in detail in Chapter 3.
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52
3
Active Packaging Systems
As mentioned in the introduction, active packaging systems are based on the

incorporation into the system of a substance, or mixture of substances, which by
themselves or by chemical reaction perform an activity that results in a beneficial
effect on the packaging content; in particular, in antimicrobial packaging systems,
an inhibitory or lethal effect on microorganisms. In this chapter, the methods of
incorporation are listed and briefly described, including the steps for preparing
the active agents and the techniques employed for their incorporation into the
packaging system.
3.1 Active Component Preparation
As described in Chapter 2, there are numerous pure compounds and mixtures that have
been described in the literature and reviewed in books which possess antimicrobial
activity. These substances exhibit very different chemical and biochemical properties,
and therefore the mode of action and conditions of activity need to be known in
order to use them efficiently in any type of application, including antimicrobial
packaging systems.
Organic acids such as acetic, lactic, propionic, sorbic and benzoic acids and their
salts are commonly used as food preservatives, and for decades they have been, and
currently are, incorporated into food formulations to increase food safety and shelf
life. They are, in general, thermally stable and can be incorporated into polymers by
melt blending during the manufacture of packaging materials. This is also the case
with several metal oxides and salts which have been used in active packaging.
Many plant essential oils are well-known antimicrobial substances and have
traditionally been used as such in cooking and home preserves, and as flavourings. This
latter use is due to the volatility of their constituents and the very low concentration
threshold of detection for the majority of consumers; characteristics which are
responsible for their valuable aroma. However, when considering their use in active
53
antimicrobial packaging systems, this volatility must be controlled in order to facilitate

incorporation of the active substances and to reduce loss of the substances during
packaging production and storage. High losses may mean not only economic loss but
also low or zero efficiency in the protection of the packaged product.
However, volatility is not the only reason for manufactured antimicrobial materials,
with pure or blended agents, exhibiting low efficiency. Degradation caused by
reactions with other packaging components, thermal abuse and mechanical stress
are other potential hazards that may result in loss of the active molecule and
subsequent reduction of antimicrobial activity. Enzymes and bacteriocins are
polypeptide macromolecules whose antimicrobial activity is highly dependent on
their three-dimensional (3D) structure. In many cases, exposure of these substances
to conventional thermomechanical procedures would lead to breakage of the
molecule or at least conformational changes that result in loss of antimicrobial
characteristics.
Consequently, various procedures have been developed and are used to protect the
active component during packaging manufacture, reducing the stresses it would
be exposed to in the case of direct melt blending with the polymer or evaporation
during the drying processes involved in impregnation, coating or casting techniques.
In some cases the active agent is absorbed onto the surface of porous substrates
(generally inorganic), reducing its volatility and providing partial protection from the
aforementioned stresses. In others, the agent is encapsulated in an organic environment
via incorporation into the organic matrix or inside an empty particle.
3.1.1 Use of an Inorganic Substrate
Inorganic substances are commonly added to polymeric matrices. Metal oxides,

salts, zeolites, clays and so on have been used to modify the functional properties of
polymers, including food packaging materials. Titanium dioxide is commonly used
in polymer applications as a white pigment and also as an ultraviolet (UV) radiation
shield. Calcium stearate is a lubricant used to improve the flowability of powders
and is largely used in polymers for food applications because of its approval as a
food additive (E470).
Clays broken down into microsized fillers are often incorporated into polymer matrices
to enhance their mechanical properties. These microcomposites generally exhibit
modulus improvement compared with the pure polymer, however the elongation at
break and toughness of the polymer are reduced. More recently, clay nanocomposites
have been developed with smectite-type clays, such as hectorite, montmorillonite and
synthetic mica.
54
A nanocomposite is defined as a composite in which at least one of the phases has

one or more dimensions of the nanometre order, whereas a polymer nanocomposite
consists of a polymer or copolymer possessing nanofillers dispersed in the matrix.
In the food packaging sector, nanofillers are added to polymer films in an attempt to
develop plastics for food packaging with greater performance with regard to thermal
stability, mechanical strength and gas barrier properties. The difference between these
two fillers, i.e., nanofiller and microfiller, is the particle dimension. The surface-to-
volume ratio increases exponentially as the particle diameter decreases, and the effect
of this large increase modifies the final properties of the composites. In the case of
smectite clays, which have a layered structure, the original microparticle structure is
modified by separating the constituting layers to different degrees. The final composites
exhibit enhanced mechanical properties at a low smectite clay loading percentage
without compromising toughness, and they have improved heat resistance, barrier
properties and so on.
Mesoporous silica is a highly porous form of silica which is used in many applications
because of the large surface-to-volume ratio and the high free volume percentage,
which can be used to protect compounds and/or to deliver substances. Apart from the
obvious applications in biomedicine, pharmacology, biosensing and so on, mesoporous
silica is also used in food and food packaging applications.
Other reinforcing materials commonly present in polymer composites are carbon fibres
and glass fibres, which have wide applications in industry (vehicles, sports items and
so on), including food containers, but with rare applications in the packaging sector.
However, carbon nanotubes are being explored as reinforcing materials because they
have high flexibility, low mass density and a large aspect ratio, and there are ongoing
studies to use them in food packaging.
Inorganic/polymer composites can be prepared via numerous manufacturing

processes, including sheet forming, fabric thermostamping, compression moulding,
injection moulding and extrusion moulding. Since most polymer packaging materials
are produced via injection or extrusion processes, the preparation of filler/polymer
composites for packaging applications is typically carried out by melt blending in
extruders or similar equipment. In some cases they are combined with a polymer at
a high concentration to produce a masterbatch that can then be diluted with virgin
polymer to achieve the required concentration.
Other procedures commonly used in film manufacturing, such as film coating

and printing technologies (gravure, flexography), are also available for composite
manufacture. In these procedures, as described below, a solution or dispersion
of the components is produced and the final composite is obtained by solvent
evaporation.
55
As a step prior to film extrusion or packaging injection moulding, these inorganic

substances can be used to protect the antimicrobial agents from evaporation or
degradation. Liquid antimicrobial substances such as plant extracts or essential oils
can be exposed to absorbent materials. The oil components can wet the surface of
the inorganic filler or can enter the pores of these materials, reducing their vapour
pressure which facilitates the feeding of film manufacturing equipment. Since this
effect takes place mainly at the surface of the filler, materials with a high surface-to-
volume ratio are preferred. In this way, the final load of the filler in the polymer can
be reduced. Nanoparticulate materials, clay platelets, mesoporous silica and carbon
nanotubes are particularly interesting in this context.
3.1.1.1 Clay Nanocomposites
Nanocomposites based on the incorporation of nanometric inorganic materials loaded

with active molecules into a polymer film is a promising area of research with potential
applications in the development of active food packaging. Nanostructured polymer
matrices could be employed in the design of antimicrobial food packages capable of
releasing antimicrobials into the food matrix or to the package headspace surrounding
the food. Antimicrobials are usually introduced during the processing of the polymer,
but this step has several potential drawbacks because the active molecule may be
inactivated, degraded or lost by evaporation during processing. Nanotechnology
can deal with these deficiencies by loading the nanofiller with the active molecule
before it is incorporated into the polymer film. In addition, nanostructured polymers
could also modulate the release of the active compounds in order to enhance their
effectiveness when they reach the food product. It is expected that these possibilities
will be exploited in the near future.
In general, nanomaterials are classified as nanolayers, nanofibres or nanoparticles

(NP) in accordance with their dimensional structure. Owing to their large aspect ratio,
nanofibrous and nanolayered materials are usually employed as reinforcing fillers in
most polymer nanocomposites. Layered clays such as montmorillonite are currently
the most important for the production of polymer nanocomposites and a large number
of commercial developments have been derived from intense research in this area.
Various considerations should be taken into account when using naturally occurring
clays as systems for the delivery of active molecules; in particular, the kind of clay and
the physico-chemical properties of the active agent, such as the presence of functional
groups, polarity, size and chemical structure. Depending on the presence and type of
functional groups and the polarity of the molecule, the sorption of the active molecule
to the clay may be chemical (cationic exchange, ligand exchange, protonation) or
physical (van der Waals forces). Thus, active molecule-clay interactions will determine
56
the release pattern of the former [1], whereas the size and molecular geometry of the
active molecule will determine its ability to intercalate into layered structures or to
be entrapped in hollow tubular structures.
However, little work is currently focused on the incorporation of active molecule-clay

systems in polymer matrices for use in antimicrobial food packaging. Recently, Liu [2]
studied the intercalation of benzoate and gallate anions in layered double hydroxides
and their incorporation into polyhydroxyalkanoate polymer films for antimicrobial
food packaging applications. Intercalation was achieved by immersion and stirring
the solid in an aqueous solution of sodium salts. Halloysite clay nanotubes are used
for encapsulation and sustained release of active agents. Furthermore, it has been
demonstrated that the sustained delivery efficiency of halloysite is better than other
encapsulating materials, such as mesoporous silica NP or polymer microcapsules [3].
The empty lumen of nanotubes can be loaded by immersion of the nanotubes in a
concentrated solution of the agent in a solvent compatible with the clay (polar),
making a vacuum to remove the trapped air and re-establishing pressure so that the
agent enters the tube lumen, after which the particles are washed and dried.
Although there are a considerable number of studies regarding its use to improve the
mechanical strength, barrier properties and adhesion of polymer films and coatings,
more studies need to be performed regarding the incorporation of halloysite loaded
with active agents into polymer matrices to modulate and extend their release. For
pharmaceutical purposes, tetracycline-loaded halloysite has been incorporated into
polyvinyl alcohol (PVA) and polymethyl methacrylate films and the release of the
active agent was studied [4]; however only a few studies have investigated the use of
halloysite-polymer nanocomposites for active food packaging applications.
Besides acting as carrier systems, inorganic materials have also been incorporated
into polymer matrices for the purpose of modifying the release of active compounds.
In this regard, bentonite clay, an aluminium phyllosilicate consisting mostly of
montmorillonite, has been incorporated into polymer and biopolymer matrices to
modify the release of antimicrobial molecules. It has been shown that bentonite can
slow the release of carvacrol from ethylene-vinyl alcohol (EVOH) copolymer films
[5], or herbicides from alginate and other synthetic polymer matrices [6, 7]. Halloysite
has also been employed to control the release of opioids added to microcrystalline
cellulose pellets and antiseptic brilliant blue incorporated into poly(e-caprolactone) [3].
It is worth noting that the great capacity of smectites for cation exchange, in particular
montmorillonite and saponite, has been used to replace Na+ cations with metallic
ions that have antimicrobial capacity, such as Ag+, and embed them in biopolymer
matrices for application in antimicrobial food packaging [1]. Zeolites are naturally
occurring hydrated aluminosilicate minerals that have a porous structure. Zeolite ions
57
exchanged with silver, copper and zinc cations, which possess antimicrobial activity
against a wide spectrum of microorganisms, are capable of slowing the release of
these metal ions into a liquid medium and have been incorporated into materials
where antimicrobial properties are required [8]. Other inorganic materials, such as
zirconium phosphate and glass, have been tested as carriers and release systems for
antimicrobial metal ions. Silver nanorods have been synthesised inside the lumen of
halloysite nanotubes, by the thermal decomposition of silver acetate embedded in the
particles from an aqueous solution, and then incorporated into polyurethane paint;
the resulting inhibition of bacterial growth was due to the slow release of silver ions
from the nanotubes [9].
3.1.1.2 Metal Nanoparticles
NP are widely studied and employed in the manufacture of polymer nanocomposites

for the development of novel materials with specific properties (optical, conductive,
antimicrobial and so on). With respect to the design of antimicrobial nanocomposites
for active food packaging, it is worth mentioning that metal NP exhibit antimicrobial
properties via various mechanisms of action. In this regard, silver metal NP have
been proved to be a reservoir of metal ions; the silver metal of the NP surface is
oxidised to silver ions in an aqueous medium, and over time the NP dissolve, releasing
antimicrobial ions into the liquid environment. Thus, immobilising NP in a polymer
matrix capable of releasing oxidised silver ions is another way of creating antimicrobial
polymer nanocomposites. There are many studies, patents and commercial applications
regarding silver NP embedded in polymer matrices. However, their application in
the development of antimicrobial packaging for food is limited owing to legislative
issues regarding the amount of silver that migrates into food.
It is worth pointing out that the antimicrobial activity of polymer nanocomposite

films incorporating antimicrobial ions will depend on several factors which affect
their release, such as polymer crystallinity and water swelling capacity, as the polymer
needs to be in an aqueous medium for it to become plasticised and facilitate diffusion.
Other factors are related to the type of nanomaterial used to hold the ions, i.e., the
release of the ions from zeolites is different to release from metal NP; for example,
silver NP offer more stability and slower silver ion release rates than silver-impregnated
zeolites, whereas the latter have a superior antimicrobial effect for short periods. The
size and shape of the particle also affect the antimicrobial activity. Smaller particles
have a larger relative surface area for silver ion release and spherical silver NP are
highly reactive, owing to their high atomic density, which allows the faster release of
ions [10]. There are several procedures for obtaining silver NP. In most cases, solutions
of silver salt are used to impregnate polymer films in the presence of a reducing agent.
Stable and uniformly distributed silver NP have been obtained via the in situ reduction
58
of silver salts in the presence of sodium borohydride as a reducing agent and using
hydrogel networks as nanoreactors. In other cases, aqueous solutions containing
both the polymer and silver salts are used and the formation of silver NP is due to
the ability of the polymer to act as a reducing agent, as occurs with PVA or chitosan
(CS) [10, 11]. In some of these procedures, temperature treatments or irradiation with
UV or ultrasound are used to start the reaction. Evaporation of the solvent results
in films containing the NP, as shown in Figure 3.1. Another procedure for obtaining
antimicrobial materials based on silver is by the use of silver-impregnated zeolites
[12]. Silver ions are incorporated into zeolite pores by ion exchange and then zeolites
can be incorporated as a solid in any matrix.
0.5% Ag 1.0% Ag
Figure 3.1 Image of CS films containing silver NP at different concentrations

showing film transparency and colour, and transmission electron
microscopy image showing the size of the NP
Similarly, copper NP act as a reservoir for the sustained release of copper ions
generated by the oxidation of copper metal on the NP surface. Copper NP, copper
oxide NP and copper ion-impregnated nanofillers have been incorporated into various
polymers of both synthetic and natural origin [13, 14]. Cioffi and co-workers [14]
electrochemically manufactured colloidal copper NP and the NP were then dispersed
in a polyvinyl chloride (PVC) solution.
Zinc oxide NP have been added to several polymers, including polypropylene (PP).
Their mechanism of action relies on the generation of hydrogen peroxide when
stimulated by visible light in conjunction with the release of zinc ions. Titanium oxide
NP are used in food processing industries to decontaminate food contact surfaces
and to prevent biofilm formation. The antimicrobial activity of these NP is related
to the photocatalytic generation of reactive oxygen species when illuminated with
UV light, but also with visible light irradiation. Recent research has been devoted
to studying the viability of using these NP for antimicrobial packaging applications.
For this purpose they have been incorporated into the polymer matrix or applied as
a coating on the polymer film surface to inhibit or reduce microbial growth on the
59
surface of solid foods. Recently, zinc nanocrystals have been embedded into polymer
matrices for antimicrobial packaging purposes. Other metal and metal oxide NP with
potential applications in the development of antimicrobial food packaging include
gold, aluminium oxide, cadmium selenide/telluride and magnesium oxide [15, 17].
3.1.1.3 Mesoporous Silica
Mesoporous silica NP are characterised by a large surface area and pore volume, with
a pore size range of 2 to 50 nm. The applications of these materials include catalysis,
adsorption, sensing and polymer reinforcement, and they are also good candidates for
encapsulation purposes [18]. They can accommodate a great volume of molecules,
increasing the solubility of poor water-soluble molecules, protecting them against
external factors or working as carriers and controlled release systems for active
compounds. Mesoporous silica NP are widely studied as controlled release systems.
Research on mesoporous NP is focused on their morphology, pore size and chemical

surface functionalisation. These nanomaterials can be synthesised with different
pore morphologies and shapes (sphere, rod, fibre, doughnut and so on), and can
be functionalised to modify their interaction with encapsulated molecules and their
release pattern [19].
A great amount of research is focused on the application of mesoporous silica NP as

carriers and controlled release drug-delivery systems [20, 21]; however, only a small
amount of investigation has been devoted to the study of bioactives [22], naturally
occurring antimicrobials allyl isothiocyanate and menthol [23, 24], herbicides [25],
antimicrobial silver ion-exchanged mesoporous silica [26] and silver nanocrystals
encapsulated in mesoporous silica as antimicrobials whose activity is induced through
their oxidative dissolution [27].
The use of mesoporous silica NP for the design of polymer nanocomposites is

currently receiving attention, and many properties of novel nanocomposites have
yet to be elucidated [28]. In this context, studies regarding the use of these materials
as active agent carriers and release systems for the development of active food
packaging are scarce. Heirlings and co-workers [29] used mesoporous silica SBA-15
for the development of antioxidant films for active food packaging applications; in
order to achieve this the antioxidant α-tocopherol was adsorbed in the material and
incorporated into low-density polyethylene (LDPE) and ethylene-vinyl acetate (EVA)
to protect it from polymer extrusion and facilitate extended agent release. Recently,
Buonocore and co-workers [30] compared the sustained release properties of SBA-15
and amino-functionalised SBA-15 loaded with tocopherol and incorporated into a
LDPE matrix.
60
Besides silica NP, other mesoporous inorganic materials, such as aluminium

oxide functionalised with hydrophilic and hydrophobic groups, titanium dioxide
nanospheres and nanosized hydroxycarbonate apatite, are being studied as carriers
and release systems for drugs [19].
Hollow silica and mesoporous hollow silica have been synthesised and their potential
in catalysis, as supports for immobilising molecules, as carriers and controlled release
drug-delivery systems is being studied. Their application in the development of polymer
composites and active packaging is expected to increase over the next few years.
3.1.2 Encapsulation in an Organic Substrate
Encapsulation enables the immobilisation of a compound or mixture of compounds

in a material that coats it or in which it is dispersed. The substance to be encapsulated
can be a solid or liquid compound or even a gas. Using encapsulation it is possible
to achieve various interesting effects, such as: isolating compounds that might react,
absorbing liquids into solids to facilitate their handling, protecting the encapsulate
from the environment around it, improving product manipulation, masking odours,
colours and flavours, and also increasing the effectiveness of active compounds
through controlled release or release sustained over time. Encapsulation processes now
occupy an important position in various areas including medicine, pharmaceuticals,
cosmetics, textiles, agrochemicals, food and, of course, packaging.
The first studies involving encapsulation and its possible application in the
pharmaceutical area began with the preparation of microcapsules of gelatin by
means of coacervation and it subsequently spread to other fields, such as inks in the
manufacture of tracing paper, and aromas and flavours in the food industry. In the
1950s the American Chicle company asked the Atlantic Gelatin Division of General
Foods to develop a method to prolong the mint flavour in its chewing gum, for which
purpose they encapsulated tiny droplets of mint oil in gelatin capsules; thus the mint
flavour was released slowly. Since then, microencapsulation has found interesting
applications in a variety of fields.
Pharmaceutical companies have led the applications of microencapsulation [31],

pursuing objectives such as masking unpleasant flavours in various drugs, protecting
them against pH in their passage through the stomach, controlling the rate at which
drugs are released and reducing differences in the drug concentration in an organism
between doses.
Some enzymes, animal and plant cells and microorganisms have been immobilised
in microcapsules. The substrates pass through the coatings and are processed by the
61
enzymes or cells inside the capsules, and the final products are released outside the
capsule. One of these applications has been the immobilisation of detoxifying enzymes
in semipermeable membranes to act as an artificial kidney [32].
The encapsulation of bacteria has also been an object of study. Early work in this area
focused on lactic acid bacteria, which have great importance and many applications in the
food industry, such as the encapsulation of lactic acid bacteria in alginate gels [33]. The
encapsulating polymer must incorporate nutrients in the capsule to keep the bacteria alive.
The processes of encapsulation in the food industry began in the 1940s and 1950s
with the microencapsulation of fish oils, essential oils and aromas [34]. Traditionally,
microencapsulation has been mostly applied in the stabilisation of aromas to avoid
their evaporation during the processing, transport and storage of the product in which
they are integrated. There are various food additives and ingredients that are capable
of being encapsulated, such as oils, enzymes, acidulants, colourings, sweeteners,
vitamins, minerals, bacteria and preservatives.
The benefits of using microencapsulation include: reduction of the reactivity of an

active molecule with other components in the food matrix or external environment,
reducing the evaporation of the active molecule to the external medium, controlled
release of the active molecule and easier dispersion of compounds that are not
very soluble. Despite all these benefits, encapsulation has not advanced as rapidly
in the food industry as in the pharmaceutical and chemical industries because it is
a costly and highly specific technology, as well as being limited by a lack of food-
grade encapsulating materials. In recent times, the demand for functional foods
with greater added value has become the main driving force in the application of
microencapsulation in the food sector [35].
As has been mentioned regarding the applications of microencapsulation in the

pharmaceutical and food fields, many antimicrobial packaging developments require
the active agent to be protected from interaction with the polymer matrix or oxygen in
the environment, its evaporation during manufacture of the packaging must be reduced
and it must be possible to control the rate at which it acts on the food. Therefore the
microencapsulation methods of both industries tend to be the same, with the additional
advantage that the encapsulating agents must be valid for food use but do not necessarily
have to be food additives. This condition would only be necessary in active packaging
in which the capsule is released into the food and becomes part of it.
3.1.2.1 Criteria for Antimicrobial Agent Encapsulation
The purpose of using and incorporating antimicrobial agents is to inhibit

microbiological growth, but this function can be performed in various ways,
62
e.g., by the release and action of the active agent on microorganisms, by retention
of some compounds needed for growth (such as water or oxygen) or via the
release of a product resulting from a reaction or decomposition of the principle
agent (chlorine or carbon dioxide). The mechanism of action is the first criterion
for defining the form of incorporating the agent into the packaging. It is then
necessary to determine whether it needs to be encapsulated to carry out its function
correctly. Therefore the purpose of encapsulating a particular ingredient must
be clearly defined so that, in this way, the encapsulation process can be designed
properly. However, it is also important to be aware of the possible techniques for
fabricating the packaging system so that the encapsulation system is compatible
with them.
The main criteria that must be borne in mind when encapsulating an ingredient are:
• Knowledge of the effect that the encapsulated agent exerts on the final product.
• Determination of the concentration of the agent required for it to be effective and

thus estimation of the concentration of the ingredient in the encapsulant.
• The agent must not deteriorate during microencapsulation and must not react
with the encapsulating material, so that the functionality is not affected.
• Knowledge of the size of particle required and the final structure desired.
• Knowledge of the conditions to which the encapsulated agent will be subjected

in order to ensure its stability before it performs its function.
• Knowledge of how and when the active compound must be released and in
response to what stimulus (pH, temperature, pressure and so on).
The cost of the polymer coating and, in general, of the microencapsulation process
must be justified in terms of improved performance [36].
3.1.2.2 Matrices for Encapsulation
The selection of the most suitable encapsulating material and process depend
upon the characteristics of the compound to be encapsulated. The particle
loading capacity and the encapsulation efficiency depend largely on the physico-
chemical properties and interactions between the encapsulating material, the
encapsulated compound, the medium that surrounds them, the presence of
excipients, and also the encapsulation process that is selected and its corresponding
processing parameters.
63
Any material employed in the encapsulation of antimicrobial agents must be recognised

as safe for food contact or as food ingredients [generally recognised as safe (GRAS)
substances] by the US Food and Drug Administration or the European Food Safety
Authority. Other characteristics to be borne in mind when selecting the encapsulating
material are its physico-chemical characteristics, such as molecular weight (MW),
solubility, crystallinity and diffusivity [37]. Most encapsulants are, therefore, food
additives, which we can classify as lipids, sugars or proteins.
A wide range of lipids (natural fats and oils, mono-, di- and triglycerides,
phospholipids, glycolipids and waxes) can be used as encapsulation matrices.
These highly apolar lipids are the main carriers of lipophilic molecules in emulsions
[38]. The physical and chemical properties of fats determine their microstructural
characteristics, colloidal stability, thermal properties [such as melting temperature
(Tm)], and rheological and moisture barrier properties. Shortening the length of
the hydrocarbonated chain or increasing the degree of unsaturation of the fatty
acids lowers their Tm and reduces the moisture barrier properties; for example,
active molecules can be incorporated into the core of a matrix of solid fat, being
released when there is an increase in temperature. Fats have good moisture barrier
properties and therefore are used as encapsulating materials for compounds that
are sensitive to moisture. In this case the more saturated fats (fats that are solid
at room temperature) have a more crystalline structure and consequently a lower
moisture permeability.
Attention must also be drawn to the existence of polar lipids (monoglycerides,

phospholipids, glycolipids), which have amphiphilic properties and can therefore be
used as active coatings and to stabilise emulsions that contain the active molecule.
These lipids interact with water and ensure the stability of liquid supramolecular
crystalline structures; this mechanism can be used for the protection of sensitive
molecules and to achieve controlled release of the active molecule [37].
The second group of encapsulating materials is composed of polysaccharides (starch

and its derivatives, dextrins, CS, cellulose and its derivatives, cyclodextrins and so on),
which have the ability to form solids in the glassy state and link specific molecules;
in addition, they can act by immobilising the active molecules in dehydrated food
formulations [39]. When sugars are used in combination with proteins in dehydrated
food formulations, the sugar also stabilises the proteins during rehydration through
the formation of hydrogen bonds.
The third group of encapsulating matrices consists of proteins, including proteins

of soy, milk (caseins and whey), egg and corn (zein), and their hydrolysates. Their
properties are influenced by the amino acid composition, conformation, load and
denaturation temperature. The aggregation and gelification of proteins allows the
64
development of networks in which active ingredients can be included. Depending

on the method of protein aggregation (modification of pH, heat treatment and
so on), different types of bonds are formed (covalent bonds, hydrogen bonds,
hydrophobic interactions, electrostatic interactions, van der Waals interactions)
and their relative contribution gives rise to a great variety of 3D networks or
structures [37]. One of the greatest advantages of proteins is that their properties
can be modified physically, chemically and enzymatically, which affect their
functionality as encapsulants. The hydrolysis of proteins alters their emulsifying
properties, with the effect depending on the degree of hydrolysis. Heat treatments
bring about conjugation between proteins and carbohydrates via the Maillard
reaction, which modifies their emulsifying properties.
3.1.2.3 Encapsulation Methods
Encapsulation processes produce particles of different sizes, depending on the

technique employed. When the diameter is less than 1 µm they are called NP and
when it is greater they are microparticles. They can be spherical but it possible for
them to have an irregular shape.
They can be classified broadly, according to their structural characteristics, into spheres
and capsules. Spheres are matrix or monolithic systems in which the encapsulated
agent is dispersed homogeneously or dissolved in the matrix, although the agent
may also be adsorbed or conjugated on the surface of the particle. Capsules are
reservoir-type systems in which the encapsulated agent is surrounded by a coating
or shell of another material of variable thickness. Figure 3.2 shows the various types
of encapsulation structures.
Simple Double-coated Polynuclear Spherical Irregular

capsule capsule capsule matrix matrix
Figure 3.2 Types of encapsulation structures
There are other, intermediate structures and structures of greater complexity; for
example, there are polynuclear capsules, double-coated capsules and capsules that
in turn contain other capsules, hence a sequence of several encapsulation processes
is required [40]. There are also other encapsulation systems, such as liposomes,
micro- and nanoemulsions, micellar systems and molecular inclusion systems with
cyclodextrins [36].
65
Numerous techniques have been developed for the preparation of microcapsules

and some of them are used for the encapsulation of food ingredients. Table 3.1
presents some of the main encapsulation methods that are used in the food industry
[36, 37, 40].
Table 3.1 Main encapsulating methods in the food industry

Physico-mechanical methods Particle Chemical and physico- Particle
size (µm) chemical methods size (µm)
Atomisation and spray drying 5–2,000 Interfacial polymerisation 0.5–1,000
Cool spray 20–200 Coacervation 2–1,200
Extrusion 250–2,500 Liposome entrapment –
Fluidised bed coating 20–1,500 Molecular inclusion 0.001
Electrostatic encapsulation – – –
• Molecular inclusion
Molecular inclusion or complex inclusion employs macrocycles, usually cyclodextrins,

and in particular β-cyclodextrin, for the entrapment of molecules. Cyclodextrins are
cyclic oligosaccharides with a hollow truncated cone shape, which have a hydrophobic
inner wall and a hydrophilic outer wall. Cyclodextrins form complexes by inclusion
or by host-guest interactions.
The encapsulation method using cyclodextrins involves the formation of

complexes in an aqueous solution by the intrusion of molecules, in which the
apolar molecules replace the water or gases in the cavity of the cyclodextrin
molecule or by mixing with the pure agent. The stability of these complexes
depends on the structure, hydrophobicity of the molecule, pH, organic solvent,
temperature and cyclodextrin concentration. Cyclodextrins protect flavours
and other heat-sensitive ingredients that are added to extruded foods, e.g.,
garlic oil, onion and the liposoluble vitamins A, E and K are complexed by
cyclodextrins [41–44].
• Interfacial polymerisation
In this method, a monomer polymerises at the interface between two immiscible

substances, forming a continuous membrane which constitutes the microcapsule
wall. Three steps can be distinguished: dispersing an aqueous solution of the
antimicrobial in an organic phase (oil) producing a water-in-oil emulsion,
polymeric membrane formation upon the addition of a monomer soluble in the
66
organic phase and separation of the microcapsules by centrifugation [45, 46]. This
process has been used to encapsulate proteins, enzymes, antibodies and cells [47].
• Coacervation
In a colloidal solution the charges can be oriented, forming bonds that bring about
a decrease in the solubility of the colloid. Consequently, part of the colloid can be
separated into a new phase, converting it into a two-phase system. The colloid-rich
phase is in a dispersed state that appears as drops of amorphous liquid, which are
known as coacervate droplets.
Coacervation can be initiated in various ways: changes in pH or temperature, or

addition of a second substance such as an ionic salt, a method which is more efficient
but more expensive. Microencapsulation by coacervation requires the active material
to be encapsulated and the matrix or coating to be mixed so that the coating is
deposited on the active material. Generally a change in pH, temperature or ionic
strength causes a phase separation or coacervation of the matrix and entrapment of
the dispersed active material. Finally, the matrix is solidified by thermal or chemical
treatment (crosslinking).
The advantages of this method are the possibility of making microcapsules as small as
4 µm, spherical in shape, which contain approximately 90% loading of the active
material to be encapsulated. Furthermore, the microcapsules provide good protection
against oxidation and loss by volatilisation. The encapsulated material can be released
by mechanical pressure or in hot water [42].
• Liposome entrapment
Liposomes are capsules which exhibit more versatile properties and are less fragile
than capsules made of fat. They are used for the release of vaccines, enzymes and
vitamins in the body. They are composed of one or more layers of food-grade lipids
whose size and composition affect the permeability, stability, surface activity and
affinity of the liposome.
Liposomes are hollow vesicles of 50–500 nm with a membrane consisting of a double

layer of phospholipids, and it is possible to produce multilaminar structures consisting
of several layers of active agent and membrane [48]. The layers of phospholipids are
permselective to cations. Release of the active agent is brought about by diffusion
through the bilayer, the vesicle is then destroyed by means of a critical concentration
of calcium ions or a change in pH. Cholesterol and tocopherols can be incorporated
to reduce the permeability of the membrane and increase the stability of the lipids in
the bilayer. Liposomes are used successfully in the encapsulation of enzyme systems;
however, the use of organic solvents limits their use in food applications [42].
67
• Spray drying and atomisation
Spray drying is widely used in the food industry because it is an economical and effective
method for food preservation, and in particular, it is employed in the dehydration of
milk. Modified starches, maltodextrins and gums are used as the matrix. The material
to be encapsulated is first homogenised with the matrix, the mixture is spray dried
by means of a nozzle or disc, and the capsules are then collected [37].
• Cool spray or freeze spray
This is a variant of spray drying which consists of cooling or freezing. The active
agent to be encapsulated is mixed with the encapsulating material and atomised by
means of cold air against hot air. The microcapsules are produced by nebulisation of
the emulsion or suspension which contains the matrix and the solid or liquid active
substance. Since these capsules are insoluble in water, as they are covered with lipids,
the procedure is especially indicated for encapsulating hydrosoluble compounds such
as enzymes, hydrosoluble vitamins and acidulants [37].
• Extrusion
The encapsulation of substances by extrusion starts by forming a low-moisture

(5–10%) carbohydrate melt (110–130 oC) composed of maltodextrin, simple sugar
and/or a starch. Under vigorous agitation of this melt, an emulsifier and the active
substance to be encapsulated are added, and the final molten emulsion is forced
through a die into a cold bath, where it is converted into solid amorphous pellets
which are mechanically broken apart into smaller particles [49]. Flavouring and
vitamins are among the food components most commonly encapsulated by extrusion.
• Fluidised bed coating
This technique involves suspending solid particles in air at high speed in a chamber
with controlled temperature and humidity, where a solution of the coating polymer
is atomised. It is generally a process that is carried out in batches, so the thickness
of the coating depends on the residence time [50]. The technique is applicable to
matrices that are soluble (starches, maltodextrins and so on) and the compounds to
be coated are usually pure solids or mixed to achieve the final formulation of the
product. Citric, ascorbic and sorbic acids have been encapsulated using this process.
• Electrostatic encapsulation: electrospray or electrohydrodynamic atomisation
This technique is based on the use of an electric field for the dispersion of a liquid in
order to obtain particles. The action of an electric field on the interphase of a liquid
was highlighted in 1600 by William Gilbert, who described the formation of a conical
68
meniscus when a piece of electrified amber was placed near a small drop of water
(Gilbert, 1628). Four hundred years later, as a result of work on the balance between the
electrical forces perpendicular to the surface of the meniscus and the capillary pressure,
Geoffrey Ingram Taylor explained the formation of a conical electrified meniscus, which
was named a Taylor cone in his honour (Taylor, 1969). To achieve electrohydrodynamic
atomisation or electrospray, i.e., the phenomenon just described, a conductive liquid
is injected very slowly through an electrified needle, producing an electrified meniscus
within the needle that acquires the form of a stationary Taylor cone, resulting in a spray
of charged drops as it emerges from the needle. If the liquid acts as a solvent of the
encapsulating polymer matrix and the compound to be encapsulated, after evaporation
of the drops particles of polymer are obtained in which the agent to be encapsulated
is dispersed. With this technique it is possible to achieve a wide range of particle
sizes, from tens of nanometres to hundreds of microns, as well as great uniformity of
particle size. It is also possible to obtain core-shell structures by making the polymer
and the compound to be encapsulated flow through two coaxial needles [51]. There
are other methods based on the electrospray technique, such as bipolar coagulation,
which involves the generation of drops containing the encapsulating material and the
agent to be encapsulated, differentiated by being generated at different electrodes and
with opposite charges so that they generate the particles when they collide.
3.2 Independent Devices
Once the antimicrobial agent is ready for its application to the food packaging system,
either because it is in a form that can be readily used or after undergoing one of the
above-mentioned preparation procedures, as described in Section 3.1, the next step is
to decide the best method of inclusion. This decision should be based primarily on the
efficiency of the substance, but economic and marketing issues should also be considered.
In the early stages of active packaging technology, the agent responsible for the
beneficial activity on the food was included in an independent device, such as bags,
strips or labels, which were incorporated into or attached to the inside of the package,
but the device was manufactured as a separate item and the food container was a
conventional passive package. This procedure is still used in many active packaging
technologies.
The main advantage of designing independent devices is that the antimicrobial agent
can be used in a pure form, mixed or encapsulated, as required for its protection and
function, and incorporated in a container without undergoing any severe processing
treatment. The activity of the substance is based on the exchange of gases and vapours
through the walls of the independent device, and therefore this procedure is valid for
volatile antimicrobials that exert their action in the vapour phase or for substances
69
that are able to remove compounds from the package headspace which are vital for
the target microorganisms. Examples of the former are active packages based on
carbon dioxide, ethanol, chlorine dioxide, sulfur dioxide, plant extracts or essential
oils; oxygen and water scavengers are examples of the latter.
Ethanol-releasing devices are used with great success in bakery products, confectionery
and dry or semidry food. Ethanol at low concentrations in the headspace acts
effectively to prevent growth of both bacteria and moulds. Since pure ethanol is
a very volatile liquid, its application in active packaging requires a pretreatment
step, such as any of those listed in Section 3.1. One of the simplest methods is its
absorption by an inorganic porous absorbent solid such as silica, zeolite or clay. In this
way the agent is handled as a solid mixture. In some devices the ethanol is released
when the absorbent is exposed to humidity, i.e., the water activity (aw) of the food
builds up a humid headspace which enters the independent device and reaches the
porous absorbent, where water and ethanol molecules are exchanged. The ethanol
molecules then leave the device through the walls, exerting their antimicrobial activity
by accumulating inside the food package. Obviously, the package device has to be
appropriately designed to allow this mass transport.
There are many different procedures for including powdered ethanol in an

independent device, although the most usual one is in the form of a sachet or
small bag. A great variety of materials are employed for manufacture of the bag,
including paper-based structures with a suitable coating to provide heat-sealing,
paper-polyethylene (PE) laminates and paper-like materials obtained from plastic
fibres such as the one marketed by DuPont under the trade name Tyvek. The partially
porous nature of the structure allows adequate passage of water and ethanol through
the structure, and this mass transport can be controlled by the suitable selection
of materials. Once the material has been selected, 4-seal bags are formed, filled
and sealed in horizontal Form/Fill/Seal (FFS) equipment such as the one presented
in Figure 3.3. In this machine, a web roll provides the film structure, which goes
through the feeding rolls and is then folded longitudinally at the centre and heat
sealers produce the lateral and optional bottom seals, after which the pouches are
cut off and the agent is dosed and added in one or several filling steps; finally, the
top seal is formed.
A similar procedure has been patented for a device that releases chlorine dioxide [52],
in which the concentrated gas is absorbed in a porous substrate, such as zeolite or
clay, and immediately sealed in a 4-seal sachet until its use. In this case the gas tends
to rapidly desorb from the sachet, and to avoid loss the sachet structure includes a
gas barrier layer, either an EVOH layer or an aluminium film or coating. When the
content of the sachet is required, it is punctured and the number and size of the pores
provide a method of release control.
70
Lateral seals and Filling steps

pouch cut off Top seal
Drive roller
Optional
bottom seal
Tensioning
control
Figure 3.3 Diagram of a horizontal FFS machine
The size and capacity of the sachet containing the ethanol-releasing agent will depend
on the weight of food, aw, type of food and shelf life required. Examples of such
devices are marketed by Freund in Japan [53]. The advantage of this method is that
the device can contain various agents with synergic or diverse activity; an example
of such a system is a device containing an oxygen scavenger and powdered ethanol.
Chlorine dioxide has also been tested as an antimicrobial agent in the sanitary
treatment of several products, especially minimally processed fruits and vegetables.
There is increasing interest in the application of gas treatment during storage. Since
the gas can be generated by mixtures of chlorate salts and acids, there have been
various attempts to make an active pouch, such as the work of Wang and co-workers
[54], in which sachets containing a powdered form of chlorine dioxide were used to
improve the stability of strawberries. The manufactured sachets release the gas in the
presence of very humid environments.
Another well-known antimicrobial gas is sulfur dioxide. This gas can be generated
by exposing sodium metabisulfite, contained in paper-based sachets, to a humid
environment. The release of the gas can be controlled by the permeability of the
structure and the size of the metabisulfite granules [55].
Besides sachets, other independent devices can be designed to provide antimicrobial

activity to the food packaging system; absorbent pads are a good example. The porous
structure of cellulose has been employed in the formation of silver NP of regular shape.
Cellulose fibres have also been immersed in a solution containing silver nitrate, which
was reduced upon the addition of borohydride and deposited the antimicrobial agent
71
in the fibres. These pads reduce the microbial load of the exudate from fish or meat
[56]. Cellulose-based materials can also be used as carriers of volatile substances
such as ethanol, e.g., a cellulose pad or cellulose paper can be immersed in ethanol or
sprayed with the agent and then incorporated into conventional packaging. Higueras
and co-workers [41] added carvacrol, an antimicrobial component of oregano
essential oil, to a CS-based film via immersion and the resulting device showed strong
antimicrobial properties.
Antimicrobial pads similar to those just described can easily be added to conventional
packaging. However, the presence inside the packaging of elements that are foreign to
the natural composition of the food may lead to rejection by the consumer. Although
the device can be attached to a pressure-sensitive tape and adhered to a specific location
in the package where its presence is not so obvious, as mentioned in the introduction
(Chapter 1), the incorporation of such a device involves an extra operation on the
packaging line; in addition the sachet or pad does not avoid interaction with, and
the mass transfer of, substances in the case of liquid products. The alternative is the
incorporation of the active agent into the package structure. This procedure is now
extensively used for many technologies and will become more widespread in the future.
The methods for developing antimicrobial active materials are described in Section 3.3.
3.3 Incorporation into Packaging Structures
This is a more attractive method for consumers, who thus do not find any strange
matter inside the packaging that might attract their attention and cause them to doubt
the safety of the food that they are about to consume, in addition it simplifies the
packaging technology by eliminating the operation of inserting the active system in
the packaging and it is applicable to all kinds of products [57].
There are several standard polymer packaging manufacturing techniques that can
be used to produce active packaging systems. Since these technologies are based on
incorporation of the active agent into a polymer matrix or onto a polymer surface,
the active packaging system actually becomes an active package. In this section,
the various methods commonly used to prepare active materials are classified as
thermomechanical methods, wet-coating methods and surface-anchorage methods.
3.3.1 Incorporation into Packaging Structures by

Thermomechanical Methods
The most common technique for moulding and shaping polymers into appropriate
structures for packaging is extrusion. The process of continuous extrusion through a
72
die, in which an Archimedean screw rotates within a cylindrical barrel, is the technique
most utilised to prepare polymeric materials, including tubes, pipes, fibres and sheets,
and coatings for other laminar materials. This technique is employed in combination
with other extrusion-based procedures, such as blow moulding, thermoforming and
heat sealing, in the preparation of films, bags, bottles, jars, trays and many other
packaging containers.
Basically, the polymer (pure, mixed or combined with additives) in the form of powder,
beads, flakes, pellets or a combination of these is fed into the hopper of the extruder,
as can be seen in Figure 3.4. The particles enter the barrel, where the Archimedean
screw pushes the particles forward. The polymer mixture is heated and gradually
converted into a viscous melt which finally enters the extruder die. Different dies can
be used to obtain flat film or sheet, blown film, like the one shown in the figure, blow
moulding or extrusion coating, but basically the molten polymer enters the die from
the barrel and flows through the die until it reaches the die opening, which can be
linear, annular or of any required shape. Once out of the extruder line, the polymer
cools down and solidifies to take the final shape. This cooling process is controlled
by auxiliary equipment to improve the final characteristics of the extrudate.
Vertical view of the feeding equipment

Side extrusion
feeder
Gravimetric Collapsing frame
Hopper
feeder Liquid feeder Winder
Hopper
Free-flowing Heaters Melted polymer Blown film

Barrel die
particles
Screw
Figure 3.4 Basic design of an extruder on a blown film line
The design of an extrusion line varies greatly, depending on the characteristics of

the raw materials and final product. The basic design shown in Figure 3.4 contains
73
a single feeding section but, as the inset highlights, there are several types of feeders
that can be distributed at different locations along the barrel. In addition to the
hopper, which is located at the feed section of all extruders, other gravimetric feeders
can be distributed along the extrusion processing line. In this way it is possible,
for instance, to feed a second polymer or substance once the first polymer is in a
partially or completely molten state, facilitating its incorporation and/or reducing its
residence time at high temperature. Furthermore, a second extruder can incorporate
a molten material at any point of the barrel, allowing the melting of two polymers
with different thermal profiles and reducing the melt blending required at the final
part of the extruder barrel. In addition, liquid substances (or even gases) can be fed
into the barrel by different feeding lines.
The barrel/screw design can also vary. The profile of the screw and the use of a single
screw, or corotating or contra-rotating double screw modify the thermomechanical
process applied to the melting materials as well as the shearing and homogenisation rates.
Extruders can also be used for compounding. In packaging applications, the

compounding process is the preparation of polymer particles, containing appropriate
additives for a specific application, which are fed into an extrusion machine. The final
product obtained using this process is pellets, solid cylindrical particles which are
mixtures of polymers, pigments and/or additives in order to facilitate manufacturing
or provide improved performance, such as antioxidants, plasticisers or antifogging
substances. A list or classification of the wide variety of polymer additives that have
been studied is beyond the scope of this guide; however, the information is available
in reviews and books [58].
3.3.1.1 Preparation of Active Materials by Compounding
Compounding equipment is basically an extruder which has several feeding ports, with
a variety of screw-barrel architectures, and a simple die that produces one or several
polymer rods which are solidified by immersion in a refrigeration fluid and then dried
and cut off into pellets of the required length. These machines are very flexible and
can be adapted to the requirements of almost any product, the format change being
relatively fast, especially in comparison with industrial extrusion machines.
Compounding has been used to manufacture pellets containing antimicrobial

substances. These pellets can contain the agent at the concentration required in the
final packaging system or they can be a concentrated mixture to be adjusted in the
final packaging production process by converters. In this way, active materials can be
manufactured by the film or packaging converter, using more or less their standard
processing conditions with minor adjustments.
74
As a result of compounding, a prehomogenisation of the antimicrobial agent in

the polymeric material is achieved, which reduces the time required to optimise the
distribution of the agent in the polymer matrix. Vartiainen and co-workers [59]
developed antimicrobial packaging material for foodstuffs by incorporating traditional
food preservatives such as sodium benzoate, sodium nitrite, potassium sorbate or
sodium lactate into several synthetic plastics: LDPE, poly(maleic acid-co-olefin),
polystyrene and polyethylene terephthalate (PET). A pilot compounder was used to
carry out the mixing of agents and polymers, and the concentration of the agents was
increased up to 15% of the initial polymer mass. The active pellets obtained were
converted into sheets by injection into a mould or film via cast extrusion.
Metal oxides have been employed as polymer additives to provide colour and/or
promote opacity in polymeric food packaging. Their excellent thermal stability
allows easy thermomechanical processing. Pellets of microparticulated polyolefins
(PO) loaded with titanium dioxide are prepared by extrusion compounding and
are commercially available. Some metal oxides have recently been found to possess
antimicrobial activity, especially when employed as nanometric particles. However,
it is not easy to obtain a good dispersion of nanoparticulated oxides in polymers by
simple extrusion or compounding. Impregnation of titanium dioxide or silver NP on
attapulgite or montmorillonite helps to provide an excellent dispersion in PO during
the extrusion process. However, good dispersion can be obtained by the simple
extrusion of composite pellets prepared by compounding anatase and rutile with PP,
and the final film is prepared by consecutive compounding extrusion processes and
exhibits antimicrobial activity via a photocatalytic process [60, 61].
Stable thermal substances, such as organic acid salts or metal oxides, are excellent
antimicrobial substances to undergo repetitive extrusion processes (first compounding
and then film extrusion or a similar process) as the agent is homogeneously distributed
in the final product with practically no depletion of activity. However, there can be other
reasons for the use of a prior compounding process. Cerisuelo and co-workers [62]
developed an antimicrobial structure containing carvacrol as the antimicrobial substance.
Owing to the difficulty of incorporating a liquid into regular industrial extruders and the
accumulation of the organic compound in the atmosphere, caused by its high volatility
at the temperature of the extrusion process, a masterbatch of carvacrol in EVOH was
prepared in a compounder. The pellets obtained were then used to feed the central
extruder of a coextrusion line employed to manufacture a PP/EVOH/PP active film.
3.3.1.2 Preparation of Active Materials by Extrusion
Direct extrusion can also be used to obtain active antimicrobial films. In this case,
the pure agent, the agent previously treated by any of the procedures described
75
in Section 3.1 or the pellets prepared by previous extrusion compounding can be

mixed with the polymer, and any other adjuvant substance, in the extruder either by
previous mixing in the extruder hopper or by using the various feeding ports of the
extrusion equipment.
As mentioned in the previous section, metal oxides have been employed as

polymer additives to provide colour and/or promote opacity in packaging. Pellets
of microparticulated PO loaded with titanium dioxide are prepared by extrusion
compounding and are commercially available. Since it is not easy to obtain a good
dispersion of nanoparticulated oxides in polymers by simple extrusion, the dilution
of a concentrated masterbatch is preferred. Other methods include the previous
impregnation of oxides and metals in clays to facilitate their dispersion during the
direct extrusion process [60, 61, 63].
A biodegradable film was produced by blown extrusion using thermoplastic starch,

poly(butylene adipate-co-terephthalate) and 4.5% of potassium sorbate as an
antimicrobial agent. In this case, the thermal degradation of the polymer mix made
it advisable to prepare the final film using a single thermal process [64].
To study the effect of extrusion on antimicrobial agents, Del Nobile and co-workers
[65] produced pellets of various polymers compounded with plant extracts, thymol
and lemon extract, and lysozyme, and the extracts obtained were diluted with pure
polymer during cast extrusion. The study proved that thermal stress during film
preparation plays a key role in the efficiency of the active film, and polymers with
higher processing temperatures performed worse. When the agents were compared,
the enzyme exhibited the least degradation, whilst most of the thymol and lemon
extract was lost by volatilisation. Therefore it is recommended to prepare the final
film in a single step.
In studies to improve homogeneity of the final product and product dosing, some
authors observed better results when the plant extract was mixed with smaller polymer
particles, followed by grinding of the polymer pellets, impregnation of the resulting
powder with the antimicrobial agent and film extrusion [66]. In another study, the
active agent was diluted in glycerol (a plasticiser) to improve dispersion and fed, using
a liquid port, to the blown film extruder. To improve the efficiency of the film it was
produced via coextrusion, the antimicrobial agent being present in a very thin outer
layer in order to be in contact with the food [67]. Other plasticisers are also used to
improve the dispersion of agents, such as the addition of polypeptides during extrusion,
which immediately increases their diffusivity in the polymer and ultimately improves
release of the agent into the food [68]. Indeed, nisin is deactivated at temperatures
higher than 120 °C and therefore cannot be extruded with most commonly used
polymeric materials. Liu and co-workers [69] successfully incorporated nisin into
76
polylactic acid by modifying the profile of the extrusion heaters. The polymer was
melted with plasticisers at 160 °C and then the molten blend was cooled down to
120 °C before adding the antimicrobial agent.
Loss of active agent by volatilisation is problematic and has often been reported, as
the concentration depletion is considerable and can result in inactive film and high
economic losses [70]. To reduce volatilisation and improve retention of the organic
compounds responsible for the antimicrobial activity of plant extracts, several
authors have successfully tested incorporation of the extract with various clays [5,
71]. Another procedure is complexation of the volatile agent with cyclodextrins, as
explained in Section 3.1, which improves retention of volatile substances during film
manufacture [72].
CS is a cationic polymer that has been used as an antimicrobial agent in many food
products. Its main antimicrobial activity is obtained when the amine groups are
positively charged. Since CS is not a thermoplastic material, CS films are obtained by
solution and solvent evaporation, as mentioned in Section 3.3. However, CS has been
melt blended with other thermoplastic polymers to provide them with antimicrobial
characteristics. To enhance its efficiency, CS can be dissolved and cast to obtain
chitosonium acetate in order to convert it into an antimicrobial polymer. Massouda
and co-workers [73] obtained CS/silica powder by spray drying, the powder was
then mixed with pellets of various ethylene copolymers and melt blended in the
barrel of an extruder; CS was the component with the lowest concentration in the
final product at 4%.
3.3.1.3 Extrusion Coating
This is a process in which an extruder forces the molten polymer through a horizontal
die with a linear aperture, producing a curtain of liquid polymer which coats the
surface of a moving web of material (plastic, paper or aluminium foil), which produces
a coating. The substrate normally gives mechanical strength to the resulting structure
and the extrudate provides a gas, moisture or grease barrier or protection. In many
of these applications it is a requirement that the extruded material should exhibit
good adhesion to the web material surface. The receiving surface is often pretreated
using corona or flame treatment, or by impregnation with an adhesion-promoting
primer. After passing under the extruder, the coated web is pressed and heated by a
calender to a suitable pressure and temperature to improve adhesion and provide a
polished surface.
This process has been standardised to add PO to paper, board and other structures,
the most common coating resins being LDPE, EVA, ethylene-methacrylic acid
77
copolymers and ionomers. These polymers provide protection, a moisture barrier

and heat-sealing capacity.
Although extrusion coating is not the preferred processing technology in the

preparation of active packaging materials, Johansson and co-workers [74] made use
of this technique to manufacture oxygen scavenger structures based on coating paper-
based materials with polymers loaded with glucose oxidase. In addition, Nerin De
La Puerta and co-workers [75] patented an active packaging development involving
the application of an essential oil containing wax onto the surface of various web
materials, including paper.
3.3.2 Wet coating
Due to the difficulty of incorporating active substances into polymeric materials via
extrusion processes without loss or degradation and with good dispersion of the agent,
other procedures have been attempted. The preparation of polymer solutions and
dispersions is a common procedure in many fields of polymer application, including
adhesives, paints, lacquers, coatings and inks. Polymers and additives are dissolved or
dispersed in a solvent or solvent mixture, the solution is applied and spread onto an
object and the solvent is allowed to evaporate to obtain the required polymeric film
or coating. The various technologies that make use of a liquid dispersion or solution
of the polymer which is converted into a film by solvent removal are included in this
section on wet-coating technologies.
Wet-coating technologies have several interesting characteristics which should be

evaluated in order to choose the most suitable technique to produce active packaging
for a particular product. First of all, the polymer should be dissolved in a suitable
solvent and the solution obtained should have the appropriate viscosity for coating
and sufficient volatility to facilitate the drying process. Although this first step appears
to be an easy one, not all polymers dissolve easily; for instance, the most common
polymers in plastic applications, and particularly in packaging design, PE and PP,
are not easily soluble. PE and PP can only be dissolved in aromatic organochloride
solvents at high temperatures, as the boiling temperatures of these solvents are close
to or above 200 °C. In addition, the ability of these solvents to form a solution may
vary with members of the PO family, depending on branching, crystallinity, average
MW and tacticity. This issue can be even more complicated for food packaging
because of the potential solvent residues which may be present in the dry coating
and could be transferred by migration to the food, causing unacceptable organoleptic
modifications in the product or even induce toxicity.
Once the polymer and potential solvents or solvent mixtures have been selected, it is
necessary to consider the antimicrobial agent that is to be dispersed, and preferably
78
dissolved with the polymer in the same solvent. If either of the two solutes (or both)
is not dissolved but dispersed, then the result will probably be a heterogeneous
distribution of the antimicrobial agent in the polymer matrix along with phase
separation, which can be clearly observed and is dependent upon the particle sizes
within the dispersion and the drying process. If the polymer and antimicrobial agent
are soluble in the same pure liquid, they are homogeneously distributed in the solution
and the coating or film obtained at the end of the process will probably also be
homogeneous. However, the solution can become unstable during drying, resulting
in dispersion of one or both solutes due to: a) the two solutes (polymer and agent)
are concentrated in the solvent during drying and the solubility limit of one or both
of them has been reached; b) the components of the solvent mixture evaporate at
different rates, resulting in destabilisation of the solution; c) the temperature rise
during drying changes the stability of the solution; or d) the two solutes are soluble
in the solvent but mutually incompatible, promoting phase separation during drying.
After the agent and polymer have been dissolved or dispersed in the liquid mixture,
the solution should be spread on the surface of a film or 3D package and immediately
dried to produce a continuous film or layer. This can be achieved by several industrial
processes which are described below and basically include liquid coating and
spraying. Both technologies are fully established and equipment is available at many
converter facilities.
3.3.2.1 Coating
The surface coating of films is a common process which serves many functions
including: to improve the oxygen barrier characteristics of a film, e.g., the application
of a polyvinylidene chloride copolymer coating on a biaxially oriented PET or PVC
film, the application of a primer to modify the polarity or chemistry of a surface to
improve adhesion to other materials, the addition of an adhesive to produce tape,
and to fill or impregnate porous surfaces and so on.
Coating technologies using polymer-based solutions or dispersions are very widely

used and are economically attractive when the materials to be employed in the coating
processes can: be applied from high-speed rolls, be spread evenly and quickly over the
width of a very fast-moving web substrate, provide a fast drying or curing process,
and the final layer maintains good cohesion and sufficient adhesion to the coated
surface. They are especially interesting when the coating layer is applied at very low
thicknesses (0.5–25 µm).
Commodity polymers in the molten state cannot be included in this technology;

however, they can be applied as a coating via extrusion coating, as described in
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Section 3.3.1. Coating technologies use polymer emulsions such as those obtained
directly by emulsion polymerisation or those prepared specifically (for instance,
using the high-shear mechanical process developed by Dow Chemical, which yields
aqueous dispersions of spherical PO particles about 1 µm in diameter, stabilised in
water with the aid of anionic surfactants of low MW [76]), or polymer solutions in
organic solvents, such as aqueous solutions or emulsions.
The coating is usually applied to a roll of film. This process requires unwinding the
film, applying the coating, drying and then rewinding the finished coated web. The
types of equipment used for coating and web handling are too numerous and varied
to be described or even listed exhaustively in this section, but several monographic
books are available for consultation on this particular subject [77, 78]. However,
this equipment always consists of three main components: a coating head, which
applies a specific amount of coating to the surface and evenly distributes it over
the surface, a dryer to remove the liquid solvent from the solution or emulsion
and web handling equipment, which includes web drivers, winders, edge guides
and web controls. Additionally, a surface treatment device is required to improve
adhesion between the web material and the coating material, especially when the
web surface to be coated is made of apolar polymers such as PO. These devices
modify the surface energy level, producing or increasing the number of bonding
or anchoring positions available for adhesion by the coating medium. There are
various ways of achieving this objective, including gas plasma, gas flame, etching
with chemicals and, mostly commonly used, corona discharge, which uses a voltaic
arc to electrically charge the web surface, producing among other changes, oxidation
of the polymer surface. The treatment gradually dissipates over time, and therefore
most coating machines include a corona discharge device in-line, immediately
before the coating head.
There are basically two main types of coating heads: roll coaters and knife, blade or bar
coaters. Roll coaters are the most widely used and include direct roll coaters, reverse
coaters and gravure coaters. A diagram of these coaters is presented in Figure 3.5.
Metering Applicator Backing roll

roll roll Metering
roll
Doctor Engraved
blade cylinder
Applicator
roll
Backing roll Backing roll
Direct roll coater Reverse roll coater Gravure coater
Figure 3.5 Basic design of roll coaters used for packaging materials
80
Using direct roll coaters, the substance is deposited on the applicator roll and
is applied onto the substrate surface, the coating solution being split between
the substrate and the applicator roll surface without controlling the amount of
deposition. The thickness of the coating can be modified by the speed of the rolls
(a faster roller speed leads to a thinner coating) and the viscosity of the coating
liquid (a lower liquid viscosity leads to a thinner coating). After the deposition, rolls
covered with elastomers or similar devices are set in-line to improve the homogeneity
of the coating.
Using reverse roll coaters, the applicator roll rotates in the opposite direction to
that of the substrate material and transfers practically all the coating material
by wiping. These coaters can apply thicker coatings, from 25 to 300 g/m2 of wet
coating, which can also have a wide range of viscosities, and the resulting coating
is more uniform than using direct roll coaters. The amount of coating is controlled
by the gap between the metering and the applicator roll, and by the speeds of
the applicator and backing rolls. The backing roll normally runs faster than the
applicator roll.
Gravure coaters are very accurate coaters for thin coating applications. Their main
advantages are the precision of the amount of substance applied onto the web and the
homogeneity over the width. An engraved chrome-plated copper roll is wetted with
the coating, excess is removed by a doctor knife (scraper) and the coating remaining
in the engraved cells is transferred to the web. The engraving cells can be of any
shape, although common cells are pyramidal, quadrangular (truncated pyramid) and
trihelical. The amount of coating depends on the liquid volume that fills the cells
(cell volume and density). The coating solution must be sufficiently fluid to allow
the transfer of the coating from the cells to the web, and latexes and solvent-based
and water-based coatings are commonly applied using this technique. The amount of
coating deposited typically ranges between 0.5 and 25 g/m2. Gravure is also one of the
preferred printing technologies. The difference between coating and printing is that
during coating the coating solution is applied over the whole surface of the material,
while during printing the deposition is limited to the printing areas, i.e., discontinuous
coating. Gravure printing can be used successfully in active packaging because the
active agent and the polymer coating used as the vehicle can be applied where it is
needed, the substrate surface being responsible for other packaging requirements,
such as heat sealability.
Flexography, or flexo, is the other type of printing process most commonly used
in packaging. In flexo, a coating metering roll transfers the coating solution to an
intermediate rubber printing plate and finally to the web material, which can be of
any kind of substrate, e.g., plastic film, foil, paper or corrugated board. The main
advantage is the low cost of the printing plates and the precise coating transfer.
81
Once the coating solution or emulsion has been deposited on the substrate, it must
be dried very rapidly by evaporation of the solvent to obtain a solid cohesive layer.
There are various methods for performing this process including convection drying
with heated air, infrared (IR) heating and conduction heating. During the convection
drying method, the wet web goes through a hot air tunnel in which a stream of
heated air flows in the same direction as the web or in the opposite direction. In some
equipment the coated web is heated by IR light combined with convection drying to
accelerate solvent release. In both cases, convection drying and IR heating, the higher
temperature is at the upper surface of the coating layer and therefore the coating
dries from top to bottom.
In conduction drying, the web rolls are heated and they raise the temperature of the
substrate. This heating increases the temperature of the deposited coating which
promotes solvent release, but in this case drying takes place from bottom to top.
Drying has to be optimised because incomplete drying could result in problems of loss
of transparency with whitening or haziness, presence of residual solvent that might
lead to later migration issues or loss of adhesion to the substrate, and overdrying could
result in substrate degradation and increased cost; therefore drying is a fundamental
step in coating technologies.
During the preparation of active coatings, the active agent is dissolved in the polymer
solution. Since polymeric materials require more heating and stirring time than the
antimicrobial compound to obtain a good dissolution, in practice the active agent
is added once the processing of the polymer solution is complete and cooled down
(unless high temperature is necessary). This incorporation requires additional stirring
time to homogenise the final active agent/polymer/solvent mixture. Any kind of
incompatibility in this ternary or multiple system may lead to problems of solubility.
If the active substance or any of its components exhibit limited solubility in the
solvent mixture, inadequate incorporation of the active substance might take place,
along with phasing and heterogeneous distribution which, although stirring may
seem to solve the problem, could reappear during storage or later during the coating
and drying processes. The active substance might also be polymer immiscible and
its addition could cause solution cloudiness or even polymer precipitation. Solution
adjuvants such as surfactants or alternative solvent systems can provide a suitable
answer. Otherwise, the incompatibility may necessitate a different procedure of active
material manufacture or even a different active agent/polymer system.
There has been considerable effort focused on the development of polymer films
using natural polymers extracted from biological resources. Groups worldwide have
considered proteins, polysaccharides, waxes and their mixtures as potential substitute
materials for petroleum-derived polymers; however, their lack of gas barrier, low
82
mechanical resistance and high water sensitivity have been the main arguments
against them. The chemical and physical modifications of biodegradable oil-derived
polymers, along with irradiation, crosslinking or blending have been the main areas
of research. In parallel, edible food coatings have also been considered, reducing
the mass transport of gases, water vapour and aroma from and to the food, and
subsequently reducing the requirements and complexity of packaging.
In those studies, edible coatings were not considered solely as barrier layers to
reduce processes such as transpiration or respiration in fresh vegetables or oxidation
in nuts; biopolymer coatings were also thought of as matrices to carry interesting
compounds that increase the nutritional content of a product (vitamins, iron, calcium)
or reduce the incidence of microbial spoilage by the addition of antimicrobial agents,
or both [79]. Thousands of reports describe the manufacture of antimicrobial films
obtained from the prior dissolution of polymers and antimicrobial agents. Natural or
modified hydrocolloids, including animal proteins and hydrocolloids (gelatin, whey,
CS), and vegetable proteins (wheat gluten, corn zein, starch, cellulose) are water- or
alcohol/water-soluble and therefore it is easy to obtain hydroalcoholic or aqueous
biopolymer solutions containing many of the antimicrobial agents described in
Chapter 2. By spreading these solutions on a flat non-adherent surface and allowing
evaporation of the solvent mixture, cast self-standing films can be obtained. Many
documents describe these processes and characterise the mechanical, optical or
thermal properties of the resultant films. Moreover, their antimicrobial activity has
been tested against pathogenic and spoilage microorganisms, using in vitro and in
vivo tests. A representative selection of these studies is referenced in the introductory
description of potential antimicrobial agents in Chapter 2 or is mentioned in the
description of antimicrobial assays in Chapter 5. However, the use of these films
as effective antimicrobial packaging systems, either alone or incorporated into a
packaging structure, has not been investigated.
Piergiovanni and co-workers studied the development of a coating based on gelatin [80,
81]. An aqueous solution of gelatin was adjusted to a suitable viscosity to be applied
as a coating on a PP web. To develop the coated material, a pilot gravure machine was
used. The PP film was previously surface treated with an in-line corona discharger,
immediately coated by gravure with the gelatin solution, dried by heated air convection
and rewound. In this study no active components were incorporated, because the
applicability and potential use of gelatin coatings were the main objectives of the
initial studies, although the group was involved in their application as antimicrobial
structures (personal communication).
Antimicrobial coated packaging for food does not necessarily require the use of natural
polymers. As mentioned above, the only requirement is that a solution which combines
the polymer and agent should be obtained. Many polymers and agents are readily
83
soluble in water, alcohols, ketones and their combinations, and solvents accepted for
the preparation of materials for food contact, although some of them require drying
to ensure the absence of residuals that might reduce food quality or safety.
Gavara and co-workers developed antimicrobial coatings using EVOH as the

polymer matrix and an alcohol/water mixture as the solvent [82–84]. Essential oils
were incorporated in the polymer solution and the final solution was used to apply a
coating by gravure printing on PP and PET bioriented films. To improve anchorage
of the coating to the substrate surface, the bioriented film was first corona treated
and then gravure coated with a polyethylenimine primer. The final materials showed
antimicrobial activity and were used to manufacture modified atmosphere packaging
bags for salad. Ensuring the increase in barrier caused by the EVOH copolymer coating
did not lead to any unacceptable permeation into the salad and subsequent anoxia
was achieved by printing only partial areas of the bag surface.
3.3.2.2 Spraying
Spraying is an alternative method for coating a surface. One standard procedure

makes use of an airless spray system, in which a polymer solution, pressurised at
30–300 bars, is atomised into small droplets after exiting from a nozzle. In others,
pressurised air is used to atomise the polymer solution.
An airless spray is applied using a spray gun which includes a spray nozzle designed
to give the desired spray pattern, a needle valve and a gun trigger to turn the spray
on and off. This technique is commonly used in the application of coatings onto two-
piece metal cans. The spray application time is short (0.1 s) and during application
the can rotates very quickly to produce a uniform coating. The amount of coating
deposited is large and normally requires a drying and curing step.
Air-spray coating is a technique in which a spray gun or device with one or more
nozzles sprays a coating through the air onto a surface, using compressed air (or
other gases) to atomise it into particles and directing them towards the surface
to be coated. The air gun has a nozzle, paint basin and air compressor, and when
it is in operation the varnish is mixed with a compressed air stream, releasing
fine particles. The gun moves back and forth to ensure a continuous coating.
Alternatively, the nozzles can be mounted in a fixed position and the substrate
is moved under the sprayed particles. As the solvent travels from the gun to the
surface, part of it evaporates, reducing the time required for drying and providing a
cohesive coating. Air-spray coating is applied to cover flat polymer film surfaces and
the inside surface of plastic containers (bottles, jars). A diagram of this technique
is presented in Figure 3.6.
84
Figure 3.6 Air-spray coating application on a film surface or the inner

surface of a bottle
Artibal has developed antioxidant and antimicrobial varnishes for application by

air-spray coating to bottles of perishable fluid food products. The activity of these
varnishes is based on the incorporation of plant extracts and essential oils into a
polymer matrix which is dissolved in a highly volatile organic solvent [85, 86].
3.3.3 Surface Anchorage
As mentioned in the introduction of Section 3.3, it is also possible to manufacture

active materials that do not release or absorb an antimicrobial agent. Indeed,
biomolecules such as enzymes or bacteriocins can be immobilised on the polymer
surface of the package, exerting their activity by direct contact with the food
product: more specifically, with the microbial cells. Since biomolecules do not
migrate, their activity is limited to the contact surface only, so application of these
active materials is restricted to food/packaging systems where there is good (close)
contact at the food/packaging interphase, such as the packaging of liquid goods,
or skin packaging or slice separators of solid foods, always on the understanding
that their activity is restricted to the area of contact between the packaging system
and the food.
The first step in the manufacture of an antimicrobial film surface is the selection of
an appropriate polymer with the properties required for the end use. Commodity
polymers commonly used in the protection and marketing of foods in the form of
a simple film or as the innermost layer of a structure (inner surface of the package)
are generally hydrophobic and provide a good barrier to water, are heat-sealable
and should be inert with regard to the food, i.e., they are of a very different nature
to the food to reduce food/packaging interactions (in active packaging, obviously,
films are not completely inert). However, this inertness should be altered to provide
anchoring sites for the chemical or physical bonding of the active biomolecule, hence
packaging films usually undergo surface functionalisation prior to the attachment
of a biomolecule.
85
The immobilisation of these substances on conventional PO films may present some

difficulties, because their low surface energy, which results in poor printability and
coating properties, is also responsible for their low capacity to bond with biomolecules.
The selected polymer material should be cleaned prior to surface modification, to
improve the process. Surface modification techniques are then chosen depending
upon the desired functional groups (interacting chemistry) to be introduced onto
the surface. After the surface preparation, covalent bonding of the biomolecule is
attempted, which might require the use of bonding substances or linkers to obtain
the final functionalised polymer surface, as shown in Figure 3.7. Lastly, after washing
to remove any residue from the treatment (including unbonded biomolecules), the
surface changes and the activity of the films must be verified after each step in order
to validate the efficacy of the selected method [87]. In the following paragraphs, these
procedures are described as they are reported in scientific papers and patents, and it
must be understood that, as yet, they are laboratory procedures and further efforts
are still needed to make it possible to introduce these procedures at an industrial level.
Polymer surface
Bioactive compound Reactive functional groups
Figure 3.7 Covalent attachment of biomolecules to the polymer surface
3.3.3.1 Sample Preparation
Although this procedure can be performed on any type of polymer surface (polymer
pellets, powder, devices), the finished films are cleaned by immersion and sonicating
in various solvents to remove dirt, debris, grease or any other residue present on the
surface. This washing step is usually performed sequentially with an organic solvent
(isopropyl alcohol, acetone and so on) and deionised water. The cleaned samples
should be dried and any solvent residue which has sorbed into the polymer must be
eliminated before surface modification. The film structure, and especially its surface,
should not be altered after the treatment
86
3.3.3.2 Surface Modification
Surface modification has been implemented to retain favourable polymer bulk

properties while changing the surface to achieve a variety of objectives. Surface
modification is not only desirable, it is also essential when used to modify physical,
chemical, mechanical or biological characteristics in order to control subsequent
surface interactions and responses for any particular application [88]. The purposes of
these techniques include increasing adhesion, improving wettability, reducing friction,
reducing susceptibility to harsh chemical or environmental agents, and printing on
the polymer surface by introducing a variety of polar groups. Surface modification
should affect only the topmost layer, since treatments affecting deeper areas may
undesirably alter bulk properties and not promote any particular adherence to the
surface. Many different methods for polymer surface modification have evolved, the
most common of which are now described.
• Wet chemical oxidation treatments
In this technique the material surface is exposed to liquid reagents which generate
reactive functional groups on the surface. Wet chemical oxidation treatments increase
wettability or adhesion and do not require specialised equipment, hence can be
performed in most laboratories [89].
Treatments using polymeric materials such as PE, PP and polyester (PET) with wet
chemical reagents such as chromic acid, nitric acid or potassium permanganate
generally lead to oxidation, and the formation of carbonyl groups, hydroxyl groups
and carboxylic acid groups on the polymer surface. Sulfuric acid and sulfonates also
introduce functional groups (sulfonation), increasing the polarity and the potential
for hydrogen bonding, which improves wettability and adhesion.
It is also worth mentioning that these wet chemical methods have some disadvantages.
Overexposure to the treatment conditions tends to damage the polymer surface or can
introduce surface flaws that reduce bulk mechanical properties such as tensile strength.
Moreover, they are non-specific methods, producing a wide range of functional groups.
The degree of surface functionalisation may not be repeatable between polymers of
different MW, crystallinity or tactility. The need to use corrosive solutions makes this
process unsuitable for industrial implementation [90].
• Active gas treatment: plasma treatment
Plasma treatment involves a mixture of reactive species such as free radicals, electrons
and heavy particles, which makes it a very interesting tool for surface modification.
Plasma is defined as a gaseous environment composed of charged and neutral species
with an overall zero charge density. It offers numerous advantages over the conventional
87
chemical process, because it can modify the top nanometre of a polymer surface without
using solvents or generating chemical waste and being a clean, dry process it causes
less degradation and roughening of the material than wet chemical methods [90].
Although some interesting applications of plasma include surface cleaning by exposing

materials to plasma of inert gases such as argon, the most relevant application
(especially interesting for this work) is the introduction of functional groups onto
the polymer surface. The type of functionalisation imparted can be varied by the
selection of the plasma gas employed: water, oxygen, nitrogen, ammonia and so on.
Air, oxygen and water vapour plasma introduce oxygen-containing functional groups.
Nitrogen and ammonia introduce nitrogen-containing functional groups. Another
application of this treatment is plasma-induced polymerisation or grafting, which is
carried out using various polymerisation gases and precursors such as fluorocarbons,
hydrocarbons and silicon-containing monomers.
These methods do not require hazardous chemicals; they have the capacity to modify
the surface while imparting less degradation and roughness compared with wet
chemical surface modification methods. Plasma treatment is a good option for the
attachment of active agents because it is a less corrosive method.
• Corona treatment
This method is a continuous process in which an electrically induced stream of

ionised air bombards the polymer surface. Corona discharge results in oxidation
of the polymer surface, which is used to improve printability and adhesion of inert
polymers or to attach biomolecules to the surface.
• Ultraviolet irradiation
Irradiation of polymers causes the modification of properties which is currently the

basis of major applications. Exposure of polymer surfaces to UV light generates reactive
sites which can become functional groups; for example, EVOH films can be placed
under a vacuum UV Xe excimer lamp with 6 W at 172 nm (UV-Consulting Peschl
España S.L., Valencia, Spain) in an air atmosphere in an open glass Petri dish to oxidise
and create carboxylic acid functional groups on the film surface (unpublished work).
3.3.3.3 Attachment of Biomolecules to the Surface
As explained above, polymers usually have inert surfaces, so the surface must be
functionalised prior to attaching the bioactive molecule to it. The bioactive compound can
be attached by electrostatic interaction, ligand-receptor interactions or covalent attachment;
covalent linkage is the most common method as this linkage is often the most stable.
88
Essential oils have been incorporated onto the surface of PO films by impregnation
after corona treatment. This process increases the polarity of the surface, an effect
that is used to improve its wettability by essential oils and diffusion into the film [91].
However, the absence of chemical attachment produced a film whose mechanism of
action was the release of the active agent.
The specific functionality imparted to the surface must be compatible with the reactive
sites on the compound to be covalently attached to that surface. Crosslinkers might
be required in order to link the bioactive compound directly to the functionalised
substrate or to introduce a spacer of several angstroms. Selection of the crosslinkers
must be based on the conditions needed for the attachment, as the active organic
compounds differ in pH value, temperature and solubility. The blocking of certain
functional groups may be required in order to ensure coupling between targeted
functional groups. Amide bonds are formed between an amino group and a carboxylic
acid group; other common linkages are esters and thioesters, formed by interactions
between carboxylic acid and alcohols [87].
For example, as described by Hermanson [92], carbodiimide compounds provide the

most popular and versatile method for labelling or crosslinking to carboxylic acids. The
most readily available and commonly used carbodiimides are water-soluble 1-ethyl-
3-(3-dimethylaminopropyl)carbodiimide (EDC) for aqueous crosslinking and water-
insoluble 1,3-dicyclohexyl carbodiimide for non-aqueous organic synthesis methods.
EDC crosslinking is most efficient in acidic (pH 4.5) conditions and must be performed
in buffers devoid of extraneous carboxyls and amines. 4-Morpholinoethanesulfonic
acid (MES) buffer is a suitable carbodiimide reaction buffer. Phosphate buffers and
neutral pH (up to 7.2) conditions are compatible with the reaction chemistry, but
result in a lower efficiency; increasing the amount of EDC in a reaction solution can
compensate for the reduced efficiency. N-hydroxysuccinimide (NHS), or its water-
soluble analogue (sulfo-NHS), are often included in EDC coupling protocols to
improve efficiency or create dry-stable (amine-reactive) intermediates.
In order to immobilise active agents on the modified surface of polymers, films are
immersed in a buffer solution prepared with 5 × 10–2 M EDC and 5 × 10–3 M NHS in
0.1 M pH 5.2 MES buffer. The concentrations represent molar excesses of 100× and
10× for EDC and NHS, respectively. The films are shaken in the MES conjugation
buffer and then the active agents are added. The treated films are finally rinsed with
deionised water and dried.
Succinimidyl succinate active esters, and their derivatives, are other possible
reagents and have been extensively employed for polyethylene glycol (PEG)
conjugation to peptides and proteins. They react with free amine groups to form
stable amide linkages.
89
Occasionally it is necessary to use a polymer spacer molecule between the biomolecule

and the polymer, as some compounds such as enzymes may lose their activity when
linked directly to the surface. A spacer also helps to reduce steric hindrance to the
activity of the attached compound. Many different oligomers can be used as spacers,
although the most common is PEG. The main considerations in selecting a spacer for
a food packaging applications are: it should not disrupt the structure of the bioactive
compound, it must be approved for food contact use, and suitable chemistry must
exist to couple it to both the polymer backbone and the bioactive compound [93].
3.3.3.4 Surface Analysis and Determination of Film Activity
The surface changes and film activity must be verified after each step in order to
validate the efficacy of the selected method. Different methods can be used to analyse
the surface of the modified polymer. X-ray photoelectron spectroscopy is the most
widely used analytical technique to monitor the surface chemistry of solid materials
because of its simplicity, flexibility and sound theoretical basis.
IR spectroscopy is another very useful technique. IR absorption occurs if the IR frequency

coincides with the vibrational frequency of a bond. By monitoring the transmitted or
absorbed IR light, information about the molecular structure can be obtained. Attenuated
total reflectance IR spectroscopy is the method most commonly applied for the analysis
of surfaces as it reflects the chemistry of the material surface. A comparison of reflectance
and transmittance spectra provides information on the modifications introduced.
The biological activity should also be tested. The protocol selection will depend
on the biomolecule examined, i.e., enzymes, peptides, antimicrobial agents and so
on. Immobilisation of bioactive compounds may reduce their activity, so it is also
important to compare the activity of the substance anchored on the surface of the
film with the free activity of the compound alone.
It is also advisable to test for modification of the surface activity in each step and to
develop various migration tests to ensure that the bioactive compound is not released
from the film into a liquid medium or food simulant.
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4
Modelling and Optimisation of Active
Packaging: Parameters Related to
Antimicrobial Efficiency
The previous chapters have described the various antimicrobial agents and
combinations that are available, together with their mechanisms of action, i.e., the
methods by which the molecule inhibits microbial growth or leads to non-viability
or death of the cell (Chapter 2). That chapter also details important parameters
such as mechanisms of activation and the concentration range at which their
presence impedes microbial proliferation. Antimicrobial agents are incorporated
into packaging systems either in an independent device, in a conventional package,
or as a component of the formulation of the packaging structure, whereby the
packaging itself becomes active. The methods for manufacturing these antimicrobial
packaging systems are described in Chapter 3. In this chapter, the packaging
parameters governing the activity of the system are introduced and discussed,
including procedures for improving its final efficiency, the evaluation of which is
described in Chapter 5.
Food preservation technologies, including active food packaging, aim to place safer
products on the market. From a microbiological point of view, their objective is to
alter and impede key microbial functions. Heat, irradiation, pH and water activity
(aw) are some of the factors often involved in food processing. In the case of the
antimicrobial agents described in this book, they usually interact by chemically
modifying the environment surrounding the microbial cell or interact directly with
the cell membrane.
Some antimicrobial compounds are able to inhibit synthesis of the cell wall, a structure
that is critical for the survival of bacteria. For instance, lysozyme hydrolyses the
β-1,4-glycosidic bonds between N-acetylmuramic acid and N-acetylglucosamine
present in peptidoglycans, which are the main constituent of the cell wall of Gram-
positive bacteria. Others, such as essential oils, damage the cell membrane, modifying
97
the flow of substances between the cell and environment, mainly as a result of their
hydrophobicity. In both cases, the mechanism of agent action modifies the ability of
microbe to interact with the environment.
Several types of antimicrobial agents target bacterial protein synthesis by binding to

the subunits of ribosomes. This activity disrupts the normal metabolism of the cells
and leads to microbial death or inhibition of its multiplication. The latter can occur
by introducing metal ions as they bind to components involved in the synthesis of
bacterial deoxyribonucleic acid or ribonucleic acid, limiting normal cellular processes
and disrupting bacterial multiplication and survival, thus affecting the function to
reproduce.
With a few exceptions in which the antimicrobial agent remains in the packaging
and the targeted microorganisms are the mobile object which approach the agent, as
described in Section 3.3.3, it is necessary for the agent to be released into the food
or for a product of the reactions, between the agent and components of the system,
to be released. Alternatively, components of the food or environment are sorbed
into the packaging materials and react with or are absorbed by an antimicrobial
substance. Thus, mass transport processes between the active material, the food
and the surrounding atmosphere are involved in the mechanism of activity of the
active material.
4.1 Mass Transport
With the exception of packaging devices, active packaging systems in which

the agent is incorporated into the packaging material make positive use of the
mass transport phenomena that take place in polymeric materials [1], i.e., the
permeation, migration and sorption processes described in Chapter 1. The driving
force for these processes is the lack of chemical equilibrium between various
substances in the product/packaging/environment system. At the interphases of
this complex system, substances are sorbed into or desorbed from the packaging
material. The extent of these mass exchanges depends on the affinity of a given
substance for the two media which are in contact. Within the polymeric packaging
structure, substances diffuse from sites of high concentration to sites of lower
concentration, with the difference in concentration acting as a driving force.
Figure 4.1 indicates the interactions that occur when a food product, packaging
and the environment are placed in contact. Some of the possible interactions
are: (i) permeation of volatile components such as gases, e.g., oxygen, carbon
dioxide, water vapour and aroma components; (ii) sorption of food components
by the packaging material (i.e., aroma scalping); and (iii) migration of packaging
components into the food [2, 3].
98
Modelling and Optimisation of Active Packaging
Environment Packaging Food
M
M Headspace
S
S
M: Migration S
S: Sorption P
P: Permeation Food matrix
P
Figure 4.1 Mass transport phenomena in the food/packaging/environment system
Antimicrobial active food packaging systems can be divided into three categories:
absorbing systems, releasing systems and other systems. Absorbing systems remove
undesired compounds present in the food or in the packaging headspace, such as oxygen
or water. Releasing systems actively add or emit compounds to the packaged food or into
the packaging headspace, such as carbon dioxide or preservatives. Other systems are those
where the active material is not directly involved in mass transport processes, such as
surface-treated packaging materials (Section 3.3.3) or those that produce antimicrobials
in the environment after irradiation (titanium dioxide-containing materials).
Releasing systems are by far the most common. In these systems, the antimicrobial
compound inhibits microbial growth via its release from the polymeric material
onto the food surface or into the surrounding headspace. The antimicrobial agent
is mixed with the polymer either by dissolution in a common solvent system from
which the film is formed or by melt blending of the agent within the barrel of an
extrusion or equivalent machine (Section 3.3). In both cases, the active compound
is homogeneously distributed in the polymer matrix when the polymer and agent
are chemically compatible, or heterogeneously when the polymer and agent are less
chemically compatible, forming a multiphase matrix where sites enriched in the
agent can be distinguished. From this matrix, the agent diffuses to the surface of the
packaging and, at the interface with the food or headspace, it is partitioned between
the packaging and the food or headspace, respectively. The mechanism of action
differs depending on the volatility of the agent.
Volatile compounds can be incorporated directly into the polymer solution if they
are soluble, and from this solution an active film or structure can be obtained by
99
evaporation of the solvent. Depending on the volatility of the agent, the drying process
might result in considerable loss of the agent, limiting the efficiency of the incorporation
procedure. Alternatively, the compound can be incorporated by melt blending in a
mechanical-thermal process such as extrusion. In this case the agent is heated to the
melting temperature of the polymer, with potential losses by both degradation and
evaporation during film cooling. Whatever the procedure, the final polymer film acts as
a reservoir for the antimicrobial agents which will be released into the headspace and
from the headspace to the food product, especially during storage. The volatility of the
compound, its solubility in the food system and in the packaging walls determine the
final concentration of the compound in the food, which should always be higher than
the minimum inhibitory concentration (MIC) to be efficient [4]. Controlled delivery of
the volatile is desirable in order to reach a suitable concentration during product shelf
life, and restriction of release before packaging is advisable in order to reduce exhaustion
of activity during film storage. The use of volatile antimicrobial agents is especially
recommended for irregularly shaped solid foods which cannot be vacuum packaged, i.e.,
where good contact between food and packaging material is not possible (Figure 4.2).
Sulfur dioxide, essential oils, natural herbs and spice extracts are some of the
antimicrobial agents widely used. Sulfur dioxide is commonly used as an antifungal
agent to extend the shelf life of postharvest foods such as fruit and vegetables [5–7].
Essential oils or their components, such as thymol from oregano essential oil (OEO) or
cinnamaldehyde from bark cinnamon essential oil, have been widely incorporated into
many different matrices, either biodegradable (zein [8]) or synthetic {polypropylene
(PP) [9]}. Cinnamaldehyde has been incorporated into gliadin films to extend the
shelf life of bread and cream cheese [10, 11].
Antimicrobial
Packaging wall packaging layer
Volatile agent
Packaging
headspace
Figure 4.2 Antimicrobial packaging with a volatile agent
100
Non-volatile compounds can be incorporated either by mixing into the film-forming

solution or by melt blending during extrusion procedures. The non-volatility of the
compound reduces losses during processing despite potential degradation due to
interaction with the polymer matrix or the temperature required for film manufacture.
The main difference in comparison with the previous group of agents is that, owing
to their non-volatility, direct interface contact between the active film and food is
required for release and, therefore, for any antimicrobial effect on the packaged
product. Furthermore, in order to exhibit efficient activity, the non-volatile compound
concentration in the food portion, where the action is required, should remain higher
than the MIC during the shelf life. Non-volatile compounds are transported directly
from packaging materials to the food surface and by diffusion into the food matrix,
and are used effectively for semisolid or liquid foods where an excellent food/packaging
contact exists; the antimicrobial is quickly distributed in the food, exerting its action
throughout the whole product (Figure 4.3).
Organic acids and their salts, enzymes, bacteriocins, natural extracts and so on,
have been used as antimicrobial compounds in packaging applications; for example,
natamycin, a powerful antifungal compound, has been incorporated in different
biopolymers [12, 13], and nisin, a polypeptide produced by Lactococcus lactis, has
been widely used in packaging films [14–16].
Figure 4.3 Antimicrobial packaging containing a non-volatile agent
Volatile and non-volatile antimicrobial agents can be homogeneously distributed

in the polymer packaging wall or concentrated in an ‘active layer’ which can be
positioned as the innermost layer, i.e., in direct contact with the food, or sandwiched
101
between passive barriers which modify the extent and kinetics of the release of the
compounds. Knowledge of the extent and kinetics, and adequate characterisation
of food/packaging interactions are essential tools for designing the proper structure
which will deliver the agent at a suitable rate and concentration, and will limit the
exchange of undesired components.
The release of the antimicrobial agent is controlled by a combination of mass transport

phenomena that involve equilibrium and kinetic processes in the food, headspace and
packaging material [17]. Every mobile component in the food/packaging/environment
system is partitioned in all the phases that constitute the packaged product, including
the antimicrobial agent, as shown in Figure 4.4. Thus, the concentration ratio of the
agent at the interfaces is given by the partition coefficient between the packaging
material and the food (KP/F) as seen in Equation 4.1:
CP
KP/ F = (4.1)
CF
where CP and CF are the concentrations of the agent at the surface of the packaging
material and at the food surface, respectively.
For volatile agents, the substance is partitioned not only at the food/packaging interface
but also at the packaging/headspace interface and at the food/headspace interface.
In these cases the partition coefficients at the packaging/headspace interface (KP/HS)
and at the food/headspace interface (KP/HS) can be calculated as:
CP C
K P / HS = K = F
C HS and F / HS C HS (4.2)
where CHS is the agent concentration in the headspace. The accumulation of agent
in the packaging headspace is often measured as partial pressure (PHS), and then the
equilibrium at the interface can be written as the solubility coefficient of the agent in
the packaging material and in the food (Henry’s law):
CP C
SP / HS = S = F
PHS and F / HS PHS (4.3)
Equations 4.2 and 4.3 express the concentrations of a substance which is partitioned
between the two phases at the border of the two contacting phases, i.e., they represent
the equilibrium or the thermodynamics of the process. These equations can also be
helpful to determine the limit of the maximum extent of mass transport, as occurs when
a system achieves its chemical equilibrium. Both partition coefficients and solubility
coefficients are thermodynamic parameters expressing equilibrium and therefore are
102
commonly considered constant for a substance and two matrices but dependent on
temperature following Van’t Hoff’s law:
   
K = K0 × exp − ∆ H  or S = S0 × exp − ∆ H  (4.4)
 RT   RT 
Since the partitioning process can be exo- or endothermic, the molar enthalpy (∆H)
can be positive or negative, and from Equation 4.4 it can be stated that a temperature
increase can result in an increase in the extent of release of a substance or the contrary.
However, equilibrium is not always achieved within the marketing period of food
packaging. This is the basis of high-barrier packaging structures, which limit the
entrance of oxygen into the headspace of oxygen-sensitive products, thus making it
possible to market them. The second factor limiting or involved in mass transport
are the process kinetics.
Substance exchange between two phases is not immediate. Mass exchange at the
interface between the packaging and food or packaging and environment or food and
environment takes time. However, the limiting factor in food packaging containing
polymeric materials is commonly the mass exchange throughout the polymer matrix,
which is governed via molecular diffusion and, to a lesser extent, via the homogeneous
concentration within the food product.
The molecular diffusion of substances in a chemical system is assumed to follow

Fick’s well-known first and second laws, Equations 4.5 and 4.6:
J = −D × ∂c (4.5)
∂x
and
∂ c = − ∂ J = ∂  D ∂ c 
∂t ∂x ∂x  ∂x  (4.6)
The first law describes the flow of a substance (mass per unit of time and surface
area) within a homogeneous material in which a concentration gradient (∂c/∂x) has
been established. In Equation 4.7, D is the diffusion coefficient of the substance in
the matrix, commonly accepted to be constant at a given temperature (T) but it does
exhibit an Arrhenius dependence.
 E 
D = D0 × exp − a  (4.7)
 RT 
103
Since the activation energy for diffusion, as for every process, is always positive,
molecular diffusivity is always faster in warmer environments.
The second law describes the change of concentration in a polymer matrix as a

function of both concentration and time.
From the aforementioned description it is obvious that many factors are involved in
the rate of release of the antimicrobial agent from antimicrobial packaging, which
is schematically presented in Figure 4.4. Besides the influence of the thermodynamic
parameters S and K at each interface, the kinetics effects described by Fick’s laws
are also relevant. In the figure, the diffusion coefficient of the agent within the
packaging material (DP), within the food (DF) and in the headspace (DHS) have also
been plotted because they are key variables in the control of the release process.
DP is particularly important, as the mobility of the agent molecules within the
polymer matrix is usually the slowest process, and thus is the process that governs
the release mechanism.
Packaging Packaging Headspace

material material
SP/HS DHS
SF/HS
Dp DF Dp
DF
Kp/F Food matrix Kp/F Food matrix
Figure 4.4 Modes of action of antimicrobial food-packaging materials

and mass transport phenomena. A) Packaging material that releases the
antimicrobial agent directly onto the food surface in contact and B) packaging
material that releases the antimicrobial agent into the headspace and onto the
food surface in direct contact
Although K, S and D are assumed to be constant for a given agent/polymer/

environment, experimentally it has been demonstrated that in many cases this is a
mistaken assumption. Changes in polymer chain mobility, such as those produced by
plasticisers or crosslinking agents, and modifications of the tortuosity of the diffusion
path, e.g., by means of inclusion of particles in the polymer matrix, are among the
methods used to alter the values of these coefficients, and can be used for controlled
release of a substance or as a triggering mechanism for release.
104
4.2 Determination of Mass Transport Parameters
It is necessary to evaluate the parameters governing the mass transport processes that take
place in an active food/packaging system in order to optimise the efficiency of the system.
Since there are a large number of variables affecting these processes, as shown in the
previous section, and each process is influenced by others that are occurring simultaneously,
it is advisable to determine each parameter by isolating each factor or process.
Equilibrium coefficients expressing the concentration ratio for a substance between

two adjacent phases are parameters that give a good idea of the extent of each mass
transfer process taking place in a chemical system. In theory, the chemical potential
of a substance in a multiphase chemical system at equilibrium should be equal in
each phase of the system. However, chemical potentials, activities and fugacities
are variables which are difficult to measure, and chemists prefer, when possible, to
work with other variables which express the accumulation of a substance, such as
concentrations or partial pressures (for gaseous substances). Thus, the equilibrium of
a substance can be expressed as partition coefficients (K), when the preferred variables
involved are concentrations, or as solubility parameters (S), when the solute is volatile
and one of the phases involved is gaseous.
It is difficult to make an exhaustive list of the procedures used to determine KP/F values
in a food/polymer system because they vary with the characteristics of the system,
and therefore it is more pertinent to understand the most widely used methodologies.
When the solute can be obtained with high purity and it is liquid, a common
procedure involves putting the pure solute in contact with the food product and with
the polymeric material in different closed vials. At certain times, the concentration
of the solute in the food and in the polymer is measured using a suitable analytical
technique, which normally involves an extraction procedure and quantitative analysis
(spectroscopy, chromatography and so on). Several consecutive analyses providing
the same results are indicative that equilibrium has been achieved; the ratio of the
concentration of the solute in the polymer (cP) over the concentration in the food
(cF) provides the value of KP/F as written in Equation 4.1. This procedure was used to
determine the sorption capacity of carvacrol in composite chitosan (CS) films [18].
CS films were immersed in pure liquid carvacrol and the sorbed concentration of this
antimicrobial in the film was determined by analysing pieces of film using thermal
desorption coupled to a gas chromatograph.
This simple method presents some problems which are worth noting:
• The concentration of a solute which is absorbed by a solid depends on the

concentration to which it is exposed, and very frequently this dependence is not
105
proportional. This dependency makes K concentration-dependent, and therefore

it is recommended to measure it at concentrations approaching that of the actual
food/packaging system.
• The exposure of food or polymers to pure solid agents may be difficult, resulting
in a very low or slow mass exchange, which complicates the achievement of system
equilibrium and its measurement.
One commonly used alternative is substitution of the food by liquid food simulants
and, more specifically, the food simulants listed in national and international
regulations related to food/packaging migration processes. The agent can be
dissolved in the liquid simulant at concentrations in the range of the MIC or
minimum lethal concentration (MLC) and then a polymer sample is immersed in
this solution or alternatively the agent could be incorporated into the polymer
sample by casting or extrusion procedures and placed in the liquid simulant. The
simulant/polymer system is analysed from time to time until the concentrations
remain constant, and the partition coefficient value is estimated by means of
Equation 4.1.
Lopez-de-Dicastillo and co-workers [19] used extrusion to make ethylene-vinyl

alcohol copolymer (EVOH) films containing several flavonoids at various
concentrations and the resulting films were immersed in various liquids selected as
food simulants, i.e., water, alcohol/water mixtures and isooctane. The release of the
agents from the films was monitored by analysing the simulants using a previously
calibrated ultraviolet-visible spectroscope. The results showed that the partition
coefficient decreases upon the decreasing chemical compatibility of the agent and
the food simulant employed.
Solubility coefficients can be estimated by exposing a polymer or a food product to

a gaseous environment containing the volatile solute at a known partial pressure. In
many reports, the volatile agent is deposited in a liquid state in an open vial located
inside a hermetic container in which the food or polymer, or both, have been placed.
When equilibrium has been reached, the SF/HS or SP/HS values are estimated from the
concentrations of the agent in the food and the polymer phases, and by measuring the
partial pressure of the solute in the container headspace, as indicated by Equation 4.3.
From these values, the partition coefficient of the volatile agent between the polymer
and food can be estimated from the ratio of solubilities:
C P C P PHS SP / HS
KP/F = = × = (4.8)
C F PHS C F SF / HS
106
Equation 4.8 or a similar expression can be used to determine the partition coefficient
values between two phases of a complex system from partition or solubility coefficient
values obtained experimentally in simple two-phase systems, such as those mentioned
in the preceding paragraphs, provided that they have been obtained at the same
concentration and temperature, i.e., without large variations in the variables affecting
equilibrium.
This simple procedure has been used to determine the partition coefficients of
complex systems. Cerisuelo and co-workers [20] determined the solubility coefficient
of carvacrol in PP by sealing a sample of a PP film containing approximately 5%
of carvacrol, previously prepared by extrusion, in a hermetic vial equipped with a
sampling port. The carvacrol concentration in the headspace was monitored by gas
sampling with a syringe and analysis via chromatography until equilibrium, and then
the remaining concentration of carvacrol in the PP sample was analysed using thermal
desorption coupled with gas chromatography. The solubility coefficient for EVOH was
determined using a similar set-up. Since this material is hydrophilic, the hermetic vial
contained a small quantity of salt-saturated solution to maintain constant humidity
in the vial. The solubility coefficient values as a function of humidity were obtained
in accordance with the procedure described previously. From the results obtained for
carvacrol in PP and EVOH, the partition coefficient for carvacrol between PP and
EVOH was estimated using Equation 4.8.
In another report, the partition coefficient value for carvacrol between salmon and
EVOH was determined by exposing both solid materials to the vapour pressure of
carvacrol at saturation [21]. The antimicrobial agent, carvacrol, and salmon fillet
samples were enclosed in a hermetic vial, which contained a metal support to avoid
direct contact between the food and the agent. At equilibrium, small pieces of fillet
were cut and analysed using thermal desorption-assisted gas chromatography. The
solubilities in PP and EVOH were determined by the same procedure, and using all the
acquired data the partition coefficient values of carvacrol between the various pairs of
materials were calculated. These values were then used to describe the antimicrobial
activity of an active PP/EVOH/PP film to protect salmon fillets.
The evaluation of diffusion coefficient values is more complex and time consuming.
The procedure consists of monitoring the change of the concentration in the
polymer, or in the food, during sorption of the solute from a fluid medium in
which the solute is maintained at a fixed concentration, or analysing the increase
in solute concentration of a fluid caused by the release of a solute from a food or
polymer in which it was originally at a homogeneous concentration. From any of
these methods, values of the concentration in the food or polymer as a function
107
of time are collected. These data are then fitted to the appropriate solutions of
Fick’s first and second laws (Equations 4.5 and 4.6), which are readily available
in the literature [22].
When the boundary conditions of the experiments are that the film is homogeneous
with respect to the concentration of the agent and it is exposed on both sides to
an environment with a constant concentration of the agent, the solution to Fick’s
equations provides the change of substance concentration throughout the thickness
of the film [c(x,t)] which is a very complex function:
 D 2 n + 1 2 π 2t 
(−1) ( )  cos (2n + 1) π x
∞ n
4c
c ( x, t ) = c0 − 0
π ∑ 2n + 1
exp −
 4l 2  2l (4.9)
n =0  
In this expression, the centre of the film is at x = 0 and the film surface is located at
x = ±l. To compare the results of this equation to experimental values, the method
should include obtaining the concentration profile throughout the film thickness
at different exposure times. Then, by comparison with the theoretical c(x,t) values
obtained using Equation 4.9, the value of D can be obtained. Determining the change
of concentration in a single film with the dimensions of a packaging structure is
not simple. In addition to the complexity of the analytical procedure to precisely
determine the concentration at different film locations along the thinnest of the
three-dimensions of the materials, e.g., a tray sealed with a film lid, there is also the
difficulty of stopping the migration process during testing, which inevitably requires
an ‘in situ’ analytical procedure, such as spectroscopy. In 1980, this issue was solved
by Moisan [23]. A set of homogeneous films is stacked one on top of the other, using
a mechanical press to improve surface contact and eliminate trapped air which might
distort the migration process. Films containing a high concentration of a substance
are located at the centre or at both ends of the film stack. After a fixed time, the press
is opened and the films forming the stack are separated and analysed, providing the
profile of substance concentration along the x-axis at a given time. By repetition
using different time periods, a sufficient number of c(x,t) values like those plotted in
Figure 4.5 are collected and fitted using Equation 4.9. This procedure was used to
analyse the diffusion coefficient of antioxidants in films of various polyolefins using
the Moisan cell [23, 24].
A very similar procedure was used to characterise the diffusion of carvacrol in salmon
meat. A metal tube was filled with salmon fillets about 1 mm thick, exposed on one
side to liquid carvacrol. At specific intervals, the fillets were removed from the tube,
separated and analysed individually. Finally, the set of c(x,t) values was fitted using
Equation 4.9 and the D value was calculated.
108
Figure 4.5 Example of results obtained using a Moisan cell containing 40 films
with a thickness of 50 µm in which there is a substance of D = 5 × 10–14 m2/s at
4× exposure. A concentration of 1 corresponds to that achieved at equilibrium
However, the experimental data usually obtained are the average concentration in the
film or the total amount of substance present in the contact media. Some solutions
to Fick’s law include integrating the concentration of agent across the film thickness.
When the solute is not partitioned between the two phases, then the change of
concentration with time is given by Equation 4.10:
  
c (t )  DP (2 n + 1) π 2t 
∞ 2
= 1−
c (∞)  π 2
8
∑ 1 
exp −
 
(4.10)
n =0 (2 n + 1)
2
l2
  
where c(t) and c(∞) are the solute concentration at time t and at equilibrium,
respectively, and l is the film thickness. When the fluid volume is limited and the
solute is actually partitioned, then the equation describing the concentration change
is given by Equation 4.11:
c (t )  2 α (1 + α )  4 D q 2t 
∞
VS
c (∞) 

= 1− ∑ 2 2
exp − P n 
2
 ; α =
 VPK P / HS (4.11)
 n =1 1 + α + α q n  l
109
where qn are the positive solutions of tan (qn) = – αqn. Although Equation 4.11 includes
Equation 4.10 when K values approach zero, for the sake of simplicity partitioning
processes are ignored and D values are calculated assuming total extraction (or release) of
the agent. This assumption, however, is not always valid, especially when the food volume
is not high or the partial pressure at saturation is very low, and then the calculated D values
are smaller than the real values because the partitioning process is reflected as a slowing
of the diffusion process. Therefore we suggest performing a prior measurement of the
partition coefficient value (which is very simple) before attempting the analysis of D values.
Lopez-de-Dicastillo and co-workers [25] developed active packaging films by extrusion,

based on EVOH and polyphenol-enriched green tea extract. The film was immersed in
four food simulants and the release of the relevant extract components was monitored
using liquid chromatography. First, as suggested in the previous paragraph, the
partition coefficient values between the film and the simulants were calculated for the
various components under investigation, and large differences were observed in the
release percentage, caused by the affinity of the tea components for the food simulant.
A description of the release kinetics was then attempted, using both Equation 4.9, which
considers full extraction, and Equation 4.10, which takes into account the achievement
of a partition equilibrium. In this study, a portion of the catechins present in the tea was
covalently bonded to the polymer or polymerised during extrusion, impeding their release.
Thus, although the release did not reach total extraction, Equation 4.9 described the
release of components better and therefore allows more precise calculation of the D values.
4.3 Release Control
The simplest active packaging material is one in which the active compound is
homogeneously distributed in a single-layer film. The film is used to package a food
product which is in contact with the whole internal surface of the package (there is
no headspace) and the food is liquid, that is, any agent molecule reaching the food
is immediately distributed so that the agent concentration in the food can always be
considered to be homogeneous. In this system, schematically presented in Figure 4.6,
the active substance is desorbed by the active film surface until equilibrium is reached.
Figure 4.6 Diagram of the release of an active agent from a single-layer active film
110
However, desorption by the external surface may also occur, resulting in part of the
agent being released into the atmosphere, as shown in Figure 4.6, which is ineffective.
The desorption of an agent into the environment will depend on the value of the
solubility coefficient SP/E. When the value of this parameter is very low, it indicates
that the agent is very volatile, while the contrary indicates low or zero volatility, as is
the case with many of the so-called non-volatile antimicrobial agents (metal, oxides,
peptides and so on).
A B
Exposure time
Agent concentration
Agent concentration
Position along film thickness Time of storage
Figure 4.7 Diagram showing the concentration change of a non-volatile agent

in the film (A) and in the food product (B) for a food/packaging system like the
one represented in Figure 4.6
Figure 4.7 represents the release into a food product of a non-volatile agent initially
present homogeneously in a single-layer film. As can be seen, the non-volatile nature
of the agent results in an SP/E value that tends to infinity, and therefore no loss of
agent is expected to occur through the external film surface in contact with the air
in the surrounding environment. In contrast, the agent concentration at the film/
food contact surface diminishes immediately, causing a concentration gradient
that makes the agent diffuse towards the food, resulting in the concentration
profiles represented in the figure. With time, the average concentration in the film
decreases and the agent accumulates in the food, which should lead to the desired
antimicrobial activity. If the system reaches equilibrium during marketing of the
product, only a partial amount of the agent will be released, with the film acting
as a reservoir of agent which will be released to replace any agent degradation or
loss that might take place.
A different process occurs when the agent is volatile, as the release advances
through the inner and outer surface of the film, i.e., into the headspace but also
into the environment, as Figure 4.8 shows. In this figure, the agent released through
the surface on the right (of Figure 4.8A) enters the food and accumulates, but
the agent that is transported to the environment (moving towards the surface
on the left of Figure 4.8A) is lost from the food/packaging system, and since
the environment is very large the substance does not accumulate. As the agent
111
concentration in the film starts to become exhausted (equilibrium will never be

achieved), the food now becomes the part of the system that acts as a reservoir
of the agent, and the active molecule goes from the food to the package and from
the latter to the environment.
A B
Exposure time
Agent concentration
Agent concentration
Position along film thickness Time of storage
Figure 4.8 Diagram showing the concentration change of a volatile agent in the
film (A) and in the food product (B) for a food/packaging system like the one
represented in Figure 4.6
According to the mechanism of action of these releasing systems, the efficiency

depends on whether agent concentration above the MIC or MLC is achieved at the
point where it is needed. Normally, a specific partial pressure of the headspace or
a specific concentration at the surface or in the outermost portion of the food is
needed. In other cases (liquid products), the MIC or MLC value should be exceeded
in the food bulk. These theoretical values of concentration along with the values
of KP/F and SP/E provide the minimum amount of agent that the active film should
contain. However, equilibrium is not normally achieved during the marketing of a
product in an active packaging system, and in these cases the diffusion coefficient
values are relevant.
Indeed, the rate of agent diffusion plays an important role in terms of the sustained
antimicrobial activity on the food product, especially in systems such as the one
represented in Figure 4.8. Rapid release causes a very high concentration of the agent
just after the packaging process, probably reaching an accumulated concentration
above the MIC value. After this the maximum concentration value decreases rapidly,
and the agent concentration may fall to values below the MIC before the end of
the product shelf life, which means that the packaging design is inefficient. In the
opposite case, if the release rate of the active agent from the polymer matrix is too
slow, the increase in concentration is so gradual that by the time the active agent
reaches the MIC value (if this occurs) the microorganisms have already proliferated
in the food and the food is spoiled. The rate of antimicrobial agent release depends
on several factors, such as the carrier material and its interactions with the active
substance [20, 26], swelling of the polymer matrix in food [27, 28], affinity of
112
the food for the polymer [29], solubility of antimicrobial agents in food [30] and
exposure temperature.
The loss of agent caused by its release to the environment can be avoided or reduced
by correct packaging structure design. Figure 4.9 shows an example of a correct
design for a volatile agent, and presents a bilayer packaging structure in which the
active layer is separated from the environment by a barrier layer.
Figure 4.9 Diagram of release of an active agent from a bilayer active film
There are various mechanisms by which the barrier layer represented by polymer
2 (P2) in the figure can impede or restrict mass transport of the agent towards the
environment. The solubility of the active agent in this polymer may be very high,
reducing its volatility and consequently its transition to the vapour phase. Low
transition of the agent to the vapour phase is uncommon, because the requirement
to protect against loss of agent from the active layer is normally a high partial
pressure at saturation of the agent. If SP1/E is very low and SP2/E is very high, then,
from Equation 4.8, the partition coefficient for the agent between the two polymers
will favour dissolution of the agent in P2, the consequences of this being partial loss
of agent by retention in the barrier layer and reduction of the release into the food,
with a corresponding decrease in efficiency.
The most preferable release mechanism is the use of a material whose matrix
provides very slow diffusion of the agent, i.e., DP2 << DP1. With this type of
structure the agent has restricted mobility through the barrier layer and the main
release of the agent is through the surface in contact with the food. However,
this is a barrier based on kinetics which will change as the film exhibits ageing
during storage. In fact, in the bilayer structure of the web every active layer
is surrounded by the barrier layer of the structure and by the barrier layer of
the previous layer of the web, and over time the whole web advances towards
equilibrium, reducing the concentration of agent in the active layer and increasing
the concentration in the barrier layer, as described in Figure 4.10. The lines
show the change of agent concentration for a bilayer film during web storage,
assuming that KP2/P1 = 1.
113
Figure 4.10 Change of agent concentration in each layer of a bilayer film

during web storage, assuming that KP2/P1 = 1. The curved lines show the
concentration profile for a given storage time, decreasing in the active layer
and increasing in the barrier layer
Structures of this type were prepared for active packaging of chicken fillets and salad.
Carvacrol, OEO and citral were incorporated into polymers by casting or gravure
printing technologies on EVOH and CS [31, 32]. To reduce loss by evaporation from
the packaging, bilayer structures were prepared with biaxially oriented polypropylene,
biaxially oriented polyethylene terephthalate and aluminium foil. In all cases, the
more rapid diffusion of the agent in wet EVOH and CS resulted in release of the
agent towards the food product, as desired.
Sometimes the active component is generated from a substance or mixture of

substances, as in the preparation of some gas releasers (chlorine dioxide), because
the initial components are not acceptable for food contact. In other cases, nanoclays
are incorporated to reduce the diffusion rate of agent release, improving its control.
In these cases, besides a barrier layer to force the gas towards a preferred direction
(the food), it is necessary to add another layer in the structure to avoid migration into
the food of the particles involved in activating the packaging system, because this
migration could result in a loss of food quality or compromise food safety. This layer
is called a functional barrier in migration studies and an example of such a structure
is shown schematically in Figure 4.11.
Figure 4.11 Diagram of the release of an active agent from a three-layer active film
containing the active layer sandwiched between a barrier layer, to reduce agent
loss to the environment, and a functional barrier to reduce the risk of potential
migration of toxic or unacceptable substances
114
Although EVOH and OEO are valid materials for food contact, Cerisuelo and
co-workers [21] prepared a PP/EVOH/PP structure by coextrusion in which the
active compound was incorporated into the central EVOH layer. The release of the
agent was activated by the presence of humidity, which plasticised the EVOH layer,
promoting and accelerating the release of carvacrol. In this structure the external PP
layer acts as a barrier to limit the entrance of water from the exterior towards the
EVOH film and at the same time it impedes loss of the antimicrobial compound via
evaporation to the atmosphere. The internal PP layer acts as a functional barrier,
since it is responsible for controlling release of the agent and extending it during the
shelf life of the product.
Structures with two or more layers have become very popular. At present,
monolayer food packaging is rare and mostly limited to bottles and trays.
Sometimes the complexity of the packaging is due to requirements of the food
products, which cannot be achieved by a single-layer structure, or to printing
and labelling which require protective layers. In other cases, the multilayer film
is due to the use of recycled materials and the need to preserve the safety of the
food product by the application of a virgin layer. In addition to this, there have
been developments in optimising the structure of active packaging to improve
its efficiency.
Such complex systems cannot be described by a single formula that includes all the
mass transport processes involved in a highly heterogeneous packaging structure.
With the aid of computer science and numerical methods it is possible not only to
describe these processes but also to optimise them.
4.4 Modelling Release of the Agent
Packaging related mass transport processes are involved in many mechanisms of food
spoilage and therefore knowledge and understanding of them is crucial to maintain the
quality and safety of foodstuffs. Permeation of gases and vapours through packaging walls
is frequently involved in processes of deterioration such as weight loss and dehydration of
high aw food, oxidation in fatty food, carbon dioxide damage or anoxia in the packaging
of fresh produce and so on; therefore, there is considerable interest in the description
and estimation of these processes. However, despite the large number of variables
affecting mass transport (polymer variables such as crystallinity, orientation or thickness,
environmental variables such as pressure, humidity and temperature, and food variables
such as composition, aw and so on), in most cases calculations are performed using a
worst-case scenario, estimating the limiting factor, applying a critical value and simply
applying the simplest solution to Fick’s laws, which is only valid in a system where all
conditions are remain constant. As is well-known, integration of Equations 4.5 and 4.6,
115
assuming that Henry’s law applies (Equation 4.3), provides Equation 4.12 which defines
the permeability (P) of a material or the permeance (℘) of a structure:
q×l q
P= or ℘= (4.12)
A × t × ∆p A × t × ∆p
which can be used to estimate the shelf life of a product by solving for ‘t’, or to decide
which type of structure is required to provide a specific shelf life by solving for ‘℘’.
Estimation of migration mechanisms has also been a major subject of attention,

because the release of additives and residues present in the packaging could be
related to an unacceptable sensory impact on product quality or even toxicological
effects on the consumer. In addition, there are many reports of efforts to describe
and provide an overall estimate of migration values for substances of interest. As
mentioned in the previous paragraph, many variables modify migration processes;
particularly relevant are the variables that affect the diffusion of residues in the
polymeric matrix, but the partition equilibrium of residues between the food and the
packaging and diffusion in the food are also important. However, since migration
is normally understood as an involuntary process, instead of thinking in terms of
partitioning, the residue is considered to be fully extracted by the food product and
diffusion into the food is considered to be very fast. Under this worst-case scenario
the estimation is based exclusively on diffusion into the packaging structure, which
provides a safe margin, so the migration in real conditions will always be less,
ensuring the safety of the product.
However, these worst-case scenario estimations are not valid for active packaging
which involves the release of antimicrobial substances. It has often been reported that
the antimicrobial effectiveness of a specific substance varies greatly depending upon
the environmental conditions or type of food (Chapter 5). Moreover, as mentioned in
Section 4.3, efficient active packaging should provide the right dose of antimicrobial
agent and maintain that dose during the shelf life of the product, achieving the
anticipated protection against microbial spoilage but reducing any side effect such
as sensory damage. Therefore an optimisation process is required, and for this task
theoretical description and modelling are crucial. With the exception of very simple
active packaging systems which can be described by analytical solutions to Fick’s
laws, such as those presented in Equations 4.10 and 4.11, the most common examples
of more complex packaging include heterogeneous packaging structures obtained
by lamination, coextrusion or coating, packaging consisting of simple or blended
polymers, and composites including inorganic fillings (nanocomposites), for which
mathematical solutions are not obvious. In addition, active packaging systems are
normally designed for a specific product on the basis of its particular characteristics,
type of microbial loading, composition and potential interaction with the active
116
agent, and therefore all these variables must be considered in the study of packaging
efficiency. Such complex systems can only be described by using computer-assisted
numerical analysis procedures. Among the many algorithms reported in the literature,
the finite difference method(s) (FDM) and the finite element method are noteworthy
because of their considerable simplicity and their demonstrated efficacy, and therefore
they are described in the following subsections. Both methods consist of solving
differential equations using partial derivatives by the previous discretisation of the
physical system into finite subspaces, which is done in such a way that the complex
and difficult expressions which cannot be solved and the heterogeneous or even
changeable boundary conditions are reduced to many simple algebraic equations or
differential expressions easily solvable by conventional analytical procedures.
4.4.1 Modelling and Description using the Finite Difference Method
Basically, the technique used in the FDM consists of the solution of differential
equations using partial derivatives by approximating the derivatives to finite
differences. Thus, finite differences approximate partial derivatives, FDM being part
of the above-mentioned discretisation methods. At present, many numerical solutions
of partial differential equations are estimated using FDM.
The method is based on the application of Taylor’s theorem to obtain the value of a
function F(x) in one position (x1 = x0 + ∆ x) if the value of the function is known in a
neighbouring position (x0) and the function is continuous and differentiable. Under
these conditions, Equation 4.13 shows the value of the function at x1 can be expressed
as a function of the successive derivatives of F (F’, F’’, …):
F ( x1) = F ( x0 + ∆ x)
F ´( x0 ) F ´´( x0 ) (3) x
= F ( x0 ) +
2 F ( 0) ∆x 3 +…
2! ( ) 3! ( )
∆x + ∆ x +
1! (4.13)
F ( ) ( x0 )
n
( ∆ x) + Rn ( x)
n
+
n!
where Rn(x) is the remainder term (or residual term) which accounts for the error
of the approximation. In this expression, the approximation is better [Rn(x) is less
significant] as the number of terms in the succession increases and as the closeness
of x1 to x0 increases. In this last condition, Taylor’s polynomial can be approximated
to the first derivative as shown in Equation 4.14:
F ´( x0 )
F ( x1) = F ( x0 + ∆x) = F ( x0 ) + ∆x (4.14)
1!
117
And by rearranging terms, the derivative can be expressed as the ratio of the function
increment over the variable increment as written in Equation 4.15:
F ( x0 + ∆ x ) − F ( x0 )
F ´(x0) = (4.15)
∆x
This approach can be used to determine the change of the concentration of a substance
over time and at each position in the packaging. The process involves dividing the
packaging structures into a finite number of small slices of thickness ∆x, and the time
of exposure into finite intervals of time ∆t, as expressed in Figure 4.12.
∆t
Time (t)
c(x,t−∆t)
c(x−∆x, t) c(x,t) c(x+∆x, t)

t
∆x
x
Position along thickness (X )
Figure 4.12 Grid representing the finite slices of thickness and time intervals
By applying the approximation of Taylor’s function (first derivative) to the change

of concentration of agent at a specific position in the film (x), the concentration at a
specific time (t + ∆t) can be estimated by the Equation 4.16:
    c ( x, t + ∆t ) − c ( x, t )
c ( x , t + ∆ t ) ≈ c ( x , t ) + δt  ∂ c  →  ∂ c  = (4.16)
 ∂ t  x ,t  ∂ t  x ,t δt
In the grid built on the position and time axes, the intervals are selected homogeneously in
such a way that a position x = n × ∆x and that any time t = m × ∆t. Moreover, the magnitude
of each time step ∆t is small enough for application of the Crank–Nicolson method [33],
which is based on the central differences of first order for each time interval and on the
central differences of second order for the position derivative at position x. With these
boundary conditions, and applying Taylor’s approximation, Equation 4.17 can be obtained:
c ( x + ∆ x, t + ∆ t ) − 2 × c ( x, t + ∆ t ) 
 
DP × + c ( x − ∆ x , t + ∆ t ) + c ( x + ∆ x , t ) 
  (4.17)
c ( x, t + ∆t ) − c ( x, t )  ( ) (
− 2 × c x, t + c x − ∆ x, t
) 

=
∆t 2 × ∆x 2
118
Although this is not an explicit equation, it provides a system of algebraic equations

which allows calculation of the concentration profile throughout the film thickness
at a time t + ∆t provided that the profile at time t and the boundary conditions at
the limits of the film are known. In this way, by calculating one ∆t after another, the
change of agent concentration can easily be obtained. The restrictions on using this
method are that all time intervals and all slice positions have to be equal and their
size requires that some conditions converge satisfactorily.
This method was used to describe the migration of caprolactam from a multilayer
structure composed of polyamide/polyethylene to cheese [34]. The authors had
previously determined the partition coefficient of the migrating agent at the various
interfaces of the multilayer structure and its diffusion coefficients in each matrix. With
these data, the FDM was applied to describe the migration, and finally, the values
were validated experimentally.
FDM were also used to determine mass transport systems in which external
conditions were varied during storage [35]. In this investigation, which was
presented at a conference, variations in relative humidity (RH) and temperature
were added to the mass transport of permeating or migrating agents. The finite
difference approach, Equation 4.15, was used to describe the change of temperature
in the system, assuming that the external temperature followed a specific profile
and applying Equation 4.15 to Fourier’s law. In this way a matrix of temperature
for certain intervals of position and time was calculated [T(x,t)]. By previously
measuring water permeability at various temperatures, a function describing
the sorption and diffusion of water within the material was obtained. The mass
transport of water was then analysed using Equation 4.17, introducing the D
value at each point of the grid using the temperature profile previously obtained.
The results explained the mass transport in a dynamic system such as a hot-filling
packaging process.
In the same conference, the effect of mass water transport on the permeation of
oxygen through a hydrophilic polymer membrane was also studied [35]. In this
work, the FDM was first used to generate a matrix showing the change of the
water profile within a PP/EVOH/PP polymer structure exposed to various water
gradients. Then, using previously obtained oxygen diffusion coefficient values as a
function of humidity, the oxygen permeation through the structure was determined
in dynamic conditions and symmetric and asymmetric structures were compared,
as Figure 4.13 shows.
119
100 1×9−10
50−90% HR
t=0
80 t = 15,000 s 100% 50−90% HR, PP/EVOH/PP 100/10/100 µm
t = 30,000 s −10 100% 50−90% HR, PP/EVOH/PP 50/10/150 µm
60 8×10
Oxygen flow (m /m × S)
t = 45,000 s
Sorbed water (kg/m3)
t = 60,000 s
40 t = 75,000 s
2
t = 21,0000 s
20 6×10−10
3
t = 36,0000 s
t = 45,0000 s
t = 60,0000 s
4×10−10
2×10−10
0 0
0 20 40 60 80 100 120 140 160 180 200 220
10 0
0
10 00
0
0
0
00
00
00
00
00
00
00
,00
00
Position throughout thickness (µm)
,0
2,
0,
1,
0,
0,
0,
0,
0,
50
20
30
40
50
60
A) B) Time (s)
Figure 4.13 Example of use of the FDM in the description and modelling of mass
transport processes in complex systems (multilayer structure) exposed to dynamic
conditions and with at least two concentration gradients (water and oxygen).
A) Change of the sorbed water in a symmetrical PP/EVOH/PP structure exposed
to 50% RH on the left surface and 90% on the right surface and B) change of the
oxygen inflow when the structure is kept at different external conditions
4.4.2 Modelling and Description using the Finite Element Method
Like the previous technique, the finite element method can be used to solve any system
of partial differential equations by first transforming them into a system of ordinary
algebraic or differential equations that can be easily treated by means of traditional
analytical methods or by other numerical methods that are easier to apply. In this
technique, the product of discretisation of the domain of the partial differential equation
also consists of a large number of nodes that form a mesh and small subdomains of
finite size, called ‘elements’, which in this case are completely interconnected and can be
treated mathematically one-by-one, forming an interrelated set of very simple models
that are easy to analyse. In this way, by applying the analysis of the finite elements to
these subdomains, an approximate value of the real solution for each node is obtained.
The greater the density of the mesh that is generated in the domain of the function
that is being simulated, the more accurate the value obtained will be.
The usual procedure for solving a partial differential equation by means of this
numerical method basically consists of the following steps [36]:
• Expression of the partial differential equation in its weak form (integral form),
separating the Dirichlet boundary conditions [related to the concentration of a
property, c(x,t)] and the Neumann boundary conditions [related to the flow of
a property, ∂c(x,t)/∂x].
120
• Discretisation of the domain into finite elements by generating a polygonal

mesh of interconnected nodes [usually three-dimensional (3D) tetrahedrons
or hexahedrons, and non-overlapping two-dimensional (2D) quadrilaterals or
triangles].
• Approximation of the functions as the interpolation of node values with shape

functions, normally n-linear or n-quadratic.
• Selection of the trial functions for verification of the weak form (e.g., those
defined in the Galerkin method, based on expression of the trial function of the
solution as a linear combination of the product of the value of each node and the
corresponding global shape function).
• Obtaining the corresponding linear system (with a non-regular matrix obtained

from the assembly of matrices associated with each element).
• Imposition of the boundary conditions.
• Solution of the matrix of the linear system by conventional methods of linear

algebra in spaces of finite dimension.
The main advantages presented by the finite element method in comparison with
the FDM lie in the possibility of using irregular and/or unstructured meshes (i.e.,
consisting of finite elements of variable size and shape), the possibility of imposing
boundary conditions systematically and increasing convergence for successively finer
divisions of the finite elements.
The finite element method is used to describe and model complex mass transport
processes such as those occurring in most active antimicrobial packaging systems.
Cerisuelo and co-workers [20] developed a method to model a 3D pillow-type bag
made of a PP/EVOH structure containing carvacrol in the EVOH layer. The system
was assumed to consist of four different material layers: environment, PP, EVOH-29
and packaging headspace; in other words, the external atmosphere and the internal
headspace were included in the system as material layers with a very fast diffusion
coefficient. To reduce the complexity of the model, the environment was considered to
be constant throughout the storage. The spatial discretisation of the system consisted
of a mesh with more than 1,000 nodes and the mass transport of water and carvacrol
within the system was calculated using a mathematical suite, COMSOL. The mass
transport of water was calculated first, since it only depends on time. Then the
carvacrol profile and change within the different layers of the system were evaluated,
assuming that the diffusion and solubility coefficients at each mesh dot were constant
(dependent only on the water concentration at that particular position and time). The
results were validated using experimental testing. Similar approaches were used to
121
describe carvacrol release in salmon fillets packaged in a PP/EVOH/PP bag containing

carvacrol in the central layer. In both studies, the model shows that the uptake of
water by the EVOH layer, which starts the moment the food is incorporated into the
packaging, actually acts as a triggering mechanism, initiating release of the carvacrol
and the antimicrobial activity on the packaged product.
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Engineering, 2012, 109, 513.
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2014, 36, 256.
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E. Almenar and M. Pilar Hernandez, International Journal of Food
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R. Gavara in Emerging Food Packaging Technologies, Eds., K.L. Yam and
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P. Hernández-Muñoz, Carbohydrate Polymers, 2013, 97, 262.
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P. Hernández-Muñoz, Journal of Agricultural and Food Chemistry,
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B. Vallejo-Cordoba, N. Gamez-Meza and A.Z. Graciano-Verdugo, Food
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124
5
Methods for the Analysis of Antimicrobial
Packaging Efficiency
Several factors must be controlled to test antimicrobial agents applied in food: strain
of microorganism, phase of growth, pH, temperature, food matrix, water activity (aw),
equipment and so on. The design phase of food packaging involves the development
and modification of conventional microbial methods to study the efficiency of the
antimicrobial material [1]. The effectiveness of the developed film can be tested using
different methods, depending mainly on the antimicrobial agent used. The antimicrobial
agent can be incorporated into the packaging via different routes including: into a layer
of a multi-laminate structure exerting its action by direct contact of the packaging wall
with the food or via its release from the packaging wall to the food or headspace; it can
be included in the polymer matrix and migrate to the surface of the food via diffusion; if
the antimicrobial agent is volatile it will be delivered in the vapour phase to the packaging
headspace; in addition, the active compound can be immobilised in the polymer and
produces its effect by direct contact with the food surface (without migration).
The methods for testing antimicrobial packaging effectiveness can be classified into
two groups: 1) in vitro methods carried out using ideal temperature and culture
medium conditions and 2) in vivo methods applied directly to a real food product
under its storage conditions.
The first step is recovery of the microbial strain using optimal conditions before
performing any in vitro or in vivo tests with the antimicrobial packaging. Strains
are usually supplied by culture collections; the main function of these organisations
is the collection, maintenance and supply of cultures. The selection of maintenance
methods that enable maximum survival levels and strain stability is very important
because non-viable strains are unacceptable in a microorganism collection. Therefore,
in order to ensure a reliable service, quality control measures are carried out routinely.
Bacteria and fungi can be obtained from the culture collections of several universities
and research institutes; some of the more important collections include: United
Kingdom National Culture Collection, German Collection of Microorganisms and
Cell Cultures, Spanish Type Culture Collection and American Type Culture Collection
(ATCC). Each strain is provided with details of technical procedures and information
125
regarding the history, isolation data, growth conditions (temperature, culture media),
risk group, tolerances and sensitivities.
The ATCC is the main source for microbial reference strains and was created in 1925.
The ATCC provides fully characterised microbial strains of the high quality necessary
for the microbiological analysis of food products to identify and prevent the spread of
foodborne disease. The ATCC bacteriology collection presents great biodiversity and
contains more than 18,000 strains in over 750 genera. The collection holds more than
3,600 type cultures of validly described species and about 500 bacteriophages. The
ATCC mycology collection has numerous filamentous fungi and yeast, representing
over 7,600 species, including 4,100 type strains and 1,500 genera. The mycology
collection also offers over 32,000 genetically modified yeast strains. This information
is available on the website: http://www.atcc.org.
Microorganisms can exhibit different growth behaviour depending on the strain

selected, and their sensitivity or susceptibility to an antimicrobial agent can vary.
However, sometimes it is possible to use a surrogate strain of a pathogen bacterium,
e.g., Listeria innocua is frequently used instead of Listeria monocytogenes. This
interchange does not introduce important differences in susceptibility between them
and a list detailing acceptable reference strains to be used for quality assurance is
available at: http://refs.wdcm.org/home.htm.
Strains are usually supplied as freeze-dried cultures. In these conditions they are
viable for at least one year from the date of shipment if the ampoules are kept in the
dark at constant temperature (from 4 to 24 °C, preferably 18 °C). The availability
of recommended media and the infrastructure needed to control all the incubation
parameters (growth temperature, anaerobic or aerobic conditions and so on) must
be checked before starting. The culture medium is usually broth or agar and has to
provide the required microbial conditions, not too dry and not too wet, sterile and
not past its expiry date. The ampoules containing the freeze-dried strain are opened
in a microbiologically protected environment by heating the tip with a flame for
5–15 s. Only the ampoule tip is heated, in order to prevent loss of strain viability.
The colour of the cotton plug inside the ampoules should not change to brown (this
indicates excessive heating). The hot tip is broken with 1–4 drops of cold sterile
water that crack the glass. Sometimes it is necessary to repeat the previous step to
break the tip. If the heating is too long the cotton plug could be sucked into the
bottom of the ampoule. This may decrease the viability of the freeze-dried material
and can complicate removal of the cotton with sterile tweezers. Finally, the strain is
rehydrated with 0.2–0.3 mL of the recommended broth medium, mixing carefully
with a pipette. The cell suspension is then inoculated into fresh liquid broth and solid
agar, and incubated at the optimum temperature for the time required. The bacterial
strains can then be stored in rich culture medium, such as tryptone soy broth with
126
Methods for the Analysis of Antimicrobial Packaging Efficiency
20% glycerol, at -80 °C until needed. For experimental use, the stock cultures are
maintained by regular subculture onto rich agar medium, such as tryptone soy agar
slants, at 4 °C and transferred monthly. Prior to the experiment, a loopful of each
strain is transferred to 10 mL of tryptone soy broth and incubated at the optimum
temperature, usually for 18–24 h, to obtain early stationary phase cells.
Fungal strains can be stored in water with 50% glycerol at -80 °C until needed. For
experimental use, the stock cultures are maintained by regular subculture on rich
culture medium, such as potato dextrose agar or malt extract agar, in plastic Petri
dishes (9 cm diameter) for 7 days at 30 °C. Conidia can be collected by flooding the
surface of the plates with sterile peptone water containing 0.05% tween 40 and gently
scraping the mycelial surface with a spatula. 10 mL of this suspension is transferred
to sterile plastic tubes, which are shaken to obtain a homogeneous suspension of
conidia. The conidial suspensions are adjusted to 1 × 106 spores/mL. Neubauer
chamber cell counting is used to determine spore concentration as the experiments
must be conducted with a known spore concentration.
5.1 In Vitro Methods
In vitro methods are applied under laboratory conditions; the microbial tests are
often carried out using ideal conditions, i.e., optimal culture medium, pH and growth
temperature, and under controlled environments. There are several methods that allow
us to test the efficacy of active packaging. In the first instance, an initial screening of
the efficiency of the active agent employed is required. Once the antimicrobial activity
of the agent has been demonstrated, it is necessary to study the effectiveness when it
is incorporated into a film matrix. At this point, the interactions and compatibility of
the agent with the polymer matrix, and the capacity to release it, must be considered.
The main requirement is that the concentration of released active agent should be
equal to or greater than the minimum inhibitory concentration (MIC).
There are five elements involved in antimicrobial food packaging systems: the material
(monolayer or multilayer structures), the food (often a complex matrix), headspace
(except in vacuum packaging), the total microbial load present in the food and the
agents that provide the antimicrobial activity. Consequently, the study of these systems
can be very complicated.
In this section, some of the tests used to study the antimicrobial effectiveness of this
kind of packaging are described in detail. However, it must be understood that not
all antimicrobial systems which have demonstrated strong antimicrobial activity
against target microorganisms under ideal conditions are equally efficient when they
are tested in real food packaging systems.
127
5.1.1 Disc Diffusion Method
The disc diffusion method has been used in microbiology laboratories for decades.
Alexander Fleming used a variant of this method to test penicillin in the 1950s. A
modification of Kirby–Bauer antibiotic test or disc diffusion antibiotic test is usually
employed to determine the antimicrobial effect of a film [2]. This method was initially
applied to study the effectiveness of antibiotics but it is possible to apply it to any
active agent.
The method is very easy and quick to perform. Petri dishes with a diameter of
9 cm are prepared filled with 15 mL of Mueller–Hinton agar medium and allowed
to cool. A known concentration of bacteria is inoculated onto the surface to provide
semiconfluent growth, usually 100 µL of 107–8 colony forming unit(s) (CFU)/mL.
A major source of error in disc diffusion testing is inoculum variation; hence the
inoculum density should be standardised to a final concentration of 1–2 × 10 8
CFU/mL according to standard methods [3].
A good alternative is to prepare the inoculum suspension by transferring isolated

colonies from an 18–24 h agar plate into broth medium to a turbidity matching a
0.5 McFarland standard. The inoculum suspension must be used within 15 min of
preparation, and this is particularly important in the case of fastidious microorganisms
that quickly lose their viability. The dried surface of the agar plate is inoculated
by streaking the entire surface and rotating the plate to obtain a homogeneous
distribution of the inoculum. Finally, sterilised paper discs are impregnated with known
concentrations of the antimicrobial agent, which are placed on the completely dry
agar surface or in wells in the agar and incubated at an appropriate temperature for
a suitable time. After incubation, inhibition zones devoid of bacterial growth can be
observed at the edges of the paper discs. Measurement of the inhibition zones gives
information regarding the efficacy of the antimicrobial agent. It is possible to study
antimicrobial packaging in the same way.
The disc diffusion method for yeast and moulds is similar to the method for
bacteria, using the same Mueller–Hinton agar medium supplemented with glucose
and methylene blue dye or another medium appropriate for microorganisms
of this kind (potato dextrose agar, malt extract agar or dichloran rose Bengal
chloramphenicol agar). Colonies with a diameter of at least 1 mm and with
similar morphology are picked, using a loop or a swab. The cells are suspended
in 5 mL of a sterile 0.85% sodium chloride solution and mixed using a vortex
for a few seconds. The turbidity of the inoculum is adjusted to the density of a
0.5 McFarland standard using a spectrophotometer at a wavelength of 530 nm.
The stock suspension in these conditions corresponds to 1–5 × 106 cells/mL and
produces semiconfluent growth.
128
Following the same method, the paper discs are replaced with discs of the antimicrobial
film that has been developed. The film must be pressed down to make immediate and
complete contact with the agar surface. Once in contact with the agar, the film must
not be moved, because this could produce a false antimicrobial effect due to scraping
of the agar surface or diffusion of the active agent from the film.
After the incubation period, usually 16–18 h for rapidly growing aerobic bacteria
or longer for slowly growing bacteria or specific resistance detection conditions, the
control plate is examined to determine if a semiconfluent and even lawn of growth has
been obtained. If individual colonies are present, the inoculum contained insufficient
cells and the experiment must be repeated because a false inhibition zone might
be considered. On the other hand, if the inoculum contained too many cells the
inhibition zone could be considered falsely small. If the lawn of growth is satisfactory
(semiconfluent), the zone diameter is measured using a ruler or sliding callipers. With
Mueller–Hinton agar, the inhibition zone is measured by turning the Petri dish over,
but for a non-transparent medium, such as blood-containing agar, the zone diameters
are read carefully from the surface of the agar. The active agent diffuses through
the agar, producing a concentration gradient which is inversely proportional to the
distance from the disc. The inhibition zone measured can be classified as susceptible,
intermediate or resistant, following tables for the microorganism under investigation
[4]. Figure 5.1 shows an inhibition zone produced by zein film containing garlic acid
as the antimicrobial agent tested against Listeria monocytogenes.
A B
Figure 5.1 Disc diffusion method. Image of Petri dishes inoculated with Listeria
monocytogenes. A) Control without film and B) active film of zein with 10%
garlic oil producing a clear inhibition zone
129
Sometimes a clearly defined inhibition halo is not seen but it is possible to observe
a reduction in growth on the Petri dish surface. This visible decrease in microbial
density on the plate agar is called a retraction zone [5].
Pieces of antimicrobial films containing volatile active agents can be stuck on the inside
lid of the Petri dish and the compound acts in the vapour phase; it is released into the
headspace and produces an inhibition zone in the agar in the same way. This variation
of the disc diffusion method is known as the microatmosphere method. Figure 5.2
shows the antifungal activity of oregano essential oil (OEO) against Penicillium spp.
using this technique. The fungus is grown on malt extract agar in plastic Petri dishes
(9 cm diameter) for 7 days at 30 °C. Conidia are then collected by flooding the surface
of the plates with sterile peptone water containing 0.05% tween 40 and gently scraping
the mycelial surface with a spatula. 10 mL of this suspension is transferred to sterile
plastic tubes, which are shaken to obtain a homogeneous suspension of conidia.
The conidial suspensions are adjusted to 1 × 106 spores/mL; a Neubauer chamber is
used to determine the spore concentration. The surface of the solid culture medium
in the 9 cm diameter Petri dishes is inoculated at three equidistant points with 3 µL
of conidial suspension.
A B C
Figure 5.2 Microatmosphere method. Antifungal activity of OEO against

Penicillium spp. A) Control; B) 3 µL of OEO; and C) 5 µL of OEO
Despite its simplicity, the disc diffusion test is based on physico-chemical principles of
antimicrobial agent diffusion into agar medium. Cooper and Woodman [6] applied
an adaptation of the formula for the diffusion of neutral particles in gases to the
diffusion of antimicrobials through agar gels as follows:
 M 
X 2 = 4 DT 2 .3 log 0  (5.1)
 M
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where:
X = zone radius.
D = diffusion coefficient.
T = critical time for zone demarcation.
M0 = antimicrobial agent concentration in the reservoir.
M = critical concentration of antimicrobial agent inhibiting the organism.
The critical time can be expressed as:
T = L + n log N (5.2)
N0
where:
L = lag period.
n = generation time.
N = critical mass of cells formed at T.
N0 = inoculum density used in the test.
To obtain meaningful results, several tests must be carried out to standardise the
method.
Disc diffusion is an easy and inexpensive method which provides qualitative results
mainly for rapidly growing aerobic bacteria. This method does not require any special
equipment, the results are easily interpreted and it has a wide-ranging selection of
discs for testing. It can be used to preliminary screen antimicrobial effectiveness and
to determine the MIC in the solid phase, but it has some limitations.
The disadvantages of the disc test are the lack of mechanisation or automation of the
test and the inhibition zone measurement can be subjective. In addition, this method
is not applicable to slow-growing or anaerobic microorganisms, and many variables
can have a direct influence leading to unreliable results (inoculum density, growth
phase, temperature, prediffusion or variations in the thickness of the agar in the
Petri dishes). However, the disc test has been standardised for testing streptococci,
Haemophilus influenzae and Neisseria meningitidis through the use of specialised
media, incubation conditions and specific interpretive criteria for zone sizes.
5.1.2 Dilution Method
Agar dilution and broth dilution are the main techniques used to determine the MIC
and minimum lethal concentration (MLC) of antimicrobial agents. As explained in
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Chapter 2, the MIC is defined as the lowest concentration of an active agent that
inhibits visible growth of a specific organism under in vitro conditions and within
a defined period of time. This value may be determined on agar or in a liquid
medium. However, the most common method for determining the MIC is the broth
dilution technique, where serial dilutions of the antimicrobial agent are incorporated
into broth medium using microtitre plates with a total volume of 0.05 to 0.1 mL
(Microdilution Broth Method) or in culture tubes with a volume >1 mL (Macrodilution
Broth Method). A complete guide to this protocol may be found in the Clinical &
Laboratory Standards Institute (CLSI) guidelines document M7-A7: ‘Methods for
dilution antimicrobial susceptibility testing for bacteria that grow aerobically’. The
MIC protocol is described as follows:
• Cells for the inoculum: isolated colonies must be selected from an 18–24 h culture
on an agar plate. The inoculum suspension must be carefully prepared. First,
select 3 to 5 well-isolated colonies from the agar plate and transfer them to
a tube containing 4–5 mL of broth medium, such as tryptic soy broth. The
tube is incubated at 35 °C until it achieves the turbidity of a 0.5 McFarland
standard, which depends on the microorganism but it usually takes 2–6 h. The
turbidity of the actively growing broth culture is adjusted with sterile saline
solution or broth medium. The resulting suspension contains approximately 1
to 2 × 108 CFU/mL. As an alternative method, the inoculum may be prepared
by direct colony suspension, making a direct broth suspension of isolated
colonies and adjusting it to achieve the 0.5 McFarland standard; this method is
recommended for fastidious organisms such as Neisseria gonorrhoeae. Within
15 min of adjusting the inoculum to the 0.5 McFarland turbidity standard, the
suspension is mixed and diluted so that the final inoculum concentration of
5 × 105 CFU/mL is achieved. Mueller–Hinton broth is the recommended medium for
antimicrobial susceptibility testing of commonly isolated, rapidly growing aerobic
or facultative organisms. This medium has demonstrated good reproducibility for
susceptibility testing and is low in sulfonamide, trimethoprim and tetracycline
inhibitors, and yields satisfactory growth for most pathogens. The broth must have
the appropriate divalent cation content which is controlled by the manufacturer
(Ca2+ and Mg2+). Cation-adjusted Mueller–Hinton broth may be purchased from
several commercial sources. The appropriate concentration of cations is 20–25 mg
of Ca2+/L and 10–12.5 mg/L of Mg2+. The pH of the medium should be between
7.2 and 7.4 at room temperature (25 °C) and must be tested with a pH meter before
it is used. For fastidious organisms, Mueller–Hinton broth can be supplemented
with 2–5% lysed horse blood.
• Serial dilutions of the antimicrobial substance are prepared. The antimicrobial

stock solutions should reach a final concentration of 10 mg/mL or 10 times the
highest concentration to be tested. Water is the recommended solvent, but some
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antimicrobial agents have limited solubility. It is possible to use a minimum

amount of other solvents, such as ethanol or dimethyl sulfoxide, and make further
dilutions with water. Each well or tube contains increasing concentrations of the
antimicrobial agent and is inoculated with a known and fixed amount of the
microorganism. Samples must be prepared in triplicate.
• Two samples containing inoculated broth without the antimicrobial agent

must be included in each assay as a control. In addition, one tube lacking the
antimicrobial agent and microorganisms must be included in the test as a check
for medium sterility.
• The positive control is plated and used to establish a baseline concentration of the
microorganisms under investigation. The presence of approximately 50 colonies
indicates an inoculum density of 5 × 105 CFU/mL.
• The wells or tubes are incubated for 24–48 h (usually overnight) at an optimum
temperature.
• Samples are measured using a spectrophotometric technique. The turbidity of

the medium increases with microbial growth; therefore a previously prepared
calibration curve relating turbidity to the number of colonies is required. The
success of this method depends on the sensitivity of the device and correct
interpretation of results. Spectrophotometers generally required log 6 to
7 CFU/mL for detection [4]. Below these concentrations, the lack of sensitivity
should not be confused with absence of microbial growth and sampling for
direct seeding is recommended. Turbidity is determined after 24 and 72 h, and
prior to a spectrophotometric reading it is recommended to shake the samples
gently. The antimicrobial-free samples must be examined for viability of the
test isolate. This is necessary to ensure that the culture conditions, such as
medium or temperature, enable growth of the microorganism. Furthermore,
the broth control tube without inoculum must be clear, because growth in this
sample indicates contamination of one of the components of the test system.
If that happens, the study is not valid and it must be repeated. If the control
tubes are correctly set up the MIC and MLC may be studied. The MIC is
determined as the lowest concentration at which no growth is observed by the
unaided eye. The MIC of each antimicrobial agent is expressed in µg/mL. To
determine the MLC, the dilution representing the MIC and at least two more
concentrated test product dilutions are plated and enumerated to determine
viable CFU/mL. The MLC may be considered as the lowest concentration at
which no growth is observed after subculturing into fresh medium [7]. All
tubes showing no growth may be subcultured onto agar medium to ensure the
non-viability of the culture.
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Bacteriostatic agents inhibit the growth and replication of bacteria; however, if

the agent is removed the cells recover the ability to multiply. On the other hand,
bactericidal agents produce irreversible damage, not only inhibiting growth of cells
but also triggering pathways within the cells that lead to cell death. For this reason,
the MLC is determined after subculturing the samples into fresh medium.
The Microdilution Method uses wells with a volume of 0.1 mL that allow
approximately 16 antimicrobial agents to be simultaneously tested in a range of 8,
2-fold dilutions in a single tray. Inoculation of panels with the standard inoculum
of 5 × 105 CFU/mL is accomplished using a disposable device that transfers 0.01 to
0.05 mL of standardised bacterial suspension into each well of the microdilution tray
or by use of a mechanised dispenser. Following incubation, the MIC are determined
using a manual or automated system. The advantages of the microdilution procedure
include the generation of MIC, the reproducibility and convenience of having prepared
panels, and the economy of reagents and space owing to the miniaturisation of the test.
Standardised protocols are required for intra- and interlaboratory reproducibility,

because results may be significantly influenced by the method employed. These
methods allow the quantification of the concentration required to inhibit growth of
a specific microorganism (bacteriostatic/fungistatic activity) or kill it (bactericidal/
fungicidal activity).
In order to study the antimicrobial capacity of packaging films, they are immersed
in 10 mL of Mueller–Hinton broth prior to inoculation with the microorganisms,
which are in an exponential phase or stationary phase. When testing an antimicrobial
film for the first time, it is recommended to use the microorganism in an exponential
phase (more sensitive) and Mueller–Hinton broth as the culture medium because
it has limited nutrients. This medium is recommended by the CLSI for testing the
aerobic bacteria most commonly found in food and clinical samples. The medium
demonstrates good reproducibility for susceptibility testing. If the bacteria are
damaged in a medium without optimal nutritional conditions, differences in growth
compared with the control can be easily seen.
The antimicrobial effect of active films can be determined using spectrophotometry or

by plating and counting colonies. A microbicide effect against a pathogen or spoilage
microorganism is not always required as usually an increased lag phase is sufficient
to protect the consumer. Study of the growth curve of a microorganism and how
the addition of an antimicrobial compound changes the kinetics of the curve is very
useful. The presence of an active agent can extend the lag phase and retard microbial
growth or even produce death of the microorganism (called the time-kill curve). This
method is similar to the broth dilution assay, but in this case the inoculated medium
is sampled at appropriate times (e.g., 0, 2, 4, 8, 12, 24 and 48 h) and the number of
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viable microorganisms is determined by plating. Figure 5.3 shows an example of this

kind of experiment, Listeria monocytogenes in exponential phase in contact with a
control film and active polymers [linear low-density polyethylene (LLDPE), ethylene
methacrylic acid copolymer (EMA) and ionomer (ION)] containing 5% carvacrol
and 5% thymol, and storage at 4 °C. Samples are plated at different times.
6
Bacterial count (Log CFU/mL)
0 1 3 6 24 48 72
Time (h)
Figure 5.3 Growth and time-kill curves of Listeria monocytogenes in contact with
active polymers. Control film (•), active films: LLDPE (▲), ION () and EMA (∆)
containing 5% carvacrol and 5% thymol
LLDPE releases the antimicrobial agent quickly, producing a bactericidal effect after
3 h. The samples with active ION and EMA show an inhibitory effect after 24 h; in
these cases the release is slow. These values allow us to describe the microbial growth
profile.
Time-kill curves can also be obtained by measuring the number of viable bacteria at
various time intervals, as shown in Figure 5.3. It is used to study the efficacy of an
antimicrobial agent against a particular bacterial strain and it is also used to determine
synergy between two or more antimicrobial agents. A standardised inoculum of
the bacterium is grown in the presence or absence of the antimicrobial compound.
Aliquots at different time intervals are removed and plated onto agar medium. Synergy
is determined as a ≥2 log decrease by the antimicrobial mixture compared with the
most active single antimicrobial. Antagonism is determined as a ≥2 log increase by
the antimicrobial mixture compared with the most active single compound.
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Several responses of the test microorganism may be encountered in this type of

analysis, including stationary-phase growth rate suppression, lag-phase increase,
decrease in the growth rate during log phase and lethality [8]. The advantages of this
method are the generation of a quantitative MIC value and important information
about the effect of the active agent on the kinetics of microbial growth. The principal
disadvantages of this method are the fact that it is more tedious and takes longer
than the agar diffusion method, the possibility of errors occurring when preparing
the antimicrobial samples, the relatively large amount of reagents used and the space
required for each test.
A way to speed up this method is the use of automated instrument systems. In addition,
the use of instrumentation may standardise susceptibility test results and provide more
precision in a shorter period than manual readings. To avoid ambiguity, it is essential
that automated instruments are standardised at regular intervals. There are 4 automated
instruments approved by the US Food and Drug Administration for use in the USA
[9]. Three of these automated instruments can produce rapid (3.5–16 h) susceptibility
test results, while the fourth automated instrument requires an overnight period [9].
The MicroScan WalkAway (Siemens Healthcare Diagnostics) is an incubator and
reader device able to simultaneously incubate and analyse 40–96 microdilution trays.
The MicroScan WalkAway uses standardised microdilution trays that are inoculated
manually and then incubated for the appropriate time. Samples are periodically read
with a photometer or with a fluorometer to study microbial growth. Gram-negative
susceptibility test panels with fluorogenic substrates can be read in 3.5–7 h. Susceptibility
tests of separate Gram-positive and Gram-negative bacteria can be obtained in
4.5–18 h using turbidimetry. The instrument includes specific software that can be used
to analyse, store and report data [9].
The BD Phoenix Automated Microbiology System (BD Diagnostics) is comprised of

an incubator and reader with a high-throughput capacity. This automated system
is able to process 99 test panels with 84 wells which must be inoculated manually.
The BD Phoenix Automated Microbiology System measures each panel periodically
(20 min) using turbidimetric and colorimetric methods. Data of microbial growth
and MIC values can be obtained in 8–16 h [9].
The Vitek 2 (bioMérieux) is a highly automated system. Samples are presented in

compact plastic reagent cards containing microlitre quantities of antimicrobials. An
Advanced Expert System is included and determines MIC values for most organisms
tested. The Vitek 2 system periodically measures bacterial growth using turbidimetry;
the incubation period is usually very short. The instrument is highly efficient and
capable of simultaneously measuring up to 240 tests. The susceptibility cards allow
testing of common, rapidly growing Gram-positive and Gram-negative aerobic
bacteria and Streptococcus pneumoniae in a short period of 4–10 h [9].
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The Sensititre ARIS 2X (Trek Diagnostic Systems) is an automated system with a

capacity to process 64 test panels. Microbial growth is determined by fluorescence
measurement after 18– 24 h of incubation. Test panels are now available for Gram-
positive and Gram-negative bacteria, Streptococcus pneumoniae, Haemophilus species
and non-fermentative Gram-negative bacilli [9].
In the agar dilution method, serial dilutions are prepared from a known concentration
of the antimicrobial agent and added directly to non-selective agar medium
before solidification. The inoculum can be applied rapidly and simultaneously to
the agar surfaces using an inoculum replicating instrument or spread after agar
solidification. Most available replicators transfer 32–36 µL inocula to each plate.
Mueller–Hinton agar is the recommended medium used for sensitivity testing of
pathogenic microorganisms, which is in accordance with the Kirby–Bauer and Ericsson
methodology. Mueller–Hinton agar is prepared from a dehydrated base dissolved in
water and autoclaved at 121 °C for 15 min. The pH of the agar must be between
7.2 and 7.4 at room temperature (25 °C). Supplemental cations must not be added
to the agar; however, it may be supplemented with 5% defibrinated sheep blood or
lysed horse blood.
Alternatively, the film-forming solution containing the antimicrobial agent or

the finely cut-up film can also be incorporated into the agar medium. The test
microorganism, diluted to around 107 CFU/mL, is added to the plates in 1 to 2 µL
spots (approximately 4.0 CFU per spot) [10] and incubated under optimal conditions
of temperature and time. The main advantage of this method is the possibility of
testing different strains at the same time. The results are reproducible and growth
is satisfactory in most non-fastidious organisms. However, the agar dilution plate
preparation is tedious and the shelf life is very short. Furthermore, this method is
normally not used to test antimicrobial films because accurate extrapolation to a
real food system is not possible.
5.1.3 Japanese Industrial Standard Method
The standard method for testing non-porous materials [International Organization

for Standardization (ISO) 22196, Japanese Industrial Standard (JIS) Z 2801] is useful
to evaluate the surface antimicrobial activity of active products of various materials,
such as textile, plastic, metal or ceramic products. This method was developed to
measure the antibacterial activity in hydrophobic materials and it provides quantitative
information about the biocidal or biostatic effects of the antimicrobial surface. It
can be applied to a wide range of microorganisms, such as bacteria, viruses, moulds,
yeast, algae and protozoa species. ISO 22196 uses Escherichia coli and Staphylococcus
aureus as model strains.
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For this method, the control and test samples are cut into small square pieces
(5 × 5 cm). All samples are inoculated in triplicate, with an additional three replicates
of the control films. Control and test surfaces are inoculated with a microbial
suspension of approximately 105 CFU/mL standardised by dilution in a nutrient broth
and then the microbial inoculum is covered with a thin, sterile film, i.e., coverslip, of
polypropylene to prevent the samples from drying out. The coverslip may adhere to
cells in the inoculum. It is not uncommon to see JIS results for antimicrobial agents
seemingly cause a reduction in log value in the order of 3 or more. Bacterial adhesion
to the coverslip will probably depend on the bacterial strain and the coverslip material;
however, if even 10% of the inoculum adheres to the coverslip, and if 10% of those
cells are recovered for enumeration, then the log reduction value is expected to be
limited to less than 2. Some researchers have avoided this problem by excluding the
coverslip, thereby providing the cells with only one solid surface, the sample surface,
but the use of a coverslip is recommended because it prevents water evaporation and
allows close contact with the antimicrobial surface.
Microbial concentrations at time zero can be determined by elution and plating at

the beginning of the experiment. Samples are incubated in a humid environment for
18–24 h. After incubation, both film surfaces are carefully transferred to a stomacher
and microbial concentrations are determined by plating and counting after incubation.
The antimicrobial effect is measured by comparing the survival of bacteria on a treated
material with that achieved on an untreated material. Results can be expressed as log
(CFU/cm2). Figure 5.4 shows a diagram of the JIS method.
The value of the antimicrobial activity of the samples tested by the JIS Z 2801 method
is determined using the following formula:
R= ( U t − U 0) − ( A t − U 0) = U t − A t (5.3)
where:
R is the antibacterial activity.
U0 is the average of the common logarithm of the number of viable bacteria, in cells/cm2,
recovered from the untreated test specimens immediately after inoculation.
Ut is the average of the common logarithm of the number of viable bacteria, in
cells/cm2, recovered from the untreated test specimens after 24 h.
At is the average of the common logarithm of the number of viable bacteria, in
cells/cm2, recovered from the treated test specimens after 24 h.
When R ≥2.0, the sample is considered to exhibit biocidal properties.
The advantages of this method include: it is quantitative and results in triplicate tend
to be reproducible when the experiment is performed carefully, without allowing the
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inoculum to spill off the target area, and a sterile film is used to cover the area to
prevent it from drying out; in addition, it is valid for determining biocidal or biostatic
properties of active materials. Microbial concentrations are standardised and the
incubation medium provides enough nutrients for the microorganisms to grow if the
active material surfaces do not exert an antimicrobial effect.
The weaknesses of the JIS Z 2801 method are that it is not necessarily representative
of actual surface contamination events, the microbial inoculum is spread over a
considerable surface area and is then kept wet (usually for a period of 24 h), whereas
in real conditions microbial contaminants dry quickly on surfaces. If contact between
the agent and the microorganism is limited in the absence of a liquid medium, the
effectiveness of the material is unknown.
Protective
film
(4 × 4 cm)
Serial dilutions
and plated
400 µL
Incubate Incubate
24 h, 37 °C 24 h, 37 °C
Stomacher bag
Active
Cell suspension film
105CFU/mL (5 × 5 cm)
Figure 5.4 Diagram of the JIS Z 2801:2000 method for antimicrobial

surface testing
5.1.4 Surface Growth Method
An innovative method involves aerosol inoculation to apply a thin film of pathogens

across the antimicrobial material. The microorganisms may be dried in place or kept
humidified. After the inoculation period, the antimicrobial surfaces and controls
are then used for growth via being in contact with agar medium or recovery for
traditional enumeration. The surface growth method is excellent for testing surface
contamination under ambient conditions and the corresponding antimicrobial
efficacy of the material. In this case, the bacteria are in very close contact with the
antimicrobial-coated substrate because the volume of liquid in contact with the
antimicrobial-coated material is particularly small; hence, assessment of the impact
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of eluted antimicrobial agents using this method is more accurate than the direct
inoculation methods described above [11].
5.1.5 Electron Microscopy
The electron microscope uses a beam of electrons to produce an image of the specimen.
It provides higher magnifications and has a greater resolving power than a common
light microscope, allowing more detailed visibility of much smaller objects. An electron
microscope is a large, expensive instrument, generally standing alone in a specially
designed room and requiring expert technicians to operate it.
There are two basic methods, scanning electron microscopy (SEM) and transmission
electron microscopy (TEM), and both use specially designed instruments. A scanning
electron microscope is relatively easy to use and the results are easy to analyse because
the specimens are shown as three-dimensional (3D) objects. On the other hand, a
transmission electron microscope generates different kinds of images produced by an
electron beam which passes through the specimen and forms an image on a fluorescent
screen [12]. The images obtained are photographed. In negative staining, a dilute
suspension of unfixed bacteria is applied on a Formvar film followed by the addition
of a dilute heavy metal salt solution (e.g., uranyl acetate, ammonium molybdate
or sodium phosphotungstate) [12]. The electrons are absorbed by the heavy metal
but they pass through the bacteria and form their images on a fluorescent screen.
This procedure is excellent for showing different parts of bacteria, such as fimbriae,
flagella and so on. Embedding bacteria in a resin and staining thin sections of the
resulting block with heavy metals, such as uranyl acetate and lead citrate, reveals the
internal structure of the bacteria. In one of the most elaborate protocols the bacteria
are rapidly frozen and fractured, the newly developed surfaces are replicated using
platinum and carbon, and finally the replicas are examined [12].
TEM involves a high voltage electron beam emitted by a cathode and formed using
magnetic lenses. The electron beam that has been partially transmitted through the
very thin (and hence semitransparent for electrons) specimen carries information
about the structure of the specimen. The spatial variation in this information (the
‘image’) is then magnified by a series of magnetic lenses until it is recorded by hitting
a fluorescent screen, photographic plate or light-sensitive sensor such as a charge-
coupled device (CCD) camera. The image detected by the CCD may be displayed
in real time on a monitor or computer [12]. Black and white images obtained by
transmission electron microscopes are two-dimensional (2D).
The sample preparation protocols recommend that the bacteria grow in a standard
medium such as nutrient broth in contact with the antimicrobial agent or film, at an
optimum temperature, usually at 37 °C. Samples lacking an active agent are incubated
140
as a control. After the incubation period, the bacterial cultures are collected, during the
exponential phase or stationary phase, by centrifugation at 5,000–9,000 rpm for 7–10 min
and are resuspended in peptone water. The samples can be fixed with 2% glutaraldehyde
in 0.1 M sodium cacodylate, pH 7.4, for 2 h at 4 °C. The cells are collected and washed
two or three times with phosphate buffer solution (PBS) buffer and they are postfixed
in 1% osmium tetroxide for 1 h at 4 °C. The cells are dehydrated using increasing serial
dilutions of ethanol, which is followed by propylene oxide treatment. Finally, the samples
are embedded, cut, and stained with 2% uranyl acetate and lead citrate.
Unlike TEM, where the electrons in the primary beam are transmitted through the
sample, SEM produces images by detecting secondary electrons which are emitted
from the surface owing to excitation by the primary electron beam [12]. During SEM,
the electron beam is scanned across the surface of the sample in a raster pattern, with
detectors building up an image by mapping the detected signals with beam position. In
principle, sample preparation for SEM consists of isolating the bacteria or trimming
the specimen in which they are present, fixing them, dehydrating in ethanol, critical-
point drying, mounting on an SEM stub, sputter-coating with gold and recording
images at an appropriate accelerating voltage [12].
Of the wide range of procedures available, SEM is one of the best suited to visualise
the external appearance of bacteria. Electron microscopy reports the morphological
changes in the tested microorganism. If the MIC and MLC of the antimicrobial agent
are known, these concentrations may be used to study its effectiveness against the
corresponding microorganism. Dilutions of antimicrobial agent are prepared in sterile
water or peptone water and added to a broth medium. The microbial inoculum, in
the exponential phase, is added and incubated overnight at an optimum temperature.
Solid specimens may be fixed in a buffered (0.1 M, pH 6.5–7.0) fixative such as 2–3%
glutaraldehyde for periods ranging from 5 min to 24 h. Occasionally postfixation
is necessary, using osmium tetroxide or ruthenium red. Fixed specimens are washed
with the corresponding buffer and dehydrated using a graded ethanol series.
In other protocols, purified bacteria are captured on filters, 13 mm in diameter (to fit the
SEM metal stubs), which have small (0.2 to 1 µm) pores. Polycarbonate ‘nuclepore’ filters,
which have relatively smooth surfaces, resistant to ethanol and liquid carbon dioxide (but
not to acetone), are suitable for alcohol dehydration and critical-point drying. Bacteria
may be captured on the filters either in live or fixed forms. Fixation and/or dehydration
are best performed in capped wide-neck vials to accommodate the filters. The specimens
are dehydrated through a graded ethanol series (20, 40, 60, 80, 95, 100, 100, 100%
ethanol) on a slowly moving, inclined rotary table. Short, 5–10 min, intervals are sufficient
to dehydrate several filters in a vial. Finally, the filters are critical-point dried.
Some less complicated protocols have been developed without fixing bacterial cells.
Untreated controls of pathogens must be prepared. The samples are centrifuged and
141
resuspended twice in a saline solution (0.8% sodium chloride) to remove the broth
medium. Each microbial suspension is filtered through a 0.2 mm membrane which
retains the bacteria. The membranes are then dehydrated in a 30, 50, 70, 90 and
100% graded alcohol series, and sputter-coated with gold under vacuum.
The effectiveness of antimicrobial films is studied in the same way. Microorganisms

are in contact with the active films and after the exposure time they are recovered
and prepared for analysis using electron microscopy. Figure 5.5 shows micrographs
of Escherichia coli. Figure 5.5A shows a control cell with a smooth surface and
characteristic rod shape, and Figure 5.5B shows the same strain, Escherichia coli,
exposed to antimicrobial films, displaying considerable morphological alterations in
comparison with control bacteria. It can be observed that the bacteria are seriously
damaged, presenting an irregular, rough surface with blisters and bubbles, and collapse
of the bacteria compared with the control.
Figure 5.5 Scanning electron micrographs of Escherichia coli cells: A) control cell
and B) cell treated with an antimicrobial agent
The main disadvantage of this technique is that electron microscopy is a high-cost

procedure and the equipment is expensive to purchase and maintain. The sample
preparation protocol must be optimised. It is essential to have trained personnel to
use the equipment and excellent sample preparation to avoid artefacts in the samples.
Nowadays, many research institutions and universities have good equipment and
qualified personnel that can help obtain good images of the microorganisms under
investigation.
5.1.6 Atomic Force Microscopy
In recent years, atomic force microscopy (AFM) has been widely used to investigate
microbial surfaces at higher resolution. The technique provides 3D images of the
surface ultrastructure at molecular resolution, in real time, under physiological
conditions, and with advantages such as minimal sample preparation. Other
techniques, such as electron microscopy, also allow the study of microbial surfaces
142
at high resolution, but they involve tedious cell manipulation prior to examination,
e.g., staining or drying, which can seriously affect the viability and validity of the
analysis. Optical and transmission electron microscopes can generate 2D images of
a microbial surface, achieving magnifications of a few hundred thousand-fold in the
case of an electron microscope. However, these techniques do not provide the vertical
dimension (z-direction) of the sample, namely the height or depth of surface features.
In comparison with electron microscopy techniques, AFM has the main advantage of
providing images of cell structures at molecular resolution and under physiological
conditions, the ability to monitor the structural dynamics of cell walls in situ in
response to various parameters, such as stress and antimicrobial agents, and the ability
to measure the localisation, adhesion and mechanics of single cell wall constituents.
AFM allows researchers to observe several cell wall components directly in live cells,
such as polysaccharides, peptidoglycan, teichoic acids, pili and flagella.
AFM measures the small forces acting between a sharp tip and the sample surface;
it is like touching the surface with a mechanical probe. Basically, an atomic force
microscope has a cantilever spring with a sharp tip at its end which is used to scan (feel)
the specimen surface (Figure 5.6). The cantilever with the tip is commonly known as
the probe or scanning probe. Most AFM probes are made from silicon, borosilicate
glass or silicon nitride, with the cantilever measuring a few micrometres and the
tip a few nanometres. When the tip is in operation, chemical forces between the tip
atoms and the sample surface atoms (van der Waals forces, dipole-dipole interactions,
electrostatic forces and so on) result in deflections of the cantilever according to
Hooke’s law. A laser spot is reflected from the top surface of the cantilever into an
array of photodiodes, as the figure shows, although other detection methods are also
common, including optical interferometry and piezoresistivity. In the imaging mode,
the cells are placed in a solution and the tip follows the cell surface to generate a 3D
cell image at molecular resolution [13].
Figure 5.6 Diagram of an AFM system
143
Bacteria are grown in a standard culture medium, such as nutrient broth, at an

optimum temperature which is usually 37 °C. The cultures are collected during the
exponential phase by centrifugation at 9,000 rpm for 7–10 min and are resuspended
in peptone water. The bacterial cells are collected by centrifugation at 9,000 rpm for
7 min, washed with 10 mM PBS, pH 7, and resuspended in PBS. The cell suspensions
can be deposited on a sterilised mica surface and dried at room temperature. At that
point, the sample is ready to be analysed by AFM.
AFM is a technique capable of providing an image of the surface topography

with extremely high magnifications, up to 1,000,000×, in three-dimensions.
The principal disadvantages are the requirements for qualified personnel and
equipment cost.
5.1.7 Flow Cytometry
Flow cytometry is a powerful tool for the differentiation and functional analysis
of eukaryotic cells as well as microorganisms. Flow cytometry is a technology that
simultaneously measures and analyses physical and chemical characteristics of single
particles, usually cells, as they flow in a fluid that passes through at least one laser.
Cell components are fluorescently labelled and excited by the laser to emit light at
different wavelengths. Size, relative granularity, viability, fluorescence intensity or
internal complexity can be measured, among other properties. These characteristics
are determined using an optical-to-electronic coupling system that records how the
cell or particle scatters incident laser light and emits fluorescence. The fluorescence
can be measured to report various properties of single particles, which are usually
cells. Thousands of particles per second can be analysed as they pass through the
liquid stream. A flow cytometer is composed of three main systems: fluidics, optics
and electronics. The fluidics system transports the single particles in a stream of fluid
to the laser beam. Particles with a size of 0.2–150 µm can be analysed, so cells from
animals, plants, bacteria, yeast and algae can be measured. Sometimes the particles
studied have natural fluorescence, but generally fluorescent labels are required to tag
the samples. The part of the fluid stream containing the particles is called the sample
core. The optics system consists of lasers which illuminate the particles. Optical filters
send the resulting light signals to the appropriate detectors. Finally, the electronics
system converts the detected light signals into electronic signals that can be processed
by a computer. The data are analysed to obtain information about a large number of
cells over a short period. Information regarding heterogeneity and subpopulations
can be identified and measured.
As explained earlier in this chapter, MIC-based broth microdilution and macrodilution

systems need an incubation period of approximately 18–24 h before antimicrobial
144
activity can be quantified. Moreover, unless the bacterial inoculum is quantified, only
the bacteriostatic effect can be assessed as determination of the MLC requires bacterial
culture dilutions and subsequent subcultures of known concentration. In addition,
neither MIC nor MLC quantifications consider the heterogeneity of bacterial culture
dilutions and subcultures [14]. Flow cytometry is a very powerful technique that
allows the study of morphological and physiological changes [membrane potential,
cell size, deoxyribonucleic acid (DNA) and so on] in individual cells and their
distribution within large cell populations in a short period of time. Flow cytometry
can also be a reliable approach for susceptibility testing, producing results in terms of
the bactericidal and/or bacteriostatic effects. Morphological changes can determine
the viability, or non-viability, of the cells tested. This method is widely employed
in clinical and experimental investigations of eukaryotic cells and also looks very
promising in the case of bacteria. Microorganisms in contact with an antimicrobial
agent can be studied using flow cytometry in order to ascertain the morphological
and physiological damage caused by exposure to the active agent or antimicrobial
film. Bacterial cells are incubated in the absence or presence of the antimicrobial
agent for a few hours, and the antibacterial effect is detected by measuring the
variations in fluorescence due to dead cells. This technique provides information
about heterogeneity by determining the presence of subpopulations that are less
susceptible to the antimicrobial agent.
There are numerous labels or fluorophores that can be employed in flow cytometry.
Two major categories of fluorochromes have been used to study antimicrobial effects:
DNA and ribonucleic acid (RNA)-labelling dyes and metabolic or protein-binding
probes, such as membrane potential-sensitive dyes [14]. Mithramycin containing
ethidium bromide was the first probe used for susceptibility testing by flow cytometry
and is still used today. Mithramycin allowed the determination of the ploidy in
organisms undergoing filamentation. Propidium iodide (PI) is a fluorochrome that
intercalates into double-stranded nucleic acids; this reagent is a more appropriate stain
than ethidium bromide for susceptibility testing by flow cytometry [14]. PI is a non-
permeable dye that is excluded by viable cells, hence it is used to measure the loss of
viability caused by antimicrobial agents. PI provides information about the integrity of
the outer membrane and it allows differentiation among antimicrobial agents able to
damage the cell wall or outer cell membrane. Other widely employed fluorochromes
are SYTO-13 and SYTO-17, which label both DNA and RNA. Acridine orange stains
DNA and RNA at the same time, but with different wavelength emissions, and can
be used to measure alterations in plasma membrane permeability produced by the
action of antimicrobial compounds [14].
Bacterial cells are able to decrease or increase their membrane potential when they
are exposed to antimicrobial agents. Rhodamine 123 and oxonol are employed as
membrane potential probes to study antimicrobial effects using flow cytometry. The
145
accumulation of these stains inside bacterial cells depends on the difference in charge
between the two sides of the plasma membrane. Rhodamine 123 is a cationic lipophilic
dye used only for Gram-positive bacteria, whereas oxonol is an anionic lipophilic
dye that can only enter the cell when the membrane has become depolarised and is
useful for Gram-positive and Gram-negative bacteria [14].
The flow cytometry assay has been proposed as an excellent alternative method to
study antifungal activity. The results obtained with flow cytometry are similar to results
obtained using standard methods such as broth dilution. The results are available
in 24–32 h or even in 9 h, and the sensitivity and reliability is potentially greater
than standard broth dilution methods. Using PI or rose Bengal, the time required for
susceptibility results decreases to only 9 h.
The protocol employed to prepare the samples is quite simple. The MIC is the
recommended concentration to treat the microorganism before flow cytometry analysis.
However, if the MIC has not been previously determined, different concentrations of
the antimicrobial agent can be used to study its effectiveness against the microorganism
by flow cytometry. Dilutions of antimicrobial agent are prepared in sterile water or
peptone water and added to a standard broth medium or saline solution, in order to
avoid the excess proteins present in the broth medium. The microbial inoculum (in a
concentration range of approximately 5 × 105 to 9 × 106 CFU/mL), in the exponential
phase, is added and incubated for a few minutes, hours or overnight, depending on
the antimicrobial effectiveness. The samples are then centrifuged and resuspended
2 or 3 times in saline solution (0.8% sodium chloride) to remove the broth medium.
The cells can be stained with the cell-permeating double-stranded DNA fluorochrome
SYTO-13 and PI solution. SYTO-13 stains the nucleic acids in all cells, while PI
stains the nucleic acids in cells which have a damaged membrane. Each cell can
be characterised by several optical parameters: side scatter, forward scatter, green
fluorescence for SYTO-13 (excitation light 497 nm and emission light 520 nm) and
red fluorescence for PI (excitation light 493 nm and emission light 630 nm). The
results can show cell death in the control due to different phases of the cell cycle.
Figure 5.7 shows the results obtained with this protocol. Figure 5.7A shows a histogram
corresponding to a control sample of Listeria monocytogenes in which the grey dots
are the viable cells; it can be seen that the main population is viable. However, the
cells treated with antimicrobial agent (Figure 5.7B) are divided into two populations,
with grey dots representing the viable bacteria and black dots the damaged cells. The
main advantages of the flow cytometry technique are versatility, simplicity, reliability
and speed. It is also possible to obtain qualitative and quantitative results. However,
the introduction of this methodology into microbiology laboratories is limited by the
cost of the instrumentation and the requirement for well-trained operators.
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105 P2 105 P2
104 104
SYTO-13
SYTO-13
103 103
102 102
P3 P3
101 101
101 102 103 104 105 101 102 103 104 105
PI PI
Figure 5.7 Dual-parameter SYTO-13/PI fluorescence histograms obtained

by staining Listeria monocytogenes: A) control samples, B) cells after
treatment at the MIC with an antimicrobial surfactant. SYTO-13 stains
the nucleic acids in all cells (grey dots) and P1 stains the nucleic acids
in cells with a damaged membrane (black dots); P2: viable population
and P3: dead population
5.2 In Vivo Methods
The results obtained when working under in vitro conditions using a standard
growth medium and optimum temperature often cannot be confirmed in tests with
real food/packaging systems owing to interference from the food matrix or food
components. The intrinsic properties of the food (proteins, lipids, cations, pH, salt
and so on) are not the only aspects that are relevant in these results; extrinsic factors
such as temperature, packaging material and so on can also influence the sensitivity
of bacteria. The use of antimicrobial agents as food preservatives has usually been
restricted because high concentrations are needed to achieve sufficient antimicrobial
activity. Furthermore, the antimicrobial potency of the active agents also depends
on pH, temperature and the initial level of microbial contamination present in the
food matrix. Therefore, the extrapolation of results from in vitro tests to real food is
difficult and a lower level of activity must be expected; for example, cilantro oil had
significant antibacterial activity at 0.018% in vitro, but when the essential oil was
applied to a ham model even 6% cilantro oil had no demonstrated antimicrobial effect
[15]. Films with oregano oil and citral showed total inhibition against Escherichia
coli, Salmonella enterica and Listeria monocytogenes when placed on agar in Petri
dishes. However, in a study with real foodstuffs – packaged salads – lower effects
were observed, which can be attributed to partial sorption of the agent into the food,
with a subsequent reduction of concentration in the vapour phase [5].
147
Therefore it is useful to examine how antimicrobial agents, or their constituents,

interact with food components under in vitro conditions before they are added to
food products. Food matrix interactions with antimicrobial agents can be studied
by measuring the growth of microorganisms in a culture medium modified with a
range of concentrations of added fat, protein or starch, simulating the composition
of the food.
Some authors have developed food model media that are intermediate between a
laboratory culture and real food [16]; for example, a lettuce leaf model medium
is prepared with 50 g of iceberg lettuce (Lactuca sativa sp.) and 100 mL of sterile
deionised water. The mix is shaken for 1 min and the suspension is filtered and
adjusted from pH 5.6 to 7.2 with potassium phosphate buffer. Finally, the suspension
is autoclaved at 121 °C for 15 min [16]. A meat-based model medium can be prepared
using autoclaved beef extract consisting of 12% protein. A milk model medium can
be prepared by mixing skimmed milk powder with an agar solution [16]. Another
example is carrot broth, which is prepared by washing, peeling and mashing carrots
with water.
In studies conducted with active packaging, the first step is to test the antimicrobial
capacity of the developed films, using any of the methods described in Section 5.1,
and the second step is to confirm the antimicrobial effectiveness of the packaging
system by applying it to real food.
No standard protocol has been established for antimicrobial assays involving food,
although some points are common in the literature [17]. To study the effectiveness
of an antimicrobial film against pathogenic bacteria, it is necessary to introduce the
pathogen into the food as an inoculum [17]. For many foods, 10–15 µL of bacterial
suspension containing approximately 104 CFU/mL can be distributed over 1 cm2,
after which it is necessary to wait 20 min for it to dry before packaging.
To study the efficacy of the developed packaging it is necessary to produce a prototype

and simulate real storage at room or refrigeration temperature, depending on the
requirements of the food product. At various storage times, food samples are analysed.
For this purpose, food samples are transferred to a bag containing buffer or peptone
water and homogenised in a stomacher. Finally, serial dilutions are prepared and
seeded on an agar medium to determine the number of viable microorganisms and
the reduction compared with control samples packaged in non-active films. Many
studies have demonstrated different antimicrobial effects over time. Usually the log
reduction in growth on the test sample increases with increased exposure time, but in
about one-third of the experiments there is less difference between the cell count for
the control and test samples [17]. This is due to active films causing inhibition in the
first stages of the storage period, when the concentration of the agent in the packaging
148
is higher and the microbial content is lower. A polymer film is able to act as a carrier
for the antimicrobial agent, allowing slow, extended release of the active compound
over time and maintaining its effect; for example, chitosan (CS) films containing a
higher silver concentration exerted their antimicrobial activity for longer due to the
larger reservoir of silver ions in the form of nanoparticles [18].
5.2.1 Total Microbial Load
Food products undergo deterioration as a result of physical, chemical and biological

processes, including microbial contamination, which is mostly caused by bacteria, yeast
and moulds as a result of a low amount or lack of additives. The intrinsic microbial load
of food is also closely related to the production of undesirable effects in foodstuffs, such as
changes in odour, colour, taste, texture and so on [19]. The set of microorganisms present
in a particular food is called the total microbial load. The presence of microorganisms
in foods not only affects the sensory quality but also limits their shelf life. To prevent
the growth of spoilage or pathogenic microorganisms, the food industry uses various
preservation techniques. Sometimes these methods involve aggressive treatment and
the products may be considered sterile until opened. The use of this treatment extends
the shelf life but it can change the organoleptic properties, the high nutritional value or
the quality of the food. Nowadays, consumers demand minimally processed products
without chemical additives. The new trends in nutrition and modern lifestyle are
increasing the demand for natural products without any processing.
Antimicrobial packaging technologies can provide safe products and extend shelf life.
The use of antimicrobial packaging can provide healthy products without altering
their properties and at the same time control the microbial load. Unprocessed food
carries a standard load of microorganisms, which may be higher or lower, depending
on how it is handled. The presence of these microorganisms can alter and damage
the product, making it unfit for consumption. The use of antimicrobial packaging
can limit the growth of these microorganisms, either by inhibiting their growth or
eliminating them completely, and it can improve the microbiological safety of food
products after package opening.
An in vivo antimicrobial study requires testing the effectiveness of active films against
the total microbial load of the food. Samples of food are coated or packaged with
the antimicrobial films and stored under suitable storage temperature and humidity
conditions; at the same time, samples of control films without the active agent are
coated or packaged too.
Analyses should be performed on different days to check the effect during the
entire storage of the food product. At least 2 samples should be analysed at each
149
time interval during storage; however, analysis in triplicate is always preferred.

The number of samples and replicates must be increased if the variability is high.
The duration of the antimicrobial study must be at least as long as the shelf life
of the product, or longer if the sensory quality is maintained by the antimicrobial
packaging.
The analysis is usually performed with 25 g or 25 mL of food product. The

samples are homogenised in buffer or peptone water in a stomacher for
5–10 min. A 10-fold dilution series is made using the same buffer or peptone water
solution and plated onto an agar medium. Study of the natural microorganisms
present in food involves the use of selective media and specific temperatures and
incubation times in order to count the microbial population in the food: total
aerobic, enterobacteria, psychrotrophic, lactic acid bacteria, yeast and moulds
and so on (Table 5.1). The efficacy is calculated as the difference between the
log 10 count of microorganisms in a control film sample and the log 10 count of
microorganisms in the test sample. The results are expressed per unit of surface
area or per gram of food product.
Table 5.1 Incubation conditions of microorganisms usually present in food

Microorganism Culture medium Temperature Time
Total aerobic Nutrient agar 30 °C 48 h
Total aerobic Nutrient agar 10 °C 10 days
psychrotrophic
Yeast and moulds Malt dextrose agar 25 °C 5 days
Enterobacteria Violet red bile dextrose agar 37 °C 48 h
Lactic acid bacteria Man, Rogosa and Sharpe agar 25 °C 5 days
Pseudomonas King agar 25 °C 48 h
Numerous studies have shown the antimicrobial effect of various active films
against the total microbial load in perishable food; for example, the antimicrobial
effect of ethylene-vinyl alcohol (EVOH)/epsilon-polylysine films applied in the
preservation of surimi gives a shelf life of approximately 48 h after opening [20].
EVOH films with 10% epsilon-polylysine showed an antimicrobial effect against
the total microbial load of surimi sticks which extended the shelf life [20]. EVOH
films with OEO and citral have been used to pack fresh salads [21]. These films
reduced enterobacteria growth by 1.38 log and 2.13 log, and yeast and mould
growth by about 2 log. The total aerobic counts were reduced by 1.08 log with
OEO and by 1.23 log with citral. The reduction of lactic acid and psychrotrophic
bacteria was 2 log [21].
150
Bioactive materials composed of edible CS and casein polymers have been used to
package cheese and salami, significantly inhibiting the growth of mesophilic bacteria,
psychrotrophic bacteria, yeast and moulds [22].
However, to increase the food safety of ready-to-eat products during storage or after
opening it is necessary to control the growth of potential pathogenic microorganisms
as well as the total microbial load.
5.2.2 Deliberate Contamination
Antimicrobial packaging provides an additional, final barrier that can prevent the
growth of foodborne pathogens. Many food products, such as various types of cheese,
meat, poultry, fruit and vegetables, are highly susceptible to microbial spoilage.
Pathogenic microorganisms can originate from postpasteurisation cross-contamination
or could survive the pasteurisation process. Listeria monocytogenes can grow at
temperatures of 4 to 45 °C, and in this case refrigeration alone is not enough to prevent
its growth in foods [23]. Sometimes the goal is to study antimicrobial activity against a
specific spoilage or pathogenic bacterium, such as Listeria monocytogenes, Escherichia
coli O157:H7 or Salmonella, because they could cause foodborne illness; for example,
Clostridium botulinum might be involved in modified atmosphere packaged (MAP)
products or Staphylococcus aureus in foods with a not very competitive total microbial
load and food products with low aw. Therefore, various specific strains may be included
in the challenge study. Occasionally a cocktail or mixture of several strains may be
used in the antimicrobial assay in order to allow for potential strain variation. In
these cases, the food should be previously inoculated with known concentrations of
the microorganisms, in the stationary or exponential growth phase, and packaged
with active film and control samples prepared with the same film but without the
antimicrobial agent. A competitive microbial load can affect the growth of pathogenic
microorganisms in a food product. As the inoculated product could contain a
competitive microbial load, the freshest product possible should be used, i.e., within
the first 10% of its shelf life. A number of factors should be considered in the design of
the microbiological study: the selection of pathogens or surrogates for microorganisms,
the inoculum preparation and concentration, the method of inoculation, the duration
and storage conditions of the study, and the sample analyses. The interpretation of the
results is critical when evaluating the antimicrobial effects [24].
5.2.2.1 Inoculum Preparation
Collections of bacteria and fungi are available from various universities and research
institutes, as explained at the beginning of this chapter. For experimental use, the
151
stock cultures are maintained by regular subculture on tryptone soy agar slants at
4 °C and transferred monthly. First a loopful is transferred into 10 mL of rich liquid
medium, such as tryptone soy broth, and incubated at an optimum temperature
(30–37 °C) for 18 h to obtain early stationary phase cells. Cell cultures of each
microorganism in the stationary phase with an optical density of 0.9 at 600 nm, can
be diluted and incubated until an optical density of 0.2 at 600 nm (105 CFU/mL) is
reached in order to obtain exponential phase cells. The cells should be washed to
remove culture medium and suspended in an appropriate carrier (diluted peptone
water) to inoculate the food.
Incorporation of the microorganisms should simulate the contamination that

could realistically occur under manufacturing or storage conditions. The intrinsic
and extrinsic characteristics of the food product, such as pH and aw, have to be
maintained. Table 5.2 shows aw values for some foods usually employed in these
assays.
Table 5.2 aw values of representative food products

Food product aw
Fresh meat, poultry, fish 0.99–1.00
Cheeses 0.95–1.00
Egg yolks 0.97
Bread 0.95
Raw vegetables 0.99
Raw fruits 0.98
Jams, marmalades, jellies 0.75–0.80
Dried spices, milk powder 0.20–0.60
Biscuits, chocolate >0.60
Previous studies should verify that the temperature, pH and aw of the matrix do
not adversely affect the pathogen viability [25]. The concentration of hydrogen
ions in the food affects the growth and survival of numerous microorganisms.
Although microorganisms may survive in a pH range of 4.6–9.0, the most
favourable pH is usually close to 7. Moulds and yeast may withstand a lower
pH and they are the primary spoilage organisms present in acidic food such as
tomatoes or orange juice.
Control of the water content in food is one preservation strategy. Microorganisms

need available water to be present in food in order to grow. The water requirements
of microorganisms are established in terms of aw which is defined as the ratio of the
152
water vapour pressure of the food substrate to the vapour pressure of pure water at
the same temperature [26]:
p
a 0w = (5.4)
p0
where:
p = vapour pressure of the solution.
p0 = vapour pressure of the solvent (the solvent is usually water).
The aw of pure water is 1.00 and the aw of a completely dehydrated food is 0.00.
Food preservation can be achieved by controlling the aw as microbial growth
requires available water in food products. The aw of a food involves the degree
to which water is bound in the food, its availability to participate in chemical
reactions and its availability to be used for microbial growth. Microorganisms
need certain aw values, below which growth is not possible. Water is required in
many biochemical reactions (proteases and lipases) and without sufficient available
water cellular metabolism will stop. The absence of water produces inactivation of
enzymes involved in the destruction of biological structures; therefore, in adverse
conditions, e.g., without water, microorganisms do not die instead they exhibit
resistance until favourable conditions resume. The aw level that limits growth of
the most pathogenic bacteria is 0.90, for spoilage moulds it is 0.70 and the lower
limit for all microorganisms is 0.60.
It must be borne in mind that fresh food such as meat, vegetables and fruit have
aw values that are close to the optimum growth level of most microorganisms,
approximately 0.97–0.99 aw. Thus aw is a critical factor in the shelf life and quality
of food products. This parameter can be manipulated in foods by adding various
solutes, such as salt or sugar, removing water by drying or baking, or employing
suitable packaging. Critical upper and lower aw levels can be determined with respect
to acceptable microbial growth, flavour, texture, appearance and nutritional quality
of food products. The selection of packaging with the right barrier properties can
control the critical aw values and optimise the quality and shelf life of food.
Furthermore, all microorganisms have a temperature range in which they grow and
this parameter is crucial to selecting the proper storage conditions for a food product.
Bacteria may grow over different temperature ranges, but most grow at 16–45 °C.
The temperature at which most rapid multiplication occurs is the optimum growth
temperature (Figure 5.8) and it is the point at which the generation time is the shortest.
It is not just one temperature, because this parameter can change with chemical and
physical factors and it is not the optimal temperature for all cellular activities. The
lowest temperature at which growth occurs is called the minimum growth temperature
153
(Figure 5.8); this temperature is difficult to determine because physiological activities

gradually decrease until they can no longer be detected by conventional methods. The
generation time may increase from minutes to days or weeks. The highest temperature
at which growth can take place is the maximum growth temperature (Figure 5.8).
Microorganisms have an optimal growth temperature, but refrigerator temperatures
do not destroy spoilage or pathogenic microorganisms as microbial growth on
food can occur slowly at lower temperatures. Perishable food will deteriorate, even
at refrigerator temperatures, owing to spoilage microorganisms or oxidation and
enzymatic processes.
Table 5.3 shows the classification of prokaryotic microorganisms according

to growth temperature. Mesophilic bacteria and fungi have optimal growth at
temperatures of 25–40 °C; the vast majority of human pathogens are mesophilic
as the human body temperature is within this range. Thermophilic or heat-
loving microorganisms grow at temperatures greater than 45 °C; Bacillus
stearothermophilus is an example of a thermophilic microorganism and can be
found in hot springs. Psychrophilic microorganisms grow at temperatures below
20 °C; Listeria spp. is a psychrophilic microorganism and can be isolated from
ice cream and other dairy products.
The antimicrobial effect of active agents is significantly enhanced by increasing

the ambient temperature and using high agent concentrations, as release from the
packaging film has been shown to increase as the assay temperature increases. The
study of antimicrobial packaging film usually begins with tests conducted at optimum
growth temperature in a culture medium appropriate for the microorganism. After
verifying the antimicrobial efficacy of the film, the experiment must be performed
at refrigeration or room temperature, simulating real storage of a food product.
Finally, the film is usually applied to food, so the study is carried out at the storage
temperature of the product.
Figure 5.8 Microbial growth in relation to temperature
154
Table 5.3 Classification and temperature range for prokaryotic microorganisms

Type of microorganism T minimum (°C) T optimum (°C) T maximum (°C)
Psychrophilic -5 to +5 12–15 15–20
Psychrotrophic -5 to +5 25–30 30–35
Mesophilic 5–15 30–45 35–47
Thermophilic 40–45 55–75 60–90
The inoculum level used depends on whether the objective of the assay is to determine
growth or inactivation of a pathogenic microorganism. An inoculum level of between
102 and 103 cells/g of product is used to determine microbial growth and stability
of the food formulation. On the other hand, log-reduction validation protocols use
an inoculum level of 106–7 CFU/g. The inoculum volume should be no more than
1% of the volume of the food, or even less. The inoculum volume can be reduced
by concentrating the microbial suspension via centrifugation and resuspending in a
minimum volume prior to the inoculation process. If the microorganism grows on a
solid medium, the inoculum is picked off the surface.
5.2.2.2 Method of Inoculation
There are a variety of inoculation methods available and the method employed
depends on the properties of the food product. In liquid matrices (sauces, juices,
milks and so on) with high aw (>0.96) the inoculum is added directly with mixing,
using a minimal amount of sterile water or buffer as a carrier. If the antimicrobial
agent is not water-soluble, it is possible to use another solvent to prepare the stock,
but subsequent dilutions should be prepared with water or dimethyl sulfoxide to
improve the microbial viability; ethanol or a similar solvent can affect the viability
of the microorganism.
For individual package or pouch-type applications, the inoculum can be incorporated

using a sterile syringe through a package wall containing a septum. The inoculum is
applied using a needle and is spread over as large an area as possible in order to reduce
the concentration of microorganisms in a specific area. In solid matrices (meat, salads,
cooked pasta and so on) with high aw (>0.96) the inoculum is added using an atomiser.
An atomiser sprays the inoculum, prepared in sterile water or buffer, over the surface
of the product. The spraying method requires protective equipment to avoid possible
worker contamination [25]. For some applications, a surrogate microorganism can be
used instead of pathogenic bacteria. An ideal surrogate retains all the characteristics
of the pathogen except its virulence; the susceptibility must also be similar to that
of the target pathogen. Clostridium sporogenes, Listeria innocua and Escherichia
155
coli are used as surrogates for Clostridium botulinum, Listeria monocytogenes and
Escherichia coli O157:H7.
Finally, alternative methods which can be applied to solid matrices consist of

immersing the food product in the inoculum suspension or dipping or spreading it
over the food surface. These methods do not guarantee homogeneous distribution
of the microorganisms over the surface.
Food products with aw<0.92 can be inoculated using the spray method with a minimal
volume of water or buffer. Sometimes a dried, lyophilised culture may be added
aseptically to the test product and agitated to obtain homogeneous distribution of
the inoculum.
The food product should always be checked to ensure that the final product has
not changed (pH, aw, volume and so on) after the inoculation. The viability of the
microorganisms should also be checked and the population in the inoculated food
should be enumerated to obtain a zero time count. The assays should be performed in
triplicate, and inoculated and uninoculated samples of the control film must be prepared.
5.2.2.3 Storage Conditions
After inoculation, the food product is packaged, simulating commercial production

(bags, under vacuum, modified atmosphere and so on). The storage temperatures
used should be representative of the expected temperature range that the product
will be exposed to during commercial distribution and storage; for example, salad
bags could be stored under refrigeration conditions at 4 °C during the first days of
its shelf life and at an abusive temperature (8 °C) during the last days, simulating
possible consumer manipulation [5].
The duration of the antimicrobial study must be at least as long as the shelf life of
the product. At various storage times, quantitative determination of the pathogenic
microorganisms should be performed and the results compared with samples from
a control package. It is important to have a minimum of 6–7 data points in order
to have a good indication of inoculum growth. If the shelf life is measured in days,
the analysis is performed daily or even several times a day. If the shelf life is longer
(weeks or months), the test frequency is typically no less than once a week. Whatever
the length of the test it must start by counting the microorganisms at time zero, i.e.,
directly, after inoculation. Sometimes it is recommendable to monitor the physico-
chemical parameters of the food product during its shelf life to ascertain the possible
changes or influences on microbial growth. Factors such as aw, pH, moisture and
MAP gas concentration could affect the microbiological stability of the food product.
156
Figure 5.9 shows an example of cream cheese that has been inoculated with Penicillium
expansum and packaged with LLDPE, ION and EMA films containing 5% carvacrol
and 5% thymol. The samples have been stored at 4 °C for 30 days. In this case the
active compounds are volatile and are released from the films into the headspace.
The surface of the cheese shows a reduction in growth of Penicillium expansum in
the presence of the antimicrobial films after 30 days of storage.
Figure 5.9 Cream cheese inoculated with Penicillium expansum. The antimicrobial
films retard fungal growth after 30 days of storage at 4 °C
5.2.2.4 Data Interpretation
The results obtained should be interpreted by expert microbiologists able to consider

the main factors. Bacterial counts could change owing to inherent variations in
sampling and enumeration procedures, particularly when foods contain antimicrobial
agents that limit microbial growth. Sometimes it is difficult to discern whether
changes are real or due to analytical variability. The first consideration is that the
control samples should show microbial growth during the study (minimum 0.5
to 1 log). This depends on the food product, inoculum and enumeration method.
Statistically significant differences may not always be biologically important. An
expert microbiologist using available data obtained from previous analyses should
decide whether the differences are relevant or not.
Mathematical models enable predictions for a wide array of microorganisms and

growth factors (pH, temperature, aw and so on), and the pathogen modelling program
(PMP) and ComBase Predictor are potent tools for modelling food microbiology. Both
programs are also based on real data collected primarily in the laboratory. ComBase
Predictor is based on observations made in culture media and comprises a set of 20
growth models, 7 thermal death models and 2 non-thermal survival models [27].
PMP is a predictive microbiology application able to estimate the effects of multiple
variables on the growth, inactivation or survival of foodborne pathogens, based on
experimental data of microbiological behaviour in liquid media [27].
157
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159
6
Review of Antimicrobial
Packaging Systems
Since the early 1990s, the development of active antimicrobial packaging technology
has been the focus of attention of numerous research teams, particularly during the
last 10 years. A substantial amount of published research has approached this issue
from diverse points-of-view in an attempt to develop novel packaging materials and
specific packaging technologies to control and reduce the microbial spoilage of food.
In Table 6.1 we have compiled a large number of these publications. This table is not
intended as an exhaustive review, which would be an enormous task of limited value,
but as a representative review in which priority has been given to reports focusing on
the development of packaging materials and coatings. With a few exceptions, edible
coatings (which is another line of research that is receiving considerable attention
worldwide), have not been included.
Each table entry includes the antimicrobial agent used, the targeted microorganism(s),
the packaging material in which the agent was incorporated and the methodology
used for that inclusion, as well as the test medium and practical applications. For
further information, see the relevant entry in the reference section.
From this table, several interesting conclusions can be drawn. The list of antimicrobial
agents is very long, but the substances most often used are: a) benzoic, sorbic and
propionic acids; and their salts; b) metals and metal oxides, such as copper, silver
and titanium dioxide; c) polypeptides, mainly nisin, lysozyme and natamycin; and
d) essential oils, mainly oregano, thyme, clove, tea, garlic and some of their basic
components, such as carvacrol and cinnamaldehyde.
These agents have been selected to control the growth of a wide range of microorganisms
which constitute the intrinsic microbial load of food products, but also, in particular,
to reduce the presence of pathogens, such as Escherichia coli, Listeria monocytogenes,
Salmonella spp., Bacillus cereus and Staphylococcus aureus.
161
A diverse range of polymeric materials has been proposed for the introduction of the
antimicrobial agent, although the preferred group of polymers has been biopolymers,
especially polylactic acid (PLA) and chitosan, but also whey protein, soy protein,
cellulose, corn zein and caseinate. To a lesser extent, probably owing to the technical
difficulty involved in their development, conventional (commodity) thermoplastics
have also been proposed, such as polyethylene (PE), polypropylene (PP) and an
ethylene-vinyl alcohol (EVOH) copolymer.
Most of the reported developments are tested by means of in vitro techniques with
the use of culture media and spoilage or pathogenic microorganisms in their preferred
conditions. However, in many of the listed reports the activity and efficiency of the
active materials have been tested on real food products, mainly cheese, ham, salmon,
fresh pasta, poultry and beef meats, and bakery products.
Only a few of these scientific developments have been implemented and are
commercialised. Table 6.2 contains a list of the commercial antimicrobial products
that have reached practical packaging applications.
162
Table 6.1 Developments in antimicrobial packaging systems: agents used, target microorganism(s), agent incorporation method,
target food product and report reference
Antimicrobial agent Microorganism(s) Polymer matrix Incorporation Food References
method
Organic acid and salts
Acetic acid Lactobacillus sakei Chitosan Casting Cooked ham [1]
Propionic acid Serratia liquefaciens Bologna
Lauric acid Pastrami
Cinnamaldehyde
Benzoic anhydride Aerobic and anaerobic counts PE Extrusion Tilapia fillets [2]
Lauric acid Listeria monocytogenes Corn zein Casting Culture medium [3]
Nisin Salmonella enteritidis
EDTA
Lauric acid Lactobacillus plantarum Corn zein Casting Culture medium [4]
Nisin
Lauric acid Listeria monocytogenes Soy protein Compression Turkey bologna [5]
Nisin moulding
Galic acid Escherichia coli Chitosan Casting Culture medium [6]
Chitosan Salmonella typhimurium
Listeria innocua
Bacillus subtilis
Malic acid Listeria monocytogenes Whey protein Casting Culture medium [7]
Natamycin Penicillium commune Cheese
Nisin Penicillium chrysogenum
163
Review of Antimicrobial Packaging Systems
Table 6.1 Continued
Propionic acid Staphylococcus aureus Chitosan Casting Pastry dough [8]
164
Salmonella spp.
Candida spp.
Penicillium spp.
Propionic acid Rhizopus stolonifer PE Extrusion Cheese [9, 10]
Benzoic acid Penicillium spp. Culture medium
Sorbic acid Aspergillus toxicsrius
Sorbic acid Staphylococcus spp. PE Compression Pastry dough [11]
Filamentous fungi moulding
Sodium lactate Listeria monocytogenes Cellulose-based Casting Cold smoked [12]
Sodium diacetate salmon
Potassium sorbate
Sodium benzoate
Nisin
Sodium lactate Listeria innocua Chitosan coating Coating Ready-to-eat meat [13]
LAE Listeria monocytogenes PLA film
Sorbic acid Salmonella typhimurium
Sodium benzoate Penicillium notatum Chitosan Casting Culture medium [14]
Potassium sorbate Rhodotorula rubra MC
Potassium sorbate Yeasts LDPE Extrusion Food products [15]

Sodium propionate Staphylococcus aureus Cellulose acetate Casting Bread slices [16]
Sarcina lute
Proteus vulgaris
Lactobacillus plantarum
Potassium sorbate Escherichia coli Sweet potato Casting Culture medium [17]
Chitosan Staphylococcus aureus starch
Potassium sorbate Yeast and mould counts Starch Blow extrusion Fresh pasta [18]
Poly(butylene
adipate-co-
terephthalate)
Potassium sorbate Escherichia coli Chitosan Casting Culture medium [19]
Garlic oil Staphylococcus aureus
Nisin Listeria monocytogenes
Bacillus cereus
Potassium sorbate Mould growth Chitosan Casting Butter cake [20]
Vanillin
Potassium sorbate Mould growth Chitosan Casting Garlic bread [21]
Garlic oil
Potassium sorbate Debaromyces hansenii Alginate Casting Culture medium [22]
Natamycin Penicillium commune Chitosan
Penicillium roqueforti
Potassium sorbate Candida spp. EVA Extrusion Culture medium [23]
Pichia spp. LDPE Cheese cubes
Trichosporon spp.
Penicillium spp.
Sorbic acid Aspergillus niger Poly(ethylene- Extrusion Culture medium [24]
Benzoic acid Penicillium spp. co-methacrylic
acid)
165
Table 6.1 Continued
166
Metals
Copper-cellulose Saccharomyces cerevisiae Cellulose Casting Culture medium [25]
composite Pineapple
Melon
Copper-PLA Pseudomonas fluorescens PLA Casting Culture medium [26]
nanocomposite Pseudomonas putida
Pseudomonas spp.
Copper Staphylococcus aureus Chitosan Casting Culture medium [27]
nanoparticles Salmonella enterica
Copper Escherichia coli Chitosan Casting Culture medium [28]
nanoparticles Salmonella choleraesuis
Salmonella typhimurium
Staphylococcus aureus
Copper Escherichia coli Chitosan Casting Culture medium [29]

nanoparticles Salmonella choleraesuis
Copper Staphylococcus aureus Cellulose Casting Culture medium [30]
nanoparticles Klebsiella pneumoniae
Copper Saccharomyces cerevisiae Polyvinyl methyl Casting Culture medium [31]
nanoparticles Escherichia coli ketone
Staphylococcus aureus Polyvinyl
Listeria monocytogenes chloride
Polyvinylidene
fluoride
Silver nanoparticles Escherichia coli Chitosan Casting Culture medium [32]
Bacillus subtilis
Silver nanoparticles Staphylococcus aureus Sodium alginate Casting Culture medium [33]
Escherichia coli
Silver nanoparticles Microbial growth Cellulose Casting Culture medium [34]
Fresh-cut melon
Silver nanoparticles Pseudomonas spp. Cellulose Casting Beef meat [35]
Enterobacteriaceae (absorbent pads)
Silver nanoparticles Staphylococcus aureus Cellulose Casting Poultry exudate [36]
Escherichia coli (absorbent pads)
Silver nanoparticles Staphylococcus aureus PLA Casting Poultry exudate [37]
Escherichia coli
Silver nanoparticles Bacillus subtilis Cellulose Casting Culture medium [30]
Klebsiella pneumoniae
Silver nanoparticles Escherichia coli Cellulose Casting Culture medium [38]
Silver nanoparticles Escherichia coli Chitosan lactate Casting Culture medium [39]
Silver nanoparticles Escherichia coli Chitosan- Casting Culture medium [40]
Staphylococcus aureus cellulose
Bacillus cereus
Silver nanoparticles Staphylococcus aureus Cellulose acetate Electrospinning Culture medium [41]
Escherichia coli
167
Pseudomonas aeruginosa
Table 6.1 Continued
168
Silver nanoparticles Staphylococcus aureus HPMC Casting Culture medium [42]
Escherichia coli
Silver nanoparticles Salmonella typhimurium Chitosan Casting Culture medium [43]
Escherichia coli
Listeria monocytogenes
Bacillus
Silver-zinc oxide Some bacteria, moulds and Chitosan Casting Culture medium [47]
yeasts
Silver nanoparticles Staphylococcus aureus Cellulose Casting Culture medium [48]

Titanium dioxide Escherichia coli microfibril
Chitosan
Zinc
Silver nanoparticles Staphylococcus aureus Zein Casting Culture medium [49]
Escherichia coli
Silver nanoparticles Escherichia coli LDPE Injection Chicken breast [50]
Zinc oxide Pseudomonas aeruginosa moulding
Titanium dioxide Staphylococcus aureus Cellulose Casting Culture medium [51]
nanoparticles
Titanium dioxide Staphylococcus aureus Gelatin Casting Culture medium [52]
nanoparticles Escherichia coli
Titanium dioxide Pseudomonas spp. LDPE Extrusion Culture medium [53]
nanoparticles Rhodotorula mucilaginosa Fresh pears
Titanium dioxide Escherichia coli Whey protein Casting Culture medium [54]
nanoparticles Zein
Titanium dioxide Staphylococcus aureus Chitosan Casting Culture medium [55]
nanotubes Escherichia coli
Salmonella enterica
Zinc Some Gram-negative and Gram- Chitosan Casting Culture medium [56]
positive bacteria
Zinc oxide Salmonella typhimurium Calcium alginate Casting Poultry meat [57]
nanoparticles Staphylococcus aureus
Zinc oxide Escherichia coli Cellulose acetate Casting Culture medium [58]
nanoparticles
Polypeptides
Dermaseptin S4 Microbial growth Corn starch Casting Cucumber [59]
coating on PE
shrinkable film
Enterocin Listeria monocytogenes Alginate-zein- Casting Cooked ham [60]
PVOH
Epsilon-polylysine Listeria monocytogenes EVOH Casting Culture medium [61]
169
Table 6.1 Continued
170
Epsilon-polylysine Microbial growth Whey protein Casting Fresh beef [62]
Sodium lactate
Epsilon-polylysine Microbial growth PVOH Casting Culture medium [63]
Epsilon-polylysine Microbial growth Whey protein Casting Fresh beef [64]
Oregano oil
Sodium lactate
Glucose oxidase Escherichia coli PA Casting Culture medium [65]
Lysozyme Pseudomonas fluorescens Ionomer
Lactobacillus helveticus
Listeria ivanovii
Listeria innocua
Glucose oxidase Escherichia coli BOPP Plasma Culture medium [66]
Bacillus subtilis activation
Lactocin Lactobacillus plantarum Commercial Casting Wiener [67]
Listeria innocua multilayer film

Wheat gluten
Lactoperoxidase Salmonella enterica Whey protein Casting Roasted turkey [68]
Escherichia coli
Lactoperoxidase Escherichia coli Alginate Casting Culture medium [69]
Listeria innocua
Pseudomonas fluorescens
Lactoperoxidase Salmonella enterica Whey protein Casting Culture medium [70]
Lactoferrin Escherichia coli
Lysozyme
Lysozyme Listeria monocytogenes Whey protein Casting Culture medium [71]
Smoked salmon
Lysozyme Listeria innocua Sodium alginate Casting Culture medium [72]
Nisin Escherichia coli κ-carrageenan
Grapefruit Salmonella enteritidis
EDTA Staphylococcus aureus
Lysozyme Listeria monocytogenes Calcium alginate Casting Smoked salmon [73]
Nisin Salmonella anatum
Lysozyme Brochotrix thermosphacta Pea starch Extrusion Culture medium [74]
Lysozyme Micrococcus lysodeikticus Cellulose Casting Culture medium [75]
triacetate
PVOH
Nylon 6.6
Lysozyme Bacillus amyloliquefaciens Cellulose acetate Casting Culture medium [76]
Escherichia coli
Lysozyme Escherichia coli Fish-skin gelatin Casting Culture medium [77]
Bacillus subtilis
Streptococcus cremoris
Lysozyme Brochothrix thermosphacta Gelatin Casting Ham [78]
Nisin Escherichia coli Bologna sausage
EDTA Lactobacillus sakei
Leuconostoc mesenteroides
171
Table 6.1 Continued
172
Lysozyme Escherichia coli Starch Casting Culture medium [79]
Bacillus subtilis
Lysozyme Escherichia coli Chitosan Casting Culture medium [80]
Streptococcus faecalis
Lysozyme Listeria monocytogenes Chitosan Casting Mozzarella cheese [81, 82]
Escherichia coli
Lysozyme Listeria monocytogenes EVOH Casting Culture medium [83]
Lysozyme Listeria monocytogenes PP Metal- Culture medium [84]
chelating
Lysozyme Bacillus subtilis Zein Casting Culture medium [85]
EDTA Lactobacillus plantarum
Lysozyme Lactobacillus plantarum Zein Heat-press Culture medium [86]
Casting
EDTA Escherichia coli Soy protein

Nisin
Lysozyme Escherichia coli Staphylococcus Fish gelatin Casting Culture medium [87]
Catechin aureus
Listeria innocua
Saccharomyces cerevisiae
Lysozyme Micrococcus lysodeikticus LDPE Extrusion Culture medium [88]
Lemon extract Pseudomonas spp.
Natamycin Debaromyces hansenii Alginate Casting Culture medium [22]
Potassium sorbate Penicillium commune Chitosan
Penicillium roqueforti
Natamycin Penicillium roqueforti Cellulose Casting Gorgonzola cheese [89]
Natamycin Staphylococcus aureus Cellulose Casting Culture medium [90]
Nisin Listeria monocytogenes Sliced mozzarella
Penicillium sp. cheese
Geotrichum sp.
Natamycin Aspergillus niger Wheat gluten Casting Fresh kashar cheese [91]
Penicillium roqueforti MC
Natamycin Listeria monocytogenes Whey protein Casting Culture medium [92]
Nisin Penicillium commune Cheese
Malic acid Penicillium chrysogenum
Natamycin Alternaria alternata Chitosan Casting Hami melon [93]
Fusarium semitectum PE wax
Nisin Salmonella typhimurium Calcium alginate Casting Poultry skin [94]
Nisin Brochothrix thermosphacta PE Extrusion Beef carcass [95]

Nisin Listeria monocytogenes Sodium caseinate Casting Culture medium [96]
Sodium lactate
Potassium sorbate
Nisin Listeria monocytogenes Sodium caseinate Casting Cheese [97]
Nisin Micrococcus luteus MC Casting Culture medium [98]
HPMC Heat-pressed
κ-carrageenan
Chitosan
173
Table 6.1 Continued
174
Nisin Lactococcus lactis Cellulose Casting Sliced cheese [99]
Lacticin Listeria innocua PE Ham
Staphylococcus aureus PA
Nisin Listeria monocytogenes Bacterial cellulose Casting Culture medium [100]
Frankfurters
Nisin Listeria monocytogenes Gelatin Casting Culture medium [101]
Turkey bologna
Nisin Brochothrix thermosphacta Gelatin Casting Ham [78]
Lysozyme Escherichia coli Bologna sausage
EDTA Lactobacillus sakei
Leuconostoc mesenteroides
Nisin Listeria monocytogenes Wheat gluten Casting Turkey bologna [102]

Nisin Listeria monocytogenes MC Casting Hot dogs [103]
HPMC
Nisin Listeria monocytogenes MC/HPMC Casting Hot dogs [104]
coating LDPE
film
Nisin Listeria monocytogenes PLA Casting Culture medium [105]
Pectin Orange juice
Liquid eggs
Nisin Listeria monocytogenes PLA Casting Culture medium [106]
Escherichia coli Orange juice
Salmonella enteritidis Liquid eggs
Nisin Lactobacillus plantarum PLA Extrusion Culture medium [107]
Pectin
Nisin Escherichia coli PLA Extrusion Culture medium [108]
EDTA
Nisin Listeria monocytogenes Soy protein Casting Culture medium [109]
Organic acids Escherichia coli O157:H7
Salmonella gaminara
Nisin Lactobacillus plantarum Zein Heat-press Culture medium [86]
EDTA Escherichia coli Soy protein Casting
Lysozyme
Nisin Lactobacillus plantarum Corn zein Casting Culture medium [4]
Lauric acid
Nisin Listeria monocytogenes Whey protein Casting Turkey frankfurter [110]
GSE Escherichia coli
Malic acid Salmonella typhimurium
EDTA
Nisin Listeria monocytogenes Whey protein Casting Culture medium [7]
Organic acids
Nisin Escherichia coli Chitosan Casting Culture medium [19]
Potassium sorbate Staphylococcus aureus
Garlic oil Listeria monocytogenes
175
Bacillus cereus
Table 6.1 Continued
176
Nisin Listeria monocytogenes Corn zein Casting Culture medium [3]
Lauric acid Salmonella enteritidis
EDTA
Lauric acid
Nisin Salmonella enterica PLA Coating Shell eggs [111]
Allyl isothiocyanate Chitosan
LAE
Wheat gluten Heat-pressed
Nisin Some Gram-negative and Gram- Zein Casting Culture medium [113]
positive bacteria
Pediocin Listeria monocytogenes Cellulose coating Casting Turkey breasts [114]
in barrier bags Hams

Boneless beef
Pediocin Listeria innocua Cellulose Casting Sliced ham [115]
Salmonella sp.
Sacacin A Listeria monocytogenes Pullulan Casting Turkey deli meat [116]
Essential oils/plant extracts
Allyl isothiocyanate Salmonella enterica PLA Coating Shell eggs [111]
Nisin Chitosan
LAE
Allyl isothiocyanate Bacillus cereus Soy protein Casting Alfalfa sprouts [117]
Cinnamaldehyde Listeria monocytogenes coating on BOPP/ Broccoli sprouts
PE film
Garlic oil Enterobacter sakazakii Radish sprouts
Rosemary oil
Allyl isothiocyanate Salmonella spp. Chitosan Coating Cantalupo [118]
Nisin
Bergamot Escherichia coli Gelatin Casting Culture medium [119]
Lemongrass Staphylococcus aureus
Carvacrol Some Gram-negative and Gram- Gelatin Coating Culture medium [120]
positive bacteria
Carvacrol Escherichia coli Sodium caseinate Casting Culture medium [121]
Staphylococcus aureus Calcium
caseinate
Carvacrol Escherichia coli Soy protein- Coating on Culture medium [122]
Cinnamaldehyde Botrytis cinerea starch coating on paper
paper
Carvacrol Salmonella typhimurium Chitosan Casting Culture medium [123]
Escherichia coli
Carvacrol – EVOH in PP/ Coextrusion Fresh salmon [124]
EVOH/PP film
Carvacrol Escherichia coli LDPE/clay Extrusion Culture medium [125]
Listeria innocua composite
177
Alternaria alternata
Table 6.1 Continued
CEO Listeria monocytogenes Chitosan Casting Culture medium [126]
178
Escherichia coli
Lactobacillus plantarum
Lactobacillus sakei
CEO Escherichia coli Chitosan Casting Culture medium [127]
Lemon essential oils Staphylococcus aureus
Thyme essential oils
CEO Aspergillus niger Chitosan Casting Culture medium [128]
Botrytis cinerea
Rhizopus stolonifer
CEO Presence of moulds Patented – Bakery product [129]
formulation
coating PP films
CEO Some bacteria, moulds and Patented – Culture medium [130]
OEO yeasts formulation

coating PP or
Clove essential oil
PE/EVOH film
CEO Alternaria alternata Paraffin coating Coating Culture medium [131]
OEO on paper Cherry tomatoes
Clove essential oil
Cinnamon extract Colletotrichum musae Chitosan Coating Culture medium [132]
Piper extract Fusarium spp.
Garlic extract Lasiodiplodia theobromae
Escherichia coli
Bacillus cereus
Cinnamaldeyde Some bacteria and yeasts MC Coating Culture medium [133]
Eugenol
Cinnamaldeyde Penicillium expansum Gliadin Casting Sliced bread [134]
Aspergillus niger Cheese spread
Cinnamaldeyde Penicillium spp. Gliadin Casting Sliced cheese [135]
Natamycin Alternaria solani
Colletotrichum acutatum
Cinnamaldehyde Pseudomonas putida Whey protein Coating Precooked shrimp [136]
Thyme oil Soy protein
Cinnamaldehyde Lactobacillus sakei Chitosan film Casting Cooked ham [137]
Acetic acid Serratia liquefaciens Bologna
Propionic acid Pastrami
Lauric acid
Cinnamaldeyde Escherichia coli PLA Casting Culture medium [138]
Bacillus cereus
Citral Campylobacter jejuni Kafirin Coating Culture medium [139]
Quercetin Listeria monocytogenes
Clove essential oil Some Gram-negative and Gram- Sunflower Casting Culture medium [140]
positive bacteria protein Sardine patties
Eucalyptus extract Staphylococcus aureus Tapioca starch Casting Culture medium [141]
Eugenol Bacillus cereus Zein Casting Culture medium [113]
Succinic acid Salmonella enterica
179
Citric acid
Table 6.1 Continued
180
Garlic oil Listeria monocytogenes EVOH Extrusion Ready to eat beef [142]
Escherichia coli loaves
Brochothrix thermosphacta
Garlic oil Escherichia coli Chitosan Casting Culture medium [19]
Potassium sorbate Staphylococcus aureus
Nisin Listeria monocytogenes
Bacillus cereus
Garlic oil Brochothrix thermosphacta Hake by- Casting Culture medium [143]
Clove oil Escherichia coli products
OEO Listeria innocua

Pseudomonas putida Salmonella
typhimurium
Garlic oil Mould growth Chitosan Casting Garlic bread [21]
Potassium sorbate
Ginseng extracts Some Gram-negative and Gram- Alginate Casting Culture medium [144]
positive bacteria
GSE Listeria monocytogenes Whey protein Casting Turkey frankfurter [110]
Nisin Escherichia coli
Malic acid Salmonella typhimurium
EDTA
GSE Listeria monocytogenes Barley bran Casting Culture medium [145]
Escherichia coli protein Gelatin Salmon
GSE Listeria monocytogenes Gelidium Casting Pork loins [146]
Escherichia coli corneum-gelatin
GSE Listeria monocytogenes Gelidium Casting Culture medium [147]
Thymol Escherichia coli corneum-gelatin
nanocomposite
GSE Listeria monocytogenes Gelidium Casting Culture medium [148]
Escherichia coli corneum Fish paste
Salmonella typhimurium Whey protein
isolate
GSE Listeria monocytogenes Red algae Casting Cheese [149]
Escherichia coli Bacon
GSE Some bacteria and moulds LDPE Extrusion Culture medium [150]
Coating Ground beef
GSE Listeria innocua Sodium alginate Casting Culture medium [72]
Lysozyme Escherichia coli κ-carrageenan
Nisin Salmonella enteritidis
EDTA Staphylococcus aureus
Trans-2-hexenal Alternaria solani PLA Extrusion Culture medium [151]
Aspergillus niger Casting
Botrytis cinerea
Colletotrichum acutatum
Penicillium spp.
Linalool Listeria innocua LDPE Extrusion Culture medium [152]
Methyl chavicol Escherichia coli
Linalool Escherichia coli LDPE Extrusion Culture medium [153]
181
Methyl chavicol EVA

Table 6.1 Continued
182
2-nonanone Botrytis cinerea Independent – Strawberries [154, 155]
device
Olive leaf extract Staphylococcus aureus MC Casting Kasar cheese slices [156]
Escherichia coli
OEO Escherichia coli Milk protein Casting Beef muscle [157]
Pimento oils Pseudomonas spp.
OEO Listeria monocytogenes Chitosan coating Casting Kashar cheese [158]
Clove essential oil Escherichia coli on PP
OEO Listeria monocytogenes Alginate Casting Culture medium [159]
Escherichia coli
Salmonella enteritidis
OEO Bacillus cereus Starch Extrusion Culture medium [160]

Escherichia coli Chitosan
OEO Some Gram-negative and Gram- Quince seed Casting Culture medium [161]
positive bacteria mucilage
OEO Pseudomonas spp. Whey protein Casting Fresh beef [162]
OEO Listeria innocua Whey protein Casting Culture medium [163]
Sage essential oil Staphylococcus aureus Cellulose
OEO Listeria monocytogenes Chitosan Casting Bologna slices [164]
Anise essential oil Escherichia coli
Basil essential oil
Coriander
essential oil
OEO Brochothrix thermosphacta Chitosan Casting Chicken breast [165]
Enterobacteriaceae
Pseudomonas spp.
OEO Rhizopus stolonifer Chitosan Casting Culture medium [166]
Aspergillus niger Grape
OEO Salmonella enterica PP Casting Minimally processed [167, 168]
Citral Listeria monocytogenes EVOH salads
Escherichia coli
Rosemary essential Listeria monocytogenes Chitosan Casting Culture medium [169]
oil Pseudomonas putida
Streptococcus agalactiae
Escherichia coli
Lactococcus lactis
Tea essential oil Listeria monocytogenes Chitosan Casting Culture medium [170]
Penicillium italicum
Tea essential oil Listeria monocytogenes Chitosan Casting Culture medium [171]
Bergamot Escherichia coli HPMC
Lemon Staphylococcus aureus
183
Table 6.1 Continued
184
Tea extract Some bacteria, yeasts and Chitosan Casting Culture medium [172]
moulds Pork sausages
Green tea extract Penicillium expansum EVOH Casting Culture medium [173]
OEO Listeria monocytogenes
Escherichia coli
Thyme oil Pseudomonas putida Whey protein Coating Precooked shrimp [136]
Cinnamaldehyde Soy protein
Thyme essential oils Listeria monocytogenes Chitosan Casting Culture medium [174]
Clove essential oils Staphylococcus aureus
CEO Salmonella enteritidis
Cinnamon Some yeasts and moulds Cassava starch Casting Bread slices [175]
Clove
Thyme essential oils Some Gram-negative and Gram- Quince seed Casting Culture medium [176]
positive bacteria mucilage
Thyme essential oils Escherichia coli Chitosan Casting Culture medium [177]
Thyme essential oils Some bacteria, yeasts Chitosan Spray Chicken-pepper [178]
and moulds kebab
Thymol Bacillus cereus Zein Casting Culture medium [179]
Candida lusitaniae
Pseudomonas spp.
Streptococcus thermophilus
Vanillin Escherichia coli Chitosan Casting Fresh-cut cantaloupe [180]
Saccharomyces cerevisiae MC Pineapple
Zataria multiflora Escherichia coli Carboxymethyl Casting Culture medium [181]
essential oil Bacillus cereus cellulose
Others
LAE Listeria monocytogenes PLA Casting Cooked cured ham [182]
LAE Salmonella enterica PLA Coating Shell eggs [111]
Allyl isotiocianate Chitosan
Nisin
LAE Listeria innocua PLA Coating Ready-to-eat deli [183]
Nisin turkey meat
Chitosan
LAE Listeria innocua PLA Coating Ready-to-eat meat [13]
Sodium lactate Listeria monocytogenes
Sorbic acid Salmonella typhimurium
Chitosan
185
Table 6.1 Continued
186
LAE Some bacteria, yeasts and fungi Chitosan Casting Chicken breast fillet [184]
LAE Escherichia coli Patented Coating Culture medium [185]
OEO formulation Sheep cheese
coating PP or
polyethylene
terephthalate
films
LAE Escherichia coli EVOH Coating Surimi sticks [186]
Listeria monocytogenes Chicken stock
Imazalil Penicillium spp. LDPE Coating Cheddar cheese [187]
Aspergillus toxicarius
BOPP: Biaxially oriented polypropylene

CEO: Cinnamon essential oil
EDTA: Ethylenediaminetetraacetic acid
EVA: Ethylene-vinyl acetate

GSE: Grapefruit seed extract
HPMC: Hydroxypropyl methyl cellulose
LAE: Ethyl lauroyl arginate
LDPE: Low-density polyethylene
MC: Methyl cellulose
OEO: Oregano essential oil(s)
PA: Polyamide(s)
PVOH: Polyvinyl alcohol
Table 6.2 Selected commercial antimicrobial materials

Trade name Manufacturer Active agent
Negamold® Freund Industrial Co., Japan Ethanol
Ethicarp®
Oitech™ Nippon Kayaku Co. Ltd., Japan Ethanol
Fretek® Fretek Co. Ltd., Japan Ethanol
AglON™ AgION Technologies Inc., USA Silver
Ageless SE Mitsubishi Gas Chem., Japan Silver
Alesta AM
®
Axalta Coating Systems, USA Silver
Novaron ®
Toagosei Co Ltd., Japan Silver
Apacider® Sangi Co. Ltd, Japan Silver
MicroFree® DuPont, USA Silver
Bactekiller Kanebo Co., Japan Silver zeolite
Microban ®
Microban International Ltd., USA Triclosan
WasaOuro ®
Lintec Corp., Japan Allyl isothiocyanate
Nisaplin ®
Integrated Ingredients, USA Nisin
Citrex Citrex Inc., USA Citric acid extract
FreshPax® Multisorb Technologies, USA Carbon dioxide
Verifrais SARL Codimer, France Carbon dioxide
Microsphere ®
Bernard Technologies, USA Dichlorine monoxide
Sanitized ®
Sanitized AG., Switzerland Triclosan
Silver
MicroGarde® Jinjiang Huawin Chemical Co., Ltd, Antimicrobial agent HW101
China
Grape Guard Química Osku S.A., Chile Sulfur dioxide
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M-L. Garcia-Lopez, Food Control, 2014, 42, 296.

187. Y-M. Weng and J.H. Hotchkiss, Journal of Food Protection, 1992, 55, 367.
199
7
Case Study: Active Packaging of Minimally
Processed Vegetables
7.1 Description of the Product
Obesity is increasing in our society owing to fast food habits and a sedentary
lifestyle. However, most of the population is aware of this problem and tries to
overcome it by regularly participating in sport and eating more healthily. Nowadays
there is considerable demand for fresh, natural, healthy products, including
fruits and vegetables, which contain high levels of vitamins and minerals and are
essential in human nutrition to form part of a balanced diet; a good example is the
Mediterranean diet.
Another characteristic of the modern lifestyle is the lack of time for cooking, which
has increased the demand for ready-to-eat products. Minimally processed salads
are widely requested in present-day society because they satisfy the desire for fresh,
natural, healthy products and the demand for ready-to-eat foodstuffs.
Fresh salads do not require additional processing or washing prior to consumption, so

it is essential that these foods meet high microbiological quality standards. However,
foods of this kind are subjected to considerable handling, increasing the possibility of
contamination by foodborne pathogens, especially psychrotrophic bacteria which are
able to grow at refrigeration temperatures. Food products must be microbiologically
safety and of high quality.
If food and food packaging producers, manufacturers and distributors follow their
good practice guidelines, i.e, good agricultural practice, good manufacturing practice
and good distribution practice respectively, it is possible to be confident that these
minimally processed ready-to-eat vegetable products can be consumed with a minimal
risk of contamination.
The implementation of good sanitation is necessary to ensure the safety of minimally

processed fruits and vegetables. Despite the constant advances that have been made
to reduce the risk of contamination, these horticultural products have been involved
201
in some cases of foodborne illness. Vegetables are produced in a natural environment,

so it is not possible to expect products which lack a microbial load. Even if there
are no pathogenic bacteria, alteration of the organoleptic quality of food is usually
related to excessive microbial growth of spoilage microorganisms naturally present
on the product, which ultimately limits its shelf life.
Fresh vegetables may be injured during harvesting, transportation and handling,

resulting in the release of nutrients which are excellent substrates for the development
of microorganisms present on the surface of the vegetables [1]. In addition, the
processing of fresh salad, which includes prewashing, peeling, washing, slicing, cutting,
shredding, straining and packaging, may alter or increase the number and type of
spoilage microorganisms or pathogens present on the surface of the product [1]. Since
minimally processed vegetables such as salads are ready-to-eat products, they must
be carefully washed and sanitised to remove dirt, residues, pesticides, insects and
microorganisms that cause quality loss and product decay. However, the sanitation
process cannot ensure complete asepsis of vegetables such as is obtained when a thermal
treatment is applied.
Foodborne illnesses can be produced by microorganisms and/or their toxins, such

as Salmonella, Escherichia coli O157:H7, Bacillus anthracis, Mycobacterium spp.,
Brucella spp., Listeria monocytogenes, Yersinia enterocolitica, Clostridium perfringens,
Klebsiella spp. and Mycobacterium avium subspecies paratuberculosis [2]. The
method of vegetable cultivation may largely account for pathogenic contamination.
The incidence of foodborne outbreaks produced by contaminated salads and fresh
vegetables has increased in recent years. In 1998, the World Health Organization
(WHO) detected the presence of the following pathogens in fruits and vegetables,
which were eaten raw: Salmonella spp., Shigella spp., Escherichia coli, Campylobacter,
Yersinia enterocolitica, Listeria monocytogenes, Staphylococcus aureus, Clostridium
spp., Bacillus cereus, Vibrio spp., viruses (Norwalk-like, hepatitis A) and parasites
(Cryptosporidium or Cyclospora) [3]. Fruits and vegetables grow in the natural
environment and are susceptible to contamination from several sources such as
soil, water irrigation, manure, wild and farm animals, humans, equipment, and
postharvesting treatment and distribution.
The primary sources of contamination are considered to be bad handling in food

service establishments and farm-derived pollution [4]. The increasing demand for
fresh vegetables has resulted in agricultural sites being located close to animal farms,
increasing the risk of cross-contamination. Escherichia coli is a bacterium which
colonises our intestines, the most dangerous type being Escherichia coli O157:H7,
which causes bloody diarrhoea, kidney failure and even death. This bacterium
produces Shiga toxin, which is responsible for haemolytic uraemic syndrome,
destroying red blood cells and causing kidney injury. Haemolytic uraemic syndrome
202
Case Study: Active Packaging of Minimally Processed Vegetables
requires intensive hospital care and transfusions. Escherichia coli O157:H7 has been
found in plant tissues and has been associated with various procedures focused mainly
on soil contamination. Contaminated irrigation water or manure may be related to
the presence of Escherichia coli O157:H7 in fresh vegetables.
Listeria monocytogenes is a Gram-positive bacterium and an important foodborne

pathogen with a high impact on public health and the economy. Listeria monocytogenes
causes septicaemia, meningitis and meningoencephalitis, usually in children or
elderly people and people with immunosuppression induced by drugs or diseases.
In pregnant women it can cause abortion or premature death of the foetus. Stephan
and co-workers [5] described a nationwide outbreak of listeriosis which occurred in
2013/2014 owing to contaminated ready-to-eat salads. Listeria monocytogenes can
be found in smoked fish or mussels, sushi, sashimi, sausages, milk, cream, cheeses,
raw vegetables, drinks made from fresh fruits or vegetables, and preprepared or
prepackaged fruit and vegetable salads, among other products.
Salmonella is heavily involved in foodborne illness produced by contaminated

products. The bacterium lives in the intestinal tracts of animals and humans which
have been infected. Salmonella produces an illness called salmonellosis and causes
diarrhoea, abdominal cramps and fever within 8 to 72 h after ingestion of the
contaminated food.
In view of the danger of contamination by these pathogens, the food safety authorities
have included strict limits in national and international regulations on the acceptable
microbial load in food, with special provisions for some ubiquitous pathogens [6].
National microbiological guidelines have been published for ready-to-eat food in

the UK [7], Spain [8], France [9], Germany [10] and Japan [11]. The European
Commission has published a new regulation (No.2073/2005, Official Journal of
the European Union, 2005, L 338), which establishes a common process to ensure
the food safety and hygiene criteria for food in the European Union (EU) countries,
including precut fruit and vegetables, and sprouts [12]. According to this regulation,
the presence of Escherichia coli in ready-to-eat precut fruit and vegetables must be
≤100 colony forming unit (CFU)/g.
In the USA, a Consumers Union report published in 2010 established the need for
the US Food and Drug Administration (FDA) to set safety standards for the growing
and harvesting of produce classed as high-risk food, including bagged salads. There
are standards for key microorganisms, such as Escherichia coli, Enterococcus and
total and faecal coliforms in meat, drinking water and milk, but there are no federal
standards for these microorganisms in salads. However, random analyses of salads
usually show a percentage of samples with a level of Enterococcus and coliforms
which is unacceptable in bagged salads [6].
203
The UK, Ireland and Germany have chosen a generic microbe, Escherichia coli, as
an indicator and set the limit at 100 CFU/g for ready-to-eat leafy greens. Switzerland
has established an Escherichia coli limit of 10 CFU/g. France and Brazil have set the
limit for faecal coliforms at 1,000 CFU/g and 100 CFU/gm, respectively. Israel has
established a limit of 100 CFU/g for total coliforms in salads. Spain has a maximum
limit of 100 CFU/g for Escherichia coli and Listeria monocytogenes and absence of
Salmonella in 25 g of product.
7.2 Decontamination Technologies and Antimicrobial Agents

Applied to Fresh Vegetables
Several decontamination technologies are applied to cut, washed and prepared salads.
In the following subsections some of the methods of sanitisation most commonly
employed and the antimicrobial agents applied to salads are described.
7.2.1 Chlorine
Among the various decontamination technologies that can be applied to this food
category, the most common method of decontaminating fresh vegetables and ready-
to-eat salads is based on the application of chlorine. This reagent is delivered as a
sodium hypochlorite solution at pH 6.5, but for chlorine to be effective it must be
used in concentrations of 50–200 ppm at a pH <8 and it must be in contact with the
product for 1 min or longer. The WHO [3] reported the efficacy of chlorine against
pathogenic bacteria such as Salmonella, Escherichia coli O157:H7 and aerobic
mesophilic bacteria in lettuce leaves washed with 200 ppm for 10 min. Other authors
found 1 log microbial reductions in lettuce washed for 3 min with 100 ppm chlorine
at 47 and 4 °C [13]. Zhang and Farber, using 200 ppm chlorine for 10 min at 4 and
22 °C, obtained log reductions of Listeria monocytogenes of 1.3 and 1.7 on lettuce
[14]. Similar results were obtained against Escherichia coli 0157:H7 with a treatment
of 200 ppm chlorine for 5 min [15]. As these results show, even with recommended
or greater chlorine concentration, pH or immersion time, the log reduction is
<2 logs. Typically, therefore, fresh vegetables have a high microbial load when they
are marketed. Giusti and co-workers [16] showed the presence of aerobic mesophilic
counts on ready-to-eat vegetables of approximately 106–107 CFU/g 24 h after packing,
increasing to 109–1010 CFU/g after 7 days.
There is industrial and consumer demand for natural alternatives to a chlorine wash
for decontamination of fresh products because, in addition to its limited antimicrobial
activity, some authors warn of the potential formation of carcinogenic compounds
in the water that is used [17]. There is no way to achieve the complete elimination
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of pathogenic microorganisms on fresh vegetables without affecting product quality;

consequently, the number of documented outbreaks of human illness related to the
consumption of ready-to-eat vegetables has increased considerably in recent decades [2].
7.2.2 Chlorine Dioxide
Chlorine dioxide is an alternative treatment to hypochlorite which is gaining attention

in the industry because it is more effective owing to the fact that chlorine dioxide is
2.5-fold more oxidative than chlorine and less reactive to organic compounds [18].
On the other hand, chlorine dioxide is unstable and can be explosive when it is
concentrated. In the USA, 5 ppm is the maximum permitted concentration of chlorine
dioxide for use in the disinfection of whole fresh fruits and vegetables [3].
Wu and Kim [19] employed aqueous chlorine dioxide for the decontamination of
blueberries. The aqueous form offers advantages compared with traditional gaseous
chlorine dioxide for the decontamination of vegetables and fruits, because it is not
necessary to use a special treatment chamber. These authors reduced the growth
of Listeria monocytogenes, Salmonella typhimurium, Pseudomonas aeruginosa,
Staphylococcus aureus and Yersinia enterocolitica. Although the antimicrobial effect
was good, the level of chlorine dioxide was higher than the previously mentioned
maximum concentration established by the regulations.
7.2.3 Ozone
Ozone is widely used to kill pathogenic bacteria in the treatment of drinking water.
In the USA, the FDA has approved the use of ozone as an antimicrobial treatment to
be applied to food products such as minimally processed fruits and vegetables. Ozone
has proved to be effective in 20–30 min of treatment at room temperature. However,
ozone is very unstable, must be generated on site and decomposes quickly in water.
The effectiveness of ozone treatment has been shown against Listeria monocytogenes,
Escherichia coli and aerobic and psychrotrophic microorganisms. The log reduction
of ozone varies in the 0.8–1.8 range, which is very similar to chlorine (2 log). Ozone
has a strong oxidising power, which could reduce the life of processing equipment
in the food industry by corroding metal surfaces. Moreover, ozone can change the
organoleptic characteristics of food, such as colour, and reduce product shelf life.
Ozone generated by an inpackage device has been used as an alternative to chemical

sanitisers for tomatoes, reducing the microbial growth of Escherichia coli O157:H7
and Salmonella spp. [20]. However, further research is needed to adapt this technology
for the food industry.
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7.2.4 Irradiation
Gamma irradiation (4 Gy) has been approved by the FDA as a treatment for
fresh foods to control foodborne pathogens and extend the shelf life of lettuce
and spinach [21]. The antimicrobial effectiveness of gamma irradiation has been
shown against Escherichia coli O157:H7 in coriander [22], and against Listeria
monocytogenes in cabbage, tomatoes, broccoli and mung bean sprouts [23].
A reduction in microbiological growth was observed during the treatment time, but
microbial growth during storage, distribution and shelf life was not investigated.
It is not possible to know whether the microbiological reduction was maintained
during the marketing of the product [21]. Villagómez and co-workers [24] reported
that the antimicrobial effectiveness of gamma irradiation against Salmonella on
cilantro decreases with storage time after irradiation; however, Salmonella was able
to grow after irradiation. These results suggest that irradiation can be used as a
postharvest treatment, not alone but as one of several combined hurdle technology
approaches [21]. Further research is needed to ensure the safety and health benefits
of this treatment: moreover, nowadays negative consumer perception of irradiated
food is common.
7.2.5 Modified Atmosphere Packaging
Packaging is perhaps the main technology for reducing or delaying the physical,
chemical and microbiological changes that take place in fruits and vegetables after
harvesting. Fruits and vegetables are highly perishable foods and require the protection
of appropriate packaging to maintain product safety and quality during the successive
stages of product progression from the field to the table. In recent decades, changing
the composition of the package headspace has become the most widely used method
to improve the preservation of fresh produce and is one of the preferred methods
for other food categories. An appropriate composition of the packaging atmosphere
can reduce microbial growth, the development of internal chemical reactions and
gases, and vapour exchanges between the food and the headspace and between the
headspace and the ambient environment.
Modified atmosphere packaging (MAP) technology involves replacing the ambient

air with a mixture of gases, normally nitrogen, oxygen or carbon dioxide, in certain
initial proportions that may vary over time, depending on the nature of the product,
the packaging characteristics and the storage conditions [25]. In fresh fruit and
vegetables, MAP can decrease the respiration rate and metabolic activity, reduce
moisture loss and retard or prevent microbial growth, thus diminishing the loss of
quality and acceptability during distribution and marketing, as well as protecting
against mechanical damage during commercial handling [26].
206
MAP must satisfy the unique demands for each product and for its particular
marketing conditions. Firstly, the composition of the packaging atmosphere is not the
same for all products because it has to counter various mechanisms of deterioration.
Secondly, the packaging structure must take into account the permeability of the
relevant gases and vapours present in the headspace. This is especially critical when
the packaged product exchanges gases and vapours with the surrounding atmosphere,
since the atmosphere will evolve as a result of the balance between the uptake and
emission of gases by the product, and the gas permeation through the packaging
material. When packaging high-respiration fruit and vegetable products, it is more
economical to package them with air as the initial atmosphere because the composition
rapidly advances towards equilibrium. However, the application of an atmosphere
composition close to the one finally reached at the stationary state could significantly
increase product shelf life.
As for the marketing of minimally processed products, the basic aims of MAP
technology are to change the surrounding atmosphere in order to reduce the respiration
rate and ethylene emission, two aspects that are related to a reduction in product
metabolism, inhibition of microbial growth and retarding enzymatic deterioration.
The atmosphere modification involves a reduction in oxygen concentration and an
increase in carbon dioxide concentration, because this reduces the respiration rate of
fruit or vegetable products as well as all aerobic activity. Carbon dioxide also works
as a bacteriostatic and fungistatic agent, delaying and reducing the multiplication of
aerobic microorganisms. Therefore the appropriate atmosphere has to be carefully
selected to limit all undesirable activities and to extend product shelf life. It is obvious
that the best gas composition will depend on product characteristics and production
and storage conditions.
During storage, an equilibrium modified atmosphere is created as a consequence of

the balance between the concentration of gaseous substances, which are involved in
product metabolism, and the rates at which the substances are sorbed or desorbed
and the rates at which they permeate through the packaging material [27], i.e., when
the carbon dioxide generation and oxygen consumption rates are equal to the transfer
rates through the packaging for a given temperature. An incorrect composition of the
equilibrium exposes the product to partial pressures of oxygen and carbon dioxide
beyond the product’s tolerance limits. Thus the use of low permeability packaging
results in product anoxia and high carbon dioxide concentrations, promoting the
development of fermentative metabolites (ethanol, acetaldehyde, ethyl acetate and
other volatiles), the softening of fruit and vegetable tissues, the physiological reactions
which result in product deterioration and the growth of anaerobic microorganisms
[27]. On the other hand, very permeable packaging provides atmospheres with
high concentrations of oxygen and low concentrations of carbon dioxide, reducing
product shelf life.
207
Optimal equilibrium modified atmosphere compositions vary according to the fruit

and vegetable species, its maturity stage and storage conditions. In general, the
concentration of oxygen should be below that of the composition of air but never
below 1% as this could result in anaerobic respiration. The quantity of carbon dioxide
is often high, to suppress ethylene synthesis and aerobic microbial growth, although
the sensory characteristics of some products can be severely modified by sorption of
this gas and product acidification. As has been mentioned, suitable selection of the
packaging materials with appropriate permeability to these gases is crucial to obtain
the desired equilibrium composition [28].
The flexible packaging industry has become increasingly responsive to the

specific gas requirements of fresh produce. Plastic materials with a wide range of
permeabilities from 0.001 to 10,000 cm3.mm/m2.day.atm are available to achieve
suitable equilibrium modified atmospheres. Those most commonly used in the
MAP of fresh produce are low-density polyethylene (LDPE), flexible polyvinyl
chloride and polypropylene (PP) [25], materials with permeabilities in the
100 cm3.mm/m2.day.atm range, which allow the industrial manufacture of bags
with permeation rates close to the product respiration rate. For products with
very high respiration rates, porous or microperforated materials are increasingly
used to provide greater gas exchange through the packaging material. By selecting
the quantity and diameter of the pores, the oxygen, carbon dioxide and ethylene
concentrations inside the package can be controlled, hence preventing undesired
gas accumulation in the headspace [29]. In order to reduce the impact of possible
cold-chain breaks a new generation of films is being developed which can adjust their
gas permeability depending upon the temperature. These materials present glass
transition temperatures which are slightly above refrigeration temperature, which
results in a great increase in their gas permeability [28]. Fluctuating temperatures
are often encountered during handling, transportation, storage and marketing.
These changes are a particular problem in MAP design as the rate of respiration
is dependent upon temperature and has to be matched to the permeability of the
packaging film [30].
The successful industrial application of MAP for fresh fruits and vegetables is
accomplished with an appropriate choice of packaging materials and working
conditions. In industrial practice, the optimisation of packaging systems is
typically achieved by ‘trial-and-error’ tests. Different materials and working
conditions are tried out and the evolution of the packaging atmosphere
composition and various freshness characteristics are monitored over the storage
period. Additionally, considerable attention and effort are being directed into
modelling studies from which predictions of package/packaged product system
behaviour under real conditions can be derived, permitting adequate optimisation
on a more scientific basis.
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To understand the effect of headspace composition and to optimise it, the product to
be packaged is stored under a controlled atmosphere and the evolution of freshness
indicators is monitored [31]. Other variables that need to be measured are the
exchange rates of relevant substances between the product and the headspace, i.e.,
respiration rate, transpiration, ethylene generation and any other relevant gas or
vapour that is considered critical for successful commercialisation. Normally the
production rates of carbon dioxide and ethylene and the consumption of oxygen
are interdependent and vary with the packaging atmosphere composition. Various
expressions derived from the Michaelis–Menten model are commonly used to describe
the respiration rate. The expression most successfully used in MAP modelling is one
that includes the inhibitory effect of carbon dioxide accumulation on the consumption
of oxygen [32, 33]:
r ( O 2 ) max × p ( O 2 )
r (O 2) = = – r (CO 2 )
[K MO 2,O 2 + p ( O 2 )] × 1 + p (CO 2 ) 
(7.1)
 K inhCO 2,O 2 

As can be seen in Equation 7.1, the maximum respiration rate [r(O2)max] can be
reduced by decreasing the partial pressure of oxygen and increasing that of carbon
dioxide, KMO2 ,O2 and KinhCO2 ,O2 being the Michaelis–Menten inhibition constant for
the consumption of oxygen due to the presence of oxygen and the inhibition constant
due to the presence of carbon dioxide, respectively. The effect of temperature on the
various constants of Equation 7.1 follows an Arrhenius dependence [32]. Very similar
equations have been derived for the production of ethylene:
r (C 2H 4 ) max × p ( O 2 )
r (C 2H 4 ) =
[K MO 2,C 2H 4 + p ( O 2 )] × 1 + p (CO 2 ) 
(7.2)
 K 
 inhCO 2,C 2H 4 
K MO2 ,C2H4 and KinhCO2 ,C2H4 being the Michaelis–Menten inhibition constant for the
production of ethylene due to the presence of oxygen and the inhibition constant due
to the presence of carbon dioxide, respectively.
Transpiration is another key process that must be controlled to achieve a longer shelf
life, since it is responsible for product dehydration processes. Most models consider
water loss to be proportional to the difference in water activity between the product
and the environment [34].
r(H2O) = k × [p(H2O)sat – p(H2O)hs] (7.3)
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Since fresh products are high water activity products, their weight loss should be
delayed by packaging, although storage at high relative humidity (RH) also helps.
The next step is to measure the gas and vapour exchange through the packaging. The
permeability coefficient is one of the most commonly reported parameters used to
characterise polymeric materials. However, most of the materials used today in food
packaging are combinations of materials, mainly multilayer structures, consisting
of layers of materials with different permeability values; therefore the preferred
parameter for measuring the barrier characteristics of a film (mono- or multilayer)
is permeance, defined as:
mi
℘i = (7.4)
A × t × ∆ pi
Thus the permeance of gas ‘i’, (i = carbon dioxide, oxygen, water, ethylene …) is the
quantity of ‘i’ (mi , which can be expressed as volume for permanent gases) that goes
through a given film per unit of packaging surface (A), in a given time (t) where the
partial pressure difference between the environments separated by the film is given by
(∆pi). From this expression, the gas exchange rate through a film can be estimated as:
mi
r ( i ) permeance = = ℘i × A × ∆ pi (7.5)
t
Therefore, mass transfer via package permeation increases as the partial pressure
gradient increases, the package surface and the film’s permeance to gas ‘i’.
As mentioned above, the use of discontinuous films and porous, microperforated or

macroperforated lids and bags is becoming popular. In these materials, gas exchange
occurs through the polymeric matrix by a sorption/diffusion mechanism and as a result
of the continuous movement of molecules in the gas state which are able to enter or
exit the packaging through the pore space. Thus, in addition to the exchange rate
caused by permeance, given by Equation 7.5, gas can also move through the pore
channel, an amount that is often described by Einstein’s diffusivity law [35]:
Apore × ∆ pi
r (i ) pore = n pores × k × (7.6)
l
where the film thickness (l) and the area of the pore (Apore) describe the pore geometry,
and k is a constant of proportionality.
Once the gas exchange within the environment/package/product system has been
characterised, mass balance can be used to predict the evolution of the gas composition
210
during storage. For instance, Equation 7.7 combines the exchanges due to respiration,
permeation and porosity for oxygen:
 A 
rO2 ( t ) =  n pores × k × pore +℘O 2 × A × ∆ pO2
 l 
r ( O 2 ) max × p ( O 2 ) (7.7)
−
[K MO 2,O 2 + p ( O 2 )] × 1 + p (CO 2 ) 
 K 
 inhCO2,O 2 
With these, it is also possible to predict the equilibrium modified atmosphere and
the weight loss in a given package from information on respiration rates, ethylene
production and film permeance [35].
In general, the MAP of minimally processed vegetables increases product shelf life
by reducing weight loss due to transpiration, reducing microbial metabolism mainly
by the depletion of oxygen and partially inhibiting microbial growth due to the
accumulation of carbon dioxide. Although MAP technology in combination with
refrigeration can delay the deterioration of a fresh fruit and vegetable product,
it is not always sufficient to maintain product quality for the desired marketing
period and the incremental microbial count during storage is the principal cause
of product quality loss. As an alternative, active packaging offers an effective,
economical way of increasing the shelf life of a fresh product during transport
and marketing.
7.2.6 Hurdle Technology
Combined technologies are a powerful tool for achieving the effective microbiological
decontamination of fresh vegetables; for example, it is possible to combine refrigeration
with MAP or low irradiation with chlorine treatment and so on. Foley and co-workers
[36] achieved a 5.4 log inhibition of Escherichia coli O157:H7 growth on shredded
iceberg lettuce by combining chlorination and low dose irradiation (0.55 KGy).
Treatment with 7.5 ppm of ozone and 150 ppm of chlorine reduced aerobic microbial
growth on shredded lettuce by approximately 1.4–2.5 log [37].
Sodium benzoate and potassium sorbate, as an antimicrobial, and irradiation

(0.43 KGy) were applied to fruit salad packaged in a high-barrier film [38]; the
results showed that irradiation of the fruit salad was not enough to replace the use
of chemical preservatives. The effectiveness of combined technologies is better than
treatments applied alone.
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7.2.7 Novel Technologies
Various technologies are being explored as alternative treatments to reduce microbial

contamination in fresh produce. As an example, electrospraying of antimicrobial agents
to coat the inner part of food packaging is being tested. Malic acid, lactic acid and grape
seed combinations were tested against Salmonella typhimurium inoculated on spinach
[39]. The log reductions were higher when the antimicrobial agents were applied using an
electrospray technique. Electrostatic spraying can increase the antimicrobial effectiveness
of active agents, achieving sustained release during the shelf life of the product.
Washing iceberg lettuce with electrochemically generated silver (5 ppm) and hydrogen
peroxide (0.4 ppm) significantly reduced the total aerobic bacteria, Pseudomonas,
enterobacteria, yeast and mould. However, the use of silver in water disinfection must
be controlled because of the potential toxic effects. In 2003, the WHO investigated its
effect and determined that, for a human, 10 g is the maximum oral intake for life [40].
Slightly acidified electrolysed water was applied on ready-to-eat vegetables to reduce

aerobic mesophilic bacteria [41]. The results obtained showed a similar antimicrobial
effect to the chlorine washing treatment; however, the nutritive impact and oxidation
processes must be studied carefully. Moreover, the organoleptic and consumer
acceptability need to be investigated further [21].
Intense pulsed light has been applied to reduce the microbial growth of Listeria
innocua and Escherichia coli on fresh watermelon [42]. This emerging technology
must be studied in more detail to ascertain the possible impact on the nutritive value
of the product, especially on water-soluble vitamins which are known to be sensitive
to ultraviolet (UV) light [21].
On the other hand, many of the antimicrobial agents described in Chapter 2 are
currently used as decontamination agents on fresh vegetables and salads. Bacteriocins
have been used as preservatives in meat and dairy products, and new isolates of lactic
acid bacteria have been applied to iceberg lettuce, reducing cell counts of Salmonella
typhimurium, Escherichia coli and Listeria monocytogenes [43, 44].
Silver-montmorillonite antimicrobial nanoparticles have been applied as a packaging

system for fresh fruit salads, extending the shelf life without changing the quality and
organoleptic properties of the food product [45]. Silver-montmorillonite nanoparticles
efficiently inhibited microbial growth.
7.2.7.1 Active Packaging
Many different types of active packaging have been proposed for controlling food
quality loss or deterioration problems, as stated in Chapter 1. They include controlling
212
the gases (oxygen, carbon dioxide, ethylene and so on) and moisture within the
package, adding chemical preservatives or aromas, removing off-flavours and
undesirable substances, and controlling microbial contamination. Many of them have
applications in fruit and vegetable packaging, especially those aimed at controlling
package atmospheres or microbial contamination.
One of the main focuses of attention in the development of packaging for fresh produce
is the control and modification of the headspace composition, especially the content
of oxygen, carbon dioxide, humidity and ethylene.
As mentioned in Section 7.2.5, oxygen is required for respiration (Equation 7.1),

the synthesis of ethylene (Equation 7.2) and most of the metabolic and biochemical
processes involved in the ripening of fruit and vegetable products, and consequently
these processes can be slowed down by reducing its concentration [46]. The use of
oxygen scavenger systems is one of the best ways of controlling the oxygen present in
the packaging headspace, because it can reduce the oxygen concentration to very low
levels (even less than 0.01%) which are impossible to achieve using gas flushing or
vacuum packaging. However, as fresh produce cannot be packaged under anaerobic
conditions, oxygen scavengers should not be used to eliminate oxygen but to reduce
the concentration of this gas to suitable levels. The application of oxygen absorbers
in combination with MAP can give excellent results for the packaging of fruit and
vegetables; studies carried out with tomatoes in LDPE bags with commercial iron-based
oxygen absorbers stored at 20 °C reduced the oxygen in the headspace by half [32].
As mentioned previously, the accumulation of carbon dioxide inside the packaging

is beneficial for the preservation of certain fruits and vegetables because it enhances
texture and delays the development of bacteria, moulds and yeasts. Carbon dioxide
emission to maintain concentrations of 20% or more is used to suppress microbial
growth [47]. Sachets containing sodium bicarbonate [48] can be placed in packages
to immediately increase the carbon dioxide content, although for fruits and vegetables
with high respiration rates the use of carbon dioxide absorbers, such as calcium
hydroxide or perforated materials, is required to avoid excessive carbon dioxide
concentrations inside the packaging and the consequent development of off-odours,
colour changes, tissue rupture and so on.
Ethylene is a plant hormone produced during the ripening of fruits and vegetables
which promotes chlorophyll breakdown, giving rise to a series of physiological
consequences such as brownish stains in lettuce, yellowing of peas, a bitter taste in
carrots, sprouting of potatoes, hardening of asparagus and so on [49]. The addition of
ethylene absorbers to the packaging of fruit and vegetable products has shown good
results in extending product shelf life, thus facilitating marketing and export. These
absorbers, the most commonly used of which is potassium permanganate (4–6%)
embedded in an inert substrate, such as silica gel, alumina, perlite, vermiculite and
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so on [48], are currently placed inside a sachet located on the top of whole fruit
packaged pallets during in transportation.
Activated carbon, clays and zeolites are introduced into films via extrusion processes to
extend the shelf life of fruits and vegetables [29, 50]. There is some controversy concerning
why these films are successful; it has been proposed that instead of ethylene scavenging
by the active polymer, the real reason could be quick release of the active agent from
the package through the pores or areas of the film containing a higher/lower level of the
active agent. Nevertheless, they are being increasingly used in commercial applications,
where the combination of an optimum gas mixture (achieved using MAP) with the use of
ethylene absorbers is required to achieve the maximum shelf life of fruits and vegetables.
As a result of the transpiration of the fruit or vegetable product, water vapour

accumulates inside the package and permeates outward into the environment through
the packaging walls. Depending on the product, this may bring about undesirable
changes such as hardening as a result of desiccation, absorption of surface moisture,
generation and accumulation of liquid water which condenses on the packaging
material. The resulting effect on the appearance of the product may lead to rejection
by the consumer [28]. Various systems for avoiding the drawbacks associated with
the transpiration of fruits and vegetables currently exist. They include moisture
control or buffer systems which can achieve the desired RH values in the packaging
headspace [47], others, such as moisture absorbing systems, simply absorb the water
given off by the product [51], finally, antifogging systems prevent condensation into
water droplets on the inner packaging wall [52].
Microbial growth is the main cause of deterioration in fresh fruit and vegetable
products. Harvested fruits and vegetables may be contaminated by spores and
microorganisms which remain latent until the environmental conditions (humidity,
oxygen, temperature) are favourable for their development. Antimicrobial active
packaging technology involves incorporating an active agent into the packaging which
is subsequently released in order to maintain a minimum inhibitory concentration
(MIC) on the surface of the product for a given time. The active agent can be
introduced into the polymer material itself during processing or applied as a coating on
the packaging wall as described in Chapter 3. Despite this wide range of possibilities,
not many commercial applications of antimicrobial polymer materials are available
for fresh produce, although they are the focus of considerable research.
7.3 Antimicrobial Active Packaging
The method most frequently employed to ensure the safety and quality of fresh-cut
salad is a combination of storage at low temperature and MAP. Both treatments reduce
214
the rate of respiration and the metabolic and biochemical activities of the fresh product.
As mentioned in the MAP section, minimally processed vegetables have a limited shelf
life, which is usually 7 or 8 days when stored at refrigeration temperature with an
adequate headspace composition. However, when the package is opened the shelf life
shortens in the following 48 h, microbial decay being the main spoilage mechanism.
In this case study, a combination of MAP with antimicrobial packaging is presented

as an approach to improve the quality, safety and shelf life of fresh-cut salads. As
mentioned in Chapter 4, the use of volatile antimicrobial compounds is recommended
for irregularly shaped solid food products where direct contact between the
food surface and packaging material is not possible. The novel packaging system
should resemble the conventional salad bag and therefore the use of independent
devices moving freely among the lettuce leaves was considered unacceptable. Thus
the antimicrobial volatile agent had to be incorporated into the polymer matrix
constituting the packaging, from which it would be released into the headspace
surrounding the food. Controlled delivery of the volatiles had to ensure that the MIC
was reached in the packaged salad and maintained during the product’s shelf life in
order to achieve an efficient antimicrobial packaging system.
In this context, natural extracts and essential oils were considered as possible agents
as they are natural food preservatives which exhibit a wide spectrum of antimicrobial
activity, including activity against foodborne pathogens and spoilage bacteria
associated with fresh vegetables. As mentioned in Chapter 2, essential oils of spices
and plants have shown high antimicrobial activity and can decrease the levels of
pathogenic bacteria present in foods. Essential oils are rich in volatile terpenoids
and phenolic compounds, which are responsible for their antimicrobial activity. This
volatility is also responsible for the intense fragrance of these liquids, which is easy to
perceive at very low concentrations and therefore could alter the aroma of the salad.
With regard to this important issue, the selection of the antimicrobial agent should
be based on which antimicrobial agent exhibited the most readily accepted aroma by
consumers. With this in mind, oregano essential oils (OEO) and lemongrass essential
oils were selected as natural antimicrobial agents in this case study [53, 54].
OEO is a natural antimicrobial agent classified as generally recognised as safe (GRAS)

by the FDA and as a food additive by the EU. The chemical composition of OEO is
complex and variable, depending on which part of the plant is used, the plant growth
zone, climate, season and so on, although carvacrol is one of the major components
of OEO (80% in some samples). This phenolic compound is able to break down
the outer membrane of Gram-negative bacteria, distort their physical structure and
solubilise some components such as lipopolysaccharides, increasing the fluidity and
permeability of the cytoplasmic membrane to adenosine triphosphate [55]. Citral is a
volatile monoterpenoid containing an aldehyde group with a strong lemon flavour and
215
is the main constituent of lemongrass essential oil [53]. The antimicrobial activity of
citron oil and citral has been tested on fruit-based salads, among other kinds of food.
With respect to the packaging structure, the starting point was the conventional bag
which was manufactured using a 35 µm thick bilayer bioriented PP film. This is a
permeable structure, required to maintain a balanced atmospheric composition, which
is transparent and glossy, with sufficient heat sealability to produce an easy-opening
hermetic seal, and contains an antifogging substance. Thus, as explained below, it
was decided to use this film as the substrate and apply to this base film an appropriate
coating containing the antimicrobial agent.
7.3.1 Active Material Manufacture: Laboratory Development
In the first steps of this case study, the development of a PP film containing the selected
essential oils was explored. The incorporation of the essential oils was attempted via
extrusion, using a liquid port as explained in Chapter 3. Unfortunately, the efficiency
of this incorporation was very low, with losses of about 80% due to evaporation,
similar to the values reported in other developments of active materials [56, 57].
Moreover, the extruded film constantly released the agent, resulting in rapid film
ageing and exhaustion of antimicrobial activity. Therefore we decided to consider the
development of a coating that could be applied to the conventional PP film.
An ethylene-vinyl alcohol (EVOH) copolymer was chosen as the base material for
development of the antimicrobial system. The reasons for its selection were, firstly,
that EVOH is a high-barrier material and therefore will maintain the antimicrobial
agent in its matrix for a long time, reducing or delaying film ageing. Secondly, EVOH
is a highly hydrophilic but water-insoluble material which is severely plasticised in
a humid environment, causing depletion of the barrier characteristic. Thirdly, it is a
polymer which is soluble in alcohol/water mixtures, and therefore compatible with
the selected essential oils and final food application.
Prior to any polymer development, a preliminary study was carried out on the
antimicrobial agents. First of all, the MIC of OEO and citral in the vapour phase was
determined against various critical pathogenic bacteria – Listeria monocytogenes,
Escherichia coli and Salmonella enterica – using the disc diffusion method (Section 5.1.1).
These microorganisms were selected because they are present in raw vegetables and are
involved in foodborne infections, since raw vegetables can become contaminated with
these microorganisms during growth, harvesting, postharvest handling or distribution.
The results demonstrated a reduction in growth for all three strains with the addition
of 2.5 mg of OEO or citral. A retraction zone – a visible decrease in microbial density
on the Petri dish – was observed. No inhibition was observed upon the addition of
lower amounts of the antimicrobial agents; therefore, 2.5 mg was considered the MIC.
216
Once the antimicrobial properties of OEO and citral had been studied, the two agents
were incorporated into the film-forming solution of EVOH-29 which was used to coat
the PP film by casting. The thickness of the coating was determined using a digital
micrometer and EVOH-29 had an average thickness of 3.0 ± 0.5 µm. The EVOH
active layers containing 5 and 10% OEO or 5 and 10% citral were made by casting
and were prepared on PP films. A PP/EVOH film without the antimicrobial agent
was also made as a control. Carvacrol is the major component of OEO and is mainly
responsible for its antibacterial properties. Accordingly, it was the only component
of OEO monitored in this study. The concentrations of carvacrol and citral retained
in the coatings were measured using thermal desorption-gas chromatography. The
loss of antimicrobial agent attributed to evaporation during the drying process was
estimated as 20% in the case of carvacrol and 50% in the case of citral.
The antimicrobial activity of the PP/EVOH films was also studied using the disc
diffusion method. The in vitro test presented excellent results against the selected
pathogenic bacteria, being more active against Gram-negative bacteria (Escherichia
coli and Salmonella enterica), with OEO producing an inhibition zone of 7.5 cm with
5% OEO and 8.5 cm with 10% OEO.
In another study, Petri dishes inoculated with 107 CFU/mL of Escherichia coli,
Salmonella enterica or Listeria monocytogenes were packaged in bags manufactured
with the PP/EVOH films. No antimicrobial effect was observed in samples packaged
with the control bag (PP/EVOH without agent), indicating that the gas barrier of the
hermetic bag did not show bactericidal or bacteriostatic effects. Total inhibition of
Escherichia coli and Salmonella enterica growth by bags manufactured with the four
active films (5% citral, 10% citral, 5% OEO and 10% OEO) was observed, as shown
in Table 7.1. Similar results were observed for Listeria monocytogenes in samples
stored in bags with 5% OEO, 10% OEO and 10% citral; however, with the sample
containing 5% citral only a retraction zone was observed for Listeria monocytogenes.
Table 7.1 Antimicrobial effect against Escherichia coli, Salmonella enterica and Listeria
monocytogenes of bags coated with EVOH containing 5 and 10% oregano essential oils
and citral
Coating Inhibition zone
Escherichia coli Listeria
Salmonella enterica
monocytogenes
OEO 5% Total inhibition Total inhibition Total inhibition
Citral 5% Total inhibition Total inhibition Retraction zone
OEO 10% Total inhibition Total inhibition Total inhibition
Citral 10% Total inhibition Total inhibition Total inhibition
Mechanical manipulation such as peeling, grating or shredding transforms the fresh

components of a salad (lettuce, carrot, red cabbage) from a relatively stable product into
217
a highly perishable one with a shelf life of approximately one week. Among other factors,
the product decays due to microbial spoilage, which generally results in the degradation of
colour, texture and flavour of the product. Therefore, in addition to the in vitro tests, the
films were used to pack minimally processed salads with MAP to extend the shelf life and
reduce possible microbiological risks. The initial atmosphere was 12% carbon dioxide and
4% oxygen, indicated as a standard for salad by the supplier of salads employed in this study.
Given the above, in this study salad samples were packaged under MAP combined with
antimicrobial agents (OEO and citral) in order to extend the shelf life of the products. The
samples were packed with PP/EVOH films containing OEO and citral at 5 and 10%, and
stored at 4 °C for 5 days and then at 8 °C for 3 days, simulating commercial conditions
of production, transport and marketing. The samples were subjected to microbiological
analysis on the 1st day and the 8th day of refrigerated storage. As shown in Figure 7.1,
the developed films caused significant inhibition of the microbiota commonly found in
salad. The inhibition of microbial growth was most effective during the first stage of
the storage period, when the concentration of the agent in the packaging headspace was
higher and the storage temperature lower. However, the antimicrobial effects observed
in the salad were lower than the results obtained in vitro owing to agent/food matrix
interactions, which can be attributed to partial sorption of the agent into the food, with
a subsequent reduction of concentration in the vapour phase.
Enterobacteria Total aerobic bacteria

7
Day 1 Day 1
Day 8 8 Day 8
6
5
6
Log CFU/mL
Log CFU/mL
3 4
2
2
1
0 0
Control OEO Citral OEO Citral Control OEO Citral OEO Citral
5% 5% 10% 10% 5% 5% 10% 10%
Psychrotrophic bacteria Yeasts and moulds
Day 1 Day 1
8 Day 8 8 Day 8
6 6
Log CFU/mL
Log CFU/mL
4 4
2 2
0 0
Control OEO Citral OEO Citral Control OEO Citral OEO Citral
5% 5% 10% 10% 5% 5% 10% 10%
Figure 7.1 Microbial population found in mixed salad packages: enterobacteria,

total aerobic and psychrotrophic bacteria, and yeasts and moulds on day 1 and 8
of storage. Stored for 8 days, the first 5 days at 4 °C and the last 3 days at 8 °C
218
Once the antimicrobial effectiveness of the PP/EVOH films had been assessed
on the intrinsic microbiota in vivo, trials were also carried out to check for
any effectiveness on pathogens in vivo. Salad samples were inoculated with the
pathogenic microorganisms Escherichia coli, Salmonella enterica and Listeria
monocytogenes, packaged in bags with the films that had been developed and
stored for two days at 4 °C. As shown in Figure 7.2, samples stored in the active
bags had a significant inhibitory effect against the two Gram-negative bacteria
tested. More effective activity was observed in the samples containing a higher
concentration of oils, both producing a significant reduction in bacterial counts,
especially with 10% OEO.
Figure 7.2 Antimicrobial effect against Escherichia coli, Salmonella enterica and
Listeria monocytogenes assessed after 2 days of storage at 4 °C in contaminated
packaged salad
As explained above, essential oils are excellent antimicrobials but they also
provide a characteristic fragrance. Therefore their impact on the odour of the food
product could be a limiting factor for their applicability. Accordingly, sensory
analyses were carried out to evaluate the odour, visual appearance and general
acceptability of the salad samples. The effects of the addition of various agent
concentrations and the type of agent on the sensory properties of the salad were
studied. In general, as shown in Figure 7.3, the visual appearance and general
acceptability of the samples packaged with films containing OEO had better
scores than those packaged with citral, and the samples with the lower agent
concentrations were also preferred.
219
8
Day 1
Day 8
Values of the parameters of odour

6 a
a a
ab
ab b
b
c
4
0
OEO 5% Citral 5% OEO 10% Citral 10%
8
Values of the parameters of visual appearance
Day 1
a a a Day 8
a
a
6
b b
4
c
0
8
Values of the parameters of general acceptability
a
6 a
a ab
ab
b
b
4
c
0
Figure 7.3 Values of odour acceptability, visual appearance and general acceptability:
a-c values within a column followed by a different lower-case letter are significantly
different from each other (Tukey-adjusted analysis of variance, P < 0.05)
220
On the basis of the results obtained above and the microbiological results, minimally
processed salad packaged with the OEO 5% film was selected to assess the differences
in visual appearance and odour in comparison with the salad packaged with the
control film. For this purpose, a paired comparison test was carried out. Only at the
end did the evaluation suggest that samples with 5% OEO were visually preferred
by the judges, as can be seen in Table 7.2.
Table 7.2 Sensory evaluation of differences between the odour and appearance of salad
packaged in film containing 5% OEO and the control
Day 1 Day 8
Control OEO 5% Control OEO 5%
Appearance 33 a
19 a
6a
46b
Smell 18 34 25 27
a-b
: Values within a column followed by a different lower-case letter are significantly
different from each other (Tukey-adjusted analysis of variance, P < 0.05)
7.3.2 Full Characterisation of Properties Prior to Film Scale-up
After the results obtained at laboratory scale it was necessary to scale-up the structure,
taking into account all the requirements of real packaging for fresh salad; therefore
more variables and parameters required consideration. The first issue to be studied
was the anchorage of the coating to the PP substrate. In the first trials, the results of
which are commented on in the previous section (Section 7.3.1), the EVOH solution
was spread and cast on the surface of a PP film previously irradiated with a UV lamp
in an air atmosphere. The final coating seemed to have adhered but was easy to
remove by applying a self-adhesive tape and pulling it off with a quick peeling motion.
Therefore it was necessary to improve the adhesion of the coating to the substrate.
PP films were subjected to four different anchorage treatments – a) the UV irradiation

just mentioned, b) corona discharge, c) corona discharge followed by the application
of a polyurethane (PU) primer and d) corona discharge followed by the application of
a polyethylene imine (PEI) primer and immediately coated with a solution of EVOH
29 [58]. Both technologies, UV irradiation and corona treatment, were able to create
ozone molecules via air ionisation, which could react with surface polymer chains and
break some of their covalent bonds, i.e., the aim was to reduce the surface tension
of the polymers and improve the wettability using primers or adhesiveness using the
coating. For treatments c and d, primers were applied with the use of an automatic
coating applicator and dried. After each of the four treatments, the EVOH solution
was applied on the pretreated PP sheets to yield the final structure. The final EVOH
coating layer was 1.5 µm thick; the primer layer in treatments c and d was 1 µm thick.
221
The adhesiveness of EVOH coatings to the PP substrate subjected to different

treatments was evaluated by means of the well-known Scotch tape test, a qualitative
test method based on the American Society for Testing and Materials (ASTM)
standard, D3359-09e2 [59]. The results are included in Table 7.3 and show that the
corona treatment followed by the PEI primer was the treatment that provided the
best results.
Table 7.3 Degree of adhesion between the EVOH coating and the PP substrate
obtained using the four tested treatments
Anchorage technology Adhesion
UV radiation 1
CD 3
CD + PU-based primer 0
CD + PEI-based primer 5
Scale: 0 is failure – 5 is excellent
CD: Corona discharge
The next step was to check the heat sealability. A requirement of a food package
for fresh produce is that the bag should be hermetically closed to avoid further
contamination of the product. The heat sealability of the bilayer structure is
dependent upon the sealing capabilities of its material layers and the adhesion
between them. The heat sealability of the EVOH film was successfully tested in
a previous study [60], so if there was a failure of the bilayer it would be caused
by a lack of adhesion between the coating and the substrate film. Several sheets
of each manufactured material were placed in pairs between the jaws of an
impulse heat sealer. The apparent result of the process was a good, continuous,
homogeneous sealing surface. However, when the two sealed films were pulled
apart, all samples showed delamination between the EVOH and PP layers
except for the structure containing the PEI primer. The sealing of this structure
presented good resistance to the manual peel strength test. These films were tested
following the ASTM D882-12 standard method [61]. Specifically, the PP/EVOH
films prepared using the corona treatment and PEI primer were heat sealed and
peeled off after cooling. The results were compared with the original PP film;
unexpectedly, the sealing strength of the original PP film was 8 N, while that of
the coated film rose to 20 N.
The third step was to evaluate the surface properties of the multilayer film.
As mentioned in Section 7.3.1, PP films for fresh salad include an antifogging
additive to improve the wettability of water droplets generated inside the bag
as a result of any thermal change. Therefore it was necessary to check the
222
wettability of EVOH. The surface properties of the coated and uncoated PP

films were investigated by measuring static contact angles using the sessile drop
method. Since the incorporation of the active agent could cause modification
of the film’s wettability, the tests were performed with pure EVOH and with
the two antimicrobial agents tested at 5% concentration. The results are shown
in Figure 7.4.
Figure 7.4 Contact angle values of the various materials tested
The systems that were studied were the substrate, PP, the passive coating, PP/EVOH,
and the active coating containing 5% citral, PP/EVOH citral, and containing 5%
OEO, PP/EVOH OEO. As the figure shows, the contact angles of the untreated
PP substrates are quite high, about 98°, which proves their hydrophobic nature
under the original conditions and the need for an antifogging additive; however,
when an active agent is embedded in the EVOH matrix the contact angles drop
dramatically to about 63°. This may be due to the presence of active molecules on
the material surface and to greater polarity of the polymer. With this result, the
addition of an antifogging compound to the developed packaging structure seemed
to be unnecessary.
The next important properties that had to be considered were the barrier characteristics.
Specifically, the permeance of the substrate and the passive coated film to oxygen
and carbon dioxide were investigated as a function of the RH, following standard
procedures [62, 63].
223
Figure 7.5 Permeance of the EVOH-coated and uncoated PP film to oxygen (a) and
carbon dioxide (b) as a function of RH
Given that the food packages to be developed are for marketing fresh-cut salad, the
multilayer films had to allow an adequate gas exchange between their inner and outer
atmospheres in order to properly maintain the quality and sensory characteristics
of the foodstuffs they contained. As mentioned earlier, the conventional packaging
for this type of product is a PP bag which is highly permeable to gases, permitting
the entrance of oxygen and reducing the accumulation of carbon dioxide released
by product respiration.
The application of a high-barrier coating of EVOH on the original PP films could lead
to a substantial decrease in the rate of gas transmission through the final structure,
making it invalid for its purpose. Figure 7.5 presents the measured oxygen and carbon
dioxide permeances as a function of RH. The oxygen and carbon dioxide permeances
were quite high in the PP film, but these permeance values dropped dramatically
when the film was coated with a 1 µm layer of EVOH, especially in dry conditions.
However, owing to its highly hydrophilic nature, as reported elsewhere [64, 65], this
barrier effect gradually dissipated as the RH increased, reaching permeance values
in wet environments more or less comparable to those reported for the uncoated
substrates, between 70 and 80% of the original values. Given the highly humid
conditions expected in the packaging headspace of the salad bag during the product
preservation time, the presence of a thin coating layer of EVOH on the packaging
materials would not substantially affect the gas transport; however, the depletion of
224
permeance should be considered and adjusted to the respiration rate of the packaged
foodstuffs in order to avoid deterioration of product quality or sensory characteristics
caused by fermentation processes.
Finally, the physical properties of the coated structure were also examined to check
whether the addition of the coating resulted in appreciable modification of optical or
mechanical properties. Strip samples of the coated and uncoated films were tested in a
universal testing machine. The data obtained showed that the tensile strength at break
of all samples was approximately 130 N/mm2, the deformation at break about 370%
and the Young’s modulus was near 700, without substantial differences among the
materials. This result was anticipated as, in the coated film, the EVOH coating was
so thin it made little impact upon the mechanical properties of the whole structure.
Optically, the coating did not visually modify the transparency of the PP film.
To conclude this preliminary characterisation of the active structure, the coated film did
not optically or mechanically modify the packaging requirements of the conventional
PP bag. The PP/EVOH film maintained or even improved the heat sealability of
the original film and improved the wettability. An aspect to be considered was the
improvement in barrier properties caused by the addition of the EVOH coating,
because this could result in a shortening of product shelf life due to fermentative
processes.
7.3.3 Industrial Production of the Active Film
The final structure was ready for the scale-up process as all the relevant properties
considered in the previous section were within acceptable values. The next step
was to consider the most suitable processing technology. Given that the two main
differences between the coated bag and conventional bags were that the PP/EVOH
coated material had a lower permeance, which could shorten product shelf life, and
that the sealing was stronger, which could make the bag more difficult to open, it
was decided to perform a partial coating of the bag. Instead of applying the EVOH
layer over the whole bag surface, the bag was designed with the edges and the back
part uncoated. The gas transmission (oxygen and carbon dioxide) through the
uncoated parts would be higher, reducing the impact of the active coating on the
modified atmosphere composition. The sealing areas also remained uncoated, so
the seal strength was unchanged. Consequently, printing technology was considered
appropriate for application of the coating – specifically, gravure printing.
Reels of PP and polyethylene terephthalate films were pretreated using corona discharge
and then coated with a PEI-based primer, followed by a layer of the corresponding
EVOH solution containing the active agent. The various active multilayer films finally
225
produced were: PP/EVOH containing 10% citral, PP/EVOH containing 7.5% OEO
and passive PP/EVOH films as control materials. The thickness of the PEI and EVOH
layers was estimated at about 0.8 and 1.15 µm, respectively. To avoid potential activity
losses, the active reels were stored in hermetic aluminium/polyethylene bags.
Once the films had been manufactured on an industrial scale, the active films were
used in a processing plant to package minimally processed salad under MAP. The
amount of carvacrol and citral retained in the coatings was measured using thermal
desorption-gas chromatography. Loss during film manufacture and storage until the
moment of use was 35% for carvacrol samples and 50% for citral, values comparable
to those observed in the samples produced at laboratory scale.
The evolution of the headspace atmosphere composition during storage was monitored
by the withdrawal of 20 mL gas samples and injection into a gas analyser. The
headspace composition was measured in all the samples, and, as expected, oxygen was
rapidly consumed as the product respired which was accompanied by a corresponding
increase in carbon dioxide levels. No relevant modification of this evolution as a
result of the presence of the active substances in the coating was observed during the
storage period.
Figure 7.6 Evolution of the headspace atmosphere composition in the salad bags
manufactured with the control and active films
226
The antimicrobial activity of the films was tested in vivo. Analyses were performed
on the 1st, 4th and 8th days after packaging. The enumeration of particular microbial
groups was achieved using the appropriate culture media. As shown in Figure 7.7,
the active films improved microbial stability, especially with regard to inhibiting the
growth of enterobacteria, lactic acid and psychrotrophic bacteria, and yeasts and
moulds at the beginning of storage. The active films inhibited the growth of intrinsic
bacteria in the first stages of the storage period, when the concentration of the agent
in the packaging headspace was higher and the storage temperature was lower.
Figure 7.7 Microbial population in mixed salad packages: enterobacteria, total

aerobic, lactic acid and psychrotrophic bacteria, and yeasts and moulds on the 1st,
4th and 8th days of storage
227
To conclude the assays on the effect of the active packaging on the product, a group
of consumers of ready-to-eat fresh products with no previous experience in sensory
analysis evaluated the smell, visual appearance, texture and general acceptability of the
samples by paired comparison tests. A total of three pairs of samples were evaluated
and each pair consisted of two samples. The first pair included the samples packaged
with control and OEO films, the second pair comprised the control and citral films,
and the third pair were the OEO and citral films.
Table 7.4 Number of responses of differences between the smell, appearance, texture and
general preference of control/OEO, control/citral and OEO/citral on the 1st and 8th days
of storage. The minimum number of responses for which the difference was significant
was determined to be 32
Control/OEO
Control OEO
Day 1 Day 8 Day 1 Day 8
Smell 36 22 15 29
Appearance 34 28 17 23
Texture 29 26 22 25
General 32 27 19 24
Control/Citral
Control Citral
Smell 37 32 14 19
Texture 25 22 26 29
General 35 21 18 30
OEO/Citral
OEO Citral
Smell 29 17 22 34
Texture 28 16 23 35
General 25 15 26 37
Three pairs of samples were evaluated in each session and two sessions were performed
in total: on the 1st day, to determine the initial impact of the aromatic agent, and
on the 8th day after packaging, to verify the quality and acceptability at the end
of storage. The Williams method for constructing such designs was applied to the
sensory studies [66]. The results are shown in Table 7.4. The sensory study showed
228
that release of the active agent resulted in a non-typical smell which led the judges to
prefer the control samples, whereas after long-term storage the samples in the active
bags were preferred, especially the one containing citral.
Thus the development of active films based on the application of an EVOH coating
on the conventional PP structure generally used in the packaging of fresh salad
was accomplished. The final bags maintained the functional characteristics of a
conventional bag, essentially heat sealability, transparency and mechanical strength.
The change in the oxygen and carbon dioxide transmission caused by the coating
was not relevant, probably because at the humidity of the product (aw>0.95), the
plasticisation of the thin EVOH coating severely reduces its barrier characteristics.
With respect to the effect of the active packaging system on the product, it can be
concluded that the sensory effect caused by incorporation of the essential oils is
acceptable at short exposure times and improves the general appearance on the
final days of commercialisation. With regard to the antimicrobial effect, the bags
appeared to be active during the first days, but the activity was inefficient at the
end of the shelf life. These results can be explained as a consequence of inadequate
agent release. The entry of water in the active EVOH layer results in the fast release
of most of the antimicrobial agent just after the packaging process, and from that
point its concentration decreases to levels below the MIC. In these later conditions
microorganisms are able to grow, reducing the validity of the active packaging system.
Optimisation of the packaging system by the use of mathematical models should be
considered, since they can indicate which variables are most critical.
7.3.4 Modelling and Optimisation of the Active System
An active antimicrobial package intended to preserve fresh or minimally processed

food products is a complex system which usually comprises several material layers
(food matrix, headspace, polymer layers, environment and so on) and involves the
simultaneous transport of several chemical compounds (oxygen, carbon dioxide, water,
antimicrobial agents and so on) under different and/or varying ambient conditions. All
these factors and variables make the experimental study of its behaviour and active
performance in real preservation conditions a costly and challenging task.
In this context, the mathematical modelling and simulation of the various mass transfer
phenomena taking place in the system becomes a highly useful tool, making it possible
to obtain exhaustive descriptions of all the physico-chemical processes involved
simply by performing some computerised calculations fed with a minimal inventory of
bibliographic and experimental data (geometrical parameters, transport coefficients,
physical constants and so on), thus saving cost as well as human or material resources.
These theoretical descriptions provide greater understanding of the system’s behaviour,
229
which, in turn, allows the prediction of its long-term evolution when subjected to
different and/or varying preservation conditions, as well as modification of the basic
design in order to direct its evolution towards some specific objective.
However, mathematical simulation has a limited ability to represent the mass transfer
phenomena that occur in real systems, given that higher accuracy in the predicted
results always involves a higher number of variables and equations which define the
model, as well as a lower amount of particular hypotheses which constrain it. As a
result, there is an exponential increase in the complexity of the mathematical model,
and also in the minimum size of its data inventory and the computing time required.
7.3.4.1 Modelling an Antimicrobial Package
The first step in the simulation of an antimicrobial package consists of constructing a

theoretical model featuring a set of geometrical characteristics and physico-chemical
properties as close as possible to those of the system being studied. In addition,
such a model should, at the same time, maintain sufficient simplicity to allow its
accurate solving in a short computing time with the aid of a reasonable inventory of
bibliographic and experimental data.
In the particular case of an active package for salad products, the typical system
consists of a pillow-type bag of three-dimensional (3D) structure and finite volume
(1 L) made up of five different material layers – environment, substrate polymer,
active coating, headspace and food matrix – in which there is simultaneous transport
of four different compounds: oxygen, carbon dioxide, water and the antimicrobial
agent. However, a 3D model of any system featuring five material domains and four
chemical species may be too complex to be properly addressed by this theoretical
approach. Therefore the following particular hypotheses must also be considered:
• The multidirectional mass transfer processes taking place in the real 3D package
can be accurately represented by transport phenomena of unidirectional
progression occurring in an equivalent one-dimensional multilayer sheet of finite
thickness and infinite width and length.
• The environment and the food matrix are independent of the package, and
therefore the concentrations of all chemical compounds in those layers can be
taken as constant and defined in the boundary conditions of the substrate and
headspace layers, respectively.
• The transport of oxygen and carbon dioxide through all the material layers
constituting the package is independent from, and uncorrelated with, the transport
of water and antimicrobial agent occurring in the same domains.
230
Acceptance of the above hypotheses provides substantial simplification of the system

studied, thus allowing its accurate representation by the model scheme displayed in
Figure 7.8.
Figure 7.8 Model scheme of a simplified antimicrobial package
As the figure shows, the model requires the incorporation of six diffusion coefficients
(DSa, DSw, DCa, DCw, DHSa and DHSw), together with two partition coefficients
(KC/Sa and KC/Sw) and two solubility coefficients (SCa and SCw), in order to solve six
concentration variables (CSa, CSw, CCa, CCw, CHSa and CHSw), defined for the two
chemical species [water (w) and antimicrobial agent (a)] in the three material layers
depicted [substrate (S), coating (C), and headspace (HS)], from their six corresponding
model equations.
The equations are obtained from microscopic mass balances of the two compounds
mentioned as applied to control volumes of the three material domains considered,
after assuming full compliance with the following general hypotheses:
• Solid phases (substrate and coating) and confined fluid phases (headspace) are
static (absence of net mass flux in any direction) and initially homogeneous
(isotropic properties at any point).
• Free fluid phases (surrounding atmosphere) are dynamic but perfectly agitated,
hence homogeneous too.
231
• Interfaces are in thermodynamic equilibrium (thus, chemical potentials are

identical in both phase thresholds).
• No interfacial volume (thus, mass flux densities are identical in both phase
thresholds).
• Pressure and temperature are uniform and constant in the whole system.
• The overall concentration of every chemical species is constant in the whole system
(absence of mass generation or degradation).
• The transient state lacks a period of induction.
• Unidirectional molecular mass transfer, which is well described at all times by

Fick’s law, with negligible mixing effects or competitive sorption effects.
Hence, by considering all the characteristics mentioned above and the properties
of the modelled system, as well as the particular and general hypotheses detailed
above, and by combining the law of mass conservation with Fick’s first law, the six
differential equations describing the various mass transport phenomena can finally
be obtained in Equations 7.8–7.13:
∂ C Sa ∂  S ∂ C aS 
=  Da × 
∂t ∂z  ∂ z  (7.8)
∂ C Sw ∂  S ∂ C wS 
=  Dw × 
∂t ∂z  ∂ z  (7.9)
∂ C Ca  ∂ C aC 
= ∂  DaC × 
∂t ∂z  ∂ z  (7.10)
∂ C Cw ∂  C ∂ C wC 
=  Dw × 
∂t ∂z  ∂ z  (7.11)
∂ C HS  ∂ C aHS 
a
= ∂  DaHS × 
∂t ∂z  ∂ z  (7.12)
HS
∂ CW  ∂ C wHS 
= ∂  DwHS × 
∂t ∂z  ∂ z 
(7.13)
232
These mathematical expressions are parabolic partial differential equations,

the solution of which discloses the concentration profiles of the two chemical
compounds in each material domain (CSa, CSw, CCa, CCw, CHSa and CHSw) at every
position within the layer structures (z) and at every instant of elapsed time (t).
7.3.4.2 Solving the Package Model
This type of packaging structure has already been modelled in a recent work [67], in
which the authors developed an active antimicrobial package for ready-to-eat salad
products consisting of a functional 30 µm thick PP substrate coated with a thin layer,
3 µm thick, of EVOH copolymer as the carrier material for carvacrol, a well-known
antimicrobial agent of natural origin, at a concentration of 5%. Since EVOH is a
highly hydrophilic material whose physico-chemical properties are severely affected
by its moisture content (including the mass transport coefficients), the packaging
system studied in the work involved the coupled mass transport of the two chemical
species, water and carvacrol, with interconnected concentrations. Consequently,
the propounded model required, firstly, the mathematical solution of the water
concentration profiles [Cppw (z, t), CEw (z, t) and CHSw (z, t)] in order to store the results
obtained with the aim of subsequently using them to solve the carvacrol transport
processes [Cppc (z, t), CEc (z, t) and CHSc (z, t)] as initial values for the former variables.
The relationship between the value of the carvacrol transport coefficients and the
moisture content of the carrier polymer was experimentally measured in the work,
and, together with other required constants, coefficients and parameters collected
from the literature, they were finally included in the data inventory of the developed
model. The mathematical model was finally numerically solved using the finite
element method in the Chemical Transport of Diluted Species physics interface of
the COMSOL Multiphysics 4.2 modelling suite, by applying the initial and boundary
conditions described in the above-mentioned paper, and by spatially discretising
its three physical domains with a freely distributed mesh of 1,000 edge elements
per domain.
As a result of this procedure, the simulation software yielded the solution graphs
displayed in Figures 7.9–7.13. These graphs depict the evolution with time of
the water and carvacrol concentration profiles within the structure of the three
material layers considered: PP substrate, active EVOH coating and package
headspace.
233
Max 15.755
300
250
200 t(s)
14
150
100
50
0 12
15
10
10
8
CPPw(kg/m3)
5
6
−5 4
0 ×10
3
2
2
1 z(m)
Min: 2.816e−15
Figure 7.9 Evolution with time of water concentration profiles in the PP substrate
Max:149.273
300
250 140
200 t(s)
150
100
50 120
0
150
100
100
80
CEw(kg/m3)
50 60
40
0 ×10−5
3.3
3.2
20
3.1 z(m)
Min:3.05e−17
Figure 7.10 Evolution with time of water concentration profiles in the EVOH coating
234
Max: 16.805
×10−5
5 16
4
t(s)
3
2 14
1
0
12
15
10
10
CPPc(kg/m3) 8
5
6
0 ×10−5 4
3
2
2
1 z(m)
0
0
Min: 0
Figure 7.11 Evolution with time of carvacrol concentration profiles in the PP substrate
Max: 60.953
60
−5
×10
5 55
4 t (s)
3
2 50
1
0
60 45
50
40
40
35
30 E 3
C c(kg/m )
20 30
10
25
0 ×10 −5
3.3 20
3.2
3.1 z(m)
3 15
Min: 13.118
Figure 7.12 Evolution with time of carvacrol concentration profiles in the

EVOH coating
235
−4
Max: 2.502e
−4
×10
×10−5 2.4
5
4
t (s) 2.2
3
2
1
×10−4
2
0
2.5
1.8
2
1.6
1.5
CHSc(kg/m3) 1.4
1
1.2
0.5
1
0
0.014
0.012
0.01 0.8
0.008
0.006
0.004 z(m)
0.002
0 0.6
−5
Min: 5.418e
Figure 7.13 Evolution with time of carvacrol concentration profiles in

the headspace
The development of a model that describes the mass transport processes that occur
in an antimicrobial packaging system based on antimicrobial release is very useful
for understanding the mechanism of action of the package. But, above all, this model
is very useful for optimising the efficiency of the packaging system by visualising
the effect of the diverse variables responsible for the release of the antimicrobial
agent; for example, Cerisuelo and co-workers [67] showed the effects of structure
layer thickness, environmental humidity and reel storage on the final activity of the
packaging system.
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A
bbreviations
2D Two-dimensional
3D Three-dimensional
AFM Atomic force microscopy
ASTM American Society for Testing and Materials
ATCC American Type Culture Collection
aw Water activity
BOPP Biaxially oriented polypropylene
CCD Charge-coupled device
CD Corona discharge
CEO Cinnamon essential oil
CFU Colony forming unit(s)
CLSI Clinical & Laboratory Standards Institute
CO2 Carbon dioxide
CS Chitosan(s)
DNA Deoxyribonucleic acid
EDC 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide
EDTA Ethylenediaminetetraacetic acid
EMA Ethylene methacrylic acid copolymer
EPS Expanded polystyrene
EU European Union
EVA Ethylene-vinyl acetate copolymer
EVOH Ethylene-vinyl alcohol copolymer
FDA US Food and Drug Administration
FDM Finite difference method(s)
FFS Form/Fill/Seal
GRAS Generally recognised as safe
GSE Grapefruit seed extract
H2O2 Hydrogen peroxide
H2S Hydrogen sulfide
243
HDPE High-density polyethylene

HIPS High-impact polystyrene
HPMC Hydroxypropyl methyl cellulose
ION Ionomer
IR Infrared
ISO International Organization for Standardization
JIS Japanese Industrial Standard
KSCN Potassium thiocyanate
LAE Ethyl lauroyl arginate
LDPE Low-density polyethylene
LLDPE Linear low-density polyethylene
LPS Lactoperoxidase
MAP Modified atmosphere packaged/packaging
MC Methyl cellulose
MES 4-Morpholinoethanesulfonic acid
MIC Minimum inhibitory concentration
MLC Minimum lethal concentration
MW Molecular weight
NHS N-hydroxysuccinimide
NP Nanoparticle(s)
O2 Oxygen
OEO Oregano essential oil(s)
PA Polyamide(s)
PBS Phosphate buffer solution
PC Polycarbonate(s)
PE Polyethylene(s)
PEG Polyethylene glycol
PEI Polyethylene imine
PET Polyethylene terephthalate
PI Propidium iodide
PLA Polylactic acid
PMP Pathogen modelling program
PO Polyolefin(s)
PP Polypropylene
PS Polystyrene
244
Abbreviations
PU Polyurethane
PVA Polyvinyl alcohol
PVC Polyvinyl chloride
PVDC Polyvinylidene chloride
PVOH Polyvinyl alcohol
RH Relative humidity
RNA Ribonucleic acid
SEM Scanning electron microscopy
TEM Transmission electron microscopy
Tg Glass transition temperature
Tm Melting temperature
UV Ultraviolet
WHO World Health Organization
245
I
ndex
A
Absorbing systems, 99
Active agents
antimicrobial effect, 154
from bilayer active film, 113
physico-chemical properties, 56
release, 110
from three-layer active film, 114
Active coatings, 64, 223, 225
preparation, 82
Active films
antimicrobial effect, 134
development, 229
industrial production, 225–229
Active food packaging, 56–58
antimicrobial nanocomposites, 58
Active materials, 60, 97
compounding, 74–75
extrusion, 75–77
functions, 20
Active molecule-clay interactions, 56–57
Active molecule-clay systems, 57
Active packaging systems, 98, 229
antimicrobial activity, 22
antimicrobial agents, 28–43
coating, 79–84
component preparation, 53–54
clay nanocomposites, 56–58
mesoporous silica, 60–61
metal nanoparticles, 58–60
use of inorganic substrate, 54–56
concept, 2
encapsulation in organic substrate, 61–69
forms, 19
incorporation into packaging structures, 72–78
independent devices, 69–72
packaging, 1–3
surface anchorage, 85–90
wet coating, 78–79
spraying, 84–85
Active packaging technology, 69
Active substance, 82
Active system, modelling and optimisation, 229–230
Aerosol inoculation, 139
AFM (see Atomic force microscopy)
Agar dilution method, 131–132, 137
Agent concentration, 114
Agent, modelling release, 115–117
finite difference method (FDM), 117–120
finite element method, 120–122
Airless spray, 84
Air-spray coating, 84–85
American Type Culture Collection (ATCC), 125–126
Antimicrobial active packaging, 99, 214–216
characterisation of properties, 221–225
containing non-volatile agent, 101
industrial production of active film, 225–229
material manufacture, 216–221
modelling and optimisation of active system, 229–236
technology, 3
Antimicrobial activity
films, 227
samples, 138
Antimicrobial agents, 69, 135
applied to fresh vegetables, 204–214
concentrations, 128
factors affecting properties and stability, 43–44
minimum inhibitory and lethal concentration, 25–27
minimum lethal concentration (MLC), 131–132
release, 102
solubility and reactivity, 44
used in active packaging, 28–43
Antimicrobial-coated packaging for food, 83–84
Antimicrobial-coated substrate, 139
248
Index
Antimicrobial effectiveness, 127

Antimicrobial films, 130
effectiveness, 142
manufacture, 83
Antimicrobial food-packaging materials, 104
Antimicrobial food-packaging systems
application, 57
elements, 127
Antimicrobial ions, 58
Antimicrobial packaging efficiency, 125–127, 230–233
agents, 163–187
developments, 163–187
in vitro methods, 127
atomic force microscopy (AFM), 143–144
dilution method, 131–137
disc diffusion method, 128–131
electron microscopy, 140–142
flow cytometry, 144–147
Japanese industrial standard method, 137–139
surface growth method, 139–140
in vivo methods, 147–149
deliberate contamination, 151
total microbial load, 149–151
methods for testing, 125
modelling, 230–233
volatile agent, 100
Antimicrobial packaging technologies, 149
Antimicrobial pads, 72
Antimicrobial polymer nanocomposites, 58
Antimicrobial surface (biostatic effects), 137
Arrhenius dependence, 103
Aspergillus niger, 41
ATCC (see American Type Culture Collection)
Atomic force microscopy (AFM), 143–144
Atomisation, 68
Automated instrument systems, 136
B
Bacteria
collections, 151–152
concentration, 128
Bacterial cells, 145–146
249
Bacterial strains, 126–127

Bacteriocins, 9, 28–43, 30–32, 31
Bacteriolytic enzymes, 28–29
Bacteriophages, 33–34
Bacteriostatic agents, 134
Barrel/screw design, 74
Biodegradable film, 76
Biomolecules
attachment, 88–90
covalent attachment, 86
Biomolecules to surface, 88–90
Bioplastics, 13
Broth dilution, 131–132
C
Calcium stearate, 54
Campylobacter jejuni, 35
Caprolactam, 119
Carrier systems, 57
Carvacrol
concentration profiles
EVOH coating, 235
headspace, 236
PP substrate, 235
transport coefficients, 233
Cation exchange, 57
Cationic surfactant, 34
CCD camera (see Chargecoupled device)
Cell components, 144
Cell suspension, 126
Cellulose-based materials, 72
Cellulose fibres, 71
Chargecoupled device (CCD) camera, 140
Chelators, 28
Chitosan (CS), 39–40, 77
antimicrobial effectiveness, 40
films containing silver nanoparticles, 59
Chlorine, 204–205
Chlorine dioxide, 70, 71, 205
Cinnamaldehyde, 100
250
Index
Clay, 54
nanocomposites, 56–58
Clostridium botulinum, 151
Coacervate droplets, 67
Coacervation, 61, 67
Coated structure, physical properties, 225
Coating, 79–84
EVOH, 235
extrusion, 77–78
film surfaces, 44
fluidised bed, 68
solution, 82
wet, 78–79
ComBase Predictor, 157
Commercial phage, 34
Commodity polymers, 79–80
Complex inclusion, 66
Compounding
active materials, 74–75
preparation of active materials, 74–75
Conical meniscus, 68–69
Contact angle values, 223
Contamination, 3, 19, 21, 34, 36
primary sources, 202
Conventional packaging formulations, 41
Cool spray, 68
Copolymers, 9
Corona treatment, 88
Cream cheese, 157
Cross-contamination, 202
Crystal polystyrene, 31
CS (see Chitosan)
Culture medium, 126–127
Cyclodextrins, 66
D
Degree of adhesion, 222
Deliberate contamination, inoculum preparation, 151–155
data interpretation, 157
method, 155–156
storage conditions, 156–157
251
Deoxyribonucleic acid (DNA), 33

Detergents, 28
Diet, 201
Diffusion coefficient values, 107
Diffusion of carvacrol, 108
Dilution method, 131–137
Direct extrusion, 75–76
Disc diffusion method, 128–131
DNA (see Deoxyribonucleic acid)
Drying, 82
Dual-parameter SYTO-13/PI fluorescence histograms, 147
E
EcoShieldTM, 33
Einstein’s diffusivity law, 210
Elastomers, 4
Electrohydrodynamic atomisation, 68–69
Electron microscopy, 140–143
Electrospray, 68–69
Electrostatic encapsulation, 68–69
Elements, 120
Emulsion, 82
Encapsulation in organic substrate, 61–62
bacteria, 62
criteria for antimicrobial agent encapsulation, 62–63
matrices, 63–65
methods, 65–69
structures, 65
Enzymes, 28–30
EPS (see Expandable polystyrene)
Epsilon-polylysine films, 150
Equilibrium coefficients, 105
Escherichia coli, 151, 202, 206
antimicrobial effect against, 217, 219
Essential oils, 36–39, 44, 89
Ethanol-releasing devices, 70
Ethylene copolymers, 7
Ethylene-vinyl acetate (EVA), 60
copolymers, 8–9
Ethylene-vinyl alcohol (EVOH), 107, 115, 150
copolymer, 162, 233
252
Index
Ethylene-vinyl alcohol (EVOH) film, 29, 57

application of a high-barrier coating, 224
coating layer, 224–225
heat sealability, 222
high-barrier coating, 224
permeance, 224
Ethyl lauroyl arginate (LAE), 34–36
mechanism of action, 35
EVA (see Ethylene-vinyl acetate)
EVOH (see Ethylene-vinyl alcohol)
Expandable polystyrene (EPS), 9
Expanded polystyrene, 10
Extruders, 74
design, 73
Extrusion, 68, 100
active materials by, 75–77
antimicrobial agents, 76
coating, 77–78
preparation of active materials, 75–77
F
Fats, 64
FDM (see Finite difference method)
Fick’s laws, 103–104, 109, 115, 231
Film activity, 90
Film-forming solution, 137
Film scale-up, 221–225
Film surface (coatings on), 44
Finite difference method (FDM), 117–120
Finite element method, 120–122
Flexible packaging, 13–14
industry, 208
Flexography, 81–82
Flow cytometry assay, 144–147, 146
Fluidised bed coating, 68
Fluorophores, 145
Food acidity, 40
Foodborne illnesses, 202
Food habits, 201
253
Food ingredients, 64
Food packages, 224
Food packaging
producers, 201
system (antimicrobial activity), 71
Food preservation technologies, 97
Food products, 152
Foods packaging, 1
essential oils, 37–38
plastic materials, 3
polymeric materials, 20
Food spoilage, 21
Form/Fill/Seal (FFS) equipment, 70–71
Fourier’s law, 120
Freeze-dried cultures, 126
Freeze spray, 68
Functional barrier in migration studies, 114
Fungal strains, 127
Fungi, 26, 30, 34
collections, 151–152
G
Gallic acid, 41
Gamma irradiation, 206
Gelatin, 61
Glassy material, 6
Gram-negative bacteria, 30, 34, 136
Gram-negative organisms, 32
Gram-positive bacteria, 28, 30, 34, 136
Gram-positive microorganisms, 32
Grape extracts, 39
Gravure coaters, 81
Green tea, 38
H
Haemolytic uraemic syndrome, 202–203
HDPE (see High-density polyethylene)
Headspace atmosphere composition, 226
Headspace composition, 209
Heat-resistant peptides, 31
Henry’s law, 102, 116
High-barrier polymers, 12
254
Index
High-density polyethylene (HDPE), 8

High-impact polystyrene (HIPS) styrene, 9–10
HIPS (see High-impact polystyrene)
Hollow silica, 61
Hooke’s law, 143
Hurdle technology, 211
I
Immobilisation, 86
Infrared (IR) spectroscopy, 90
Injection moulding, 15
Inoculum of bacterium, 135
Inoculum preparation, 151–155
data interpretation, 157
method, 155–156
storage conditions, 156–157
Inorganic/polymer composites, 55
Inorganic substrate, 54–56
Intelligent packaging, 2–3, 3
Interfacial polymerisation, 66–67
In vitro methods, 127
atomic force microscopy (AFM), 143–144
dilution method, 131–137
disc diffusion method, 128–131
electron microscopy, 140–142
flow cytometry, 144–147
food components, 148
Japanese industrial standard method, 137–139
surface growth method, 139–140
In vivo methods, 147–149
deliberate contamination, 151
total microbial load, 149–151
Ionomers (ION), 8, 135
Irradiation, 206
J
JIS Z 2801 method, 139
L
Lactoperoxidase (LPS), 29–30
LAE (see Ethyl lauroyl arginate)
255
Lantibiotics, 31
Law of mass conservation, 232
Layered clays, 56
LDPE (see Low-density polyethylene)
Lipids, 64
Liposomes, 67
Listeria monocytogenes, 135, 146, 151, 203
antimicrobial effect against, 217, 219
ListShieldTM, 33
Low-density polyethylene (LDPE), 7–8, 60, 208
LPS (see Lactoperoxidase)
Lysozyme, 28–29
incorporated, 29
M
MAP (see Modified atmosphere packaging)
Mass exchange, 103
Mass transfer
mathematical modelling and simulation, 229–230
mechanisms, 7
properties, 6–7
Mass transport, 98–105
food/packaging/environment system, 99
parameters, 105–110
Mechanical-thermal process, 100
Mediterranean diet, 201
Mesoporous hollow silica, 61
Mesoporous silica, 55, 60–61
nanoparticles, 60–61
Metal nanoparticles, 58–60
Metal oxides, 75
Metals, 41–43
MIC (see Minimum inhibitory concentration)
Michaelis–Menten inhibition, 209
Microatmosphere method, 130
Microbial concentrations, 138
Microbial growth, 154
Microbial population, 227
Microbiological growth, 206
Microcapsules, 61–62
Microdilution Broth Method, 25
Microdilution Method, 134
256
Index
Microencapsulation, 61
applications, 62
benefits, 62
Microorganisms, 21, 126, 152–153
growth curve, 134
incubation conditions, 150
Migration, 17–18
Minimum growth temperature, 153–154
Minimum inhibitory concentration (MIC), 25–27, 100, 112, 146
Minimum lethal concentration (MLC), 25–27
antimicrobial agents, 131–132
protocol, 132–133
MLC (see Minimum lethal concentration)
Modelling and optimisation of active packaging, 97–98
mass transport, 98–110
modelling release of agent, 115–122
release control, 110–115
Modelling release of agent, 115–117
finite difference method (FDM), 117–120
finite element method, 120–122
Modified atmosphere packaging (MAP), 206–211
industrial application, 208
Moisan cell, 109
Molecular inclusion, 66
Molecular weight (MW) organic polymers, 3
Molten plastic, 15
Mueller–Hinton agar, 137
Multilayer film (surface properties), 222–223
N
Nanocomposite, 55
Nanomaterials (classification), 56
Nanoparticles (NP)
copper, 59
gold, 42
mesoporous silica, 60–61
metal, 58–60
silver, 42
synthesis, 42
titanium, 42
zinc oxide, 42–43, 59
257
Nanotubes, 58
Natural food preservatives, 32
Natural polyphenols, 38
Nisin, 31, 76
Non-aggressive thermal methods, 44
Non-processed foods, 34
Non-volatile agent, 110
Non-volatile antimicrobials, 44–45, 111
Non-volatile compounds, 101
Novel technologies, 212
NP (see nanoparticles)
O
Odour acceptability, 220
OEO (see Oregano essential oil)
Optical properties, 5
Oregano essential oil (OEO), 100, 115, 228
antimicrobial properties, 217
Organic acids, 40–41, 44, 53, 101
Organic substrate encapsulation, 61–62
criteria for antimicrobial agent encapsulation, 62–63
matrices, 63–65
methods, 65–69
Oxidoreductase systems, 29–30
Oxygen (permeation), 119
Ozone, 205
P
Package model, 233–236
Packaging
and active packaging, 1–3
active systems (see Active packaging systems)
antimicrobial active (see Antimicrobial active packaging)
basic characteristics, 18–22
characteristics, 2
efficiency (see Antimicrobial packaging efficiency)
environment interactions, 15–18
flexible, 13–14
function, 1
injection moulding, 56
intelligent, 3
258
Index
plastic (see Plastic packaging)

plastic materials, 3–18
polymeric material structure, 17
principal plastics, 7
production technologies, 13–15
rigid, 14–15
valued characteristics, 2
Packaging structures
active materials by compounding, 74–75
extrusion coating, 77–78
preparation of active materials by extrusion, 75–77
thermomechanical methods, 72–74
Paper-polyethylene (PE) laminates, 70
Pathogen modelling program (PMP), 157
Pathogens, 140–141
PE (see Polyethylene)
Pellets, 74–75
Penicillium expansum, 157
Penicillium spp., 41
Permeability, 16–17
PET (see Polyethylene terephthalate)
Petroleum-derived polymers, 82–83
Phages, 33
immobilisation, 34
Phenolic resins, 4
Phosphate buffer solution (PBS), 141
PLA (see Polylactic acid)
Plant extracts, 36–39
Plasma treatment, 87
Plastic packaging
basic characteristics, 3
bioplastics, 13
classifications, 4
food/packaging/environment interactions, 16
general characteristics, 3–5
high-barrier polymers, 12
mass transfer properties, 6–7
mechanical properties, 5
optical properties, 5
polyamides, 12
polyesters, 11
polyolefins, 7–9
259
polystyrenes, 9–10
raw materials, 4
thermal properties, 5–6
vinyl polymers, 10–11
PMP (see Pathogen modelling program)
PO (see Polyolefins)
Polar lipids, 64
Polyaddition, 4
Polyamides, 12
Polycarbonate(s) (PC), 11
Polycondensation, 4
Polyesters, 11
Polyethylene (PE), 7, 162
Polyethylene terephthalate (PET), 11
Polylactic acid (PLA), 162
Polymer
ability, 59
additives, 74, 76
composites, 55, 61
crystalline regions, 6
films, 56, 82
matrix, 79
nanocomposites, 58, 60
pellets, 76
surface, 86
Polymeric materials, 17
treatments, 87
Polyolefins (PO), 7–9, 75
Polypropylene (PP), 9, 162
surface properties, 222–223
Polysaccharides, 39–40, 64
Polystyrenes, 9–10
Polyurethane (PU), 221
Polyvinyl chloride (PVC), 10
Polyvinylidene chloride (PVDC), 10–11
PP (see Polypropylene)
Prehomogenisation, 75
Preservation methods, 33
Probe, 143
Product dehydration processes, 209
260
Index
Prokaryotic microorganisms, 154–155

Proteins, 64–65
degree, 65
hydrolysis, 65
PVC (see Polyvinyl chloride)
PVDC (see Polyvinylidene chloride)
R
Rapid release, 112
Relative humidity (RH), 119
Release control, 110–115
Releasing systems, 99
Retraction zone, 130
Ribonucleic acid (RNA), 33
Rigid packaging, 14–15
RNA (see Ribonucleic acid)
Roll coaters used for packaging materials, 80–81
Rubbery material, 6
S
Salmonella, 203, 206
enterica, 217, 219
Salmonellosis, 203
Sample core, 144
Sample preparation protocols, 140–141
Sanitation, 201–202
Saturated fats, 64
Scanning electron microscopy (SEM), 140–141
Scanning probe, 143
SEM (see Scanning electron microscopy)
Semicrystalline thermoplastics, 6
Sensory evaluation, 221
Silica, 55
Silver acetate, 58
Silver nanorods, 58
Simplified antimicrobial package, 230–231
Solid cylindrical particles, 74
Solubility coefficients, 106
Sorption, 17
Spray drying, 68, 77
Spraying phages, 34
261
Stable thermal substances, 75

Staphylococcus aureus, 151
Streptococcus pneumoniae, 136
Substances
exchange, 103
molecular diffusion, 103
Sulfur dioxide, 71
Surface analysis, 90
and determination of film activity, 90
Surface anchorage, 85–86, 85–90
attachment of biomolecules to surface, 88–90
sample preparation, 86
surface analysis and determination of film activity, 90
surface modification, 86–88
Surface growth method, 139–140
Surface modification, 86–88
Surfactants, 34–36
T
Taylor’s approximation, 118
Taylor’s function, 118
TEM (see Transmission electron microscopy)
Thermomechanical methods (packaging structures), 72–74
Thermoplastics, 4
Thermosets, 4
Time-kill curves, 134, 135
Titanium dioxide (impregnation), 75
Total microbial load, 149–151
Transmission electron microscopy (TEM), 140–141
Transpiration, 209
U
Ultraviolet irradiation, 88
Unmodified styrene, 9
Urea-formaldehyde resins, 4
V
Van’t Hoff’s law, 103
Vinyl polymers, 10–11
262
Index
Volatility, 54, 77
agent, 112
antimicrobials, 69–70
compounds, 99–100
W
Water concentration profiles, 234
Wet coating, 78–79
spraying, 84–85
WHO (see World Health Organization)
World Health Organization (WHO), 202
Y
Yeast, 30, 34
Z
Zeolites, 57–58
ions, 58
263
Published by Smithers Pira Technology Ltd, 2015
Antimicrobial packaging systems are those that beneficially interact with food or
the surrounding environment, inhibiting or reducing microbial growth in order to
improve the quality and extend the shelf life of industrially produced foods. They have
undoubtedly become a fully accepted alternative to the direct addition of preservatives
to foods and have excellent future prospects.
The aim of this book is to develop a working knowledge and understanding of

antimicrobial packaging, including a description of the antimicrobial agents most
commonly used and their mechanisms of action, the manufacturing methods available
to fabricate the active system, the critical parameters which need to be considered
to make an effective product and the tools to optimise them, and the various in vitro
and in vivo methods for measuring the effectiveness of the antimicrobial system for
validation purposes.
This book also aims to develop an understanding of why a specific agent is selected
for a particular food product, or why a specific polymeric material and manufacturing
technology are chosen. The reader will become familiar with the different procedures
for improving the activity of the developed packaging solution and ways of testing
its efficacy. This will accelerate the formulation of the active packaging concept,
reducing development time with respect to the trial-and-error processes common in
many published reports. Finally, it will help to identify the best and most cost-effective
solutions. This book is intended to be a practical guide to antimicrobial packaging and
a quick reference for students and researchers from both academia and industry.
Shawbury, Shrewsbury, Shropshire, SY4 4NR, UK

Telephone: +44 (0)1939 250383
Fax: +44 (0)1939 251118
Web: www.polymer-books.com

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Practical Guide to Antimicrobial

Rafael Gavara, Gracia López-Carballo,

A Smithers Group Company

Shawbury, Shrewsbury, Shropshire, SY4 4NR, United Kingdom

Smithers Pira Technology Ltd

©Smithers Information Ltd., 2015

All rights reserved. Except as permitted under current legislation no part

ISBN: 978-1-91024-273-5 (hardback)

Typeset by Integra Software Services Pvt. Ltd.

The aim of this book is to develop a working knowledge and understanding of

The authors acknowledge the financial support of the Spanish Ministry of

1 Introduction to Active Packaging .................................................................. 1

1.3 Active Packaging: Basic Characteristics ............................................. 18

3 Active Packaging Systems ............................................................................ 53

3.3.1.1 Preparation of Active Materials by

4 Modelling and Optimisation of Active Packaging:

5 Methods for the Analysis of Antimicrobial Packaging Efficiency ............... 125

5.2 In Vivo Methods.............................................................................. 147

6 Review of Antimicrobial Packaging Systems.............................................. 161

References ................................................................................................. 187

7 Case Study: Active Packaging of Minimally Processed Vegetables ............. 201

7.1 Description of the Product ............................................................... 201

Abbreviations .................................................................................................... 243

Index ................................................................................................................. 247

The Packaging Group of the Instituto de Agroquímica y Tecnología de Alimentos

Gracia López-Carballo, Virginia Muriel-Galet, Josep P. Cerisuelo, Irene Domínguez,

Packaging Group, Instituto de Agroquímica y Tecnología de Alimentos (IATA, CSIC),

1.1 Packaging and Active Packaging

For some foods packaging is simply a form of presentation, a means of commercial

context, the new paradigm of packaging is smart behaviour, understood as active

Active packaging and intelligent packaging have been gradually introduced

1.2 Plastic Materials for Packaging

1.2.1 General Characteristics of Plastics

1.2.2 Important Properties of Plastics for Packaging

The properties of plastic materials are a consequence of their chemical nature,

1.2.2.1 Mechanical Properties

The behaviour of plastics in response to mechanical stress of any kind, which is of

1.2.2.2 Optical Properties

1.2.2.3 Thermal Properties

Thermoplastics are characterised by a mechanical behaviour which is affected by

The behaviour of plastics in response to temperature can be described by the following

1.2.2.4 Mass Transfer Properties

The various mechanisms of mass transfer correspond to permeability phenomena, i.e.,

1.2.3 Principal Plastics used for Packaging

Of the commercially available polymer materials, the principal families of plastics

• Polyethylene and ethylene copolymers

PE are non-polar semicrystalline thermoplastics with various degrees of branching

Low-density polyethylene (LDPE) was the first plastic to be developed as a packaging

by practically all current technologies and is most commonly applied as a flexible

High-density polyethylene (HDPE) is semitransparent, exhibits a low degree of

• Ethylene-vinyl acetate copolymers

developed, known as ethylene-vinyl alcohol (EVOH) copolymers, which are derived

The PS commonly known as crystal PS is the homopolymer of unmodified styrene.

1.2.3.3 Vinyl Polymers

Polyvinylidene chloride (PVDC) is an inert, semicrystalline polymer that forms very

1.2.3.6 High-barrier Polymers

A well-established alternative to multilayer structures containing EVOH is the

The environmental impact attributed to conventional plastic materials, because they

Biopolymers can be obtained from a wide variety of sources [9, 19]:

• From agricultural, livestock and marine products, e.g., polysaccharides such as

• By chemical synthesis from biomass monomers such as polylactic acid (PLA).

• Produced by natural or genetically modified microorganisms, such as

1.2.4 Packaging Production Technologies