43074724 (3)

Influence of some physical treatments and
natural food preservatives on the diversity of

microorganisms in certain Egyptian juices
By
Mohamed Nagah Zayed Ibrahiem Abu El-Naga

B.Sc. (Microbiology - chemistry), Fac. Sci., Al-Azhar Univ., Egypt, 2003.
THESIS
Submitted in Partial Fulfillment of the
Requirements for the Degree of
MASTER OF SCIENCE
IN
Microbiology
Department of Botany & Microbiology

Faculty of Science
Al-Azhar University
Cairo
2009
SUPERVISION SHEET
Influence of some physical treatments and

natural food preservatives on the
diversity of microorganisms in certain
Egyptian juices
By
Mohamed Nagah Zayed Ibrahiem Abu El-Naga

B.Sc. (Microbiology - chemistry), Fac. Sci., Al-Azhar Univ., Egypt, 2003.
SUPERVISION COMMITTEE
Prof. Dr. Backry Mohamed Haroun

Prof. of Microbiol., Fac. of Sci., Al-Azhar University.
Prof. Dr. Mohie El-Din Zohier El-Fouly

Prof. of Microbiol., National Center for Radiation Research and
Technology (NCRRT), Atomic Energy Authority.
Dr. Hala Ahmed Hussein

Assistant Prof. of Microbiol., National Center for Radiation
Research and Technology (NCRRT), Atomic Energy Authority.
Acknowledgment
Praise and gratitude is to ALLAH SOBHANAHU

WATAALA, the God of all creatures, for guiding me to the
right way.
I wish to express my appreciation to Dr. Backry

Mohamed Haroun, Professor of Microbiology, Botany and
Microbiology Department, Faculty of Science, Al-Azhar
University, for helpful advice, encouragement and for
sharing in the supervision.
Sincere thanks and deep gratitude to Dr. Mohie El-Din

Zohier El-Fouly, Professor of Microbiology, National Center
for Radiation Research and Technology (NCRRT), Atomic
Energy Authority, for helpful advice, encouragement,
sharing in the supervision and lively interest he showed
during the entire thesis.
I wish to express my grateful acknowledgment to Dr.

Hala Ahmed Hussein, Assistant Professor of Microbiology,
National Center for Radiation Research and Technology
(NCRRT), Atomic Energy Authority, for suggesting the
subject of study, progress of the work, helpful advice,
encouragement, sharing in the supervision and lively
interest she showed during the entire thesis.
I would especially like to thank Prof. Dr. El-Said Ghazal,

Professor of Microbiology, and the head of Botany and
Microbiology Department, Faculty of Science, Al-Azhar
University, for offering facilities, continuous encouragement
and sustainment.
Also, I would like to express my appreciation and thanks

to Prof. Dr. Bothaina M. Youssef Professor and Head of
Microbiology Department at NCRRT.
Sincere thanks to Prof. Dr. T. M. El-Mongy, Professors of

Food Microbiology of National Center for Radiation Research
and Technology, for help through out conducting my
experiments.
Finally, special thanks to Said El-Sayed Desouky,

Assistant lecturer, Botany and Microbiology Department,
Faculty of Science, Al-Azhar University, for his cooperation
and great help in this study.
Last, but not least I wish to express my grateful thanks,

to all Members of Microbiology Department, and to all my
Colleagues at Microbiology Department, NCRRT.
DEDICATION
I dedicate this work to my Parents, as well as to my Sisters and my

Brother and a special dedication for my Mother for her great
support.
Mohamed Nagah
CONTENTS
CONTENTS
Page
INTRODUCTION ................................................................. 1
REVIEW OF LITERATURE..................................... 4
1. Fruit Juices and soft Drinks .......................................... 4
2. Food additives ............................................................................... 7
2.1. Introduction...................................................................................... 7
2.2. Types of additives……………………………………………… 8
2.2.1. Preservatives................... ……………………………………… 8
2.3. Risks of additives…………………………………………… 9
2.4. Balancing risks and benefits…………………………… 11
2.5. Categories of Risk………………………………………………… 14
2.5.1. Phenyl ethylamine………………………………………………… 16
2.5.2. Azo Dyes and Benzoates…………………………………… 16
2.5.3. Sulfites ………………………………………………………………… 17
2.5.4. Nitrites……………………………………………………………………………… 18
2.5.5. Other Agents………………………………………………… 20
3. The use of natural antimicrobials……………………………………… 22
3.1. Natural preservatives................................................... 25
3.1.1. Nisin…………………………………………………………………… 25
• Mechanism of action……………………………………. 31
3.1.2. Organic acids ………………………………………………………… 36
3.1.2.1. Mode of action………………………………………………… 38
3.1.2.2. Resistance mechanisms…………………………………… 42
3.1.2.3. Citric acid ………………………………………………………… 48
3.1.2.4. Lactic Acid………………………………………………………… 49
3.1.3. Spices and their essential oils……………………… 53
3.2. Physical treatments…………………………................... 58
3.2.1. Gamma irradiation ……………………………………… 58
3.2.1.1. Types of radiation ……………………………………… 58
3.2.1.2. Living cell and radiation……………………………… 60
3.2.1.3. Dose survival curves…………………………………… 61
3.2.1.4. The decimal reduction doses (D10 value) 61
3.2.1.5. Effect of radiation on microorganisms…… 61
3.2.1.6. Direct action ………………………………………………… 62
3.2.1.7. Indirect action…………………………………………………… 63
3.2.1.8. Effect of radiation on water molecules… 64
3.2.2. Heat treatment………………………………………………………… 67
3.2.2.1. Introduction..................………………………………….... 67
3.2.2.2. Thermal technologies………………………………… 67
3.2.2.3. Pasteurization……………………………………………… 68
3.2.2.4. The decimal reduction time……………………… 68
3.2.2.5. Mechanism of action………………………………… 69
MATERIALS AND METHODS.... ………………… 71
• Materials……………………………...........…………… 71
1. Samples………………………………………………………............................... 71
2. Source of samples………………………………............................………… 71
3. Samples sorts……………………...........................…………………………… 72
4. Source of preservatives…………………….....................…………………… 72
5. Irradiation source…………………………………………………..................…… 72
6. Isolation media………………………………………………................................ 73
• Methods…………………………......................…………………………… 76
1. Microbial counts ……………………………................................……………… 76
2. Isolation of bacteria……………………………............................…………… 76
3. Isolation of yeast............................................................ 76
4. Identification……………………………………...........................……………… 77
5. Selection of most common isolates……………………………………. 78
6. Effect of some natural preservatives……….............………………… 78
7. Effect of some physical treatments……………………..............……… 79
8. Effect of the combination treatments…………...............…………… 80
9. Storage………………………………………………………….............................… 81
RESULTS AND DISCUSSION………………………..... 83
1. Isolation and identification……………………………….. 83
2. Single treatments……………………………………......................………….. 93
2.1. Physical treatments……………............………………………. 93
2.1.1. Gamma irradiation………………………………….........................… 93
2.1.2. Pasteurized temperature…….........................…………………… 99
2.2. Natural treatments…………………..........................………………………………… 105
2.2.1. Nisin……………………………………….……………… 105
2.2.2. Organic acid ………………………………………… 112
2.2.2.1. Citric acid …………………………… 112
2.2.2.2. Lactic acid…………………………… 117
2.2.3. Cinnamon……………………………………………… 124
3. Combination treatments …………………. 130
3.1. Combination with Gamma Irradiation….. 130
3.1.1. Combination of Gamma irradiation with citric acid 131
3.1.2. Combination of Gamma irradiation with lactic acid 134
3.1.3. Combination of Gamma irradiation with cinnamon 138
3.1.4. Combination of Gamma irradiation with Nisin 142
3.1.5. Comparison between gamma irradiation single and combined
treatments
147
3.2. Combination with Pasteurized temperature 149
3.2.1. Combination of pasteurized temperature with Nisin 149
3.2.2. Combination of pasteurized temperature with citric acid 152
3.2.3. Combination of pasteurized temp with lactic acid 154
3.2.4. Combination of pasteurized temp with cinnamon 156
3.2.5. Comparison between the selected natural preservatives
alone 160
and in combination with pasteurized temperature.
3.3. Combination of natural preservatives…………… 163

3.3.1. Combination of Nisin with citric acid and lactic acid……………… 163
3.3.2. Combination of Nisin with cinnamon………………………………………… 170
3.3.3. Comparison between the selected natural preservatives in single
treatment and in combination with nisin. 174
4. Combination of gamma irradiation with
177
pasteurized temperature………………………………………….
5. Storage………………………………………………………………………………… 179
6. General Discussion………………………………………………………………… 182
SUMMRAY…………………………………………………………… 188
CONCOLUSION AND RECOMMENDATION 198
REFERENCES…………………………………………………… 199
ARABIC SUMMARY……………………………………………… 1
Table LIST of TABLES Page
(a) Properties of some organic acids used as additives. 42
Screening and isolation of microorganisms

(1) 86
contaminated certain Egyptian juices.
Identification of contaminated microorganisms isolated
(2) 90
from certain Egyptian juices.
Effect of different doses of gamma irradiation on the
(3) 96
viable counts of the selected organisms.
Effect of pasteurized temperature 90 Cْ at different time
(4) 102
on selected organisms.
Effect of different conc. of nisin on the selected
(5) 109
organisms.
Effect of different conc. of citric acid on P. aeruginosa and
(6) 114
S. aureus.
(7) Effect of different conc. of citric acid on Debaryomyces sp. 115
(8) Effect of different conc. of lactic acid on Debaryomyces sp. 118
Effect of different conc. of lactic acid on P. aeruginosa and S.

(9) 119
aureus.
Effect of different conc. of cinnamon on S. aureus and
(10) 126
Debaryomyces sp.
(11) Effect of different conc. of cinnamon on P. aeruginosa 127
Effect of different doses of gamma irradiation in

(12) 137
combination with lactic acid.
(13) 138
combination with citric acid.
(14) 140
combination with cinnamon.
(15) 145
combination with nisin.
D10 - values of gamma irradiation for single and
(16) 148
combined treatments.
Effect of pasteurized temperature 90 Cْ for 30 sec in
(17) 151
combination with different conc. of nisin
(18) 153
combination with different conc. of citric acid.
(19) 155
combination with different conc. of lactic acid.
(20) 159
combination with different conc. of cinnamon.
Sub-lethal values of natural preservative alone and in
(21) 161
combination with pasteurized temperature.
Effect of different conc. of citric acid in combination
(22) 165
with nisin.
(23) 167
with Nisin 200 µg/ml on Debaryomyces sp.
Effect of different conc. of lactic acid in combination with
(24) 168
nisin
(25) 169
Nisin 200 µg/ml on Debaryomyces sp.
Effect of different conc. of cinnamon in combination with
(26) 172
nisin
Sub-lethal values of selected natural preservatives alone and in
(27) 175
Effect of storage for recovery of Debaryomyces sp.
(28) 180
Fig. LIST of FIGURES Page
(a) Structure of nisin and unusual amino acids. 28
A schematic diagram of the stress response of a yeast

(b) 47
cell challenged with weak organic acids.
(1) Percentages of isolated microorganisms. 92
Effect of increasing doses of gamma irradiation on

(2) 97
the selected organisms.
Effect of pasteurized temperature 90 Cْ at different
(3) 103
time on the selected organisms.
General model of the `barrel-stave' mechanism of pore
(5) 108
formation by peptides
Effect of different conc. of nisin on the selected
(4) 110
organisms.
(6)
Effect of different conc. of citric acid on P. 115
aeruginosa and S. aureus.
Effect of different conc. of citric acid on
(7) 116
Debaryomyces sp.
Effect of different conc. of lactic acid on Debaryomyces
(8) 118
sp.
(9)
Effect of different conc. of lactic acid on P. aeruginosa 120
and S. aureus.
A schematic impression of the stress response of a
(10) 122
yeast cell challenged with weak organic acids.
Effect of different conc. of cinnamon on S. aureus and
(11) Debaryomyces sp. 127
(12) Effect of different conc. of cinnamon on P. aeruginosa 128

(13) 136
(14) 134
(15) 146
(16) 146
combination with Nisin.
D10 - values of gamma irradiation for single and
(17) 148
combined treatments
Sub-lethal values of natural preservative alone and in
(18) 162
combination with pasteurized temperature
(19) 166
with nisin
(20) 167
with Nisin 200 µg/ml on Debaryomyces sp.
(21) 169
nisin
(22) 170
Nisin 200 µg/ml on Debaryomyces sp.
(23) 173
nisin.
(24) 173
nisin on S. aureus.
Sub-lethal values of selected natural preservatives alone and in
(25) 176
Abbreviations
ACSH → American Council on Science and Health.

AMU→ Atomic Mass Unit.
ATP → Adenosine Triphosphate.
BHA → Butyl Hydroxyanisole.
BHT → Butyl Hydroxytoluene.
CDCP → Centers for Disease Control and Prevention.
CFU → Colony Forming Unit.
CO60 → Cobalt 60.
Conc. → Concentration.
D → Dalton.
D10 → Decimal Reduction Dose.
DHA → Dehydroalanine.
DHSS → Department of Health Social Security.
Dt → Decimal Reduction Time.
E300 → Europe Number.
EDTA → Ethylene DiamineTetracetate.
FDA → Food and Drug Administration.
FPCFN → Food Protection Committee of the Food and
Nutrition Board.
GRAS → Generally Recognized as Safe.
H → Hour.
HSP → Heat Shock Protein.
IAEA → International Atomic Energy Agency.
IFT → Institute of Food Technologists.
IgE → Immunoglobulin E.
INS → International Numbering System.
kGy → kilo Gray.
LD50 → Lowest Dose (in mg/kg or g/kg of body).
LPS → Lipopolysaccharide.
mg → Milligram.
MIC → Minimum Inhibition Concentration.
Min → Minute.
MRS → de Man Rogosa and Sharpe.
NAS/NRC → National Academy of Science/National
Research Council.
OM → Outer Membrane.
PDA → Potato Dextrose Agar.
PL → Phospholipids.
PMF → Proton Motive Force.
RAST → Radio-allergo sorbent Test.
ROS → Reactive Oxygen Species.
TDT → Thermal Death Time.
W/V → Weight Per Volume.
ΔΨ → Membrane Potential.
μg → Microgram.
INTRODUCTION
Introduction
Foods begin to lose their quality from the moment they
are harvested through changes resulting from physical,
chemical, enzymatic, or microbiological reactions. Food
preservation prevents deteriorative reaction, extending the
shelf life and assuring its safety. Microorganisms and
enzymes are the main agents responsible for food spoilages
and therefore the targets of preservation techniques.
The "ideal" method of food preservation has the following
characteristics:
• It improves shelf life and safety by inactivating spoilage
and pathogenic microorganisms.
• It dose not leave residues.
• It is cheap and convenient to apply.
• It encounters no objection from consumers and
legislators.
One of the major advances in human history was the
ability to preserve food. It was the perquisite to man setting
down in one place instead of moving from place to place in
the never ending hunt for fresh food. The earliest
preservation technologies developed were drying, smoking,
chilling and heating. The use of various compounds such as
salts and spices to preserve food was also used in ancient
times.
1
Unfortunately, the gradual use of a wider range of
chemicals for preservation for long time led to several
problems and hence consumers have developed some
suspicion of the use of chemical additives. On the other
hand, consumers have fewer reservations about physical
treatments, although one of the oldest technologies smoking
is now suspected of being carcinogenic.
Another more recent physical treatment which is also
much under debate is irradiation. Many studies have shown
it to safe and it has been approved for use in food processing
in several countries because it has proved to be the best way
to kill salmonella and other pathogenic bacteria.
Recent debate about preservation techniques has focused
on not only safe but also preserves the intrinsic nutritional
and sensory qualities present in raw and fresh food.
Combined treatments are advantageous principally
because many individual treatments alone are not adequate
to ensure food safety or stability. In some circumstances
combined treatments allow a milder use of single treatments
with consequent improvement in food quality.
Combining non-thermal methods with other food
preservation techniques can enhance the lethal effect of non-
thermal processing, reduce the severity of non-thermal
treatment needed to obtain a given level of microbial
2
inactivation and or prevent the proliferation of survivors
following treatment.
So the purpose of this article is to study some successful
combinations of given thermal and non-thermal technologies
with other natural preservatives to ensure the safety of some
juices commonly produced in food Egyptian factories. To
achieve this aim, the work plane was:
1- Isolation and identification of the micro flora of some
Egyptian fruit juices (pulp – end product) before and after
pasteurization, with and without preservatives.
2- Study the effect of nisin, lactic acid, citric acid,
cinnamon, radiation and temperature on the isolated
organisms under study.
3- Study the effect of combining treatments of irradiation
with other food preservatives on isolated juice microflora.
4- Study the effect of combining treatments of
pasteurization with other food preservatives on isolated juice
microflora.
5- Study the effect of combining treatments of nisin with
other food preservatives on isolated juice microflora.
6- Study the storage of juice by use the best natural
preservatives and physical treatment obtained from the
previous experiments.
3
LITERATURE REVIEW
4
Review of Literature
1. Fruit Juices and soft Drinks:
Due to their intrinsic properties in particular their low
pH value and low nitrogen and oxygen contents, fruit juices
and soft drinks impose an adverse environment for most
microorganisms. On the other hand, these beverages are
excellent substrates for supporting growth of certain
microorganisms such as yeasts and, to a lesser extent,
molds and lactic acid bacteria (Deak, 1980). Of these
beverages, fruit juices contain the highest amounts of
nitrogenous compounds and vitamins and, hence, are most
susceptible to yeast spoilage.
The stability of fruit-based beverages and drinks is also

influenced by the type of fruit juice or concentrate used in
their formation (Deak, 1986). Low- pH fruit juices,
especially apple cider and orange juice, have been associated
with food born diseases (Birkhead et al., 1993; CDCP,
1995; CDCP, 1999). One major factor in the occurrence of
juice- related outbreaks is the lack of a kill step such as
pasteurization during juice processing. In fact, un-
pasteurized fruit juices have been reported to be the vehicle
in numerous outbreaks of Salmonellosis and Escherichia
5
coli 0157:H7 infections (Besser et al., 1993; CDCP, 1995;
CDCP, 1999).
In the past, low pH juices were not considered to pose
a risk because of the poor survival of pathogens like
Salmonella at the pH of these products (Goverd et al.,
1979). However, pathogens such as Listeria
monocytogenes and E.coli 0l57:H7 have been shown to
tolerate adverse conditions, including acidic conditions and
in the case of L. monocytogenes, low temperatures (Ita and
Hutkins, 1991; Benjamin and Datta, 1995; Conner and
Kotrola, 1995). Therefore, the avoidance of the presence of
pathogens on the surface of a fruit before juice is squeezed
from it may be important in preventing food borne disease
associated with these types of foods. The potentials for cross
contamination from the rinds to the juice may differ for
different products. Citrus juices are not exempt from cross-
contamination if the surfaces of the raw ingredient are
contaminated. However unlike the case for apple juice
processing where by apples are milled, there by providing
intimate contact between juices and peel, little contact
between peel as a significant source of microorganisms in
citrus juice (Parish, 1998).
6
Freshly squeezed or extracted fruit juices require
processing to prevent spoilage. Chilling in most commonly
used to extend shelf life of un-pasteurized products.
Murdock and Hatcher (1975) showed that the rate of
yeast growth in orange juice decreases as temperature
decreases. Zygosaccharomyces rouxii grew at
temperatures between 1.7 to 10 ْC with a generation time of
19.6 to 8.6 h. Fruit juices can also be chemically preserved,
pasteurized, frozen, concentrated, and or irradiated to
prevent spoilage.
Preservation of concentrated orange juice can be
achieved by the addition of sulfur dioxide (230 mg/ l) or by
sorbic acid (800mg/l) (lioyd, 1975).
The spoilage yeast Z. balii often exhibits resistance to
benzoic acid. Due to its pH, light pasteurization (66 ْC for 10
s) is sufficient to inactivate most microorganisms in orange
juice (Sadler et al., 1992 ) other studies have shown that
pasteurization juice may undergo spoilage due to yeast or
mold growth within five weeks of refrigerated storage
(parish and Higgins, 1989). The effect of temperature and
juice composition on survival of yeasts has been the subject
of numerous studies. Citric acid influence the thermal
resistance of spoilage yeasts. Heat resistance increases with
an increase in concentration of orange juice (Juven et al.,
7
2. Food additives
2.1. Introduction :
According to the FPCFN, food additives may be

defined as follows:
A substance or mixture of substances, other than a basic

foodstuff, which is present in a food as a result of any aspect
of production, processing, storage, or packaging. The term
does not include chance contaminants. Since prehistoric
times, chemicals have been added to foods to perform
special functions. Although basic foods contain no additives,
as foods are processed for conversion into a variety of
products, an increasing number of additives are generally
used. Technological advances in food processing have
increased the variety and use of these additives. Today, more
than 2500 different additives are intentionally added to
foods to produce a desired effect. The use of these additives
is a well-accepted practice but is not without controversy
(Branen et al., 2002).
8
2.2. Types of additives :
Additives can be divided into six major categories:

preservatives, nutritional additives, flavoring agents, coloring
agents, texturizing agents, and miscellaneous additives.
Several additives commonly serve more than one function in
foods. In Europe and other parts of the world, the E system,
developed by the European Union (formally the European
Economic Community), provides a listing of several
commonly used additives (Jukes, 2001).
Since preservatives are the main object of the present

research, some details will be given here.
2.2.1. Preservatives:
There are basically three types of preservatives used in
foods: antimicrobials, antioxidants, and antibrowning
agents. These additives are grouped under the category of
preservatives in the INS system (Branen et al., 2002).
• The antimicrobials, with E and INS numbers ranging
from 200 to 290, are used to check or prevent the growth of
microorganisms. Antimicrobials play a major role in
extending the shelf-life of numerous snack and convenience
foods and have come into even greater use in recent years as
microbial food safety.
9
• The antioxidants (INS 300–326 and E300–E326), are
used to prevent lipid and/or vitamin oxidation in food
products. They are used primarily to prevent autoxidantion
and subsequent development of rancidity and off-flavor.
They vary from natural substances such as vitamins C and
E to synthetic chemicals such as BHA and BHT. The
antioxidants are especially useful in preserving dry and
frozen foods for an extended period of time.
• Antibrowning agents are chemicals used to prevent

both enzymatic and non-enzymatic browning in food
products. Vitamin C (E300), citric acid (E330), and sodium
sulfite (E221) are the most commonly used additives in this
category. These additives are classified as either antioxidants
or preservatives in the INS system.
10
2.3. Risks of additives :
Despite the benefits attributed to food additives, for

several years there have also been a number of concerns
regarding the potential short- and long-term risks of
consuming these substances. Critics of additives are
concerned with both indirect and direct impacts of using
additives. As for many of the benefits mentioned, there is not
always adequate scientific proof of whether or not a
particular additive is safe. Little or no data are available
concerning the health risks or joint effects of the additive
cocktail each of us consumes daily (Branen et al., 2002).
The indirect risks that have been described for

additives are the converse of some of the benefits attributed
to their use. While it is accepted that through additives a
greater choice and variety of foods have been made available,
there is no question that additives have also resulted in the
increased availability of food products with a low density of
nutrients. These so-called junk foods, which include many
snack items, can in fact be used as substitutes in the diet
for more nutritious foods. Recently the food industry has
attempted to address this criticism by adding nutritional
additives to snack items so that these foods are a source of
selected vitamins and minerals (Branen et al., 2002).
11
Of greater concern than the indirect risks are the
potential direct toxicological effects of additives. Short-term
acute effects from additives are unlikely. Few additives are
used at levels that will cause a direct toxicological impact,
although there have been incidents where this has
happened. Of particular concern are the hypersensitivity
reactions to some additives that can have a direct and severe
impact on sensitive individuals even when the chemicals are
used at legally acceptable levels. Toxicological problems
resulting from the long-term consumption of additives are
also not well documented. Cancer and reproductive
problems are of primary concern, although there is no direct
evidence linking additive consumption with their occurrence
in humans. There are, however, animal studies that have
indicated potential problems with some additives. Although
most of these additives have been banned, some continue to
be used, the most notable being saccharin (Branen et al.,
2002).
12
2.4. Balancing risks and benefits :
Due to the difficulties in precisely defining the risks
and benefits of individual additives, a legal rather than a
scientific decision is commonly made regarding the safety of
a food additive. In such a decision, the potential risks must
be weighed against the potential benefits. A common
example of this balance is saccharin. Although there is no
direct evidence that saccharin, in the low amounts
consumed in foods, causes cancer in humans, risk
evaluation in rats indicates a potential for cancer in
humans. On the benefit side, saccharin is an excellent non-
caloric sweetener that is useful for diabetics and those inter-
ested in reducing consumption of calories. Many consumers
feel that the benefits of having saccharin available as a
sweetening agent outweigh the risks (Branen et al., 2002).
The potential risks of additives are well recognized, but
the beneficial role these additives play in food production,
processing, and utilization is also felt to be essential to the
maintenance of our current food systems. With the
convenient, tasty, and nutritious foods demanded, or at least
desired, by consumers and the increasing overall demand for
foods as populations increase, food additives will continue to
play an important and essential role in food production.
There will, however, continue to be concern regarding the
13
potential risks associated with long-term consumption of
small amounts of these chemicals and the possible
interactive toxicological effects. As methods improve for
evaluating these toxicological effects, some additives may be
banned. At the same time, the same information may be
used to develop safer new additives or techniques for using
existing additives in a way that will lessen risk (Branen et
al., 2002).
New technology is likely to have a profound
impact on the use of food additives in the future. Of these,
recombinant DNA biotechnology may have the greatest effect
on the future development and use of food additives.
Recombinant DNA biotechnology is already routinely used
for production of additives through bioprocessing, including
organic acids, bacteriocin preservatives, enzymes,
microorganisms, vitamins, and minerals (IFT, 2000).
Biotechnology may also decrease the need for food
additives. Plants have been produced through recombinant
DNA with increased shelf-life and nutritional value, thus
decreasing the need for a variety of additives. Although it is
expected these recombinant DNA methods will be accepted
in the future, there are currently several questions being
raised regarding the risks and benefits of these products as
well.
14
2.5. Categories of Risk
What are the risks associated with our food

supply? This can be a very confusing question because of
our inability to adequately measure the risks associated with
different components. Roberts (1981) has categorized the
major hazards associated with foods, including additives,
into five groups, ranked in order of importance: (1) food
borne hazards of microbial origin, (2) nutritional hazards, (3)
environmental contaminant hazards, (4) food borne hazards
of natural origin, and (5) food and color additive hazards.
The public perception of the risks associated with foods is
often in the reverse order of the list above (Oser, 1978).
Therefore, it is important to examine each of these areas to
gain a better understanding of the total food safety problem.
Ranked below the other four food hazards is the risk
obtained from food additives. The GRAS ingredients would
be included in this classification. Although GRAS ingredients
are not legally food additives, the public perceives no
distinction. This class includes thousands of substances.
Any potential hazard to humans from a certain food additive
depends on the toxicity of the food additive and the level at
which the additive is ingested. The four most widely used
direct food additives, which account for 93% by weight of all
the direct food additives, are sucrose, salt, corn syrup, and
15
dextrose (Roberts, 1981 and Clydesdale, 1982).
Oser (1978) and Roberts (1981) speculated that the
problem with the public perception of food ingredients is
that these food borne substances must be proven ‘‘safe.’’ It is
impossible to ensure the complete safety of any substance
for all human beings under all conditions of use. Therefore,
any uncertainty about the safety of a food additive can result
in the public suspicion of a much greater risk. The recently
approved ingredient olestra (brand name Olean), a non-
caloric fat replacer, is just one example where debate
continues about the safety of its use in foods (ACSH, 1998).
16
The categories of additive risks are:
2.5.1. Phenyl ethylamine
Ingestion of phenyl ethylamine may lead to
headaches. It is present in small amounts in many cheeses
and wines and in very low concentration also in chocolate.
Three milligrams of phenyl ethylamine provoked headaches
during 12 h. In 16 of 38 patients who had suffered from
headaches after ingestion of chocolate (Moneret-Vautrin,
1983). However, it seems unlikely that one could eat enough
chocolate to reach 3 mg of phenyl ethylamine.
2.5.2. Sodium Nitrite

Sodium nitrite is used as an antioxidant and
antimicrobial agent in various foodstuffs, such as cooked
pork meats. The authorized and widely accepted daily dose
is 0.2 mg/kg. Daily consumption commonly exceeds this
figure, particularly in children. Moneret-Vautrin et al.
(1980) have shown that an oral challenge test with 30 mg of
sodium nitrite may cause urticaria, intestinal disorders, or
headache. The mechanism by which sodium nitrite acts are
unclear. It may cause cellular anoxia and inhibit the
protective enzymatic activities of the intestinal mucosa. This
may lead to increased permeability of the mucosa to other
17
antigens. In addition, sodium nitrite may in some way or
other enhance the effect of histamine present in many foods.
2.5.3. Azo Dyes and Benzoates
In the late 1950 it was discovered that food colorants

may provoke asthma (Lockey, 1959). Of them, tartrazine
has been most studied, and its role in asthmatic reactions
was established in the 1960 (Chafee and Settipane, 1967).
It is still added to many pharmaceutical products as well as
to foods and soft drinks in several countries. Many adverse
reactions reported have been associated with medications,
pills, capsules, and elixirs. Sodium benzoate and other
benzoic acid derivatives can also produce respiratory
symptoms. The tartrazine molecule possesses similarities to
other azo compounds, benzoates, pyrazole compounds, and
the hydroxyaromatic acids, which include salicylate and
aspirin (Miller, 1985).
2.5.4. Sulfites
Sulfating agents include sulfur dioxide (SO2),
sodium sulfite, and the potassium and sodium salts of
bisulfite and metabisulfite. Sodium and potassium bisulfite
and metabisulfites are converted into sulfurous acid in
solutions and sulfur dioxide itself. Sulfites are antioxidants
that are used as preservatives in foods and drugs, especially
18
in injected and inhaled medications, and as antimicrobial
and sanitizing agents in the production of wine (Settipane,
1984). They are used on dehydrated vegetables and dried
fruits, in fruit drinks, and also in many other products
(Przybilla and Ring, 1987). Prenner and Stevens (1976)
were the first to report a patient who experienced an
anaphylactic reaction after ingestion of sulfites in foods.
After that, several reports suggesting anaphylactoid
reactions to sulfite agents were published (Stevenson and
Simon, 1981).
Inhaled sulfur dioxide may produce bronchoconstriction

at 1 ppm in asthmatics (Koenig et al., 1980). It seems that
sulfites cause adverse respiratory reactions more often than
food colorants and benzoates. These reactions are obviously
nonspecific in nature, similar to the response to metacholine
and histamine (Bush et al., 1986). Reactions to sulfites may
be severe and not readily recognized if not suspected.
3. Nitrites
Nitrite salts (KNO2 and NaNO2) have been used in meat
curing for many centuries. Meat curing utilizes salt, sugar,
spices, and ascorbate or erythorbate in addition to nitrite.
The reported contributions of nitrite to meat curing include
19
characteristic color development, flavor production, texture
improvement, and antimicrobial effects (IFT, 1987).
Nitrite has been shown to have variable effects on

microorganisms other than Clostridium botulinum. C.
perfringens growth in laboratory medium at 20°C was
inhibited by 200 µg/ml nitrite and 3% salt or 50 µg/ml
nitrite and 4% salt at pH 6.2 (Gibson and Roberts, 1986).
The mechanism of nitrite inhibition of

microorganisms has been studied for 50 years (Tompkin,
1983). The inhibitory effect of nitrite on bacterial
sporeformers is apparently due to inhibition of outgrowth
and during cell division (Genigeorgis and Riemann, 1979;
Cook and Pierson, 1983).
The lethal dose of nitrites in humans is 32 mg/kg body

weight or 2 g (Burden, 1961). Prolonged ingestion of sodium
nitrite or sodium nitrate has been shown to cause
methemoglobinemia, that is, excessive production of
abnormal hemoglobin, especially in infants (NAS/NRC,
1981).
20
2.5.6. Other Agents
‘‘Chinese restaurant asthma’’ has been suggested

to be caused by monosodium l-glutamate (Schaumburg et
al., 1969; Allen and Baker, 1981 and Allen et al., 1983).
There are, however, studies in which double-blind
provocation tests have produced no asthmatic symptoms at
all (Ghezzi et al., 1980; Grattini, 1982 and Morselli and
Grattini, 1970). Numbness of the neck, headache, facial
pressure, and chest pain have also been claimed to be
symptoms of Chinese restaurant syndrome in some
individuals. Tarasoff and Kelly (1993) made a critical
review of the glutamate literature and performed a very strict
double-blind per oral challenge test with 1.5, 3.0, and 3.15 g
per person. They found no difference between the responses
to placebo and glutamate, and they stated the ‘‘Chinese
restaurant syndrome’’ to be anecdotal. Just recently, Yang
et al. (1997) recorded headache, muscle tightness,
numbness and tingling, general weakness, and flushing
from glutamate more often than from placebo, 2.5 g of
monosodium glutamate being a threshold dose for positive
responses. The dilemma on the effects of glutamate thus
seems to continue. Occupational asthma induced by
inhalation of dust from spices such as powdered coriander,
21
curry, and paprika has been described (Toorenenberger
and Van Dieges, 1985).
Enzymes are frequent causes of IgE-mediated nasal,

bronchial, and skin symptoms. α-Amylase is a usual flour
additive which has been found to cause both immediate and
delayed contact allergies in bakers. Morren et al. (1993)
tested 32 bakers, 7 of whom reacted to a-amylase in scratch-
chamber test at 20 min. RAST was positive in five of them.
Two patients showed also a delayed reaction in the scratch-
chamber test. Larese et al. (1993) made skin prick tests
with A-amylase in 226 bakers and pastry makers. Seventeen
(7.5%) reacted to it. Rhinitis, conjunctivitis and asthma were
the most common allergic symptoms.
22
3. The use of natural antimicrobials
Introduction:
Food antimicrobials are usually chemical
compounds added to or present in foods that retard
microbial growth or kill microorganisms. The functions of
food antimicrobials are to inhibit or inactivate spoilage
microorganisms and pathogenic microorganisms. The latter
function has increased in importance in the past 10-15
years as food processors search for more and better tools to
improve food safety (Davidson, 2001). Most of the
traditional, currently approved food antimicrobials have
limited application due to pH or food component
interactions. For example, organic acids function at low
concentrations is only effective in high acid foods (generally
less than pH 4.5-4.6). This is because the most effective
antimicrobial form is the undissociated acid which exists in
majority only at a pH below the pKa of the compound. All
regulatory-approved organic acids used as antimicrobials
have pKa values less than 5.0 which means their maximum
activity will be in high-acid foods. For food products with a
pH of 5.5 or greater, there are very few compounds that are
effective at low concentrations. Another factor leading to
reduced effectiveness among chemical food antimicrobials is
23
food component interactions. Most chemical food
antimicrobials are amphiphilic. As such, they can solubilize
in or be bound by lipids or hydrophobic proteins in foods
making them less available to inhibit microorganisms in the
food product (Zeuthen and Bøgh-Sørensen, 2003).
Interest in natural antimicrobials is also driven by

the fact that international regulatory agencies are generally
very strict about requirements for toxicological evaluation of
novel direct food antimicrobials. In many parts of the world,
toxicological testing of new synthetic compounds could take
many years and many millions of dollars to obtain approval.
For some types of food additives a payback may be possible
(e.g., artificial sweeteners), but for food antimicrobials it is
less likely that obtaining approval would be profitable
(Zeuthen and Bøgh-Sørensen, 2003).
An argument often used to justify natural antimicrobials

is that they will produce `green' labels, i.e., one with few or
no `synthetic' additives in the ingredient list. While this
rationale may be true, it must be remembered that many of
the antimicrobial compounds approved for use in foods
today come from natural sources. If a truly effective
antimicrobial was discovered from a natural source, it may
24
be more economically feasible to synthesize it than to extract
it from a natural source. This justification also leads
consumers to the mistaken belief that food additives
currently in use are potentially toxic and should be avoided.
In addition to potential benefits associated with
natural antimicrobials in foods, there are a number of
potential concerns that need to be examined with respect to
food safety. For example, if an antimicrobial is to be used
exclusively to inhibit a pathogenic microorganism, it must be
uniformly effective, stable to storage, and stable to any
processes to which it is exposed. Standardized assays for
activity need to be developed to ensure that the
antimicrobial compounds retain potency. Finally, producers
and users of natural antimicrobials that make claims for
efficacy of use will be likely to be liable for any claims they
make. In short, natural antimicrobials have excellent
potential but probably will not produce miracles (Zeuthen
and Bøgh-Sørensen, 2003).
25
3.1. Natural preservatives:
3.1.1. Nisin
Nisin was first recognized in 1928 by Rogers and
Whittier and was later isolated, characterized, and named
by Mattick and Hirsch (1947). The compound is a peptide
produced by a strain of the dairy starter culture
Lactococcus lactis ssp. lactis. The structure and amino
acid content of nisin was determined by Gross and Morell
(1971). The molecular weight of nisin is 3.4 kDa (Gross and
Morell, 1967); however, it usually occurs as a dimer with a
molecular weight of 7000 AMU (Jarvis et al., 1968).
Nisin is a 34 amino acid peptide which contains the

unusual amino acids DHA, dehydrobutyrine, lanthionine,
and 0-methyl-lanthionine (Delves-Broughton and Gasson,
1994). The latter amino acids are found in other inhibitory
peptides produced by gram positive bacteria. The group of
anti-microbial peptides containing these amino acids are
called ‘‘lantibiotics’’ (Chatterjee et al., 2005). A variant of
nisin, called nisin Z, is produced by some strains of L. lactis
ssp. lactis and contains an asparagine in place of a
histidine at residue 27 (fig. a). Lantibiotics are gene-encoded
peptides that contain intramolecular ring structures,
introduced through the thioether containing lanthionine and
26
methyllanthionine residues. The overwhelming majority of
the lantibiotics shows antibacterial activity. Some
lantibiotics, e.g. nisin, are characterized by a dual mode of
action. These peptides form a complex with the ultimate cell
wall precursor lipid II, thereby inhibiting cell wall
biosynthesis. The complexes then aggregate, incorporate
further peptides and form a pore in the bacterial membrane.
Recent results show that complexing of lipid II is widespread
among lantibiotics; however, pore formation depends on the
overall length of the peptide and the lipid composition of the
test strain membrane. (Bierbaum and Sahl, 2009).
Nisin by itself has a narrow spectrum affecting only

gram positive bacteria, including Alicyclobacillus,
Bacillus, Clostridium, Desulfotomaculum, Enterococcus,
Lactobacillus, Leuconostoc, Listeria, Pediococcus,
Staphylococcus, and Sporolactobacillus (Branen et al.,
2002). It does not generally inhibit gram negative bacteria,
yeasts, or molds.
Nisin activity is affected by a number of environmental

factors, including pH, inoculum size, and interaction with
food components (Harris et al., 1991 and Ukuku and
Shelef, 1997). Activity generally increases with decreasing
27
pH and decreased initial numbers of microorganisms. The
presence of food components such as lipids and protein
influence nisin activity (Scott and Taylor, 1981). Nisin was
less active against L. monocytogenes in milk (Jung et al.,
1992) and ice cream (Dean and Zottola, 1996) with
increasing fat concentrations. This was probably due to
binding of nisin to fat globules (Jung et al., 1992); this
binding was overcome by adding emulsifiers (e.g., Tween 80).
Thomas et al. (1998) demonstrated that sucrose fatty acid
esters (sucrose palmitate and sucrose stearate) enhanced
the activity of nisin against Bacillus cereus, Listeria
monocytogenes, Lactobacillus plantarum, and
Staphylococcus aureus, but had no effect on the activity of
nisin against any gram negative bacteria.
28
Fig. (a) Structure of nisin and unusual amino acids. Primary structure of nisin as determined by Gross (1977). ABA, Amino
butyric acid; Ala-S-Ala, lanthionine; ABA-S-Ala, β-methyllanthionine. D-Stereo configuration (*) is shown for the α-carbon
29
In most cases nisin is sporostatic rather than
sporocidal (Delves-Broughton et al., 1996). Morris et al.
(1984) reported the existence of sulfhydryl groups in the
membrane of germinated B. cereus spores. Sulfhydryl
agents such as S-nitrosothiols inhibit outgrowth of
germinated spores by interacting with the sulfhydryl
groups in the membrane. At low concentrations, nisin
inhibited spore outgrowth and competed with sulfhydryl
agents for membrane sulfhydryl groups, indicating that
the membrane sulfhydryl groups may be the target for
nisin.
Morris et al. (1984) hypothesized that the
dehydroalanine (DHA) groups on nisin may react with the
membrane sulfhydryl groups. In light of the results of
Hansen (1994), where alterations of the 5-dehydroalanine
group of subtilin eliminated activity against spores, it is
becoming clearer that the DHA groups may in fact play a
very important role in the sporostatic activity of nisin.
The application of nisin as a food preservative has been

studied extensively (Hurst, 1981 and Hurst and Hoover,
1993). Nisin was first used as a food preservative by
Hirsch et al. (1951). Somers and Taylor (1987) studied
the use of nisin to prevent C. botulinum outgrowth in
30
process cheese spread formulated to have higher than
normal moisture content and/or lower salt content. Nisin
was an effective antibotulinal agent at 12.5–250 µg/g. The
higher nisin levels allowed for the safe formulation of
cheese spreads with higher moisture content and lower
salt concentration. Delves-Broughton (1990) reported
that nisin levels of 6 to 12.5 µg/g controlled non-
Clostridium botulinum spoilage in process cheese.
Nisin has been recommended for use in canned

vegetable products to prevent the outgrowth of
Clostridium botulinum when less severe sterilization
conditions are desired or required (Denny et al., 1961).
Other researchers have reported decreased survival for
pathogenic or spoilage microbes in juice products
supplemented with nisin or other antimicrobials (Kisko
and Roller, 2005 and Komitopoulou et al., 1999).
In addition, Delves-Broughton et al. (1992)

demonstrated that nisin may be used to increase the shelf-
life of pasteurized liquid whole eggs. They found that 5
µg/ml nisin added to eggs prior to pasteurization
increased the refrigerated shelf-life by 11–14 days. In
addition, nisin prevented growth of the pathogen B.
31
cereus, which grew well in pasteurized eggs with no added
nisin.
Nisin has generally been considered nontoxic. The oral
LD50 in mice is 6950 mg/ kg body weight, which is similar
to that of common salt (Hara et al., 1962). Nisin was
found to have no effect on animals in studies of sub
chronic or chronic toxicity, reproduction, or sensitization.
It has also been shown that nisin does not produce cross-
resistance in microorganisms to therapeutic antibiotics
Mechanism of action:
Nisin has no antimicrobial effect on yeasts and
filamentous fungi. These organisms each have a rigid cell
wall, a complex structure consisting of glucan cross-
linked with chitin and cell wall protein (Brul et al., 1997
and Klis et al., 1997). The processing of mannoproteins
is complex and has been partially characterized in yeasts
(Lu et al., 1994 and Kollar et al., 1997). A similar
mechanism has been suggested for filamentous fungi (Brul
et al., 1997). Because mannoproteins are generally
considered one of the key wall components which
determine cell porosity (Cid et al., 1995 and Klis et al.,
1997), they may represent a major barrier preventing free
32
Under normal circumstances bacteriocins produced
by Gram-positive bacteria do not have a bactericidal effect
on gram-negative species. However, in some cases activity
against gram-negatives can be observed on disruption of
the outer membrane as reported for nisin (Stevens et al.,
1991). It has been established that the primary target for
many of these small cationic peptides is the cytoplasmic
membrane of sensitive cells (Kordel and Sahl, 1986; Van
BelKum et al., 1991 and Moll et al., 1996). Where they
act to dissipate the proton motive force (PMF) through the
formation of discrete pores in the cytoplasmic membrane
and thus deprive cells of an essential energy source
(Montville and Bruno, 1994). The PMF which is
composed of a chemical component (the pH gradient and
pH) and an electrical component (the membrane potential;
Δψ), drives ATP synthesis and the accumulation of ions
and other metabolites through PMF – driven transport
system in the membrane. Collapse of the PMF induced by
bacteriocin action leads to cell death through cessation of
energy-requiring reactions. However, recent work has
shown that the activity of nisin is dependent on the
concentration of lipid II (undecaprentyl – pyrophosphory-
33
MurNAc – (pentapeptide- GlcNAc) in the membrane of
sensitive cells (Breukink et al., 1999 and Breukink and
de Kruijff, 1999).
It was demonstrated that in vitro nisin inhibited

bacterial cell wall biosynthesis (Reisinger et al., 1980).
Subsequently, it has been shown that nisin kills bacterial
cells by interfering with basic energy transduction
occurring at the cytoplasmic membrane (Ruhr and Sahl,
1985; Kordel and Sahl, 1986 and Sahl et al., 1987). It
was found that pores formed in the membrane by the nisin
molecules allowed the diffusion of small compounds since
no transport system for ATP has been reported (Sahl et
al., 1987 and Abee et al., 1994). The increase in
membrane permeability result in the collapse of the PMF ;
in the case of nisin, both the ΔpH and the Δψ are
completely dissipated leading to a rapid cessation of all
biosynthetic processes (Sahl et al., 1987; Okereke and
Montville, 1992; Bruno et al., 1992 and Bruno and
Montville,1993).
In contrast, the inhibition of cell wall biosynthesis is a

comparatively slow process. Thus, pore formation is
considered the primary mode of action of nisin. The
34
interaction of nisin with membrane components of
sensitive cells is considered a vital step in its mode of
action. It has been reported that even in the absence of an
energized membrane, nisin can associate tightly with lipid
bilayers through electrostatic interaction with the
phospholipids head groups (Driessen et al., 1995).
The degree of association of nisin with the

membrane is largely dependent on the type of lipids
present, and most importantly, the charge carried by those
lipids. Several groups have demonstrated that due to the
cationic nature of nisin, its activity in vitro is most efficient
when a high percentage of anionic, or negatively charged,
membrane lipids are present (Garcia-Garcera et al.,
1993; Martin et al., 1996 and Breukink et al., 1997).
More recent studies have substantiated this evidence,

confirming the ability of nisin to insert into lipid
monolayers in an anionic lipid-dependent (Demel et al.,
1996). Therefore, the composition of membranes is likely
to be an important determination in the sensitivity of
different bacterial species to nisin.
35
Breukink et al. (1997) reported that this initial
interaction with anionic phospholipids is mediated by the
C-terminal domain of the peptide, since this region
contains the bulk of positive charge carried by the nisin
molecule. It is believed that nisin also mediates inhibition
of cell wall biosynthesis, by forming a complex with the
bactoprenol-bound peptidoglycan precursor, lipid II
(Reisinger et al., 1980).
Brotz et al. (1998) suggesting that nisin may use

lipid II as a’docking molecule’ for binding to specific
membranes. Breukink et al. (1999) demonstrated that
increasing the concentration of lipid II in isolated model
membranes in the range of 0.001-0.1 mol percent
increases the sensitivity of the membrane to nisin.
36
3.1.2. Organic acids
• Introduction
Organic acids have a long history of being utilized as
food additives and preservatives for preventing food
deterioration and extending the shelf life of perishable food
ingredients (Ricke, 2003). Although the antibacterial
mechanism(s) for organic acids are not fully understood,
they are capable of exhibiting bacteriostatic and
bactericidal properties depending on the physiological
status of the organism and the physicochemical
characteristics of the external environment (Ricke, 2003).
Given the weak acid nature of most of these compounds,
pH is considered a primary determinant of effectiveness
because it affects the concentration of undissociated acid
formed (Davidson, 2001). It has been traditionally
assumed that undissociated forms of organic acids can
easily penetrate the lipid membrane of the bacterial cell
and once internalized into the neutral pH of the cell
cytoplasm, dissociate into anions and protons (Davidson,
2001). Generation of both these species potentially
presents problems for bacteria that must maintain a near
neutral pH cytoplasm to sustain functional
macromolecules (Ricke, 2003). Export of excess protons
requires consumption of cellular adenosine triphosphate
37
(ATP) and may result in depletion of cellular energy
(Davidson, 2001).
The effective use of organic acids in food products is

governed in part by the desired end product. Acidulants
contribute a variety of functional properties that enhance
the quality of food. The proper selection of an acid depends
on the property or combination of properties of the desired
acid as well as cost. Acids are most commonly used for
flavor and tartness, and the buffering ability of the salts of
some acids can modify and smooth out these
characteristics. Acidulants all share a characteristic sour
flavor; however the degree of flavor profile varies
significantly depending on the final pH of the product
(Hartwig and Mc Daniel, 1995). These differences can be
used in the formulation of foods with specific sensory
qualities that may be unrelated to any antimicrobial
effects of the individual acids.
In their synergistic capacity, acids used in conjunction

with antioxidants prevent rancidity or complex with heavy
metals that might initiate oxidation or browning reactions.
Acids stabilize color, reduce turbidity, change melt
characteristics, prevent splattering, or enhance gelling.
38
They can also act as leavening or inversion agents,
emulsifiers, nutrients, and supplements (Branen et al.,
2002).
A major use of acidulants is as an antimicrobial agent

and this use is receiving much attention today in a desire
to achieve safer food products. With many foods, the
incorporation of acids into the product at sufficiently high
levels ensures a commercially sterile product. The target
levels necessary for this purpose, however, can overwhelm
the sensory properties of the food and thereby prevent the
use of an acid. When used with other preservation
processes, such as refrigeration or heating, acids extend
shelf-life for almost indefinite periods from an
antimicrobial standpoint. The judicious use of an
acidulants for its antimicrobial properties is
counterbalanced by the desired sensory characteristics
that predicate the amount of acid that is utilized. It is in
the product development and safety testing phases that
the addition of the acid with its inherent properties can be
evaluated to produce a product desirable to the consumer.
39
3.1.2.1. Mode of action
The most common classical preservative agents are the
weak organic acids, for example acetic, lactic, benzoic and
sorbic acid. These molecules inhibit the outgrowth of both
bacterial and fungal cells. Sorbic acid is also reported to
inhibit the germination and outgrowth of bacterial spores
(Sofos and Busta, 1981 and Blocher and Busta, 1985).
The inhibiting effect of organic acid is based on their

pKa, the antimicrobial activity of their non-dissociated
form, and the specific effects of each acid. Less direct
antibacterial activities include interference with nutrient
transport, cytoplasm membrane damage resulting in
leakage, disruption of outer membrane permeability, and
influence on macromolecular synthesis (Velázquez et al.,
2009). In solution, weak acid preservatives exist in a pH-
dependent equilibrium between the undissociated and
dissociated state. Preservatives have optimal inhibitory
activity at low pH because this favours the uncharged,
undissociated state of the molecule which is freely
permeable across the plasma membrane and is thus able
to enter the cell. Therefore, the inhibitory action is
classically believed to be due to the compound crossing
the plasma membrane in the undissociated state.
40
In yeasts, it has also been proposed that the actual
inhibitory action of weak acid preservatives could be due
to the induction of an energetically expensive stress
response that attempts to restore homeostasis and results
in the reduction of available energy pools for growth and
other essential metabolic functions (Holyoak et al., 1996
and Bracey et al., 1998).
41
The principal mode of action of organic acids is the
release of protons from carboxylic groups, thus lowering
the pH of their environment. Proton release means
dissociation of the acid molecule, with the degree of
dissociation depending on the pKa value. Strong acids
tend to be fully dissociated and thus produce a strong
drop in pH, while weak acids are only partially dissociated.
This has two important consequences: (i) organic acids
with two or three carboxylic groups react on changes of pH
by changes in protonation of their carboxylic groups, i.e.,
they may `buffer', and (ii), in their undissociated state,
some of these acids can pass through lipophilic cell
membranes. Consequently, on a molar basis, the
antibacterial efficacy of weak acids is stronger than that of
strong acids. If the undissociated acid is not soluble in
lipids, it is unlikely to have any inhibitory effect on
microorganisms because the cell membrane is
impermeable to protons and to polar compounds such as
citric acid and malic acids, unless it contains specific
carrier proteins.
On the other hand, lipophilic undissociated acids
will penetrate the cell membrane and acidify the
cytoplasm. Table (a) reviews properties of selected organic
acids. Kation effects are reported also. Protons may
42
change the conformation of polar membrane proteins.
Binding metal ions (Fe3+, Cu++, Mn++, Ca++, Mg++) by
chelation is relevant only for di- and tricarboxylic acids,
especially citric acid (Stratford, 1999).
Table (a): Properties of some organic acids used as additives

(Smulders, 1987; Stratford, 1999, Saltmarsh, 2000 and
Naidu, 2000).
weak
Acid COOH* pKa Acid.** taste spec. effect****
acid'***
Membrane diffusion and

water activity
Lactic 1 3.66 2.5 No Yes
reduction(specific anion
effect)
marked
Citric 3 5.7/4.3/2.9 2.6 generic no Chelation
taste
* Number of carboxylic groups; ** pH fall of a 1% (W/V) bacteriological

peptone solution (pH 6.2), when acids (0.5% (W/V), compared on a weight
basis, Stratford, 1999) are added; this represents the strength of the
acidulant effect on `environmental pH'; *** Lipophilic character; ability to
depress intracellular pH; **** Effects other than acidulants.
3.1.2.2. Resistance mechanisms

Microbial resistance to weak organic acids can involve
various mechanisms. For bacteria, significant knowledge
exists on their intrinsic, non-inducible resistance
mechanisms against these compounds (Russel, 1991).
Gram-positive bacteria do not possess an outer membrane
43
and the exclusion limit of the cell wall of vegetative cells of
Bacillus megaterium has even been calculated to be as
high as 30,000 D (Lambert, 1983). Hence preservatives
can easily enter these cells and their intrinsic resistance is
relatively low. In gram-negative bacteria, resistance
mechanisms are more complicated since these organisms
possess an inner and an outer membrane. The latter
membrane has a clear role in modulating the accessibility
of a cell to preservatives and other small molecules (Vaara,
1992 and Helander et al., 1997); the lipopolysaccharide
layer is of crucial importance in this respect (Helander et
al., 1996). In some cases microorganisms are able to
degrade the added preservatives by making use of specific
enzymes. An example of this is the degradation of methyl
para (4)-hydroxybenzoate by P. aeruginosa (Hugo and
Foster, 1964).
Recently, inducible resistance mechanisms in micro-

organisms have been more extensively studied. For
Salmonella typhimurium, it is known that cells
encounter many potential stress factors in their ‘‘natural’’
habitat, e.g., the extremely low pH in the stomach or the
presence of large amounts of partially hydrophobic weak
organic acids in the intestine.
44
Indeed, this organism is known to possess an acid
tolerance response which consists of a complex defence
system that allows cells to survive pH values as low as pH
3 (Park et al., 1996). Interestingly enough, this stress
response also conveys cellular resistance against the weak
acids butyric, acetic and propionic acid (Baik et al.,
1996). Furthermore, in a recent study with Escherichia
coli O157:H7, resistance towards benzoic acid was
observed upon induction of an acid tolerance response
with a strong acid at pH 2.0 (Lin et al., 1996).
Also, certain gram-positive bacteria, e.g., Listeria

monocytogenes, are known to induce an acid tolerance
response at a challenge pH of 3 after prior exposure to a
mild-acid at pH 5.0 (Davis et al., 1996). No data are
currently available on whether this response gives
protection to weak organic acids commonly used in the
food industry.
In fungi, similar resistance mechanisms may be found.

The enzymatic degradation of sorbic acid to pentadien by
certain fungal species is well documented (Samson et al.,
1995). The resistance of spoilage yeasts to weak organic
acid preservatives has been extensively studied and is
45
known to depend on the H+ -pumping P-type membrane
ATPase (Holyoak et al., 1996). Studies by Kubo and Lee
(1998) have shown that compounds that inhibit the
plasma membrane H+ -ATPase synergistically enhance the
activity of sorbic acid.
Piper et al. (1997) showed that the long term stress

response of yeasts to weak organic acids also involves the
induction of an integral membrane protein, Hsp30, which
downregulates the increased activity of the membrane
ATPase. It was deduced from these observations that
Hsp30 was acting as a molecular ‘‘switch’’ downregulating
the activity of the membrane ATPase in order to conserve
cellular energy pools which would otherwise be consumed
by the enzyme attempting to restore homeostasis (Braley
and Piper, 1997).
Studies by Henriques et al. (1997) have shown that

Saccharomyces cerevisiae is able to actively extrude [C-
14] labelled benzoic acid, suggesting that there is an efflux
system, presumably membrane localised, and that
removes accumulated an-ions from inside the cell.
Supporting this, data gathered by Piper et al. (1998) has
demonstrated the existence of a multidrug resistance
46
pump, the ATP binding cassette transporter, Pdr12, which
actively extrudes preservative anions from the cell. Our
current understanding of the mechanisms that are
involved in weak acid resistance in yeasts are summarised
in Fig. (b). As indicated on the diagram, simply pumping
preservative anions out of the cell could create a futile
cycle where the anions reassociate at the lower external
pH and reenter the cell. However, this assumes that any
rate of diffusion across the plasma membrane remains the
same and that the cell makes no effort to alter membrane
composition or structure to reduce the access of the toxic
compound.
Recently, have shown that adapted yeasts reduce the

diffusion coefficient of preservatives across the plasma
membrane such that passage of weak acids into the cell is
reduced. Therefore, efflux of protons and anions by the H+
-ATPase and Pdr12 respectively would not create a futile
cycle if there is a concurrent reduction in the ability of the
compounds to diffuse across the cell membrane and enter
the cytosol (Brul and Coote, 1999).
47
Fig. (b): A schematic diagram of the stress response of a yeast cell challenged with weak organic acids (Piper et
al., 1998). Shown are, a glucose transporter, and the membrane located Pdr12 multidrug resistance pump active
against anions of acetic, sorbic and benzoic acid, and the plasma membrane P-type H + -ATPase.
48
3.1.2.3. Citric acid
Citric acid generally is not used as an antimicrobial,
but it has been shown to possess activity against some
molds and bacteria. Reiss (1976) found that 0.75% citric
acid slightly reduced growth and greatly reduced toxin
production by Aspergillus parasiticus. With Aspergillus
versicolor, growth was inhibited at the same level, but
toxin production was prevented by 0.25% citric acid. In
contrast, 0.75% citric acid did not influence the growth or
toxin production of Penicillium expansum (Reiss, 1976).
Citric acid was observed to be more inhibitory to
Salmonella than lactic or hydrochloric acids
(Subramanian and Marth, 1968). In a related study,
Thomson et al. (1967) found that as little as 0.3% citric
acid could reduce the level of viable Salmonella on poultry
carcasses. Shrimp, shrimp puree, tomato puree, and
shrimp and tomato puree acidified to pH 4.2 and 4.6 with
citric acid showed no significant growth or toxin
production by C. botulinum after 8 weeks at 26°C (Post
et al., 1985). Minor and Marth (1970) showed that
Staphylococcus aureus was inhibited 90% and 99% in 12
h at pH 4.7 and 4.5, respectively, by citric acid. Citric acid
was found to be particularly inhibitory to flat-sour
organisms isolated from tomato juice, but the inhibition
49
was pH-dependent (Branen, et al., 2002).
The mechanism of inhibition by citrate has been

theorized to be related to its ability to chelate metal ions.
Branen and Keenan (1970) were the first to suggest that
inhibition may be due to chelation, in studies with citrate
against Lactobacillus casei. Chelation was also indicated
as the reason for inhibition of S. aureus (Rammell, 1962)
and Arthrobacter pascens (Imai et al, 1970) by citrate.
In contrast, Buchanan and Golden (1994) found that
while un-dissociated citric acid was inhibitory against
Listeria monocytogenes, the dissociated molecule
protected the microorganism. They theorized that this
protection was due to chelation by the anion. The U.S.
FDA has classified citric acid as GRAS for miscellaneous
and general purpose use (Branen et al., 2002).
3.1.2.4. Lactic Acid

Lactic acid (pKa = 3.79) is a primary end-product of
the lactic acid bacteria and serves to assist in preservation
of many fermented dairy, vegetable, and meat products. It
is used as a food additive primarily for pH control and
flavoring. The antimicrobial activity of the compound is
variable. For example, in cold pack cheese formulated with
50
lactic and acetic acid and inoculated with Salmonella
Typhimurium, no increase in destruction of the organism
was found (Park et al., 1970). In contrast, lactic acid was
found to be four times as effective as malic, citric,
propionic, and acetic acids in inhibiting growth of Bacil-
lus coagulans in tomato juice (Rice and Pederson,
1954). Lactic acid inhibited spore forming bacteria at pH
5.0 but was much less effective against yeasts and molds
(Woolford, 1975). Staphylococcus aureus was
inactivated by 90 and 99% in 12 h at pH 4.9 and 4.6,
respectively, by lactic acid (Minor and Marth, 1970).
Based upon molar concentration, pH, and activity of un-
dissociated acid, lactic acid was one of the most effective
organic acids against the growth of Yersinia
enterocolitica (Brackett, 1987). Lactic acid at
concentrations between 0.3% and 4% has demonstrated to
be effective in the in vitro reduction of Y. enterocolitica
(Virto et al., 2005). Addition of 5.0% lactic acid in ground
beef patties stored at 4°C for 10 days reduced coliforms by
3.9 logs (Podolak et al., 1996). Oh and Marshall (1993)
reported that there was little interaction between lactic
acid and ethanol against L. monocytogenes. The MIC
value of lactic acid alone was 0.5%, but was lower when
1.25% ethanol was combined with 0.25% lactic acid. When
51
2.5% ethanol was combined with 0.25% lactic acid, the
extent of inhibition was not more than that of the most
active single compound alone. Different concentrations of
lactic acid alone or in combination with other chemicals
have been shown to be effective in the elimination of
bacterial pathogens (Akbas and Olmez, 2007).
Snijders et al. (1985) and Smulders et al. (1986)

advocated the use of lactic acid for surface
decontamination of fresh meats, slaughter byproducts,
and poultry. Lactic acid at 1–2% was reported to reduce
Enterobacteriaceae and aerobic plate counts by 0.3–2.7
log on beef, veal, pork, and poultry. The compound also
delayed growth of spoilage microflora during long-term
storage of products. Visser et al. (1988) confirmed the
antimicrobial effectiveness of 2.0% (v/v) L-lactic acid on
fresh veal tongues and demonstrated that the compound
decreased the growth rate of spoilage organisms during
vacuum-packaged storage at 3°C. Presumably lactic acid
applied as antimicrobial to foods functions similarly to
other organic acids and has a primary mechanism
involving disruption of the cytoplasmic membrane proton
motive force (Eklund, 1989).
52
In contrast, there has been some controversy
concerning the mechanism of lactate salts used at high
concentrations. These salts have been shown to have little
effect on product pH. Therefore, most of the lactate
remains in the less effective anionic form. Initially it was
thought that the high concentration of the salts may
reduce water activity sufficiently to inhibit microorganisms
(Debevere, 1989).
However, Chen and Shelef (1992) and Weaver and

Shelef (1993) using cooked meat model systems and liver
sausage, respectively, containing lactate salts up to 4%
concluded that water activity reduction was not sufficient
to inhibit Listeria monocytogenes. In addition,
Papadopoulos et al. (1991) reported that water activity
was not lowered in cooked beef top rounds injected with
various levels of sodium lactate. Inhibition may be due to
the presence of sufficient undissociated lactic acid,
possibly in combination with a slightly reduced pH and
water activity, to cause inhibition of some microorganisms.
Lactic acid was probably one of the first acids used in

food due to its wide distribution in nature as a product of
fermentation. The U.S. FDA has approved lactic acid as
53
GRAS for miscellaneous and general purpose usage with
no limitation upon the concentration used. It may not be
used in infant foods and formulas. The U.S. Department of
Agriculture allows lactic acid in meat products at the
lowest concentration necessary for the intended purpose
54
3.1.3. Spices and their essential oils
Spices and their essential oils have varying
degrees of antimicrobial activity. Among the spices, cloves,
cinnamon, oregano, thyme, sage, rosemary, basil and
vanillin have the strongest antimicrobial activity. The
major antimicrobial components of clove (Syzygium
aromaticum) and cinnamon (Cinnamomum zeylanicum)
essential oils are eugenol (2-methoxy-4-(2-propenyl)-
phenol)) and cinnamic aldehyde (3-phenyl-2-propenal),
respectively. Smith-Palmer et al. (1998) determined that
the 24h. minimum inhibitory concentrations of cinnamon
and clove essential oils against Campylobacter jejuni,
Escherichia coli, Salmonella Enteritidis, Listeria
monocytogenes, and Staphylococcus aureus were 0.05,
0.04-0.05, 0.04-0.05, 0.03, and 0.04%, respectively, in an
agar dilution assay. Cinnamic aldehyde or thymol (600
mg/liter of air) significantly reduced Salmonella
populations on alfalfa seeds used for sprouting and did
not affect germination (Weissinger et al., 2001). Mixtures
of cinnamon and clove oils were capable of suppressing
the growth of major spoilage microorganisms of
intermediate moisture foods (Huiyun Zhang et al., 2009).
55
Many spices, condiments, and plant extracts have
strong medicinal, preservative, and antioxidant properties
(Zaika, 1988). The antimicrobial activity of these
ingredients is attributed to their essential oils, which are
lipophilic and penetrate through the membrane to the
interior of the cell and perform the inhibitory activity at
the target site (Smith-palmer et al., 1998). Cinnamon
effectively inhibits the growth of bacteria (with gram-
positive being more sensitive than gram –negative), yeasts,
and molds (Shelef, 1983).
Bullerman (1974) determined that 1.0% cinnamon in

raisin bread inhibits growth and aflatoxin production by A.
parasiticus. Lòpez-Malo et al. (2002) confirmed growth
inhibition of the related mold, A. flavus, by eugenol,
thymol ((5-methyl-2-(1-methylethyl) phenol)) and carvacrol
((2-methyl-5-(1-methylethyl) phenol)). Smid and Gorris
(1999) reported that cinnamic aldehyde inhibited growth
of both bacteria and fungi and increased shelf life of
treated packaged tomatoes.
Cinnamon oil and clove oil are both natural
preservative and flavouring substances that are not
harmful when consumed in food products. There have
been a number of reports of substances in each of
56
cinnamon and clove oils that inhibit the growth of molds,
yeasts and bacteria. Both cinnamon oil and clove oil added
at 2% in potato dextrose agar (PDA) completely inhibited
the growth of seven mycotoxigenic molds (A. flavus, A.
parasiticus, A. ochraceus, Penicillium sp. M46, P.
roqueforti, P. patulum, and P. citrinum) for various
times up to 21 days (Azzouz and Bullerman, 1982) and
could also inhibit the growth of yeasts (Conner and
Beuchat, 1984). Suksrikarm (1987) similarly reported
that cinnamon oil and clove oil could separately inhibit
many other microbes including Lactobacillus sp.,
Bacillus thermoacidurans, Salmonella sp.,
Corynebacterium michiganense, Pseudomonas
striafaciens, Clostridium botulinum, Alternaria sp.,
Aspergillus sp., Canninghamella sp., Fusarium sp.,
Mucor sp., and Penicillium sp. Soliman and Badeaa
(2002) found that <500 ppm of cinnamon oil can inhibit
A. flavus, A. parasiticus, A. ochraceus and Fusarium
moniliforme on PDA and August (1978) reported that
high concentrations of cinnamon oil and clove oil could
also inhibit the asexual spores of fungi. Mixtures of
cinnamon and clove oils are therefore an interesting
alternative to use of other chemical preservatives and
appear well suited to use in active packaging systems.
57
The greater effectiveness of the cinnamon bark oil on
S. Enteritidis and E. coli O157:H7 in fruit juices was
associated to the media pH, being more effective in media
with lower pH. Burt (2004) reported that the bacterial
susceptibility to the antimicrobial effect of essential oils
appears to increase with a decrease in the pH of the food,
because at low pH the hydrophobicity of an essential oil
increases, enabling it to more easily dissolve in the lipids
of the cell membrane of target bacteria. Although, the
mechanism of action of cinnamon oil on the microbial cells
is still unclear, Wendakoon and Sakaguchi (1995) and
Burt (2004) have indicated that the interaction of carbonyl
group of the cinnamaldehyde, main compound of
cinnamon oil from bark, on the cell proteins embedded in
the cytoplasmatic membrane appear to inhibit the action
of the enzymes amino aciddecarboxylases, which are
necessary for the amino acids biosynthesis and
biodegradation.
On the other hand, Oussalah et al. (2006) reported

a release of the cell constituents, a decrease of
intracellular ATP concentration and a decrease in
intracellular pH due to an increase in the permeability of
cell membrane when cinnamon oil was applied up to 0.1%.
58
Nonetheless, these authors did not observe an apparent
change on the cell surface (by electron micrographs) when
cinnamon oil was added. In the same way, Gill and
Holley (2004 and 2006) reported a rapid decrease of
cellular ATP but no increase of extracellular ATP when
0.15 or 0.6% of cinnamaldehyde was used. Thereby, the
mechanism of action of the cinnamon oil and its main
compound to inactivate microorganisms according to Gill
and Holley (2004 and 2006) and Oussalah et al. (2006)
appear to be related to the cell membrane from which a
slight disruption seem to occur causing dispersion of the
proton motive force by leakage of small ions without
leakage of larger cell components, such as ATP, which are
subsequently degraded by the ATPase enzyme.
59
3.2. Physical treatments
3.2.1. Gamma irradiation
Gamma radiation is a physical phenomenon in
which energy travels through spaces as a wave motion
without the aid of traveling medium. The exposing
materials (living cells, foods, medical products) to gamma
radiation in such away that a precise and specific dose in
absorbed termed "gamma irradiation"
3.2.1.1. Types of radiation

I. Non ionizing radiation, having longer wave length
and lower energy such as: UV, visible light, infrared,
microwave and radio waves. The amount of energy
carried by UV radiated is not large and its lethal
activity is of a relatively lower order. Its penetration
ability is much smaller than gamma radiation (Alper,
1990).
II. Ionization radiation, having short wavelength and

contain enough energy to ionize the molecules in their
paths.
Ionizing radiation is a convenient tool for studying
the fundamental of life. There are no phenomena in nature
that do not experience the modifying action of ionizing
60
radiations because their energy is always superior to that
of intermolecular bonds. This is why radiobiology
inevitably reflects all the fields of biology to some extent.
Accordingly, the set of objects studies by radiobiology is
exceedingly diverse. It includes macromolecules phages,
viruses, protozoa cell tissues, plants, animals, cells tissues
humans, populations, and biogenesis (Yarmenko, 1988).
Types of ionizing radiation used in peaceful

application.
All ionizing radiation are divided in their nature
into electromagnetic and particulate one. Electromagnetic
radiation include x-rays from x-rays tube, the gamma-rays
of radioactive, and the x-rays of electron accelerators
produced when highly accelerated electrons are directed at
a special x-rays target. Visible light and radio waves also
electromagnetic radiation but they do not ionizing matter
because they are characterized by a longer wavelength
"lower frequently". The depth of penetration of an ionizing
radiation depends on one hand on the nature of the
radiation, the charge of the particles forming it and their
energy, and on the other hand, on the composition and
density of the irradiated substance (Ivanov et al., 1986).
61
Gamma radiation from (CO60) is the most widely used in
practices because of costs and high penetrating.
3.2.1.2. Living cell and radiation

The exposure of cells to ionizing radiating sets
off a chain of reaction giving rise to chemical and then to
metabolic or physiological changes. So, irradiation
presents an additional stress to the cell, which tends to
disturb their organization (Lawerence, 1971). Irradiation
effects have been shown to be occurring with proteins,
enzymes, nucleic acids, lipids and carbohydrate, all of
which may have marked effect on the cell (Habbs and
Macellan, 1975). Generally, radiation occurs over abroad
time scale which extends from his radiation easily physical
process to the very late biological effect (Edward, 1990). It
has been reported that on number of intracellular
constituents may be responsible for the high radiation
resistance in some radio resistance strains. This may
include certain chemical compounds such as mercopto-
alkyl amine (Anderson et al., 1956), sulphydryl
compounds (Bridges, 1964), amino acids (Work, 1964)
and proteins (Mareson and Stelow, 1987).
62
3.2.1.3. Dose survival curves
Dose survival curves illustrate the relationship
between numbers of surviving organisms and radiation
dose. In practice the necessary data are usually obtained
by exposing number of equal size population to increasing
radiation doses and counting the number of survivors
(Ley, 1973).
3.2.1.4. The decimal reduction doses (D10 value).

The resistance or sensitivity of microorganisms is
measured by the so-called D10 value. The D10 value is the
doses required to reduce the initial population to 90 %.
When the dose survival curve is straight line it is possible
to read the D10 value from the graph by reading off the
dose required to reduce a surviving fraction throughout
one log cycle (Erdman et al., 1961). The D10 value is
defined as the dose of ionizing radiation required to reduce
a given microbial population by a factor of 10 or 90% or by
one logarithmic cycle.
3.2.1.5. Effect of radiation on microorganisms

Ionizing radiation imparts their energy to
molecules in a manner that depends on the atomic
number of constituent atoms and not on the molecular.
63
Configuration as in the case with UV interaction of
radiation with matter occurs in a more or less random
manner in a biological material in living cell. It is to be
expected that certain sites or system will be more readily
damaged than others (IAEA, 1973 and Giusti et al.,
1988). The primary chemical changes in target molecules
can be produced both by direct and indirect effect of
radiation. The result would be ionization and or excitation
in atoms of these molecules and radical formation, with
the important biological system of which leads to its death
or inactivation (Roger et al., 1998).
3.2.1.6. Direct action

The direct effects are changes that appear as a
result of the absorption of radiation energy by the
molecules of interest. In such processes the energy by is
directly deposited in the target molecule of biological
system without the intervention of radical species derived
from water radiolysis, or other system of the environment.
The target theory states the effect of ionizing or in very
near to some particular molecules or structure is
responsible for the measured effect, the production of on
effective in the target is often called hit. The target may be
a whole cell, part of cell, or critical molecules. Generally,
64
the measured effect in a system may be cell death or
inability to grow or to divide in the simplest from of the
target theory, one hit is sufficient to product the measured
effect in associated organisms with very small radiation
doses, the number of effected targets will be directly
proportional to the amount of radiation (Roots et al.,
1985 and Roger et al., 1998).
3.2.1.7. Indirect action
The indirect effect of radiation on target molecule is

produced by way of intermediary radiation products, the
photon may interact with water, the predominant molecule
in the mammalian cell to produce free radicals, these free
radicals are relatively short- lived, they can interact with
biologically important material causing a determined
effects, or conversely can react innocently to revert to their
former state (Yuring et al., 2000). A free radical is an
electrically neutral molecule, which has unpaired electron
in the outer orbit. The steps of the formation of free
radicals could proceed through various ways the most
simple of which is as follow (Casarett, 1968). Cell death (
defined for proliferation cells as loss of reproductive
capability ) is predominatly induced by double-strand
breaks in DNA, separated by not more than a few base
65
pairs, which can generally not be repaired by the cell (
Hall and Giaccia, 2006).
Reactive oxygen species (ROS), including

hydrogen peroxide (H2O2), superoxide, and hydroxyl
radical, are toxic to cells due to their ability to damage
DNA and especially Proteins containing Iron-sulfur
clusters or sulfur atoms (Imlay, 2003). Mn (II) can act
catalytically as scavenger of either superoxide (O-2) or
hydrogen peroxide (H2O2) (Archibald and Fridovich, 1982
and Stadtman et al., 1990).
3.2.1.8. Effect of radiation on water molecules:

Note: the dot [•] means exciting (has excess energy) or
unpaired electron in the radical
.
1- H2O → H2O (excitation)
. . .
2- H2O →H + OH (splitting)
3- H2O →H2O+ + e- aq (ionization)

4- H2O + e- aq →H20-
The positive and negative free radicals of water ions are
both unstable and each dissociates to form stable ions and
free radicals.
.
5- H2O+ → OH + H+
66
.
6- H2O- → OH-+ H
The stable [H+, OH-] ions combine to form water

7- OH-+ H+ →H2O
. .
But the primary free radical [H , OH , e- aq] are very
reactive and during the course of diffusion, they distribute

the absorbed energy to solute molecules either organic or
inorganic with high efficiency and may give rise to
secondary free radical ( somewhat less reactive) and
molecules which in turn are capable to attaching
macromolecules.
.
If there is O2 in the environment [H and e- aq] react
with it.
. .
8- O2+ H → H O2
9- O2+ e- aq→ O2-

The primary free radicals can also react with each
other.
. .
10- H + H →H2
. .
11- H + OH → H2O
. .
12- OH + OH → H2O2
H2O2 may also form by:
67
. .
13- HO2 + HO2 → H2O2+ O2
The ionized water molecules [H2O+] may react with

another natural water molecule to form:
.
14- H2O+ + H2O→H3O -+ OH
The products of water radiolysis could be

summarized in the follow
. .
15- (H , OH , H3O +, e- aq, OH-, H+, H2O2)
Radiation may cause alteration in DNA:
a) Rupture of the hydrogen bonds which link the base

pairs adenine-thymine, cytosine- guanine, loss of base or
change of abase of (for example deamination).
b) Induction of break in either or both of the DNA

chains, between sugars and phosphate groups.
c) Formation of links between adjacent thymine of

residues on a chain, to from a dmire.
d) Cross-linking between the singlestrands of helix, or

between a strand and the protein (histone) associated with
DNA in the chromosome (IAEA, 1970).
68
3.2.2. Heat treatment
3.2.2.1. Introduction
The delivery of an adequately lethal thermal process is
central in the preservation of food by the application of
heat, both to prevent a risk to public health and to achieve
a commercially reliable shelf life for the food. Conventional
heat treatment systems must reliably deliver the required
thermal process and at the same time accommodate many
products, process and package variables and their
interactions with each other and the thermal process. By
its very nature, thermal processing has a detrimental
effect on some food quality attributes and the food
processor must design the thermal process to balance the
unavoidable needs of commercial sterility with the
commercial desire to present a high-quality product
3.2.2.2. Thermal technologies

The thermal process is required to achieve
commercial sterility that is to render the food free from
viable micro-organisms, including those of known public
health significance, capable of growing in the food at
temperatures at which the food is likely to be held during
distribution and storage (DHSS, 1994).
69
3.2.2.3. Psateurization
Pasteurization is the treatment of packaged foods

to temperatures below 100 ْC over a given time to eliminate
pathogenic microorganisms which may gow under certain
storage conditions (Hackney et al, 1991). It is a heat
treatment which kills pan of the vegetative microorganisms
present in the food and relies heavily on handling and
storage conditions to further minimize bacteria growth
(Karel et al, 1975).
3.2.2.4. The Decimal Reduction Time
The thermal death of specific microorganisms does

not follow a linear path with a finite time to complete
death. Rather, it follows a logarithmic relationship
(Stumbo, 1973). If a semi-logarithmic plot was made
between the numbers of one type of bacteria surviving a
single lethal heat and time, an approximately linear
relationship would be observed with a negative sloping
line. The Iength of time required to reduce the numbers of
microorganisms by one log scale, by 90%, is referred to as
the decimal reduntion time and is given the symbol D.
This is qual to the negative inverse of this semi-logarithmic
or"survivor" curve slope.
70
One D time increment at the lethal temperarure reduces
the population of microorganisrns by 90%. The next D will
reduce the remaining population by 90% and so on.
Sterilization processes usually hold the product at the
lethal heat for the equivalent of 12 D time increments
while pasteurization processes usually involve 4 - 6 D time
increments. If D has a value of 0.20 minutes then 12D, a
sterilization process, wouId be 2.4minutes, reducing 106
bacteria to 10-6, read as 1 in a million chance of 1 bacteria
surviving. This would be read as a 12 D reduction in the
target microorganism if it was held at the sterilization
temperature for 2.4 minutes (Trenholm, 1998).
3.2.2.5. Mechanism of action

Heat can damage protein, lipids, and nucleic acids and
destabilize membranes (such as oxidation of sulfydryl
groups of the membrane-binding bound proteins) (Gould,
1989). However, heat induces primarily blebbing and
vesiculation of the outer membrane, leading to bacterial
cell inactivation, and consequently increases its
permeability to hydrophobic compounds (Tsuchido and
Takano, 1988). Heating-induced membrane
destabilization sequentially initiates indirect DNA
damageas a consequence of increased nuclease activity in
71
bacteria and is often the critical injury, resulting in death
of the cell (Gould, 1989).
72
MATERIALS
&
METHODS
73
Materials
(1) Samples :
Ten juice samples from each product were tested

according to the production of the company. During two
years from summer 2006 to summer 2007 for microbial
contaminations. Five samples were tested from the end
product for the manufacture and five samples from
concentrated juices (raw materials). Complete
microbiological analysis was done (bacteriological, yeast
estimation). The raw materials was free from any
preservatives while the end product contained sodium
benzoate , citric acid with concentrations 0.1 %, 0.3 % (
wt/v) respectively also, the end product was pasteurized
at 90 ْC for five minutes, approximately.
(2) Source of samples:
The end product were taken from United Food

Industries Company – bader city(A) and sterilized by
pasteurization, while concentrated juices (raw materials)
were taken from Cairo Agro-Processing Company- Al-
Obour city (B).
74
(3) Samples sorts:
No. of
Samples Type Company Preservatives Sterilization
samples
Apple 10 Pulp A
Guava 10 Pulp A without preservatives
Pulp A
Pasteurization
Mango 10 End Sodium benzoate (0.1%) and citric acid
B
product (0.3%)
Pulp A without preservatives
Orange 10 End Sodium benzoate (0.1%) and citric acid
B
product (0.3%)
End Sodium benzoate (0.1%) and citric acid
Cocktail 10 B
product (0.3%)
(4) Source of preservatives:

• Lantibiotic nisin standard supplied from Biomedical
Company U.S.A with activity approximately 1000-
units/mg powder.
• Cinnamon (ground state) supplied from commercial
source.
• Citric acid supplied from china.
• Lactic acid (local) supplied from El- Naser Company.
(5) Irradiation source:
Irradiation was carried out using cobalt 60

irradiation source (Gamma chamber 4000 India, located at
75
National Center for Radiation and Technology) with dose
rate 8.33 KGy/min. at the time of experiment).
(6) Isolation media:
i. Isolation media for bacteria

a. Nutrient Agar (NA) medium (Barrow and Feltham,
1993):
Nutrient agar medium contained the following (g/l):

Beef extract, 3; peptone, 5; sodium chloride, 5; and agar,
15. All ingredients were dissolved in distilled water and
completed up to 1000 ml. pH was adjusted at 6.8.
b. Nutrient Broth (NB) medium (Barrow and Feltham,

1993):
Nutrient agar medium contained the following (g/l):

Beef extract, 3; peptone, 5; sodium chloride, 5. All
ingredients were dissolved in distilled water and completed
up to 1000 ml. pH was adjusted at 6.8.
c. M.R.S Agar
This medium was supplied from Oxoid (CM361) (62 g/l),

pH was adjusted at 6.8.
76
d. Violet Red Bile Agar
This medium was supplied from Oxoid (CM107) (38.5

g/l), adjusted at pH 6.8.
e. Baired-parker Agar
This medium was supplied from Oxoid. Suspend 63 g

in one litre of distilled water and boil to dissolve the
medium completely. Dispense into tubes or flasks and
sterilize by autoclaving at 121 C for 15 minutes. Cool to 50
C and aseptically add 50 ml of Egg Yolk-Tellurite Emulsion
SR54. Mix well before pouring, adjusted at pH 6.8.
f. Peptone water
This medium supplied from oxoid (15g/l), adjusted at pH

6.8.
ii. Isolation media for yeast

a. Sabauroud's Agar
Sabauroud's agar medium contained the following (g/l):

glucose, 40; peptone, 10; agar 15. All ingredients were
dissolved in distilled water and completed up to 1000 ml,
adjusted at pH 7.
77
b. Sabauroud's broth
Sabauroud's agar medium contained the following

(g/l): glucose, 40; peptone, 10. All ingredients were
dissolved in distilled water and completed up to 1000 ml,
adjusted at pH 7.
c. YM Agar( yeast extract-malt extract agar)
YM agar (yeast extract-malt extract agar) contained

the following (g/l): yeast extract, 3; malt extract, 3;
peptone, 5; glucose, 10. All ingredients were dissolved in
distilled water and completed up to 1000 ml, adjusted at
pH 6.
All previous media were sterilized at 121 ْC for 15 min.
78
Methods
(1) Microbial counts
Ten milliliters of each fruit juice was transferred into 90

ml of sterilized saline solution to get the 1:10 dilution,
which serially diluted in sterilized saline solution to 104.
0.1 milliliter volume of the dilutions was transferred onto
various Plate count agar.
(2) Isolation of bacteria
Five milliliters of juice was added by sterile pipette to

enrichment medium 50 ml nutrient broth double strength
as enrichment media for 24h. at 37 ْC . Isolates were
obtained in pure culture from enriched cultures by
streaking on a suitable medium, such as Nutrient agar,
Violet Red Bile Agar, and Baired-parker Agar, M.R.S Agar
(for this medium another layer is added to obtain
anaerobic condition.
(3) Isolation of yeast
Five milliliters of juice was added by sterile pipette to

enrichment media 50 ml sabauroud's broth (double
strength) for 24h. at 37 ْC . And after incubation period it
was cultured by added 0.1 ml over agar surface by sterile
79
(4) Identification
a- Identification of bacterial isolates
Bacterial isolates were identified according to

According to the Keys of Bergey's Manual of Determinative
Bacteriology (Buchanan and Gibbson, 1974), and Cowan
and Steel's Manual for the Identification of Medical
Bacteria (Barrow and Feltham, 1993) and Bergey's
Manual of Systematic Bacteriology, Vol. (2) (Sneath,
1986), Bergey's Manual of Determinative Bacteriology 9th
ed. (Hensyl, 1994).
b- Identification of yeast isolates
Yeast isolates were suggested to follow the next

genera according to keys of (Barnett et al., 1990),
confirmed and renamed by (Barnett et al., 2000) and
(Kurtzman and Fell, 2000).
80
(5) Selection of most common isolates:
Three isolates were selected, gram positive isolate,

gram negative isolate, and yeast isolate. Selection of most
common isolates was according to its existing percentage
of its dominance in samples, and fast growth.
(6) Effect of some natural preservatives:
Series containing different concentrations of:
• Nisin (25-50-100-200-250) µg/ml.

• Cinnamon (0.2-0.3-0.4-0.5-0.6-0.7-0.8-1-1.2-1.5-1.7-2-
3-4) % (W/V).
• Citric acid (0.05-0.1-0.15-0.2-0.3-1-1.5-2.0-2.5-3-3.5-
4.0) % (W/V).
• Lactic acid (0.001-0.01-0.05-0.1-0.15-0.2-0.3-1.5-2.5-3-
3.5-4) % (W/V).
Were added to a broth medium individually, inoculate with

the organism and incubate at 37ْc for 48h, 1 ml of each
treatment and control were serially diluted in sterile
saline, cultured over agar surface on a suitable medium.
All plates were incubated at 37 C
ْ for 72h.
81
(7) Effect of some physical treatments:
i. Irradiation
The selected isolates were irradiated at different doses

(0.25-0.5-1-2-3-4-5-6-7) kGy on broth medium.
Immediately after irradiation, 1 ml of each treatment and
control (non-irradiated spore suspension) were serially
diluted in sterile saline. The count of yeast was
enumerated on Sabauroud's dextrose agar. All plates were
incubated at 37 ْC for 72h. The growing colonies were
counted and irradiation dose was plotted against log of the
number of survivors divided by the initial count before
irradiation (log N/N0).
D10 values of the tested isolates were calculated from the

regression linear equation (Lawerence, 1971).
Y=a±bx
D10 value = - 1/b

− −
b=
∑ xy − n x y
− 2
∑x 2
−n x
Where:
a : log of microbial count when x equal zero.
82
b : regression factor.
y : log of surviving fraction.
x: dose level in KGy.
n : number of calculated points.
x-: ∑x/n
y-: ∑y/n
ii. Pasteurized temperature
The selected isolates were pasteurized at temperature

90 ْC for different times (0.25-0.5-1-3) minute. 0.1 ml of
the pasteurized cells were cultured over agar surface on a
suitable medium and incubated for 48h. at 37 ْC.
(8) Effect of the combination treatments:
The concentration of the natural preservatives which

start to give distinct reduction for the growth in single
treatment was applied in combination with physical
treatments in one treatment in broth medium. 1 ml of
each treatment and control (non treated ) were serially
diluted in sterile saline, 0.1 ml of the treated cells were
83
cultured over agar surface on a suitable medium and
incubated for 24h. at 37 ْC.
(9) Storage
The storage was performed for more resistant organism by

applying two experiments as follow:
1-experiment (1)
Was perfomed by applying the combination between the

following treatments:
a- Nisin was used in concentrations (50, 100 and 200).
b- Citric acid 2% (W/V).
c- Pasteurized temperature 90ْ C for (30, 60 and 120 sec.).
Where, the nisin was added to Sabauroud's broth (double

strength) with citric acid, then it was inoculated with 1ml
of the organism broth (fresh culture broth with count 45 x
104), then it was pasteurized, finally it incubated at 37 ْC.
This was perfomed for each conc. of nisin with its time of
pasteurized temperature and citric acid. Storage time was
for 90 days, during this period 0.1 ml of the treated cells
were cultured every week over agar surface on a double
84
2-experiment (2)
Was perfomed by applying the combination between the

following treatments:
a- Gamma irradiation (0.2 kGy).

b- Pasteurized temperature 90 ْC for 30 sec.
Where Sabauroud's broth (double strength) was inoculated

with 1ml of the organism broth (fresh culture broth with
count 45 x 104), then it was pasteurized and after that
treated withgamma rays (0.2 kGy), finally it incubated at
37 ْC. Storage time was for 90 days, during this period 0.1
ml of the treated cells were cultured every week over agar
surface on a double strength medium of Sabauroud's agar
and incubated for 24h. at 37 ْC.
85
RESULTS
&
DISCUSSION
86
RESULTS AND DISCUSSION
1–Isolation and identification
The development of food preservation process has

been driven by the need to extend the shelf- life of foods.
Several food preservation systems such as heating,
refrigeration and addition of antimicrobial compounds can
be used to reduce the risk of outbreaks of food poisoning
microorganisms. However, these techniques frequently
have associated adverse changes in organoleptic
characteristics and loss of nutrients.
The objectives of this study were divided into

three parts; the first considered the main microorganisms
contaminated certain juices usually consumed in Egypt
before and after the preservation processes. The selected
juices were apple, guava, mango, and orange. The second
was to use some physical and natural agents for
preservation the above juices instead of using chemical
agents or high temperature degree with pressure. The
physical agents were gamma radiation and pasteurized
temperature while the natural agents were nisin, citric
acid, lactic acid, and cinnamon. The third objective was to
investigate the application of combined treatments from
87
the above agents aiming to prolonging the shelf life of the
juice products and applying the minimum doses of these
agents.
As shown in table (1), ten samples of each type of

chosen juices were collected represented the raw materials
and the end product: the samples were taken during years
2006 and 2007 from United Food Industries Company and
Cairo Agro-Processing Company in Egypt. The samples
represented apples, guava, mango, orange, and cocktail
(mixture of apple pulp, mango and peach juice). The end
products of apple juices and guava juices were found to be
free from microorganisms. It worth to mention that these
end products juices were treated with sodium benzoate
combined with citric acid with the dose permissible by the
Egyptian authorities (i.e. 0.1% (W/V) for sodium benzoate
and 0.3% (W/V) for citric acid ).
On the other hand, positive results of

contamination in the end product of mango, orange and
guava were observed in spite of pasteurization and the
addition of sodium benzoate and citric acid indicating the
insufficient of this treatment.
From the same table, all guava juice and apple

juice or cider samples were free from yeast, or bacteria,
88
the lowest contamination was recorded in cocktail samples
which contained total microbial loads ranging from 12 x
10 cfu/ml to 18 x 102 cfu/ml, followed by apple pulp
samples which contain microbial load between 15 x 10 to
45 x 102 cfu/ml. While the total bacterial load for mago
pulp and orange pulp samples ranged from 104 to 105
cfu/ml. while for guava (pulp and juice), mango juice and
orange juice samples the bacterial load was ranged from
102 to 103, while yeast load was in range104 cfu/ml for the
same samples. This is agreement with Hatcher et al.,
(1992) which reported that the microbial populations of
the raw juices are within the range of 102 –105cfu/ml.
However, the high bacterial and mould counts may be
indication of improper hygiene and may perhaps be a
result of poor quality fruit being used (Lateef et al.,
2004).
89
Table (1): Screening and isolation of microorganisms contaminated certain Egyptian juices
Average total count Microorganisms

Sample Type State pH ± 0.1 cfu m-1 Bacteria Yeast
Cider*** 2.7 Nil - -
Apple Raw material**
Pulp 3.0 15 x 10 - 45 x 102 + -
Juice End product* 3.8 Nil - -
2
Cocktail Juice End product* 3.5 12 x 10- 18 x 10 + -
Guava Pulp Raw materials** 4.2 15 x 102- 52 x 103 + +
2 3
Juice End product* 4.2 15 x 10 - 52 x 10 + +
Pulp Raw materials** 3.5 23 x 104- 35 x 105 + -
Mango 2 3
Juice End product* 4.0 12 x 10 - 45 x 10 + +
4 5
Pulp Raw materials** 3.5 17 x 10 - 28 x 10 + -
Orange 2 3
Juice End product* 3.8 22 x 10 - 38x 10 + +
*End product with sodium benzoate and citric acid ***Cider = Oily product from apple
**Raw materials without any preservatives -Cocktail = apple + mango + peach
90
Fresh apple juice is expected to be safe because its
acidic pH (usually ranging from 3 to 4) affects growth and
multiplication of pathogens. However, several outbreaks
have occurred due to consumption of un-pasteurized apple
juice and cider (Goverd et al., 1979; Parish, 1997 and
Sado et al., 1998). Some outbreaks of Salmonella food
poisoning have been traced to un-pasteurized apple cider
and orange (Goverd et al., 1979).
Regarding the pulp of the previous fruits (raw

material), all of them as expected were contaminated (table
1) either with bacteria or yeast. End product of cocktail
juice samples were contaminated with yeast only. On the
other hands, the pH of previous samples was found to be
ranged from 3.0 to 4.2 as clearly shown in table (1).
Martinez – Gonzales et al. (2003) reported that,

the low pH fruit juice samples, especially apple cider and
orange juice, have been associated with food borne
diseases. Lateef et al. (2004) reported that the acidic
juices are (pH 3.0 and 3.65) a good condition for the
growth of yeasts.
91
Forty two colonies were taken from contaminated
samples according to their morphological shape, four from
apple pulp, twelve from guava pulp, five from mango pulp,
one from mango end product, ten from orange pulp, and
seven from orange end product and three from cocktail
juice.
As clearly shown from table (2), the four colonies

obtained from apple pulp were identified as two strains of
Micrococcus sp. and two strains of S. aureus. The twelve
colonies of guava pulp were; seven S. aureus, one
Staphylococcus warneri, three Debaryomyces sp., and
one Pichia sp. The five colonies of mango pulp were; two
Staphylococcus auricularis and three Staphylococcus
epidermidis while the only colony isolated from mango
end product was identified as Kluveromyces sp. The five
colonies of orange pulp were; two Bacillus sp. (Endo
spore former), six P. aeruginosa and two Citrobacter
frundii. While the seven colonies isolated from orange end
product were identified as Streptococcus pedococcus.
The three colonies of cocktail were identified as; two
Debaryomyces sp. and one Pichia sp..
92
Lateef et al. (2004) isolated strains of
Saccharomyces cerevisia, Saccharomyces sp.,
Rhodotorula sp; Bacillus cereus, subtilis, E. coli, S.
aureus, Streptococcus pyogenes and Micrococcus sp.
from orange juice. Debaryomyces hansenii isolated from
orange concentrate (Deak and Beuchat, 1996).
Debaryomyces hansenii and Zygosaccharomyces
rouxii on syrups, fruit concentrates and jams (Andrews et
al., 1997).
93
Table (2): Identification of contaminated microorganisms isolated from certain Egyptian juices
Juice micro flora
Samples Type
Total Isolated microorganisms No. of isolates
Micrococcus agilis 2
Apple Pulp* 4
Staphylococcus aureus 2
Staphylococcus aureus 7
Staphylococcus warneri 1
Guava Pulp* 12
Debaryomyces sp. 3
Pichia sp. 1
Staphylococcus epidermidis 3
Pulp* 5
Mango Staphylococcus auricularis 2
End prods. ** 1 Kluveromyces sp. 1
Bacillus sp. ( endo-spore former ) 2
Pulp* 10 Pseudomonas aeruginosa 6
Orange
Citrobacter frundii 2
End prods. ** 7 Streptococcus pedococcus 7
Debaryomyces sp. 2
Cocktail End prods. ** 3
Kluveromyces sp. 1
Total 42
* Pulp = raw juice with out any preservatives. ** End product = juice with sodium benzoate preservative.
94
Youssef et al. (2002) isolated strains of Candida
tropicalis, Hanseniaspora uvarum, Rhodotorula
glutins, Zygosaccharomyces bailii and Z. rouxii from
un-steamed and un-irradiated mango pulp.
Mean while Abd El-karem and Farag (1996)

found that Candida tropicalis, Zygosaccharomyces
rouxii and Rhodotorula graminis were occurred in
mango juice and the first was the predominant (54%
(W/V)).
Lee et al. (2002) reported that the thermo

acidophilic spore-forming Alicyclobacillus
acidoterrestris were isolated from commercial
pasteurized apple juice in the United Kingdom, Germany,
and the United States.
Dellaglio et al. (2005) isolated two strains of

gram negative bacteria, rod-shaped, non-spore-forming
bacteria; Gluconacetobacter swingsii and
Gluconacetobacter rhaeticus from apple fruit juice in
the region of the Italian Alps.
95
According to table (2) the 42 identified colonies were
divided to; 15 of genus Staphylococcus, 6 of genus
Pseudomonas, 7 of genus Streptococcus, 2 of genus
Micrococcus, 2 of genus Citrobacter, 2 of genus
Bacillus, 5 of genus Debaryomyces, 2 of genus Pichia
and one Kluveromyces. In addition fig (1) shows the
percentages of these identified organisms.
25.0
21.4
20.0
16.7
14.3
Percentages %
15.0
11.9
10.0 7.1
4.8 4.8 4.8 4.8 4.8
5.0 2.4 2.4
0.0
us . s p. is is ri sa ii p. p. p.
sp idi gil lar rne geno s s h ia s
.a ure rep. r m l u s s a cu a f r u nd
c e c ess
St e c il s ri .w ro y c y
ph pid cu au ph ae c te
r
om Pi rom
S ta h.e Ba oc h. ary
a p c r oc ap Sta eud. r oba
e b l uve
St St Ps t
Mi Ci D K
Types of microorganisms
Fig. (1): Percentages of isolated microorganisms
96
For further studies, three different strains of above
isolates were chosen. The selection was according to their
common contamination in all samples and their
characteristics; S. aureus represented gram positive
bacteria, P. aeruginosa represented gram negative
bacteria, and Debaryomyces sp. chosen to represented
yeast strains.
97
3. Single treatments
Irradiation is a non-thermal food preservation
process that has been reexamined in recent years.
Irradiation increases the shelf-life of foods and ensures
their innocuousness because most microorganisms in
vegetative form are extremely sensitive to irradiation at low
doses (Radomyski et al, 1994). However, in some
circumstances combined treatments are more satisfactory
since the dose required for complete sterilization can
induce undesirable changes in food flavor or is higher than
permitted levels (Thakur and Singh, 1995).
As shown in table (3) illustrated by fig. (2), it is

clear that the viable counts of all the selected isolates
decreased by increasing the dose of radiation.
The dose response curve of S. aureus, P. aeruginosa
and Debaryomyces sp, showed straight-line curves (type
A). The D10 values, of the tested strains were 0.75 kGy,
0.69 kGy, and 1.48 kGy, for S. aureus, P. aeruginosa,
and Debaryomyces, respectively.
It is clear from the results that S. aureus and P.
aeruginosa were more sensitive to the irradiation
98
treatment than Debaryomyces strain. Also dose levels 4
and 5 kGy was completely enough to destroy the viable
cells of P. aruginosa and S. aureus, respectively. While 7
kGy was needed to obtain the same effecting case of
Debaryomyces strain. It is worth to mention here that the
initial viable counts of the tested strains, used in the
present experiment, was ranged between 107and 108
cfu/ml.
In practice these counts never exceed than 105 cfu/ml,
hence, the dose level needed to eliminate the resistant
strain (i.e Debaryomyces strain) will not exceed 5.5 kGy.
Several investigators have reported that fungi are

typically more resistant to radiation than are bacteria
(ƠConnor and Mitchell, 1991; Monk et al., 1994). D10
values, i.e., the amount of radiation necessary to Kill 90 %
of the initial microbial count, have been reported for yeasts
and molds in the range of 1 to 3 kGy (Lescano et al.,
1991 and Narvaiz et al., 1992), as opposed to D10 of 0.3
to 0.7 kGy for pathogenic bacteria on produce and juices
(Buchanan et al., 1998; Rajkowski and Thayer, 2000
and Niemira et al., 2001).
99
Frazier and Westhoff, 1988 stated that the
lethal dose for yeasts, S. aureus and P. aeruginosa were
at 4 to 9, 1.4 to 7.0, and 1.6 to 2.3 KGy, respectively.
Lawerence (1971) mentioned that the loss of
proliferate capacity is usually covered by damage with in
DNA molecules which controlling all aspects of
metabolism, structure and development. In the mean time,
high doses of gamma radiation were proved to be
inhibitory for both growth and enzymatic activities of
microorganisms, (Sadi, 1987).
Doses KGy
0
0 1 2 3 4 5 6 7
-2
-4
Log N/No
-6
-8
-10
Staphylococcus aureus Pseudomonas aeruginosa Debaryomyces spp

-12
Fig. (2): Effect of increasing doses of gamma irradiation on the

selected organisms.
100
Table (3): Effect of different doses of gamma irradiation on the viable counts of the selected organisms*
Staphylococcus aureus Pseudomonas aeruginosa Debaryomyces sp.
Doses kGy Average Log Average Log Average Log
Log N** Log N** Log N**
count cfu m-1 N/No count cfu m-1 N/No count cfu m-1 N/No
Initial count 98 x 106 7.99 0 18 x 106 7.25 0 41 x 106 7.61 0
± 0.045 A ± 0.056 A ± 0.089 A
0.5 10.2 x 106 7.0 - 0.98 98 x 104 5.99 - 1.26 89 x 105 6.94 - 0.66
± 0.021 B ± 0.022 B ± 0.072 B
1 11.5 x 105 6.06 - 1.93 48 x 103 4.68 - 2.57 20 x 104 6.30 - 1.31
± 0.028 C ± 0.033 C ± 0.074 C
2 85 x 103 4.92 - 3.06 37 x 102 3.56 - 3.68 25 x 104 5.39 - 2.21

± 0.029 D ± 0.042 D ± 0.03 D
3 98 x102 3.99 - 4.0 15 x 10 2.17 - 5.07 10.4 x 104 5.01 - 2.59

± 0.024 E ± 0.033 E ± 0.015 D
4 12 x 102 2.07 - 5.91 -*** -*** -*** 12 x 103 4.07 - 3.53

± 0.013 F F ± 0.025 E
5 -*** -*** -*** 84 x 102 3.92 - 3.68

G ± 0.024 F
6 94 x 10 2.97 - 4.63
± 0.022 G
7 -*** -*** -***

H
D10 0.75 kGy 0.69 kGy 1.48 kGy

* This experiment was counted after 24h incubation from irradiation. *** The dash means no distinct growth.
** Means with the same letter are not significantly different.
101
In general, the gram–negative bacteria, including
most of the common food spoilage organisms (e.g.,
Pseudomonas), are more sensitive to irradiation than are
the gram-positive organisms (e.g., lactic acid bacteria and
micrococci) and yeasts are more radiation resistant than
typical spoilage bacteria (Ingram, 1975). Generally,
Pseudomonas sp. lacks the inducible error-prone DNA repair
system (Miller and Kokjohn, 1990; Karentz, 1994 and Joux
et al., 1999).
Daly et al. (2004) propose that Mn (II) accumulation

facilitates recovery from radiation injury. It was further
noted that the intracellular concentration of Mn in
S.aureus was ~50-100um (Horsburgh et al., 2002).
In yeast enhanced radiation resistance is generally

believed to be the result of an increase in the capacity or
efficiency of a cell to repair its DNA (Bryant, 1976 and
Mitchel and Morrison, 1984), DNA being the critical
target for ionizing radiation- induced lethality. The
recombination DNA repair system is considered to be the
major DNA repair pathway that confers resistance to
killing by ionizing radiation in yeast cells (Mitchel and
Morrison, 1984). Irradiation of concentrated juice in the
range of 0.5-2.0 kGy slightly altered some sensory
102
characters; these changes increased at higher doses
(Zegota et al., 1988).
2.1.2. Pasteurized temperature

Pasteurization is widely used in the food and
dairy industry both to minimize the risk from pathogens
and to extend product shelf life. To ensure a secure and
steady food supply, food needs to be preserved and
stabilized so it can be stored for extended periods.
Extending food products shelf life requires inactivation of
the spoilage or toxic microorganisms they contain and
avoiding their subsequent proliferation. Food preservation
thus implies exposure of microorganisms to hostile
conditions. Heating is one of the most practical efficient
and inexpensive methods of disinfection and sterilization
of food.
Results in table (4) and figure (3) showed that,

exposured the three organisms, Staphylococcus,
pseudomonas, and Debaryomyces to 90 ْC sharply
decreased their counts. The initial count was 98 x 105, 98
x 105, and 55 x 105 cfu m -1 for Staphylococcus,
pseudomonas, and Debaryomyces respectively. The Dt
values (the decimal reduction time i.e. that time of heating
103
Also, TDT (thermal death time) was 157.4 sec. for
Staphylococcus, 208.0 sec. for pseudomonas and 224
sec. for Debaryomyces. The lethal time was at 120 sec for
three organisms. The initial count was decreased to 1.84
log for Staphylococcus at sub lethal time 60 sec, to 2.87
log for pseudomonas at sub lethal time 60 sec, and to
3.54 log for Debaryomyces at sub lethal time 60 sec.
By comparing between three treated strains, it was

observed that Staphylococcus was highly sensitive to
pasteurized temperature followed by Pseudomonas and
then Debaryomyces.
According to Moats (1971), Cell death due to the

exposure to high degree of temperature results from the
denaturation or inactivation of numerous critical sites (or
a large number of the same site) in cells.
104
Rosenberg et al. (1971) demonstrated the
existence of a good numerical correlation between some
thermo-dynamic parameters in protein denaturation and
in death rates of bacteria yeast, viruses and drosophila.
Hurst, (1977) and Gould, (1989) summarized

some of the mechanisms by which heat may cause cellular
inactivation: (i) damage of DNA, (ii) inhibition of protein
synthesis, (iii) damage of cell membrane, and (iv)
inactivation of critical metabolic enzymes.
Heat can damage protein, lipids, and nucleic

acids and destabilize membranes (Gould, 1989). Heating-
induced destabilization, such as oxidation of sulfydryl
groups of the membrane – binding bound proteins,
resulting in cell death in addition into direct DNA damage
(Yatvin and Grummer, 1987).
105
Tim / sec.
0
0 10 20 30 40 50 60 70
-1
-2
Log N/No
-3
-4
-5
Staphalylococcus aureus Pseudomonas aeruginosa Debaryomyces spp

-6
Fig. (3): Effect of pasteurized temperature 90 Cْ at different time

on the selected organisms.
106
Table (4): Effect of pasteurized temperature 90 Cْ at different time on selected organisms
Time/Sec. Average Log Average Log Average Log
-1 Log N* -1 Log N* -1 Log N*
count cfu m N/No count cfu m N/No count cfu m N/No
Initial count 98 x 105 6.99 0 98 x 105 6.99 0 55 x 105 6.74 0

± 0.045 A ± 0.014 A ± 0.046 A
15 24 x 104 5.38 - 1.61 32 x 104 5.50 - 1.48 95 x 104 5.97 - 0.76

± 0.032 B ± 0.055 B ± 0.038 B
30 55 x 102 3.74 - 3.25 29 x 103 4.46 - 2.52 118 x 103 5.07 - 1.66
± 0.014 C ± 0.058 C ± 0.022 C
60 7 x 10 1.84 - 5.14 75 x 10 2.87 - 4.11 35 x 102 3.54 - 3.19

± 0.088 D ± 0.055 D ± 0.031 D
120 -** -** -** -** -** -** -** -** -**
E E E
Dt 13.1 Sec. 17.3 Sec. 18.6 Sec.

TDT 157.4 Sec. 208.0 Sec. 224.0 Sec.
* The same letter is not significantly different. ** The dash means no distinct growth.
TDT = thermal death time (Dt * 12).
107
Many factors influence the heat resistance of non-spore
forming microorganisms. Time and temperature of
incubation dramatically affect the heat resistance of both
gram-negative and gram-positive bacteria.
Beuchat (1978) speculated that growth at high

temperature results in the production of thermo stable
membranes, which results in increased thermo tolerance.
A factor that has received considerable attention recently
is sub lethal heat shock (Mackey and Derrick, 1986),
which induces the rapid synthesis of heat shock proteins
(Lindquist and Craig, 1988). Factors other than heat,
such as hydrogen peroxide (Morgan et al., 1986) and
glucose starvation (Jenkins et al., 1988), also induce the
rapid synthesis of heat shock protein. Evidence is
accumulating that heat shock proteins are a major
determinant of bacterial thermo tolerance (Jenkins et al.,
1988). An important factor in thermo tolerance may be the
effect of superoxide (O2-) and hydrogen peroxide (H2O2) on
the recovery of bacteria injured by heat (Knabel et al.,
1990).
108
Iandolo and Ordal, (1966) reported that heating at
55ْC caused the release of intracellular constituents from
cells of S. aureus. Release of intracellular constituents
could also occur as a result of cell lysis (Pethica, 1958).
It is believed that these temperatures trigger

physiological responses that lead to the synthesis of
special proteins Known as heat shock proteins (HSPs)
(Knabel et al., 1990 and Lindquist, 1986).
109
2.2-Natural treatments
2.2.1-Nisin
Nisin is an antimicrobial peptide produced by
lactococci and has been used in consumer products for
many years (Taniguchi et al., 1994). Although this
lantibiotic is inhibitory to microorganisms, it is harmless
to humans (Hurst and Hoover, 1993). Nisin is the first
antimicrobial peptide with generally "recognized as safe"
status in the United States for use in processed cheese; in
addition, its use in various food products is allowed in
several countries (Delves-Broughton, 1990).
As shown in table (5) illustrated by figure (4), the

effect of Nisin on S. aureus was examined, when Nisin
concentration increases the count decreased. At the sub
lethal conc. 200 ug/ml the initially bacteria count 6.17
was decreased to 1.39, the lethal conc. was at 250
ug/ml.In the same time as shown in table (5) and fig. (4),
nisin has no effect on both Debaryomyces sp and P.
aeruginosa.
110
Crandall and Montville, (1998) reported that
the lethality of nisin is due to its effect on the cytoplasmic
membrane of vegetative cells and that is the primary site
of action of Nisin. The primary mechanism of nisin
believed to be the formation of pores on the cytoplasmic
membrane which result in depletion of proton motive force
and loss of cellular ions; amino acids, and ATP. Nisin
binds electro statically to the negatively charged
phospholipids and increases the permeability of the
membrane by pore formation, resulting in rapid efflux of
essential intracellular small molecules (Abee et al., 1994;
Breukink et al., 1997; Montville et al., 1999). The
efflux of cellular constituents resulted in a complete
collapse of the proton motive force and subsequently in
cell death (Wincowski et al., 1994). Nisin uses lipid II
(undecaprenyl- pyrophosphoryl-MurNAc-(pentapeptide)-
GlcNAc) as a receptor molecule to increase its
antimicrobial efficacy dramatically (Breukink et al.,
2003). However, recent work has shown that the activity
of Nisin is dependent on the conc. of lipid II in the
membrane of sensitive cells (Breukink et al., 1997;
Breukink and de Kruijff, 1999).
111
Pore formation by the `barrel-stave' mechanism, used
by a number of cytolytic pore-forming toxins (Ojcius and
Young, 1991), has been predicted (Fig. 5). This model
involves the initial accumulation of the peptide at the
membrane surface through ionic interactions with the
phospholipids head groups. The presence of these peptides
induces significant thinning of the membrane in these
areas, due to localized displacement of the phospholipids.
On application of a ΔΨ, the molecules adopt a
transmembrane orientation. As lantibiotics are small
peptides that can span the membrane only once, so it is
assumed that several molecules associate with the
membrane to form a pore. Whether this aggregation of
molecules occurs prior to insertion, or in the membrane
after insertion is unknown. It has been demonstrated that
at high pH, nisin monomers aggregate outside the
membrane, significantly reducing biological activity
(Garcia-Garcera et al., 1993).
Hasper et at. (2006) has been reported that, nisin binds

to the pyrophosphate moiety of lipid II and removes lipid II
from its functional location, thereby inhibiting cell wall
synthesis, and it induces the formation of lipid II-nisin
hybrid pores in the cytoplasmic membrane ().
112
Fig. (5): General model of the `barrel-stave' mechanism of pore
formation by peptides (Jack et al., 1997).
Ruhr and Sahl, (1985) reported that the addition

of Nisin to susceptible cells (Staph. cohnii, Bacillus sub,
Micrococcus luteus, Strepto zymo) resulted in a rapid
efflux of small cytoplasmic compounds such as K+ and
amino acids. In addition Nisin inhibited proline and
glutamine uptake by membrane vesicles of susceptible
cells. The loss of ions resulted in a rapid decrease in the
membrane potential, ΔΨ the effects of Nisin in depleting
proton motive force components, ΔΨ and Δ pH ,were
confirmed in L. monocytogene (Bruno et al., 1992) and
C. sporogenes (Okereke and Montville, 1992).
113
Conc. u g/ml
0
0 50 100 150 200 250 300
-1
-2
Log N/No
-3
-4
-5
Staphylococcus aureus Pseudomonas aeruginosa Debaryomyces spp

-6
Fig. (4): Effect of different conc. of nisin on the selected

organisms.
114
Table (5): Effect of different conc. of nisin on the selected organisms
Conc.
μg/ml Average Log Average Log Average count Log
count cfu m-1
Log N* count cfu m-1
Log N* cfu m-1
Log N*
N/No N/No N/No
Initial 6.17 6.99 6.61
15 x 105 0 98 x 105 0 41 x 105 0
count ± 0.045 A ± 0.0 A ± 0.0 A
5.63 6.99 6.61
25 43 x 104 - 0.54 98 x 105 0 41 x 105 0
± 0.060 B ± 0.0 A ± 0.0 A
4.74 6.99 6.61
50 55 x 103 - 1.43 98 x 105 0 41 x 105 0
± 0.064 C ± 0.0 A ± 0.0 A
3.41 6.99 6.61
100 26 x 102 - 2.76 98 x 105 0 41 x 105 0
± 0.046 D ± 0.0 A ± 0.0 A
1.39 6.99 6.61
200 25 - 4.77 98 x 105 0 41 x 105 0
± 0.079 E ± 0.0 A ± 0.0 A
-** 6.99 6.61
250 -** -** 98 x 105 0 41 x 105 0
F ± 0.0 A ± 0.0 A
* The same letters are not significantly different. ** The dash means no distinct growth.
115
On the other hand, Brul et al., (1997) and Klis et al.,
(1997) reported that nisin has no antimicrobial effect on
yeasts. These organisms have a rigid cell wall, a complex
structure consisting of glucan cross- linked with chitin
and cell wall proteins. The processing of manno-proteins is
complex and has been partially characterized in yeasts (Lu
et al., 1994 and Kollar et al., 1997). Because manno-
proteins are generally considered one of the key
components, which determine cell wall porosity (Cid et
al., 1995 and Klis et al., 1997) they may represent a
major barrier preventing free permeation of Nisin through
the cell wall and thus access to the cytosolic membrane.
The inability of nisin to attack Gram-negative bacteria

is due to the protective outer membrane (OM), which
covers the cytoplasmic membrane and peptidoglycan layer
of Gram-negative cells. This asymmetrical membrane
contains Glycerophospholipids in its inner leaflet, but the
outer leaflet is built of lipopolysaccharide (LPS) molecules.
LPS, which are composed of a lipid part and a complex
heteropolysaccharide with partly anionic character, form a
tight layer endowed with a hydrophilic surface (Nikaido,
1996). As a result, OM is a penetration barrier that
excludes hydrophobic substances and macromolecule.
116
Nisin, as a hydrophobic macromolecule, is unable to
traverse a normal OM and thus cannot reach its target of
action.
2.2.2- Organic acids

Weak acid preservative are generally considered
safe antimicrobials, consistent with the long history and
widespread use of these compounds for the preservation of
foods and beverages. The preservative molecule diffuses
into the cell until equilibrium is reached in accordance
with the pH gradient across the membrane resulting in the
accumulation of anions and protons inside the cell (Booth
and Kroll, 1989).
There for, inhibition of growth by weak acid

preservative has been proposed to be due to a number of
actions including , membrane disruption (Stratford and
Anslow, 1998., Bracey et al., 1998) , inhibition of
essential metabolic reaction ( Krebs et al., 1983), stress
on intracellular pH homeostasis (Bracey et al., 1998),
and the accumulation of toxic anions ( Eklund , 1985).
117
2.2.2.1- Citric acid
From the tables (6 & 7) illustrated by figures (6
& 7), the growth curve of both P. aeruginosa and
Debaryomyces sp. were belonged to type B (i.e it has a
shoulder at the begging of the curve that means, it has
resistance to citric acid at low concentration), followed by
exponential rate death. The increase in citric acid
concentration after shoulder part was paralleled to the
decrease in the number of survival bacterial cells. While
the growth curve of S. aureus was belonged to type A (i. e.
a straight-line curve with a logarithmic decrease was
obtained). The S. aureus organism count was decreased
sharply by increasing the conc. of citric acid.
The sub lethal conc. 0.15% (W/V) decreased the

count from 6.92 log to 2.81 log; the lethal conc. was at
0.2% (W/V). On the other hand, the sub lethal conc. of p.
aeruginosa was 0.2% (W/V), while lethal conc. at 0.3%
(W/V). The initial count 7.06 log decreased to 2.44 log.
With Debaryomyces sp. the sub lethal conc. was at
conc.3.5% (W/V) where the initial count 6.57 log
decreased to 1.39 log, and lethal dose was at 4.0% (W/V)
of citric acid. By comparison between the three
organisms, it was observed that, the highest sub lethal
118
conc. was 3.5% (W/V) then 0.2% (W/V) and 0.15% (W/V)
for Debaryomyces sp., p., and S. aureus respectively.
The growth count was decreased 4.11 log cycles, 4.62 log
cycles and 4.62 log cycles for each S. aureus, p.
aeruginosa and Debaryomyces sp., respectively at the
sub lethal conc. of citric acid.
119
Table (6): Effect of different conc. of citric acid on P. aeruginosa and S. aureus
Staphylococcus aureus Pseudomonas aeruginosa
Conc.% Average count cfu Average count
Log N* Log N/No Log N* Log N/No
m-1 cfu m-1
6.92 7.06
Initial count 85 x 105 0 115 x 105 0
± 0.045 A ± 0.014 A
6.02 6.64
0.05 105 x 104 - 0.90 44 x 105 - 0.41
± 0.030 B ± 0.028 B
4.11 5.25
0.1 13 x 103 - 2.81 18 x 104 - 1.80
± 0.030 C ± 0.029 C
2.81 3.72
0.15 66 x 10 - 4.10 53 x 102 - 3.33
± 0.151 D ± 0.140 D
-** 2.44
0.2 -** -** 28 x 10 - 4.61
E ± 0.058 E
-**
0.3 -** -**
F
120
Conc. %
0
0 0.05 0.1 0.15 0.2 0.25
-0.5
-1
-1.5
-2
Log count
-2.5
-3
-3.5
-4
-4.5
S. aureus P. aeruginosa
-5
Fig (6): Effect of different conc. of citric acid on P.

aeruginosa and S. aureus
Table (7): Effect of different conc. of citric acid on

. Debaryomyces sp
Average count cfu
Conc.% Log N* Log N/No
m-1
Initial count 38 x 105 6.57 0
± 0.089 A
1 90 x 104 5.95 - 0.62

± 0.020 B
1.5 68 x 103 4.83 - 1.74

± 0.023 C
2 93 x 102 3.96 - 2.61

± 0.022 D
2.5 18 x 102 3.53 - 3.32

± 0.050 E
3 26 x 10 2.41 - 4.16
± 0.038 F
3.5 25 1.39 - 5.18

± 0.061 G
4 -** -** -**

F
* The same letters are not significantly different.
** The dash means no distinct growth.
121
Conc.%
0
0 0.5 1 1.5 2 2.5 3 3.5 4 4.5
-1
-2
Log N/N o
-3
-4
-5
-6
Fig. (7): Effect of different conc. of citric acid on

.Debaryomyces sp
122
2.2.2.2 - Lactic acid
It shown from tables (8 & 9) and figures (8 & 9),
the growth curves of S. aureus, p. aeruginosa and
Debaryomyces sp., shown a straight-line curve with a
logarithmic decrease was obtained. The growth curve of S.
aureus was decreased sharply by increasing the conc. of
lactic acid. Where the count was decreased to log 1.59 at
conc. 0.1% (W/V) of lactic acid, the initial count was 6.98
log, where 5.99 log cycles were decreased.
The sub lethal conc. was 0.2% (W/V) for P.

aeruginosa; the initial count was decreased 4.76 1og
cycles. While the Debaryomyces sp. were decreased 4.48
log cycles and the sub lethal conc. was at 4% (W/V) and
count 2.44 log, where the initial count was 6.86 log
(cfu/m-1).
By comparing between three organisms, the lactic

acid was decrease S. aureus 5.39 log cycles and P.
aeruginosa 4.78 log cycles and Debaryomyces sp. 4.48
log cycles at sub-lethal conc. 0.1% (W/V), 0.2% (W/V) and
4% (W/V), respectively. This shows that, the
Debaryomyces sp. was relatively resistant to lactic acid
followed by P. aeruginosa in comparing by S. aureus.
123
Table (8): Effect of different conc. of lactic acid on Debaryomyces sp.
Conc.% Average count cfu m-1 Log N* Log N/No

Initial 6.92
85 x 105 0
count ± 0.069 A
1.5 37 x 104 5.56 - 1.36

± 0.041 B
2 93 x 103 4.96 - 1.96

± 0.039 D
2.5 16 x 103 4.20 - 2.72

± 0.048 E
3 43 x 102 3.63 -3.29

± 0.021 F
4 28 x 10 2.44 - 4.48
± 0.045 G
4.5 -** -** -**

H
** The dash means no distinct growth
Conc.%
0
0 0.5 1 1.5 2 2.5 3 3.5 4 4.5
-0.5
-1
-1.5
-2
Log N/No
-2.5
-3
-3.5
-4
-4.5
-5
Fig. (8): Effect of different conc. of lactic acid on Debaryomyces sp.
124
Conc. %
0
0 0.05 0.1 0.15 0.2 0.25
-1
-2
Log count
-3
-4
-5
-6
Fig (9): Effect of different conc. of lactic acid on P. aeruginosa and S.

aureus.
125
Table (9): Effect of different conc. of lactic acid on P. aeruginosa and S. aureus
Conc.% Average count cfu Average count cfu
Log N* Log N/No Log N* Log N/No
m-1 m-1
Initial count 96 x 105 6.98 0 98 x 105 6.99 0
± 0.045 A ± 0.014 A
6.68
0.001 48 x 105
± 0.045 A
- 0.30 Not tested
5.64 6.46
0.01 47 x 104
± 0.042 B
- 1.31 29 x 105
± 0.048 B
- 0.52
3.06
0.05 62 x 102
± 0.082 C
- 3.18 Not tested
1.59 4.41
0.10 40
± 0.064 D
- 5.38 26 x 103
± 0.047 D
- 2.57
-**
0.15 -**
E
-** Not tested
2.23
0.20 17 x 10
± 0.098 E
- 4.76
-**
0.30 -**
F
-**
126
By comparing between the lactic acid and citric
acid and its antimicrobial activity against the treated
microorganisms it was showed that: (i) with S. aureus the
sub lethal conc. of lactic acid and citric acid was at 0.10%
(W/V) and 0.15% (W/V) respectively, lethal conc. of lactic
acid was 0.15% (W/V) and citric acid was 0.2% (W/V). (ii)
With P. aeruginosa the sub lethal conc. of lactic acid and
citric acid was the same at 0.20% (W/V), lethal conc. 0.3%
(W/V). (iii) With Debaryomyces sp. the sub lethal conc. of
lactic acid and citric acid was at 4.0% (W/V) and 3.5%
(W/V) respectively, lethal conc. of lactic acid was 4.5%
(W/V) and citric acid was 4% (W/V).
From the previous comparison, the sub lethal

concentration of citric acid was less than lactic acid with
S. aureus and Debaryomyces sp., and the same conc.
with P. aeruginosa.
127
Acid tolerance can be viewed in terms of efflux ability.
The mechanism by which organic acids inhibit
microorganisms involves passage of the un-dissociated
form of the acid across the cell membrane lipid bilayer.
Once inside the cell, the acid dissociates because the cell
interior has a higher pH than the exterior. Protons
generated from intracellular dissociation of the organic
acid and other organic acids via several mechanisms. They
use the enzyme H+-ATPase along with ATP (adenosine
triphosphate) energy to remove excess protons from the
cell. Inhibition and/or inactivation of yeasts may be due to
eventual loss of cellular energy or inactivation of critical
cellular functions due to low intracellular pH (Fig. 10). To
prevent energy depletion, a membrane protein may be
induced for decreasing ATPase activity and thus conserve
energy (Brul and Coote, 1999). To circumvent the
problem of extruded anions and protons reentering the cell
upon recombining in the extra cellular medium, adapted
yeasts apparently reduce diffusion of the weak acids, most
likely by altering cell membrane structure to reduce
passage of the acids into the cell (Brul and Coote, 1999).
128
Fig. (10) A schematic impression of the stress response of a yeast
cell challenged with weak organic acids (Piper et al., 1998).
It is clear from the above results that S. aureus was

the most sensitive of treated organisms to both citric acid
and lactic acid followed by P. aeruginosa while
Debaryomyces sp. was the highest resistant strain.
Gram- positive bacteria (such as S. aureus) do not

possess an outer membrane and the exclusion limit of the
cell wall of vegetative cells (Lambert, 1983), hence
preservatives can easily enter these cells and their
intrinsic resistance is relatively low. In gram- negative
bacteria (such as P. aeruginosa), resistance mechanisms
129
are more complicated since these organisms possess an
inner and outer membrane; the latter membrane has a
clear role in modulating the accessibility of a cell to
preservatives and other small molecules (Vaara, 1992 &
Helander et al., 1997).
The actual inhibitory action of weak acid

preservatives on yeast could be due to the induction of
energetically expensive stress respond that attempts to
restore homeostasis and results in the reduction of
available energy pools for growth and other essential
metabolic functions (Holyoak et al., 1996, Bracey et al.,
1998).
Indeed, lactic acid was recently shown to permeabilize
Gram-negative bacteria efficiently and causes LPS release
(Alakomi et al., 2000) and citric acid is metal chelating
(Verhoff, 1986).
130
2.2.3- Cinnamon
In recent years, because of negative perception
of synthetic additives, consumers are demanding natural
and fresh-like food products with guaranteed safety and
long shelf- life. For this reason, there is a renewed interest
in the use of spices, condiment and plant extracts as
alternative food processing method.
Several authors (Yousef and Tawil, 1980;
Deans and Ritchie, 1987; Baratta et al., 1998; Smith-
palmer et al., 1998) reported that cinnamon was one of
the inhibitoriest against pathogens such as Salmonella
sp.., Y. enterocolitice and S. aureus. Ground cinnamon
(0% (W/V) and 0.3% (W/V) was added to apple juice to
study the sensitivity of Salmonella typhimurium,
Yersinia Enterocolitica and S. aureus (Yuste and Fung,
2003). Yousef and Tawil (1980) and Smith-palmer et
al., (1998) found that the minimum bacteriostatic conc.
was equivalent to the minimum bactericidal conc. for S.
aureus.
Tables (10 & 11), fig (11 & 12) showed that, the
growth of Debaryomyces sp. had shoulder at the
beginning of the curve till conc. 0.6% (W/V), followed by
exponential rate death indicating the resistance of
131
Debaryomyces sp.. to cinnamon at low concentrations.
The increase in cinnamon concentration after shoulder
part was paralleled to the decrease in the number of
survival yeast cells. The sub lethal was at conc. 1.7%
(W/V).
Mean while, the growth curves of both S. aureus and

P. aeruginosa, showed straight-line curve. S. aureus was
effect by increasing the conc. of cinnamon and the sub
lethal conc. was at 1% (W/V). While, the sub lethal conc.
for P. aeruginosa was at conc. 3% (W/V).
By comparing between the treated organisms it was
found that, S. aureus was highly sensitive to cinnamon
than P. aeruginosa and Debaryomyces sp., while P.
aeruginosa was more resistant to cinnamon than
Debaryomyces sp.
132
Conc.
0
0 0.2 0.4 0.6 0.8 1 1.2 1.4 1.6 1.8
S. aureus Debaryomyces spp.

-1
-2
Log count
-3
-4
-5
-6
Fig. (11): Effect of different conc. of cinnamon on S. aureus and

Debaryomyces sp.
133
. Table (10): Effect of different conc. of cinnamon on S. aureus and Debaryomyces sp
Staphylococcus aureus Debaryomyces sp.
Conc.%
Average count cfu m-1 Log N* Log N/No Average count cfu m-1 Log N* Log N/No
7.0 5.84
Initial count 102 x 105 0 70 x 104 0
± 0.016 A ± 0.069 A
6.51
0.2 33 x 105
± 0.123 B
- 0.49 Not tested
5.62
0.3 Not tested 42 x 104 - 0.21
± 0.084 A
5.79
0.4 63 x 104
± 0.038 C
- 1.20 Not tested
4.67 5.54
0.6 47 x 103 - 2.33 35 x 104 - 0.3
± 0.017 D ± 0.044 B
2.95
0.8 9 x 102
± 0.04 E
- 4.05 Not tested
1.43 4.32
1.0 27 - 5.57 21 x 103 - 1.52
± 0.036 F ± 0.034 C
-** -**
1.2 -**
G
Not tested
3.07
1.5 12 x 102 - 2.76
± 0.026 D
2.49
1.7 31 x 10 - 3.35
± 0.036 E
-**
2 -** -**
F
134
Table (11): Effect of different conc. of cinnamon on P. aeruginosa
Average count cfu

Conc.% Log N* Log N/No
m-1
Initial 6.74
55 x 105 0
count ± 0.014 A
0.5 36 x 104 5.55 - 1.18

± 0.065 B
1 43 x 103 4.63 - 2.10

± 0.067 C
2 29 x 102 3.46 - 3.27

± 0.067 D
3 86 1.93 - 4.80
± 0.055 E
4 -** -** -**

F

** The dash means no distinct growth.
Conc.%
0
0 0.5 1 1.5 2 2.5 3 3.5 4 4.5
-1
-2
Log N/No
-3
-4
-5
-6
Fig. (12): Effect of different conc. of cinnamon on

P. aeruginosa
135
Cinnamon effectively inhibits growth of bacteria,
yeasts and molds (Smith-palmer et al., 1998). Its
essential oil fraction contains cinnamaldehyde and
eugenol as major antimicrobial compounds (Shelef,
1983).
Confirming our results Shelef (1983) and Zaika
(1988) stated that gram-positive bacteria are more
sensitive to cinnamon than gram-negative bacteria. This is
probably due to the outer membrane of gram-negative
bacteria, which is highly hydrophilic and acts as a strong
barrier (Smith-Palmer et al., 1998).
The antimicrobial activity of cinnamon due to its

essential oils, which are lipophilic and could penetrate
through the membrane to the interior of the cell and
perform the inhibitory activity at the target site (Ramos-
Nino et al., 1996 and Smith-Palmer et al., 1998).
Smith-Palmer et al. (1998) determined that the

24h. minimum inhibitory concentrations of cinnamon and
clove essential oils against Campylobacter jejuni,
Escherichia coli, Salmonella Enteritidis, Listeria
monocytogenes, and S. aureus were 0.05, 0.04-0.05,
0.04-0.05, 0.03, and 0.04% (W/V), respectively, in an agar
136
dilution assay. Cinnamic aldehyde or thymol (600 mg/liter
of air) significantly reduced Salmonella populations on
alfalfa seeds used for sprouting and did not affect
germination (Weissinger et al., 2001).
In conclusion the inhibitory effect of cinnamon
on Gram positive bacteria, Gram negative bacteria, yeast
organisms was observed, which suggests the potential use
of this spice in the food industry for enhancement of safety
furthermore, sensory attributes given by cinnamon may
appeal to consumer . These, if foods and beverages are
manufactured under sanitary conditions so that they have
relatively low microbial conditions so that they have
relatively low microbial counts, naturally occurring food
grade ingredients with antimicrobial properties can give
substantial protection and replace some artificial
preservations (Yuste and Fung, 2003).
137
3. Combination treatments
The antimicrobial effect of preservation

treatment can be optimized by combining them as hurdles
in an overall preservation strategy by placing a number of
sub-lethal stresses (i.e. hurdles) on a microbial cell, the
organisms expends energy to metabolic exhaustion and
death (Leistner, 2000). Hurdles targeting the same
elements within the cell have an additive inhibitory effect
only, where as synergistic effects may result from
disturbing several functions of the cell (Leistner, 1992;
1994).
Proper application of combined methods gives stable
products, prevents the undesired side effect of each
individual treatment, saves energy and lowers the required
concentration of added preservatives (Pokorn, 1994; Abd
El-karem and Farag, 1996).
3.1. Combination with Gamma Irradiation
According to Leistner (2000), attacking various

cellular targets will have a synergistic effect by making the
organism strain every possible repair mechanism and the
activation of stress shock proteins also, becomes more
difficult.
138
Gamma irradiation is considered the only known
technique capable of ensuring the hygienic quality of raw
food in a fresh or frozen state (Loaharanu, 1995). So
combining this treatment by other preservatives will give
synergistic effects on undesirable microorganisms
contaminating food and beverages.
3.1.1. Combination of Gamma irradiation with

lactic acid
From the previous experiments, Lactic acid was

used in conc. 0.01% (W/V), 0.1% (W/V), 1.5% (W/V) for S.
aureus, P. aeruginosa, and Debaryomyces sp.,
respectively, at this conc. lactic acid start to give distinct
reduction for the growth count as shown previously in
single treatments tables (8 & 9).
As shown in table (12) illustrated by fig (13), it is

clear that, the dose level 2 kGy combined with lactic acid
was sufficient to eliminate both S. aureus and P.
aeruginosa while Debaryomyces sp. neede 3 kGy . The
D10 values of the previous organisms were 0.27, 0.40 and
0.47 kGy for S. aureus, P. aeruginosa and
139
Debaryomyces sp., in the same sequence comparing by
0.75, 0.69 and 1.48 kGy for the same organisms in the
single treatments with gamma rays, respectively. These
results clearly indicated the synergistic effect of the
combined gamma rays by lactic acid. Also, the dose levels
needed for eliminated the tested organisms were reduced
from 5 kGy to 2 kGy for S. aureus and from 4 kGy to 2
kGy for P. aeruginosa, whiledestorying the cells of
Debaryomyces sp. neede only 3 kGy instead of 7 kGy in
the case of single treatment.
Doses kGy
0
0 0.5 1 1.5 2 2.5
-1 Staphylococcus aureus
Pseudomonas aeruginosa
-2 Debaryomyces spp.
Log N/No
-3
-4
-5
-6
Fig. (13): Effect of different doses of gamma irradiation in

140
Table (12): Effect of different doses of gamma irradiation in combination with lactic acid
Organism Staphylococcus aureus Pseudomonas aeruginosa Debaryomyces sp.
Lactic acid
0.01 0.1 1.5
conc. %
Average count Log Average count Log Average count Log
Doses kGy Log N* Log N* Log N*
cfu m-1 N/No cfu m-1 N/No cfu m-1 N/No
Initaial count 25 x 106 7.39 - 65 x 106 7.81 - 55 x 105 6.74 -

± 0.030 A ± 0.011 A ± 0.032 A
Control 6.68 4.41 5.56

48 x 105 0 26 x 103 0 37 x 104 0
lactic acid ± 0.045 B ± 0.047 B ± 0.048 B
0.25 Not tested 98 x 102 3.99 - 0.42 Not tested

± 0.022 C
0.5 11 x 103 4.04 - 2.63 26 x 102 3.41 - 1.0 13 x 103 4.11 - 1.45
± 0.050 C ± 0.037 D ± 0.05 C
1 25 x 10 1.39 - 4.28 14 x 10 2.14 - 2.26 82 x 10 2.91 - 2.65

± 0.042 D ± 0.05 E ± 0.057 D
2 -** -** -** -** -** -** 9 .95 - 4.61

E F ± 0.024 E
3 -** -** -**

F
D10 0.27 kGy 0.40 kGy 0.47 kGy

141
citric acid
Citric acid was used in conc. 0.05% (W/V), 0.1%
(W/V), 1.5% (W/V) for S. aureus, P. aeruginosa, and
Debaryomyces sp., respectively, at this conc. citric acid
start to give distinct reduction for the growth count as
shown previously in tables (6 & 7).
It was obvious from table (13) and Fig (14), a

straight-line curve with a logarithmic decrease was
obtained. The death-value was at 2 kGy for S. aureus, 3
kGy for P. aeruginosa, while Debaryomyces sp.. requires
4 kGy to destroy its cells. D10 was 0.32, 0.58, and 0.75
kGy, respectively for the same organism's combaring by
single treatments 0.75, 0.69 and 1.48 kGy, respectively.
On the other hand, by comparing between lactic

acid and citric acid in combination with gamma
irradiation, lactic acid was more active against the treated
organisms than citric acid and the D10 values where
distinct decreased from single treatment. This may due to
lactic acid has lipophilic character; ability to depress
intracellular pH. In addition to membrane diffusion and
water activity reduction ' specific anion effect', while citric
acid lack this character and acting as chelating agent
142
(Smulders, 1987; Stratford, 1999, Saltmarsh, 2000 and
Naidu, 2000)
Ross et al. (2003) reported that the lethal

effects of ionized radiation are not greatly enhanced by
mild acidification. The addition of naturally occurring
antimicrobial has proven to be an effective hurdle when
combined with non-thermal processing techniques (Ross
et al., 2003).
Doses kGy
0
0 0.5 1 1.5 2 2.5 3 3.5
-0.5
Staphylococcus aureus
-1
-1.5
Debaryomyces spp.
-2
Log count
-2.5
-3
-3.5
-4
-4.5
-5

143
Table (13): Effect of different doses of gamma irradiation in combination with citric acid
Citric acid conc.
0.05 0.1 1.5
%
Average count cfu Log Average count Log Average Log
Doses KGy Log N* Log N* Log N*
m-1 N/No cfu m-1 N/No count cfu m-1 N/No
Initial count 32 x 106 7.50 - 65 x 106 7.81 - 41 x 105 7.50 -

± 0.031 A ± 0.030 A ± 0.047 A
Control 6.02 5.25 5.95

citric acid
10.5 x 105 ± 0.030B
0 18 x 104 ± 0.029 B
0 90 x 104 ± 0.020 B 0
0.5 18 x 102 3.25 - 2.76 83 x 103 4.91 - 0.33 98 x 103 4.99 - 0.96
± 0.094 C ± 0.070 C ± 0.038 C
1 50 1.69 - 4.32 14 x 103 4.14 -1.1 18 x 103 4.25 - 1.69

± 0.064 D ± 0.058 D ± 0.013 D
2 -** -** -** 24 x 10 2.38 -2.87 78 x 10 2.99 - 3.06

E ± 0.042 E ± 0.015 E
3 -** -** -** 45 1.65 - 4.3

F ± 0.008 F
-**
4 -**
G
-**
D10 0.32 kGy 0.58 kGy 0.75 kGy

144
cinnamon
From the previous experiments it is clear that dose

level 0.75, 0.69 and 1.48 kGy was needed to reduce one
log cycle of viable cells of S. aureus, P. aeruginosa, and
Debaryomyces sp., respectively (table 3). On the other
hand, low concentrations of cinnamon did not affect the
viable counts of the tested organisms. Concentration of
0.4% (W/V) of cinnamon started to decrease the viable
counts of S. aureus indicating its sensitivity to cinnamon
comparing with the Debaryomyces sp., P. aeruginosa
whose started to be affected by cinnamon after
concentration 1.0 %(W/V) and 0.5%(W/V), respectively
tables (10 & 11). So, in the present experiment
concentration of 0.4, 0.5 and 1.0 %( wt/v) of cinnamon
were used combined with gamma rays.
As shown in table (14) and fig. (15), it is clearly that
combined treatments reduced the D10 value of the tested
organgisms to 0.31, 0.57 and 1.02 kGy for S. aureus, P.
aeruginosa, and Debaryomyces sp., respectively. Hence,
the dose levels of gamma rays needed to eliminate the
previous organisms decreased from 5 to 2 kGy, from 4 to
3kGy and from 7 to 4 kgy for S. aureus, P. aeruginosa,
and Debaryomyces sp., in the same sequence.
145
Shelef (1983) and Zaika (1988) stated that
Gram-positive bacteria are more sensitive to cinnamon
than Gram-negative bacteria. This is probably due to the
outer membrane of gram-negative bacteria, which is highly
hydrophilic and acts as a strong barrier (Smith-Palmer et
al., 1998).
The primary mode of action of ionizing radiation is via
oxygen and hydroxyl radicals (Niemira, 2003).In
suspensions with a high antioxidant capacity, these
radicals can be neutralized before doing damage to
bacterial cell membrane, protein structures, and nucleic
acid strands, thereby protecting the bacteria and reducing
the efficacy of the process (Sommers, 2003).
Doses kGy
0
0 0.5 1 1.5 2 2.5 3 3.5
-0.5
-1
Debaryomyces spp.
-1.5
-2
Log N/No
-2.5
-3
-3.5
-4
-4.5
146
Table (14): Effect of different doses of gamma irradiation in combination with cinnamon
Cinnamon conc.
%
0.4 0.5 1.0
Average Log Average count Log Average count cfu Log
count cfu m-1 N/No cfu m-1 N/No m-1 N/No

± 0.031 A ± 0.02 A ± 0.046 A
Control 5.79 5.55 4.32

Nisin
63 x 104 ± 0.038 B
0 36 x 104 0 21 x 103 ± 0.034 B
0
± 0.065 B
0.5 28 x 103 4.44 -1.35 59 x 103 4.77 -0.78 98 x 102 3.99 -0.37
± 0.03 C ± 0.064C ± 0.007 B
1 72 x 10 2.85 -2.94 98 x 102 3.99 -1.56 38 x 102 3.57 -0.74

± 0.017 D ± 0.043D ± 0.017 C
2 -** -** -** 15 x 10 2.17 -3.38 53 x 10 2.72 -1.59

E ± 0.027 E ± 0.033 D
3 -** -** -** 35 1.54 -2.77

F ± 0.030 E
4 -** -** -**

F
D10 0.31 kGy 0.57 kGy 1.02 kGy
147
nisin
From the previous experiment, application of nisin

did not affect either P. aeruginosa or Debaryomyces sp.
On the other hand nisin at concentration 25 µg/ml
decreased the viable count of S. aureus approximately one
log cycle in single treatment table (5). Several investigators
has reported about the synergistic effect ofcombined
treatments on microorganisms.the object of the present
experimentwas to study the effect of gamma radiation
combinedwith nisin on the S. aureus besides its its effect
on the cell wall of both of P. aeruginosa and
Debaryomyces sp. which may permit the nisin to enter
their cells and hence assist to destroy the cells. In the case
of S. aureus, gamma radiation was combined with 25
µg/ml of nisin. While, 200 µg/ml of nisin was used in case of
P. aeruginosa and Debaryomyces sp.
Data presented in table (15) and fig. (16) clearly

showed that, cmbined treatment of gamma rays and nisin
sharply affcte the viable count of S. aureus and dose level
3 kGy was sufficient to destroy its cells, giving D10 value
0.48 kGy, while it was 0.75 kGy in case of single
treatment of gamma rays.
148
As expected from the previous experiment, the results
clealy indicated that gamma rays affected the cell wallsof
both P. aeruginosa and Debaryomyces sp. which permit
the nisin to enter theircells andgave synergistic effect on
destroying the cellsof both organisms. In the case of P.
aeruginosa the data showed that the combined treatment
decreased its D10 values from 0.69 to 0.45 kGy and hence
dose level 3 kGy wasquiet sufficient to eliminate its cells
from themedium instead of 4 kGy in the single treatment.
The same trend was occurred in case of Debaryomyces

sp.; hence its D10 value was decreased from 1.48 kGy in
single treatment to 0.68 kGy in combined with nisin. Also,
dose level 4 kGy eleminat its cells insteated of 7 kGy in the
single treatment with gamma rays alone.
It is clear from the previous data that, the highly

resistant of P. aeruginosa and Debaryomyces sp. to
nisin become more sensitive to the natural preservative
and this may be due to the injuring by irradiation.
Irradiated bacteria may be more easily lyses than non-
irradiated bacteria (Andrej Trampuz et al., 2006).
Both direct and indirect reactions between
ionizing radiation and cellular components occur in direct
149
proportion to the amount of energy that is absorbed. Since
50 to 70% of the bacterial cell mass is water, it absorbs
much of the radiation. As a result, hydroxyl radicals and
hydrated electrons (which are important in irradiation-
induced cell inactivation) are produced (Kim and Thayer,
1996). These radicals damage cell membranes beside
protein structures and nucleic acid strands (Niemira and
Solomon, 2005). And hence increase the permeability of
the cell wall which permite to the preservative to enter the
cell.
150
Doses kGy
0
0 0.5 1 1.5 2 2.5 3 3.5
-0.5
-1 Pseudomonas aeruginosa
Debaryomyces spp.
-1.5
-2
Log N/No
-2.5
-3
-3.5
-4
-4.5
-5

combination with Nisin.
151
Table (15): Effect of different doses of gamma irradiation in combination with nisin
Nisin conc.
µg/ml
25 200 200
± 0.062 A ± 0.028 A ± 0.032 A
Control 5.63 6.99 6.61
Nisin
43 x 104 ± 0.064 B
0 98 x 105 ± 0.0 A
0 41 x 105 ± 0.0 A
0
0.5 64 x 103 4.80 - 0.82 64 x 104 5.80 - 1.18 91 x 104 5.95 - 0.65
± 0.039 C ± 0.021 B ± 0.050 B
1 65 x 102 3.81 - 1.82 64 x 103 4.80 - 2.18 26 x 104 5.41 - 1.19

± 0.034 D ± 0.026 C ± 0.053 C
2 53 1.72 - 3.9 32 x 10 2.50 -4.48 74 x 102 3.86 - 2.74

± 0.046E ± 0.058 D ± 0.006 D
3 -** -** -** -** -** -** 23 x 10 2.36 - 4.25

F E ± 0.003 E
4 -** -** -**

F
D10 0.48 kGy 0.45 kGy 0.68 kGy
152
• General Discussion
Gamma irradiation enhances the shelf-life of foods and

ensures their innocuousness because most
microorganisms in vegetative form are extremely sensitive
to irradiation at low doses (Radomyski et al., 1994).
The lethal effect of ionizing radiation is primarily due to
DNA damage, which destroys the reproductive capabilities
and other functions of the cell (DeRuiter and Dwyer,
2002).
On the other hand, 'minimal processing' is a concept

describing approaches to food safety and preservation that
are designed to retain the natural and as-fresh properties
of foods (Manvell, 1997). Hence, gamma irradiation can
be used in low doses (which cause injuring to microbial
cell) in combination with other prsevatives.
However, in some circumstances combined treatments

are more satisfactory since the dose required for complete
sterilization can induce undesirable changes in food flavor
or is at higher than permitted level (Thakur and Singh,
1995). Effective Combinations of two or more preservation
hurdles may be chosen once the modes of action and
153
cellular targets of each treatment are known (Leistner,
2000).
As mention previously, nisin, cinnamon, citric acid and
lactic acid acts on cell membrane of microbial cell. So, the
combination of these preservatives with gamma irradiation
it implies more stress will acts on the same target of the
microbial cell this refers to hurdles (by placing a number
of sub lethal stresses on microbial cell which agree with
Leistner, (2000). Hurdles targeting the same elements
within the cell have an additive inhibitory effect only,
whereas synergistic effects may result from disturbing
several functions of the cell (Leistner, 1992, 1994).
154
3.1.5. Comparision between gamma irradiation
single and combined treatments
Table (16) and Fig. (17) shows that, the D10 of gamma
irradiation in single treatment for S. aureus was 0.75 kGy
which decreased to 0.32, 0.27, 0.31 and 0.48 kGy by
combination with citric acid, lactic acid, cinnamon and
nisin, respectively.
Also, the D10 of P. aeruginosa was 0.69 kGy which
reduced to 0.58 kGy by combination with citric acid,
reduced to 0.40 kGy by combination with lactic acid,
lowered to 0.57 kGy by combination withcinnamon, and
reduced to 0.45 kGy by combination with nisin.
The D10 of Debaryomyces sp. was 1.48 kGy which
reduced to 0.75 kGy by combination with citric acid and
reduced to 0.47 kGy by combination with lactic acid. Also,
this dose was decreased to 1.02 and 0.68 kGy by
combination with cinnamon and nisin, respectively.
155
Table (16): D10 - values of gamma irradiation for single and
combined treatments
Debaryomyces
Treatment sp.
D10 value kGy
Radiation alone 0.75 0.69 1.49
Radiation and citric

0.32 0.58 0.75
acid
Radiation and lactic
0.27 0.40 0.47
acid
Radiation and
0.31 0.57 1.02
cinnamon
Radiation and nisin 0.48 0.45 0.68
1.6 1.48
Staphylococcus aureus Pseudomonas aerogenosa Debaryomyces spp.
1.4
1.2
1.02
0.69
1
0.75
D10 kGy
0.75
0.8
0.68
0.58 0.57
0.45
0.6
0.47 0.48
0.4
0.4 0.32 0.31

0.27
0.2
0
ne cid cid on nisin
on alo citric a lactic a cinnam on and
Radiati on and on and on and Radiati
Radiati Radiati Radiati
Treatment
Fig. (17): D10 - values of gamma irradiation for single and combined
treatments.
156
3.2. Combination with Pasteurized temperature
Thermal treatment leads either to

pasteurization, when only vegetative microorganisms are
destroyed or sterilization, when spores are also destroyed.
Food preservation implies exposure of microorganisms
contaminated the food to hostile conditions. Synergies are
particularly interesting because combination of processes
i.e., hurdle technology, are a promising means of
enhancing safety whilst retaining food quality.
In combination commonly pasteurized temperature,
90 ْC for 30sec was used.
3.2.1. Combination of pasteurized temperature

with Nisin
Data presented in table (17) clearly indicated

that, S. aureus was highly sensitive to both high
temperature and nisin. The initial count log 7.53 sharply
decreased to log 3.74 by temperature alone while
combined it with 25 μg/ml nisin, completely desteroyed all
the organism cells. Although both of P. aeruginosa and
Debaryomyces sp. revealed highly resistance to nisin
even at higher concentration, but combined treatment
157
with pasteurization assisted to eliminate them from the
medium. Application of 50 μg/ml of nisin combined with
pasteurized temperature (90 ْC for 30sec) completely
inhibited the cells of Debaryomyces sp. while P.
aeruginosa needed 150 μg/ml of nisin to destroy the
cells in the medium as clearly shown in table (17).
Allan et al., 1988 demonstrated HSP in P.
aeruginosa following a temperature shift from 30 to 45 ْC
that led to the synthesis of at least a new 17 proteins
within approximately 1 min.
Heat can damage protein, lipids, and nucleic acids

and destabilize membranes (Gould, 1989). Heating-
induced destabilization, such as oxidation of sulfydryl
groups of the membrane – binding bound proteins, Yatvin
and Grummer, 1987). This disturbance in the cell
especially in the cell membrane may due to the entrance of
nisin to the cell and induced its death.
158
Table (17): Effect of pasteurized temperature 90 Cْ for 30 sec in combination with different conc. of nisin
Conc. ug/ml Average Average Average

Log Log Log
count cfu Log N*
N/No
count cfu Log N*
N/No
count cfu Log N*
N/No
m-1 m-1 m-1

± 0.043 A ± 0.03 A ± 0.013 A
Control 3.74 4.46 5.07

55 x 102 0 29 x 103 0 118 x 103 0
temperature ± 0.014 B ± 0.058 B ± 0.022 B
25 -** -** -** Not tested 15 x 102 3.17 - 1.89

C ± 0.040 C
50 20 x 102 3.30 - 1.16 -** -** -**

± 0.037 C D
100 15 1.17 - 3.28

± 0.050 D
150 -** -** -**

E
159
3.2.2. Combination of pasteurized temperature
with citric acid
It is clear from table (18) that, pasteurized temperature

at 90 ْC for 30sec. greatly affected all the tested
organisms. Pasteurization treatment alone decrease the
viable count of S. aureus more than 4 log cycle while it
decrease the viable count about 2 log cycle in case of P.
aeruginosa and Debaryomyces sp..combined the
pasteurized temperature with only 0.1 % (W/V) of citric
acid, completely destroyed all the cells of S. aureus while
0.15 %(W/V) of citric acid was needed to eliminate P.
aeruginosa. The rest viable cells of Debaryomyces sp.
revealed relativily high resistance to the combined
treatment and 3 % (W/V) of citric acid was applied to
annihilate its cells.
160
Table (18): Effect of pasteurized temperature 90 Cْ for 30 sec in combination with different conc. of citric acid

Conc. % (W/V) Average Average Average
Log Log Log
count cfu Log N* count cfu Log N* count cfu Log N*
N/No N/No N/No
m-1 m-1 m-1
± 0.038 A ± 0.020 A ± 0.013 A
Control 3.74 4.46 5.07
55 x 102 0 29 x 103 0 118 x 103 0
temperature ± 0.014 B ± 0.058 B ± 0.022 B
0.05 67 1.82 -1.91 Not tested Not tested

± 0.064 C
0.1 -** -** -** 41 1.61 - 2.84 Not tested

D ± 0.031 C
0.15 -** -** -** Not tested

D
1 64 102 3.8 - 1.26

± 0.027 C
2 17 x 10 2.21 - 2.84
± 0.044 D
2.5 21 1.32 - 3.74

± 0.021 E
3 -** -** -**

F
161
3.2.3. Combination of pasteurized temp with
lactic acid
From table (19), lactic acid combined with

pasteurized temperature revealed to be more effective for
elimination the tested organisms than citric acid. In the
case of S. aureus and P. aeruginosa, 0.05% (W/V) and
0.15 %( wt/v) were sufficient to completely destroy their
viable cells while only 0.5% (W/V) of latic acid was needed
to eliminate Debaryomyces sp. compared with 3 % (W/V) of
citric acid
The data of combination of pasteurized

temperature with citric acid and lactic acid shows that, the
lactic acid more effective with low conc. this may due to
lactic acid has lipophilic character; ability to depress
intracellular pH. In addition to membrane diffusion and
water activity reduction ' specific anion effect', while citric
acid lack this character and acting as chelating agent
(Smulders, 1987; Stratford, 1999, Saltmarsh, 2000;
Naidu, 2000).
162
Table (19): Effect of pasteurized temperature 90 Cْ for 30 sec in combination with different conc. of lactic acid

Conc. %
(W/V) Average Average Log Average
Log N* Log N/No Log N* Log N* Log N/No
count cfu m-1 count cfu m-1 N/No count cfu m-1
Initial count 21x 106 7.32 - 35 x 105 7.39 - 65 x 105 6.81 -

± 0.048 A ± 0.03 A ± 0.013 A
Control 3.74 4.46 5.07

55 x 102 0 29 x 103 0 118 x 103 0
temperature ± 0.014 B ± 0.058 B ± 0.022 B
0.001 35 x 10 2.54 - 1.19 Not tested Not tested

± 0.021 C
0.01 24 1.38 -2.36 75 x 102 3.87 - 0.58 Not tested

± 0.053 D ± 0.031 C
0.05 -** -** -** Not tested Not tested

E
0.1 45 1.65 - 2.8 48 x 10 2.68 -2.39

± 0.033 D ± 0.026 C
0.15 -** -** -** Not tested

E
0.5 -** -** -**

D
163
3.2.4. Combination of pasteurized temp with
cinnamon
It is clear from table (20) that, pasteurized
temperature at 90 ْC for 30sec. greatly affected all the
tested organisms. Pasteurization treatment alone decrease
the viable count of S. aureus more than 4 log cycle while
it decrease the viable count about 2 log cycle in case of P.
aeruginosa and Debaryomyces sp.. Combined the
pasteurized temperature with only 0.5 %(W/V) of
cinnamon, completely destroyed all the cells of S. aureus
while 0.6 % (W/V) of citric acid was needed to eliminate
both of P. aeruginosa and Debaryomyces sp.. And by
comparing with single treatment of cinnamon (tables 11,
12) it showed that, the lethal concentration of cinnamon
was reduced from 1.2, to 0.5 for S. aureus while 2, and
4%(W/V) were reduced to 0.6%( wt/v) for P. aeruginosa and
Debaryomyces sp., respectively.
Shelef (1983) and Zaika (1988) stated that Gram-

positive bacteria are more sensitive to cinnamon than
gram-negative bacteria. This is probably due to the outer
membrane of gram-negative bacteria, which is highly
al., 1998).
164
According to Moats (1971), cell death results from
the denaturation or inactivation of numerous critical sites
(or a large number of the same site) in cells. Sub-lethally
injured bacterial cells are reported to become sensitive to
bactericins of lactic acid bacteria (Kalchayanand et al.,
1992 and Ray, 1993).hence the pasteurized temperature
increases the sensitivity of both P. aeruginosa and
Debaryomyces sp. to cinnamon
Hurst, 1977 and Gould, 1989 reported that heat

can damage protein, lipids, and nucleic acids and
destabilize membranes. However, heat induces primarily
blebbing and vesiculation of the outer membrane leading
to bacteria cell inactivation, and can sequentially increases
its permeability to hydrophobic compounds (Scheie and
Ehrenspeck, 1973 and Tsuchido and Takano, 1988).
Heating induced membrane destabilization

sequentially initiates indirect DNA damage as a
consequence of increased nuclease activity in bacteria and
is often the critical injury , resulting in death of the cell
(Sedgwick and Bridges, 1972; Gomez, 1977; Kadota et
al., 1978 and Gould, 1989). Because multiple changes
may occur during the inactivation of bacteria by heat,
165
thermal inactivation is not linear (Hurst, 1977 and Gould,
1989). Similarly the mechanism of inactivation of bacteria
by heat, it may also occur with yeast.
166
Table (20): Effect of pasteurized temperature 90 Cْ for 30 sec in combination with different conc. of cinnamon
Conc. % Average Average Average

count cfu Log N* Log N/No count cfu Log N* Log N/No count cfu m-1 Log N* Log N/No
m-1 m-1
6.74 6.85 6.65
Initial count 56 x 105 ± 0.049 - 72 x 105 ± 0.020 A
- 45 x 105 ± 0.118 -
A A
Control 3.74 4.46 5.07
55 x 102 ± 0.014 0 29 x 103 0 118 x 103 ± 0.022 0
temperature B
± 0.058 B
B
1.93 2.81 2.78
0.3 86 ± 0.049 - 1.8 66 x 10 ± 0.033 C
- 1.64 58 x 10 ± 0.057 -2.3
C C
0.5 -** -** -** 22 1.34 - 3.11 Not tested

D ± 0.025D
0.6 -** -** -** -** -** -**

E D
167
• General disscusion
Exposure of heat-stressed microbial cells generally
results in cytoplasmic membrane disfunction
characterized by a release of nucleic acids, amino acids,
ATP, and K+ (Beuchat et al., 1997). In the same manner
the primary action site of nisin against vegetative cells is
considered to be the cytoplasmic membrane, with nisin
acting as a voltage-dependent polarizer (Ruhr and Sahl,
1985 and Delves-Broughton and Gasson, 1994). Also,
lactic acid was recently shown to permeabilize Gram-
negative bacteria efficiently and causes LPS release
(Verhoff, 1986).
As, mentioned previously gram-negative bacteria,
are normally insensitive to lysozyme and nisin because
their outer membrane prevents access to the sites of
action of these agents. Similarly, in yeast are insensitive to
Nisin and may make adaptation to make organic acids as
mentioned previously in single treatment. Hence, the
sensitivity of P. aeruginosa, Debaryomyces to nisin,
organic acid were increased during inactivation by
pasteurized temp according to the mode of action of
pasteurized temp which mentioned previously. Effective
combination of two or more preservation hurdles may be
168
In application manner, it is feasible to produce a
safe apple cider by combining heat with pH modification
and preservative addition. Dock et al, (2000) investigated
the effect of pH and preservatives on the heat resistance of
E. coli O157:H7 in apple cider. The D- values at 50ْC of
65min in apple cider were reduced to 13.9, 13.2 and 7.0
min in apple cider with addition of 0.5% (W/V) malic acid,
0.1% (W/V) sorbate and 0.1% (W/V) benzoate,
respectively.
169
3.2.5. Comparison between the selected natural
preservatives alone and in combination with
pasteurized temperature.
1- S. aureus
From table (21) and fig. (18), The sub-lethal value of
nisin was 200 μg/ml which decreased to 25 μg/ml by
combination with pasteurized temperature, the sub-lethal
value of citric acid was 0.15 % (W/V) which reduced to
0.05% (W/V) by combination with pasteurized
temperature, the sub-lethal value of Lactic acid was 0.1 %
(W/V) which lowered to 0.01% (W/V) by combination with
pasteurized temperature, and the sub-lethal value of
cinnamon was 0.15 % (W/V) which decreased to 0.05%
(W/V) by combination with pasteurized temperature.
2- P. aeruginosa
From table (21) and fig. (18), there is no effect of
nisin on P. aeruginosa which enhanced by combination
with pasteurized temperature and the sub-lethal conc.
decreased to 100 μg/ml, the sub-lethal conc. of citric acid
and lactic acid was 0.2 %(W/V) which decreased to 0.1
%(W/V) by combination with pasteurized temperature,
also, the sub-lethal conc. of cinnamon was 3 %(W/V) and
170
by using pasteurized temperature it was decreased to 0.5
% (W/V).
3- Debaryomyces sp.
From table (21) and fig. (18), there is no effect of nisin
on Debaryomyces sp. but it enhanced by combination
with pasteurized temperature and the sub-lethal conc.
decreased to 25 μg/ml, the sub-lethal conc. of citric acid
and lactic acid was 3.5 and 4 %(W/V) which decreased to
2.5 and 0.1 %( wt/v) by combination with pasteurized
temperature, also, the sub-lethal conc. of cinnamon was
1.7 %(W/V) and by using pasteurized temperature it was
lowered to 0.3 % (W/V).
171
Table (21): Sub-lethal values of natural preservative alone and in
combination with pasteurized temperature*
Debaryomyces
Treatment sp.
Sub-lethal value
Nisin alone 200 ** **
Temperature +
25 100 25
Nisin
Citric acid
0.15 0.2 3.5
alone
Temperature +
0.05 0.1 2.5
Citric acid
Lactic acid
0.1 0.2 4
alone
Temperature +
0.01 0.1 0.1
Lactic acid
Cinnamon
1 3 1.7
alone
Temperature +
0.3 0.5 0.3
Cinnamon
* Nisin conc. = μg/ml, other preservatives conc. = % (W/V).
** There is no effect of nisin alone on P. aeruginosa or Debaryomyces
sp.
172
300 300
300 Staphylococcus aureus Pseudomonas aeruginosa Debaryomyces spp.
250 200
200
Conc. %
150
100
100
25 25
4 0.1
50 3.5 2.5 0.1 0.3
0.2 1.7
0.2 0.1 0.01 0.5
0.1 3
1 0.3
0.15 0.05
ne in ne cid ne cid ne on
alo Nis alo ca alo ca alo am
in nd cid itri c id acti on inn
Nis p.a
ric
a
nd
C
c a d L
na
m
nd
C
tem Cit p.a cti .an Cin .a
st. em La mp mp
Pa st. t te te
Pa st. st.
Pa Pa
Fig. (18): Sub-lethal values of natural preservative alone and in

combination with pasteurized temperature
173
3.3. Combination of natural preservatives
Natural antimicrobial compounds available for
use in food processing and shelf life extension include
enzymes (lactoperoxidose, lactoferrin, Avidin, lysozyme),
microbial preservatives produced from starter cultures
(Nisin, Lactacin/Lactococin/Lacticin/, natamycin,
Variacin), and plant sources (herbs, spices, extracts,
essential oils and their isolated components) (Valero and
Francés, 2006).
3.3.1. Combination of Nisin with citric and
latic acid.
From the previous experiments (single treatment),
nisin alone has no effect on both of Debaryomyces, and
Pseudomonas while it wasvery effective in case of S.
aureus.
The object of the present experiment was to investigate
the effect of nisin combined with citric acid or lactic acid,
hoping that the acids may affect the cell membrane of both
Debaryomyces, and P. aeruginosa which permite the
nisin to enter their cells. Concentration of 25 μg/ml of
nisin was used in case of S. aureus which reduce its
counts by one log cycle as previously mentioned, while 200
174
μg/ml in case of Debaryomyces, and P. aeruginosa was
used in combined with different concentration of citric acid
or lactic acid.
As shown in tables (22 & 23) and Figures (19 & 20),
only 0.05 % (W/V) of citric acid beside 25 μg/ml nisin was
sufficient to annihilate the viable cells of S. aureus, while
0.2 % (W/V) of citric and 200 μg/ml nisin were needed to
eliminate P. aeruginosa (it worth tomention that 0.3 %
(W/V) of citric acid alone was needed to destroy the cells of
P. aeruginosa in the single treatment). Regared the
Debaryomyces, 3.0 % (W/V) of citric acid was applied
beside 200 μg/ml nisin to reach its cell death (it worth
tomention that 4 % (W/V) of citric acid alone was needed
to destroy the cells of Debaryomyces in the single
treatment).
In case of lactic acid tables (24 & 25) and Figures (21 &
22), 0.1% (W/V) was used beside 25 μg/ml of nisin to
eliminate S. aureus and 200 μg/ml of nisin to eliminate P.
aeruginosa (comparing with 0.3 % (W/V) required in
single treatment). Concentration 3.5 % (W/V) of lactic acid
beside 200 μg/ml nisin were applied to eliminate
Debaryomyces (comparing with 4.5 % (W/V) required in
single treatment).
175
Conc. %
0
0 0.02 0.04 0.06 0.08 0.1 0.12 0.14 0.16
-0.5
-1
-1.5
-2
Log count
-2.5
-3
-3.5
-4

-4.5
Fig. (19): Effect of different conc. of citric acid in combination with nisin
176
Table (22): Effect of different conc. of citric acid in combination with nisin
Organism Staphylococcus aureus Pseudomonas aeruginosa
Nisin conc. µg/ml 25 200
Log
Conc. % Average count cfu m-1 Log N* Log N/No Average count cfu m-1 Log N*
N/No
Initial count 98 x 105 6.99 - 98 x 105 6.99 -

0.017 A ± 0.014 A
Control 5.63 6.99

Nisin
43 x 104
± 0.060 B
0 98 x 105 0
± 0.0 A
0.01 89 x 102 3.94 - 3.04 Not tested
± 0.032 C
2.17
0.03 15 x 10
± 0.050 D
- 4.81 Not tested
0.05 -** -** -** 48 x 104 5.68 -1.30

E ± 0.045 B
0.1 22 x 103 4.34 -2.64

± 0.094 C
0.15 56 x 10 2.74 -4.24

± 0.032 D
0.2 -** -** -**

E
177
Table (23): Effect of different conc. of citric acid in combination with Nisin
.200 µg/ml on Debaryomyces sp
Log
Conc.% Average count cfu m-1 Log N*
N/No
0 41 x 105 6.61 0
± 0.012 A
6.61
Control nisin 41 x 105 0
± 0.0 A
1.0 30 x 104 4.47 -1.13

± 0.016 B
2.0 51 x 102 4.70 -2.90

± 0.027 C
2.5 88 x 10 2.94 -3.66

± 0.020 D
3.0 -** -** -**

E
Conc. %
0
0 0.5 1 1.5 2 2.5 3 3.5
-0.5
-1
-1.5
-2
Log N/No
-2.5
-3
-3.5
-4
-4.5
Fig. (20): Effect of different conc. of citric acid in combination with Nisin 200
µg/ml on Debaryomyces sp..
178
Table (24): Effect of different conc. of lactic acid in combination with nisin
Organism Staphylococcus aureus Pseudomonas aeruginosa
Nisin conc. µg/ml 25 200
Log
Conc. % Average count cfu m-1 Log N* Log N/No Average count cfu m-1 Log N*
N/No
Initial count 121 x 105 7.08 - 98 x 105 6.99 -

± 0.011 A ± 0.014 A
Control 4.74 6.99

Nisin
43 x 104
± 0.060 B
0 98 x 105 ± 0.0 A
0
0.01 Not tested 88 x 104 5.94

± 0.062 C
0.02 25 x 103 4.39 -1.23 Not tested

± 0.030 C
0.04 38 x 102 3.57 -2.05 Not tested

± 0.062 D
0.05 Not tested 25 x 102 3.39 -3.59

± 0.025 D
0.06 150 2.17 -3.45 Not tested

± 0.050 E
0.1 -** -** -** -** -** -**

F E
179
Conc. %
0
0 0.01 0.02 0.03 0.04 0.05 0.06 0.07
-0.5
-1
-1.5
Log count
-2
-2.5
-3
-3.5
Staphylococccus aureus Pseudomonas aeruginosa
-4
Fig. (21): Effect of different conc. of lactic acid in combination with nisin
Table (25): Effect of different conc. of lactic acid in combination with Nisin
Log
Conc.% Average count cfu m-1 Log N*
N/No
Initial count 41 x 105 6.61 0
± 0.01 A
Control 6.61
41 x 105 0
nisin200 µg/ml ± 0.0 A
1.5 29 x 103 3.46 -2.15

± 0.012 B
2.5 13 x 10 2.11 -4.49

± 0.086 C
3 5 0.69 -5.91
± 0.106 D
3.5 -** -** -**

E
*The same letters are not significantly different. ** The dash means no distinct growth.
180
Conc. %
0
0 0.5 1 1.5 2 2.5 3 3.5 4
-1
-2
Log N/No
-3
-4
-5
-6
-7
Fig. (22): Effect of different conc. of lactic acid in combination with Nisin
3.3.2. Combination of Nisin with cinnamon

It is obvious from table (26) and Figures (23 &
24) that, the addition of cinnamon with nisin, the count
decreases sharply by increasing the conc. of cinnamon
and accelerate the death of S. aureus, Pseudomonas,
and Debaryomyces. The lethal-values were at 2% (W/V)
for both Pseudomonas, Debaryomyces (comparing with 4
% (W/V) and 2 % (W/V) in single treatments, respectively)
and at 0.4% (W/V) for S. aureus.
181
Cinnamon contains approximate 0.5-2.0 %
Essential oil (Snyder, 1997). Cinnamon oil contains 50-60
% cinnamaldehyde and 4-7% eugenol (Matan et al.,
2006). Although essential oils components are used as
flavoring in the food industry, nowadays they represent a
highly interesting source of natural antimicrobials for food
preservation due to their antimicrobial and antioxidative
activity (Beuchat, 1994). Cinnamon effectively inhibits
growth of bacteria, yeasts and molds (Smith-palmer et
al., 1998).
Shelef (1983) and Zaika (1988) stated that Gram-

positive bacteria are more sensitive to cinnamon than
Gram-negative bacteria. This is probably due to the outer
membrane of gram-negative bacteria, which is highly
al., 1998).
The mechanism of action of the cinnamon oil and its

main compound to inactivate microorganisms according to
Gill and Holley (2004, 2006) and Oussalah et al. (2006)
appear to be related to the cell membrane from which a
slight disruption seem to occur causing dispersion of the
proton motive force by leakage of small ions without
182
leakage of larger cell components, such as ATP, which are
subsequently degraded by the ATPase enzyme. Hence, the
antimicrobial effect of nisin combined with cinnamon is
enhanced due to the formation of pores on the cell
membrane, which may favor more easily the diffusion of
the nisin to cell inside and cause damage in the cell
functions.
183
Table (26): Effect of different conc. of cinnamon in combination with nisin
Nisin conc. ug/ml 25 200 200
Conc. % Log N* Log N* Log N*
Initial count 95 x 10 5 6.97 - 98 x 105 6.99 - 41 x 105 6.61 0
± 0.009 A ± 0.012 A ± 0.003 A
Control 4.74 6.99 6.61
Nisin
43 x 104
± 0.060 B
0 98 x 105 ± 0.0 A
0 41 x 105
± 0.0 A
0
0.2 48 x 102 2.68

± 0.015 C
-1.95 Not tested
1.07 5.78
0.3 12
± 0.079 D
-4.55 Not tested 61 x 104
± 0.003 B
-0.82
-**
0.4 -**
E
-** Not tested
0.5 65 x 103 4.3 -2.17

± 0.030 B
4.54
0.6 Not tested 35 x 103
± 0.087 C
-2.06
1 35 x 102 3.5 -3.44 26 x 102 3.41 -3.16

± 0.043 C ± 0.011 D
1.84
1.5 Not tested 7 x 10
± 0.020 E
-4.76
2 -** -** -** -** -** -**

D F
184
Conc. %
0
0 0.2 0.4 0.6 0.8 1 1.2 1.4 1.6
-0.5
-1
-1.5
-2
Log count
-2.5
-3
-3.5
-4
-4.5
Pseudomonas aeruginosa Debaryomyces spp.
-5
Fig. (23): Effect of different conc. of cinnamon in combination with nisin
Conc. %
0
0 0.2 0.4 0.6 0.8 1 1.2
-0.5
-1
-1.5
Log N/No
-2
-2.5
-3
-3.5
-4
-4.5
-5
Fig. (24): Effect of different conc. of cinnamon in combination with nisin

on S. aureus.
185
• General discussion
In Gram–positive cocci (such as S. aureus) there are
no specific receptor molecules or permeases to prevent
preservatives penetration. Consequently, preservatives
enter these cells readily and intrinsic resistance is
therefore low (Russell, 1991). In contrast, the outer
membrane of Gram-negative bacteria (such as P.
aeruginosa) plays an important role in limiting the entry
of preservatives into the cell (Nikaido and Vaara 1985).
The cell surface of smooth, wild-type gram-negative
bacteria is hydrophilic in nature; deep rough
(hepatoseless) mutants are much more hydrophobic
because of the appearance of phospholipids (PL) patches
on the cell surface. In wild-type bacteria, low molecular
weight hydrophilic molecules (such as lactic acid) cross
the outer membrane via the aqueous porins. On the other
hand, lipopolysaccharide (LPS) molecules prevent ready
access of hydrophobic preservatives to the PL (Russell,
1991).
Lactic acid was recently shown to permeabilize Gram-

negative bacteria efficiently and causes LPS release
(Verhoff, 1986). Like EDTA which destabilize and cause
186
lipopolysaccharide release from the outer membrane by
complexation of bridge-forming bivalent cations such as
Mg2+ and Ca2+ (similarly citric acid act in the same
manner) (Hauben et al, 1996). Inhibition of the growth of
the outer membrane gram-negative bacteria can be
achieved by simultaneous treatment with nisin and an
agent which modifies and chelates the outer membrane,
such as EDTA (Stevens et al., 1991). These findings are
consistent with notion that nisin acts on the cytoplasmic
membrane.
Phillips and Duggan, (2002) reported that a

compound like citric acid makes gram negative bacteria
more sensitive to Nisin. Similarly, in yeast because its
sensitivity to acids (single treatments) make it sensitive to
nisin (combined treatments).
Nisin speeded up the inactivation, with cinnamon

and generally contributing to the suppression of
pathogens. The higher of nisin dose needed to greater
killing effect (Yuste and Fung, 2003). Kalchayanand et
al, (1992) observed an increase in nisin effectiveness
when gram negative bacteria were subjected to sub lethal
stresses (Including exposure to acids). Further more, Nisin
187
is more stable and more effective of acidic pH (Deans and
Ritchie, 1987 and Ray, 1992).
In the same mechanism, the combination of Nisin

and cinnamon accelerates death of Salmonella
Typhimurium and E.coli 0157:H7 in apple juice and give
substantial protection to the product (Yuste and Fung,
2003). In a similar purpose, it may be occurring with both
Pseudomonas and Debaryomyces. This suggests the
potential use of such combination in the food industry for
enhancement of safety.
188
3.3.3. Comparison between the selected
natural preservatives in single treatment and
in combination with nisin.
1- S. aureus
As showen in table (27) and fig. (25), the sub-lethal
value of citric acid was 0.15 % (W/V) and by using nisin it
was reduced to 0.03 %( wt/v). The sub-lethal conc of lactic
acid 0.1 %( wt/v) was reduced to 0.06 %( wt/v) by using
nisin. While, cinnamon was reduced from 1 %( wt/v) to 0.3
%( wt/v).
2- P. aeruginosa
Table (27) and fig. (25) showed that, the sub-lethal
value of citric acid and lactic acid was 0.2 % (W/V) and
by using nisin it was reduced to 0.15 and 0.05 %( wt/v),
respectively. While, cinnamon was reduced from 3 %(
wt/v) to 1 %( wt/v).
3- Debaryomyces sp.
From Table (27) and fig. (25), the sub-lethal value of
citric acid and lactic acid was 3.5 and 4 % (W/V) and by
using nisin it was decreased to 2.5 and 4 %( wt/v). While,
cinnamon was lowered from 1.7 %( wt/v) to 1.5 %( wt/v).
189
Table (27): Sub-lethal values of selected natural preservatives
alone and in combination with nisin*
S. aureus P. aerogenosa Debaryomyces sp.

Treatment
Sub-lethal value %
Nisin conc. ** 25 200 200
Citric acid 0.15 0.2 3.5
Nisin + citric
0.03 0.15 2.5
acid
Lactic acid 0.1 0.2 4
Nisin + lactic
0.06 0.05 3
acid
Cinnamon 1 3 1.7
Nisin +
0.3 1 1.5
Cinnamon
* Nisin conc. = μg/ml, other preservatives conc. = % (W/V).

** There is no effect of nisin alone on P. aeruginosa or Debaryomyces sp.
190
4
4 Staphylococcus aureus
3.5 Pseudomonas aerogenosa
3.5 Debaryomyces spp.
3 3
3
2.5
2.5
Conc. %
2 1.7
1.5
1.5
1 1
0.2 0.2 0.05

0.15 0.3
0.5 0.15 0.1 0.06
0.03
one acid e id lone n

id al alon c ac on a amo
c ac citric acid lacti Cinn
Citri in and Lactic s in and Cinnam s in and
Nis Ni Ni
Treatment
Fig. (25): Sub-lethal values of nisin alone and in combination with the
selected natural preservatives.
191
4. Combination of gamma irradiation with
pasteurized temperature
A combination of irradiation has been proposed

for required lethality while preserving food quality, there
by reducing the detrimental effects of heat or irradiation
alone on food while the inactivation effect of heat
treatment followed by irradiation is additive (Kim and
Thayer, 1996), a synergistic effect has been observed
when heat treatment is applied after irradiation or when
both treatments are simultaneously applied (thermo
radiation).
This experiment was performed by heating

suspension of yeast cells in broth medium to pasteurized
temperature 90 ْC for 15 sec followed by gamma irradiation
at dose 0.25 KGy, and results showed, nil count in all
dilutions, at control 98 x 104cfu ml-1.
The results suggest that irradiation enhanced heat

sensitivity of Debaryomyces sp. originated from oxygen-
independent intracellular damages such as DNA damages
at radiation doses of 0.2 to 1.0 KGy as suggested by
previous experiments.
192
However, the denaturation or dissociation of broken
DNA strands from the cellular membrane by heating
following irradiation makes them unrepairable.
Alternatively, (i) heating may alter DNA polymerase and
topoisomerase I as in yeast cells (Borehan et al., 1990),
or (ii) irradiation may induce functional changes in the
cytoplasmic membrane in addition to the heating-induced
desbilization, such as oxidation of sulfydryl groups of the
membrane-binding bound proteins, resulting in cell death
in addition to direct DNA damage (Yatvin and Grummer,
1987).
Also, it is possible that heat and radiation act on

different sites but also share some targets such as DNA to
cause additive or synergistic effect on cells (Bridges et al.,
1969 and Yatvin et al., 1986). Alternatively, heated cells
could be more sensitive to irradiation either because the
heat-labile recovery capacity of bacterial cells as affected
(Bridges et al., 1969) or because recovery from heating-
induced damage by irradiation is abolished, preventing
protein synthesis as a consequence of DNA damage in
vegetative bacterial cells (Mukherjee and Bhattacharjee,
1970)
193
Pallas and Hamdy (1976) observed that the D10-
value (dose inactivating 90% of population) of S. aureus
decreased from 0.098 t 0.053 KGy, while the irradiation
temperature increased from35 to 45 ْC .
5. Storage
The Debaryomyce sp. was selected for storage, due
to it was more resistant to physical and natural
treatments than Staphylococcus and Pseudomonas, two
experiment were performed as follow:
5.1. Combination between nisin and citric acid and

As shown in table (28), the nisin was used in

concentration 200 µg/ml in combination with temperature
90 ْC for 60, 120 sec. and at citric acid concentration
2.0%, it was more effective for more than 90 days without
any growth. While, when the time of temperature was
decreased to 30 sec., the Debaryomyces can be recovery
and give count 12 x 10 cfu ml-1. Also, when the conc. of
nisin was decreased to 100 or 50 µg/ml at 2 % citric acid
and pasteurized temperature 90ْ C for 30 or 60 or120 sec,
194
Table (28): Effect of storage for recovery of Debaryomyces sp.*
Pasteurized temperature90ْ C for

time/sec
Nisin conc. Citric acid
Storage period 30 60 120
μg/ml conc. %
Average count (cfu/ml)
6 days 50 45 x 104 78 x 103 28 x 102
6 days 100 2.0 33 x 103 46 x 102 38 x 10
90 days 200 22 x 10 Nil Nil
*Treated with pasteurized temperature, nisin and citric acid.
5.2. Combination between gamma irradiation and

It was performed by heating broth medium
inoculated with yeast cells to pasteurized temperature 90
ْC for 30 sec followed by gamma irradiation (0.25 kGy),
results showed, nil count in all serial dilutions and it was
effective for more than 90 days at control 98 x 104cfu ml-1.
195
This synergistic effect of thermoradiation has also been
observed in the inactivation of microorganisms in several
foods. It has been reported that combining low-
temperature (50 ْC) heating with low-dose irradiation is
highly effective for controlling spoilage organisms in apple
juice (Urbain, 1986).
196
SUMMARY
197
Summary
1. Isolation and identification

From forty two isolates from different juice
samples, it was select three isolates (Staphylococcus
aureus, Pseudomonas aeruginosa, and Debaryomyces
sp.) to study the effect of physical treatment and natural
treatments as follow:
2. Single treatments
The lethal dose for S. aureus was 5 kGy; P.
aeruginosa was more sensitive to gamma irradiation than
S. aureus. The lethal dose was at 4 KGy, while
Debaryomyces sp. was highly resistant to irradiation
treatment and more survive than Pseudomonas
aeruginosa or Staphylococcus aureus where, the lethal
dose was at 7 kGy.
2.1.2. Pasteurized temperature (90 ْC)

The lethal time for S. aureus was reached to
120 sec. Dt 13.1 sec., while the lethal time of P.
aeruginosa was reached to 120 sec. and Dt was 17.3 sec.,
198
Debaryomyces sp. was more survival and sub-lethal time
reached to 120 sec. and Dt was13.1 sec.. Also the TDT was
157.4, 208.0 and 224.0 sec., respectively for the previous
organisms.
2.2. Natural treatments

2.2.1. Nisin
The lethal conc. for S. aureus was at 250
µg/ml, While, nisin alone has no effect on P. aeruginosa
or Debaryomyces sp.
2.2.2. Citric acid

The lethal conc. of S. aureus was 0.2 % (W/V), the
lethal conc. of P. aeruginosa was 0.3 % (wt/v), while For
Debaryomyces sp. was the most resistant to citric acid
than P. aeruginosa or S. aureus. Where, the lethal conc.
reached to 4.0 % (W/V).
2.2.3. Lactic acid

The lethal conc. of S. aureus was 0.15 % (W/V), the
lethal conc. of P. aeruginosa was 0.3 % (W/V), while
Debaryomyces sp. was highly survive to lactic acid, where
the lethal conc. was 4.5 % (W/V).
199
2.2.4. Cinnamon
The lethal conc. of Staphylococcus aureus was 1.2
% (W/V), the lethal conc. for P. aeruginosa was 4.0 %
(W/V), While, for Debaryomyces sp.. the lethal conc. was
at 2.0 % (W/V).
3. Combination treatments
3.1. Combination with Gamma rays
The lethal dose for S. aureus was 5 kGy; this dose was
reduced to 3 kGy by combination with nisin 25 µg/ml. The
lethal dose decreased to 2 kGy by combination of gamma
irradiation with citric, lactic acid and cinnamon at conc.
0.05% (W/V), 0.01% (W/V) and 0.4 % (W/V), respectively.
P. aeruginosa was more sensitive to gamma

irradiation than S. aureus. The lethal dose was at 4 KGy,
and by combination with nisin (200 µg/ml) or citric acid
0.1 % (W/V) or with cinnamon 0.5 % (W/V), it was
decreased to 3 kGy. The lethal dose was highly reduced to
2 KGy by combination with lactic acid 0.1 % (W/V).
Debaryomyces sp. was highly resistant to

irradiation treatment and more survive than
Pseudomonas aeruginosa or Staphylococcus aureus
200
where, the lethal dose was at 7 kGy which decreased to 4
kGy by combination with nisin 200 µg/ml or with citric
acid 1.5 % (W/V) or cinnamon 1 % (W/V). Also, this lethal
dose lowered to 3 kGy with lactic acid 1.5 % (W/V).
3.1. Combination with pasteurized temperature

3.1.1. Combination with Nisin
The lethal conc. for S. aureus was at 250 µg/ml, which
highly reduced to 25 µg/ml by combination with
pasteurized temperature 90 ْC, 30 sec. While, nisin alone
has no effect on P. aeruginosa or Debaryomyces sp., by
combination with pasteurized temperature 90 ْC 30 sec,
lethal conc. effective at 150 µg/ml for P. aeruginosa and
to 50 µg/ml for Debaryomyces sp.
3.1.2. Combination with citric acid

The lethal conc. of S. aureus was 0.2 % (W/V), which
lowered to 0.1 % (W/V) by combination with pasteurized
temperature 90 ْC 30 sec.
The lethal conc. of P. aeruginosa was 0.3 % (wt/v),
which highly decreased to 0.15 % (W/V) by combination
with pasteurized temperature 90 ْC 30 sec.
For Debaryomyces sp. was the most resistant to citric
acid than P. aeruginosa or S. aureus. Where, the lethal
201
conc. reached to 4.0 % (W/V). This conc. decreased to 3.0
% (W/V), by combination with pasteurized temperature 90ْ
C for 30 sec.
3.1.3. Combination with lactic acid

The lethal conc. of S. aureus was 0.15 % (W/V), by
combination with pasteurized temperature 90ْ C 30sec, the
lethal conc. of lactic acid reduced to 0.05% (W/V).
The lethal conc. of P. aeruginosa was 0.3 % (W/V).
This conc. decreased to 0.15% (W/V) by combination with
pasteurized temperature 90ْ C for 30 sec.
For Debaryomyces sp. was highly survive to lactic
acid, where the lethal conc. was 4.5 % (W/V), which
lowered to 0.5 % (W/V) by combination with pasteurized
temperature 90 ْC 30 sec.
3.1.4. Combination with cinnamon
The lethal conc. of S. aureus was 1.2 % (W/V) and by

combination with pasteurized temperature 90 ْC for 30 sec.
it was reduced to 0.4 % (W/V).
The lethal conc. for P. aeruginosa was 4.0 % (W/V), by
combination with pasteurized temperature90 ْC for 30 sec.,
it was reached to 0.6 % (W/V).
202
While, for Debaryomyces sp. the lethal conc. was at
2.0 % (W/V), by the combination with pasteurized
temperature 90 ْC for 30 sec, the sub-lethal conc. of
cinnamon decereased to 0.6 % (W/V).
3.2. Combination of Nisin with nature

preservatives
3.2.1. Combination with citric acid
decreased to 0.05 % (W/V) by combination with nisin (25
µg/ml).
The lethal conc. of P. aeruginosa was 0.3 % (wt/v),
which decreased to 0.2 % (W/V) by combination with nisin
200 µg/ml.
For Debaryomyces sp. was the most resistant to
citric acid than P. aeruginosa or S. aureus. Where, the
lethal conc. reached to 4.0 % (W/V). This conc. decreased
to 3.0 % (W/V), by combination with nisin 200µg/ml.
3.2.2. Combination with lactic acid

reduced to 0.1 % (W/V) by combination with nisin 25
µg/ml.
203
The lethal conc. of P. aeruginosa was 0.3 % (W/V),
which reduced to 0.1 % (W/V) by combination with nisin
200 µg/ml.
For Debaryomyces sp. was highly survive to lactic
acid, where the lethal conc. was 4.5 % (W/V), which
decreased to 3.5 % (W/V) by combination with nisin 200
µg/ml.
3.2.3. Combination with cinnamon
The lethal conc. of S. aureus was 1.2 % (W/V) and by

combination with nisin 25 µg/ml, it was lowered to 0.5 %
(W/V).
The lethal conc. for P. aeruginosa was 4.0 % (W/V)
and by combination with nisin 200 μg/ml it was decreased
to 2 % (W/V).
While, for Debaryomyces sp. the lethal conc. was at
2.0 % (W/V) and by combination with nisin 200 μg/ml the
lethal conc. redeuced to 2.0 % (W/V).
5. Storage
The Debaryomyce sp. was selected for storage due to
it was more resistant to physical and natural treatments
than Staphylococcus and Pseudomonas
204
5.1. Nisin was used in concentration 200 µg/ml in
combination with temperature 90 ْC for 60, 120 sec. and at
citric acid concentration 2.0%, it was more effective for
more than 90 days without any growth.
5.2. Pasteurized temperature 90 ْC for 30 sec followed
by gamma irradiation (0.25 kGy) were performed, results
showed, nil count in all serial dilutions and it was effective
for more than 90 days.
205
CONCOLUSION
&
RECOMMENDATION
206
CONCOLUSION AND RECOMMENDATION
The best results obtained from our experiments for

preserving the fruit juice were occure by combing the
pasteurized temperature 90 ْC for 30 sec. with gamma
irradiation (0.2 kGy). Or using 200µg/ml of nisin in
combination with 2 % (W/V) of citric acid and pasteurized
temperature90 ْC for 30 sec.
207
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Influence of some physical treatments and

natural food preservatives on the diversity of

Mohamed Nagah Zayed Ibrahiem Abu El-Naga

Department of Botany & Microbiology

Influence of some physical treatments and

Mohamed Nagah Zayed Ibrahiem Abu El-Naga

Prof. Dr. Backry Mohamed Haroun

Prof. Dr. Mohie El-Din Zohier El-Fouly

Dr. Hala Ahmed Hussein

Praise and gratitude is to ALLAH SOBHANAHU

I wish to express my appreciation to Dr. Backry

Sincere thanks and deep gratitude to Dr. Mohie El-Din

I wish to express my grateful acknowledgment to Dr.

I would especially like to thank Prof. Dr. El-Said Ghazal,

Also, I would like to express my appreciation and thanks

Sincere thanks to Prof. Dr. T. M. El-Mongy, Professors of

Finally, special thanks to Said El-Sayed Desouky,

Last, but not least I wish to express my grateful thanks,

I dedicate this work to my Parents, as well as to my Sisters and my

3.3. Combination of natural preservatives…………… 163

Screening and isolation of microorganisms

(8) Effect of different conc. of lactic acid on Debaryomyces sp. 118

Effect of different conc. of lactic acid on P. aeruginosa and S.

Effect of different doses of gamma irradiation in

(a) Structure of nisin and unusual amino acids. 28

A schematic diagram of the stress response of a yeast

Effect of increasing doses of gamma irradiation on

(12) Effect of different conc. of cinnamon on P. aeruginosa 128

Effect of different doses of gamma irradiation in

ACSH → American Council on Science and Health.

The stability of fruit-based beverages and drinks is also

According to the FPCFN, food additives may be

A substance or mixture of substances, other than a basic

Additives can be divided into six major categories:

Since preservatives are the main object of the present

• Antibrowning agents are chemicals used to prevent

Despite the benefits attributed to food additives, for

The indirect risks that have been described for

What are the risks associated with our food

2.5.2. Sodium Nitrite

2.5.3. Azo Dyes and Benzoates

In the late 1950 it was discovered that food colorants

Inhaled sulfur dioxide may produce bronchoconstriction

Nitrite has been shown to have variable effects on

The mechanism of nitrite inhibition of

The lethal dose of nitrites in humans is 32 mg/kg body

‘‘Chinese restaurant asthma’’ has been suggested

Enzymes are frequent causes of IgE-mediated nasal,

Interest in natural antimicrobials is also driven by

An argument often used to justify natural antimicrobials

Nisin is a 34 amino acid peptide which contains the

Nisin by itself has a narrow spectrum affecting only

Nisin activity is affected by a number of environmental

The application of nisin as a food preservative has been

Nisin has been recommended for use in canned

In addition, Delves-Broughton et al. (1992)

It was demonstrated that in vitro nisin inhibited

In contrast, the inhibition of cell wall biosynthesis is a

The degree of association of nisin with the

More recent studies have substantiated this evidence,

Brotz et al. (1998) suggesting that nisin may use

The effective use of organic acids in food products is

In their synergistic capacity, acids used in conjunction

A major use of acidulants is as an antimicrobial agent