Classical and emerging food

processing technologies: effect on

food-borne pathogens
Physical methods for food preservation:
 Temperature
 Low: refrigeration, freezing
 High: pasteurization, sterilization, cooking
 Water activity: drying
 Radiation: gamma rays, X-rays or accelerated
electrons
 Pressure: high hydrostatic pressure
 Others:
 Light (UV, visible)
 Electric fields
 Ultrasounds…
Chemical methods for food preservation

 Salt and/or Sugar (Aw reduction)

 Organic acids:
 Addition: Marinades
 Fermentation: microbial action
 Smoke
 Additives: preservatives and
antioxidants
 Curing: a combination of everything
According to their effect on the microorganisms...
 Microbiocides: (totally or partially) eliminate the
contaminating microbiota
 They need protection against re-contamination: packaging
 Recontaminations may allow growing without competence

 They may require additional storage conditions:

 Cooling/freezing
 Modified atmospheres (CO2)

 Microbiostatics: inhibit replication of microbiota (transiently

or permanently)
 They need to maintain adverse conditions permanently
Microbial hazards in food: F. Untermann/ Food Control 1998 9 (2)
Effect of temperature on bacterial growth
 All microorganisms have maximum, minimum and
optimum temperatures of growth
 Depending on their optimum temperature:
 Thermophiles
 Mesophiles
 Psychotrophs
 Psychophiles
 Optimum, minimum and maximum growth temperatures of
food-borne pathogenic bacteria

Microorganism Temperature (ºC)

Min. Op. Max.

A. hydrophila 0-4 28-35 42-45
B. cereus 4 30-40 55
C. jejuni 32 42-43 45
Cl. perfringens 12 43-47 50
E. coli 7,8 35-40 44-46
L. monocytogenes - 0,4 37 45
Salmonella spp. 7 35-43 46,2
S. aureus 7 35-40 48
Y. enterocolitica - 1,3 25-37 42
Cl. botulinum Grup I 10-12 30 48
Cl. botulinum Grup II 3,3 25 45
Source: ICMSF (2006)
 Effect of low temperatures on m.o.
 Cooling:
 Reduces membrane fluidity:
 Affects nutrients transport processes and
proton gradient
 Increases viscosity of cytoplasm
 Weakens hydrophobic bonds of proteins
 Consequences:
 Inactivation of enzymes
 Inhibition of ribosome activity
 Decreases growth rate
 Affects cellular functionality
 Effects of freezing
 Reduction of Aw
 Relatively low case-fatality rate:
 Two components:
 Immediate effect: depends on how fast sample gets frozen
 Long-term effect: depends on the time the surviving bacteria
remain in a frozen state
 The area of greatest cellular damage occurs between
-1 ºC and -10 ºC
 Slow freezing increases cell damage:
 Cytoplasm freezes more slowly:
 Cell losses inner water: Increases concentration of solutes
 Protein denaturation
 Increases the oxidative stress of bacteria
 Formation of large and irregular ice crystals: mechanical
Freezing chamber/tunnel damage to the cell wall and membrane
Effect of heat:
pasteurization/sterilization/cooking

 High temperatures cause…:

 Denaturation (and inactivation) of
proteins
 Essential enzymes
 Fusion of membrane lipids
 Collapse of the cytoplasmic
membrane
 Lysis of the bacteria
 Fatality rate depends on the
type of microorganism: D & z
Thermal treatments applied to milk
 Decimal reduction time (D) of pathogens in milk

D-values (the time required to reduce the number of microorganisms by one log cycle)
Source: S. Sarkar , 2015. Microbiological Considerations: Pasteurized Milk. International Journal of Dairy Science, 10: 206-218.
Effect of the matrix characteristics on the thermo-
resistance of microorganisms
 Water:
 Absence of water increases thermo-resistance: slows down protein
denaturation
 Fat:
 Thermoprotective effect (?)
 Carbohydrates:
 Increase resistance when reducing the Aw
 Proteins:
 Increase resistance
 pH:
 Deviations from the optimum pH increase thermosensitivity
 Age of microorganisms:
 The older the culture is (stationary phase), the more resistant
Driying

 Reduction of Aw to a level that does not

allow microorganisms to grow: needs
extraction of “bound water”
 Natural: action sun + natural air stream
 Forced
 Drying chambers (Fans + hot air)
 Vacuum evaporators
 Drying drums
 Spraying facilities
 Aw ≤ 0.70 is the target for long-life foods
 Alarm water contents can range from 8% in
milk powder to 25% in dehydrated fruit
Effect of reducing Aw on microorganisms
 Prolongs the lag phase, but lethality rate is low
 D values measured as days
 Lethality may be caused by:
 Increasing the intracellular concentration of salts:
 Toxic effects on cellular functions
 Protein denaturation
 Damages due to oxidation

 Low Aw protects microorganisms during heat

treatments
 Re-hydration of the medium can cause the recovery
of the microorganism
Radiation

 May cause changes in the molecular structures affecting the

viability of microorganisms.  Units:
 Types of radiation:  July / kg or Cm2; Rad and Gray
 Ultra-violet radiation  1 J / kg = 1 Gy = 100 rads
 Rotgen radiation (X)  Treatments:
 Gamma radiation  Radiation
 Accelerated electrons  Radicidation
 Radappertización
 Effects of ionizing radiation
 High radiation doses: direct lethal effect
 By direct impact on DNA: irreparable injuries
 Breaks in both DNA chains
 Crosslinking between the DNA chains
 Lack of gene expression
 Lack of enzyme synthesis

 Low radiation doses:

 Mutagenic effect:
 Minor damage to DNA: can be repaired by error-prone mechanisms
 Indirect lethal effect: the most important
 Radiolysis of water: causes generation of free radicals
 Hydroxyl (OH-): a powerful oxidant
 Hydrogen (H · +): a powerful reducer
 If exposed to oxygen, they cause autooxidation reactions promoting the
formation of highly destructive peroxides and epoxides.
 H· + O2 ------> ·HO2
 2 ·HO2 ------> H2O2 + O2
 They cause breaks in DNA chains
 Sensitivity of microorganisms to radiation
D value in irradiation treatments

Source: Alimentos seguros: microbiología. S.J.Forsythe, Acribia

High Hydrostatic (or isostatic) Pressure (HHP)

 Applications of pressures of 100-

1000 MPa (1000-10.000 bar)
 Pressure is transmitted through an
aqueous (hydrostatic) medium in a
uniform (isostatic) way to all
points of food (packed)
 Application times range from a
few minutes to a few hours
 Wide range of temperatures: -20ºC
to +90ºC
Mechanisms of microbial inactivation by HHP

 Membrane and cell wall:

 Loss of functionality
 Interference in transport processes
 Increase in the viscosity of the
cytoplasm:
 Decrease in the ability of enzymes
to bind to their respective substrates
 Effects on cellular enzymes
 Effects on genetic mechanisms
 Damage to ribosomes
 Interference in protein biosynthesis
Relative resistance of microorganisms to API
_

 Moulds and yeasts

 Virus

Resistencia
 Gram-negative bacteria
 Gram-positive bacteria
 (cocci> bacilli)
 Bacterial spores
+

•There are remarkable differences within the same species

•Bacteria are more resistant in the stationary phase of growth

Addition of Salt and/or Sugar
 Its purpose is to increase the osmotic pressure:
 Reduction of Aw (≈ to drying)

 The lethal effects are similar to those described for

drying
 Depending on the ability to survive at different
concentrations of salt or sugar: halophiles or
osmophiles
 Acidification
 Most bacteria can grow within a variable range of
environmental pH while maintaining constant their optimum
internal pH (ion transport mechanisms)
 Regarding optimal pH range of growth can be classified as:
 Neutrophils: between pH 5.5 and 8
 Acidophilus: between pH 0 and pH 5.
 Alkalophilus: between pH 8.5 and pH 11.5
 pH outside these ranges may cause lethality:
 Affects transport through membrane
 Inhibit enzymes activity
 Denaturalization of DNA
Optimal, minimum and maximum pH values of foodborne
pathogenic bacteria

Microorganism pH
Mín Op. Màx

A. hydrophila <4,5 7,2 -

B. cereus 5,0 6,0-7,0 8,8

C. jejuni 4,9 6,5-7,5 9,0

Cl. Perfringens 5,5-5,8 7,2 8,0-9,0

E. coli 4,4 6,0-7,0 9,0

L. monocytogenes 4,4 7,0 9,4

Salmonella spp. 3,8 7-7,5 9,5

S. aureus <4,3 6-7 -

Y. enterocolitica 4,2 7,2 -

Cl. botulinum Group I 4,6 7,0 9,5

Cl. botulinum Group II 5,0 7,0 9,5

Bactericidal effect of organic acids
 Organic acids can be added to the food or generated by
fermentation processes
 Acetic acid is the most used and most effective
 Others:
 Lactic ac., citric ac.: natural sources
 Benzoates, sorbates, propionates:
 Authorized as food additives
 Greater effect as mycostatics
 Bactericidal effect of organic acids
 Antimicrobial activity of organic acid is
due to the undissociated molecules
 More soluble in cell membranes (lipophilic)
 Are more effective at higher
concentrations and lower pH
 The dissociated form of the acid is less
frequent
 Once inside the cell it is ionized due to
the higher internal pH
 Lowers intracellular pH
 Effect:
 Decoupling of transport of substrates and
transport of electrons from oxidative Source: Beales, 2004. Comprehensive Rev. Food Sci.
phosphorylation and Food Safety, 3:1-20

 Acidification of cellular content

 Bacteria can not get energy and die
Wesche et al. , 2009. Journal of Food Protection, 72(5):1121–1138
What happens if the injuries caused are reversible?

 “Viable but not culturable“ Microorganisms

 May recover under environmental favorable conditions
Stress adaptation
 Pathogens can sense changes in their surroundings and
can respond by altering gene expression
 A protective response may follow that increases
tolerance to one or more stresses: stress adaptation
 Allows survival of pathogens in food products and in the food
processing environment
 Stress adaptation may alter the virulence properties
and can contribute to survival in vivo during infection
 Recommended literature:
 Beales, 2004. Comprehensive Rev. Food Sci. and Food Safety, 3:1-20
 Wesche et al. 2009. Journal of Food Protection,72(5): 1121–1138
 Begley and Hill 2015. Annu. Rev. Food Sci. Technol. 6:191–210
Adaptation to low temperatures (psychrophiles /
psychotrophs)
 Cooling:
 Synthesis of “Cold Shock proteins"
 Enzymes more resistant to cold
 Transport systems adapted to low temperatures
 Increased proportion of unsaturated, polyunsaturated and short-chain fatty
acids in membrane phospholipids
 Lower melting point
 Membrane remains in a semifluid state
 Freezing:
 Activation of genes that encode protective enzymes against oxidation:
 Superoxide dismutase: catalyzes the dismutation of superoxide into
oxygen and H202
 Catalase: catalyzes the decomposition of hydrogen peroxide (H202) in
oxygen and water
 Increase in carbohydrate reserves: glycogen, trehalose
 Cryoprotectant effect
Mechanisms of adaptation to high temperatures

 Biochemical adaptations to high temperatures in

bacterial vegetative cells:
 Synthesis of "Heat Shock proteins"
 Participate in repair of damaged proteins
 Destroy non-functional denatured proteins (proteases)
 Heat-resistant enzymes and ribosomes
 Very hydrophobic molecular interior
 Membranes rich in saturated fatty acids
 High melting points
 Allow stronger hydrophobic bonds
 Reaction to changes on osmotic pressure
 Bacteria tend to increase internal
osmolarity to reduce loss of water:
 Pumping ions and other
substances into the cell
 Potassium
 Glycine
 Carnitine
 Synthesizing osmotically active
organic molecules
 Proline
 Glutamate
 Glutamine
 Trehalose Source: Beales, 2004. Comprehensive Rev. Food Sci. and
Food Safety, 3:1-20
Consequences of an adverse pH
 Control mechanisms:
 Synthesis of "Acid Shock Proteins"
 Helps to repair other proteins or DNA

 Activation of H+ transport coupled to e- the transport of

the respiratory chain (aerobic)
 H+/K+ antiport system: exchanges protons with potassium
ions neutralizing the interior pH and maintaining a
membrane potential
 Proton translocator ATPases (expel H+ to the outside)
 Decarboxylases: consume H+ in the process
Stress Cross-Adaptation
 Cell response to an specific stress may include the activation of many of
the proteins involved in responses and can protect against multiple
stresses
Link Between Stress Adaptation and Virulence
 foodborne pathogens reencounter many of the same stresses they
experienced in food matrices
 Many of the genes required for tolerance and survival in foods are also
likely necessary for survival within the host