Microbiology of Food

Preservation

Alejandro Castillo
Texas A&M University

Methods of food preservation

• Cold storage
• Heat processing
• Reduced aw
• Acidification
• Atmosphere modification
• Addition of chemical
preservatives
• Ionizing radiations

Cold Storage
•Freezing
•Refrigeration

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 Freezing
• Needs previous
conditioning
• Freezing is applied to
previously packaged
foods

Freezing
• Vegetables
– Selecting/sorting
– Washing
– Cutting (if any)
– Blanching
– Packaging
• Meats, poultry, seafood, eggs etc.
– Trimming
– Packaging

Freezing
• Blanching
• Enzyme inactivation
• Fixing green color in some vegetables
• Easing packaging of leafy vegetables
(by wilting)
• Displacing of air from plant tissues
• Reduction of microbial counts

2
 Freezing
• Fast freezing
– Lower temperature to -20°C within 30
min
– Blast freezing or immersion
• Slow freezing
• Lower temperature to -20╖C within
3-72 h
• Home freezers

Freezing
• Effects of freezing on microorganisms
• Sudden mortality immediately on
freezing
• Continued mortality rate decreases
gradually during storage
• The lower the temperature the lower
the bacterial decline

Refrigeration
• The lower the temperature the
lower the growth rate

• How can psychrotrophs grow at

refrigeration temperatures?

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 Psychrotrophs and psychrophiles
• Greater content of unsaturated fatty acids
• Slower metabolic rate
• Better membrane transport
– Still active at low temperatures
• Production of larger cells
– Additional RNA
• More efficient flagella synthesis
• Favored by aeration
• Greater heat sensitivity
• Usually do not grow above 35°C
– Inactivation of respiratory enzymes
– Leakage of intracellular constituents

Heat processing
•Pasteurization
•Sterilization

Pasteurization
Types of milk
Acronym Treatment
pasteurization

Low temperature
LTLT 63°C/30 min
long time

High temperature
HTST 72°C/15 s
short time

90°C/0.5 s
Ultrahigh
UHT 94°C/0.1 s
temperature
100°C/0.01 s

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 Pasteurization
Milk pasteurization
• Originally designed to target
Mycobacterium tuberculosis
• Coxiella burnetti is currently the
target organism

Pasteurization
• Thermoduric organisms
–Organisms capable of surviving
pasteurization
–Examples: lactobacilli,
micrococci, streptococci,
lactococci
–Pathogens are not thermoduric

Factors affecting heat resistance of

microorganisms
• Water
• Fat
• Salts
– Salts that reduce aw (Na+, K+)
more protective than those that
increase aw Ca+2, Mg+2)
• Carbohydrates
• pH

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 Factors affecting heat resistance of
microorganisms (cont.)

• Proteins
• Numbers of microorganisms
• Age or microorganisms
– Old cells (stationary phase) more
resistant that younger cells (log phase)
• Growth temperature
• Inhibitory compounds
• Time and temperature
• Ultrasonic effect

Canning
• Processing of food products in
hermetically sealed containers
– Metal
– Glass
• Canned products are commercially
sterile

Spoilage organisms associated with

canned products

Mesophilic
• Putrefactive anaerobes
– Clostridium botulinum
• Butyric anaerobes
– C. butyricum
• Aciduric flat sour
– Bacillus polymyxa
• Lactobacilli
• Yeasts
• Molds

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 Spoilage organisms associated with
canned products (cont.)
Thermophilic
• Anaerobic sulfide producers
– Desulfotomaculum nigrificans
• Flat-sour spores
• B. stearothermophilus, B. coagulans
• Anaerobic non-sulfide producers
– Thermobacterium thermosaccharolyticum

Canning
pH category Examples of foods Spoilage

Low acid Meats, seafood, •Thermophilic flat-

> 4.6 milk, some sour group
vegetables •Sulfide spoilers
•Gaseous spoilers
•Mesophilic spoilers –
Putrefactive
anaerobes
Acid Tomatoes, pears, Thermophilic spoilers
3.7-4.0 to 4.6 figs

High acid Fruits and fruit / Non-sporeforming

<4.0-3.7 vegetable mesophiles (yeasts,
products molds, lactic acid
bacteria

Reduced aw
•Drying
•Concentration

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Foods of low and intermediate
moisture
• Low moisture
– Dry, dessicated
– <20% moisture
– aw 0.00 – <0.60
• Intermediate moisture
– 15-50% moisture
– aw 0.60 – 0.85

These foods are shelf stable

Effect of drying on microorganisms

Time to spoilage
Microorganisms
aw at room
involved
temperature
>0.9 Bacteria Hours

0.80-0.85 Molds and yeasts 1-2 weeks

>0.65 – 0.7 Molds and yeasts Months

0.60-0.65 Molds and yeasts >2 years

No microbial
<0.60 N/A
spoilage

Drying

•Freeze drying
–lyophilization
•Hot vacuum drying
•Hot air drying
•Sun drying

Drying depends on temperature and

pressure conditions

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 Effect of the aw adjustment
method
Ways of adjusting the aw of a food
• Adsorption
– Adding water to dry foods to adjust aw
• Freeze dried foods
• Egg beaters
• Desorption
– Eliminate water from the food system
• Hot vacuum dried or sun dried foods
• At similar moisture content (% water)
desorption tends to produce lower aw

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 Concentration
• Adding solutes to the food system
– Salting (dry fish)
– Adding sugar (jellies, syrups)
• Adding solutes and eliminating water
water
– Caramelized condensed milk (cajeta)
• The solute plays an important role in
aw reduction

Growth of Staphylocoucus aureus in mocha cake

stored at 25°C as affected by moisturizing agent
9

8
20% Sucrose
7
20% Glucose
6
Log CFU/g

0
0 3 6 9 12 15 18
Hours of storage

Acidification

•Low pH
•Organic acids

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 Acidification
• Fermented foods
• Acidified foods
–Acidic foods vs.
acidified foods

Atmosphere modification

•Controlled atmospheres
•Modified atmospheres

Controlled atmospheres
• Substitution of air by a gas mixture
• Controlling gas concentration during storage
• Usually includes CO2
– Greater inhibition of microorganisms by CO2 at
refrigeration temperatures
– Lower APC in steaks during storage in 100% CO2
compared to 100% N2 or air
– Shelf life of lean beef inoculated with PMA and
stored at 5°C in 85% O2 + 15% CO2 was 9 days
longer than beef stored in air.
• Reducing O2 concentration may affect food
quality
– Red meats

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 Effect of CO2 on microorganisms in
meat packs
• Microbiota is shifted
• Predominant PMA (aerobic, CO2
sensitive) to lactic acid bacteria (LAB,
facultatively anaerobic, CO2 resistant)
• PMA are putrefactive, LAB are
fermentative
– Some sensorial change is still accepted
by consumers

Modified atmosphere packaging

(MAP)
• Substitution of air by a gas mixture
• Vacuum packaging
– 80% of fresh beef in the U.S. is vacuum
packaged
– Not all O2 is removed
– The O2 remaining is consumed by the
microbiota and the food
– CO2 is also produced by metabolic activity
• Inhibitory for microorganisms
• Careful selection of packaging film

Spoilage of vacuum packaged

meats
• Long-term refrigeration spoilage
associated with Brocchothrix
thermosphacta and lactobacilli
• Clostridium laramie is also associated
with gassy spoilage in vacuum
packaged meats

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 Food safety concerns
• Some researchers believe that MAP
results in a selection of potential
pathogens by inhibiting competitive
biota
– Aeromonas hydrophila
– Listeria monocytogenes
• A. hydrophila survives and grows
better in meat under air packaging
compared to vacuum

Addition of chemical
preservatives

GRAS substances
• Chemical additives must be listed as
GRAS by FDA regulations
– GRAS = Generally Recognized as Safe
• There is a maximum tolerance for
their concentration in foods
• Different preservatives target specific
organisms
• The effect of some preservatives is
affected by food characteristics such
as pH

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 Benzoates and parabens
Effect depends
HOOC COOH
on pH

Effect does not Benzoic acid

depend on pH

HO COOH

p-Benzoic acid

Parabens

Parabens

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 Other organic acid derivatives
• Sorbates
– Used as sorbic acid or as calcium or potassium
sorbate (salts more soluble than acid)
– Widely used as a preservative
– One half the toxicity of NaCl
– pH dependant
– No effect above pH 6.0
– May change the taste of foods if added in
excess (usually no more than 0.2%)
– Effective against spoilage and pathogenic
bacteria
• Propionates
– Lower tendency to dissociate
– Effective in low-acid foods

SO2 and sulfites

• SO2 used as a gas or in liquid form
• Used as preservative in wines, dried fruits,
juices, molasses
• Effect greatly favored by low pH
• Bacteriostatic against Acetobacter and LAB
– Juices and beverages
• SO2 can change to sulfurous acid derivatives
SO3-2 then to sulfites, bisulfites and
metabisultfites
• Sulfites are used as preservatives in juices and
beverages
– Also to prevent melanosis in shrimp

Nitrites and Nitrates

• Used in curing formulas for meat

• Stabilize red color of meat
• Inhibit spoilage and pathogenic
organisms
• Contribute to flavor development
• NO2 and NO3 change to HNO2
then to NO

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 NO2 and Clostridium botulinum

• Prevents germination of spores

of C. botulinum
• Also effective against S. aureus
• Added to cheeses to prevent
gassy spoilage caused by
clostridia

The Perigo factor

• Question:
– Why is C. botulinum almost totally absent in
cured, canned and vacuum packaged meats?
• Perigo and Roberts (1968) found that NO2
was 10 times more effective if added
before heat treatment
– A chemical factor is thought to be produced
during heating that enhances the inhibitory
effect of NO2
• Several researchers have reported that the
antibotulinal effect is interdependent with
NO2, with other curing ingredients, pH,
heat treatment, and temperature/time of
storage

Antimicrobial mechanism
• NO2 inhibits C. botulinum by
interferring with iron-sulfur enzymes
thus preventing ATP synthesis
– Ferredoxin
• Bacteria that lack ferredoxin are
resistant to NO2
– LAB lack ferredoxin
– Lactobacillus and other LAB spoilage in
cured meats is reported
• Green ham (Sam I am)

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 Other antimicrobials
• Ethylene oxide
– Highly toxic, but no residues
• Antibiotics and bacteriocins
– Nisin, subtilin, tylosin
• Antifungals
– Natamycin, benomyl, thiabendazole

Ionizing radiations
•Electron beam
•X-rays
•Gamma rays

Food irradiation

“Food Irradiation is a process that

exposes food to a carefully controlled
amount of ionizing energy to
damage the DNA of parasites, fungi,
and bacteria and eventually destroy
these organisms.” (IFST, 1999)

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Radioactivity vs. Irradiation
Ý Radioactivity:
Spontaneous emission of particles,
radiations or both

Ý Irradiation:
Transmission of energy (in the form
of waves) through space

Food irradiation in the U.S.

In the United States, ionizing

irradiation of food is considered a
food additive under current law
and thus must be approved by
FDA

Ionizing radiations of interest in

food preservation

• Electrom beam
• Gamma rays
• X rays

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 Electromagnetic spectrum

Characteristics of different forms

of radiation applicable in foods
ÝElectron Beams:
– Effective at converting electrons into high energy
beams such as X-rays
– Easily applicable on different types of foods,
climatic conditions and harvest seasons
– Can turn on and off with ease
– Less penetrating than gamma rays
– More energy than gamma rays
– Electron beams generated by linear accelerator can
generate 103-106 Gy/seg, whereas the dose generated by
60Co is 1-100 1-100 Gy/min

Characteristics of different forms

of radiation applicable in foods
Ý Gamma rays:
– Electromagnetic radiations emitted from
excited nuclei (60Co or 137Cs )
– Relatively inexpensive byproducts of
atomic fission
– Greater penetration power
– Half-life of 60Co: 5.27 years
– Half-life of 137Cs: ~30 years

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Characteristics of different forms
of radiation applicable in foods
Ý X rays:
– Produced by linear accelerator
– Essentially the same as electron beam
but after passing through a heavy metal
target
– Otherwise, essentially similar to gamma
rays

Forms of radiation
(from Jay, 1998)

Types of treatments
• Radappertization
– 30 - 40 kGy
– Commercial sterility
• Radicidation
– 2.5-10 kGy
– Pathogen reduction
• Radurization
– 0.75 – 2.5 kGy
– Reduction of bacterial populations
– Enhancement of food quality

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 Fundamentals of bacterial
destruction by irradiation
ÝGram positive bacteria more resistant than Gram
negative bacteria
ÝMolds less resistant than bacteria (especially G+)
– Some yeasts are highly resistant
Ý Types of microorganisms
– Sporeformers are more resistant than non-sporeformers
– Some highly resistant non-sporeforming bacteria
– Deinococcus, Deinobacter, Rubrobacter, Acinetobacter
Ý Most highly resistant bacteria
– Sporeformers
– Paenibacillus larvae
– type A Clostridium botulinum
– Non-sporeformers
– Enterococcus faecium R53
– Homofermentative lactobacilli

Fundamentals of bacterial
destruction by irradiation
Factors affecting irradiation
performance
Ý Number of microorganisms
Ý Composition of the food
Ý Presence or absence of oxygen
Ý Physical state of the food
Ý Age of microorganisms

D-values (D10) for selected bacteria

Organism D10 (kGy)
C. botulinum, type E Beluga 0.80
C. botulinum type A, toxin production in meat 36.08
Escherichia coli 0.20
Listeria monocytogenes 0.42 - 0.55

Salmonella typhimurium 0.50

Staphylococcus aureus 0.16
Yersinia enterocolitica, beef at 25°C 0.195
Yersinia enterocolitica, beef at -30°C 0.388

Adapted from Jay, 1998

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 D10 values for selected molds
Organism D10 (kGy)

Aspergillus flavus spores 0.66

Aspergillus flavus 0.055-0.06

Aspergillus niger 0.042

Penicillium citrinum, NRRL 5452 0.88

Penicillium sp. 0.42

Adapted from Jay, 1998

D10 value for Salmonella Poona on cantaloupe

cubes treated by electron beam irradiation

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8
7
M e an log 10 C F U /g

6
5
4
3
2
D10 = 0.203 kGy
1
0
0

1
1

1
05

15

25

35

45

55

65

75

85

95

05
0.

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1.
0.

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Dose (kGy)

D10 values for selected viruses

Type of virus D10 (kGy)

Adenovirus 4.1 - 4.9
Coxsackievirus 4.1 - 5.0
Echovirus 4.4 - 5.1
Herpes simplex 4.3
Poliovirus 4.1 - 5.4
Adapted from Jay, 1998

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 Effect of irradiation on
microorganisms

ÝAntimicrobial mechanism
ÝBreaking of DNA strands
ÝIrradiation breaks 6 chemical
chains out of every 10 million
chains present in a cell

Uses in the United States

• Control insects in wheat, wheat flour
• Inhibit sprouting in white potatoes
• "Sterilize" Trichinella in fresh pork (0.3-1 kGy)
• Inhibit mold decay, control insects in fresh
fruits and vegetables (0.3-1 kGy)
• Microbial disinfection of spices and herbs (up
to 30 kGy)
• Radicidation of fresh poultry
• Recent approval for red meat, shell eggs

Foods approved by WHO for

irradiation
Type of food Objective Dose (kGy)
Potatoes Sprout inhibition 0.1-0.15
Oatmeal flour Insect elimination 0.2-0.75
Dried fruits Insect elimination 1.0
Poultry Radicidation 7.0 max.
Fresh fruits Radurization 2.5
Fresh meats Radurization 6.0-8.0
Shrimp Radurization 0.5-1.0

Adapted from Jay, 1998

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Effect of irradiation on
quality

Water radiolisis

3H2O H + OH + H2O2 + H2

Consumer Acceptance
• Most consumers have little
knowledge of the process or it’s
advantages.
• Concern about foodborne illness has
created interest in irradiated foods
• After receiving educational
information market trial consumers
were more likely to purchase
irradiated foods

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 Packaging
• Most foods are irradiated in their
packaging
• Irradiated foods are clearly marked
– Radura symbol
– “irradiated for your food safety”
• Packaging must be approved for
irradiation use

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