FOOD MICROBIOLOGY

I. Relationships Food-Microorganisms-Consumers

François Lyumugabe
PhD in Agro-Food Industry and Brewing Engineering
Associate Professor of Biotechnology, CST-UR
1
 CONTENTS

1. Introduction
2. Primary sources of microorganisms
3. Food spoilage
4. Factors affecting the growth and survival of
micro- organisms in foods
5. Food shelf life determination and prediction
References:

- R Martin Adams and O Maurice Moss, 2008., Food microbiology (Third Edition),
Published by The Royal Society of Chemistry, Cambridge, UK. .
-Bibek Ray , 2004., Fundamental of food microbiology (Thirt ed.), CRC press, New
York,
- Juneja VK, Sofos JN, 2002., Control of foodborne microorganisms, Marcel
Dekker.,New York:
-Valero A, Carrasco E. and García-Gimeno R.MA., 2012. Principles and
Methodologies for the Determination of Shelf–Life in Foods, In: Eissa AA (ed) Trends
in vital food and control engineering. Intech, Rijeka 2
 I.1. INTRODUCTION

Food consists basically of plants and animals or product derived them. In

other terms, food can be raw materials (processed or formulated)
consumed by human and animals to produce energy, promotes growth,
and to maintain good health.

In most cases, there are no limitations on food consumption. However,

the excessive consumption of certain types of food such as fat, sugar and
salt, may be harmful to health.

Chemically, food products consist of water, fat, carbohydrates, protein

and small amounts of organic compounds and minerals. Thus, it will
promote the growth of microorganisms since all these compounds are the
energy sources for microbial grow.

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 it is understandable that our food can contain microorganisms in
interaction with food.

• The presence of microorganisms will cause 2 possibilities:

1˚) Deterioration of food quality.

2˚) Interactions between microorganisms and food give beneficial for
human.

• On microbiological plan, food quality can be defined in 2 ways:

- Marketing quality: is essentially concerned with organoleptic

characteristics and is reflected in the attitude of consumers. The
nutritional and technological characteristics of the food contribute to this
quality. Its economic impact is crucial for the food industry.

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•All our foods can be the site of microbial growth. The proliferation of
microorganisms depending to nutritive value of food and environmental
conditions.
•The proliferation is characterized by changes in appearance (color, silt),
texture and flavor (odor and taste).

Orange juice after seven days of

refrigerated storage.
1. Unprocessed juice is
completely spoiled due to
yeasts and has segregated by
enzymatic activity.
2. Heat pasteurized orange juice is
stable for more than five
weeks.

1 2
5
•Thus, most of foods (not subjected to antimicrobial treatments) have
microbial loads between 104 and 106/g.
•The microorganisms frequently encountered in foods belong to the
genus of Pseudomonas, Acinetobacter, Moraxella, Alcaligenes,
Aspergillus, Rhizopus, Clostridium and Flavobacterium.

Apple spoilage by Aspergillus flavus

Bread spoilage by Aspergillus niger

6
Food spoilage is not always microbiological origin; a
product may become unacceptable as a result of damage
caused by insects or rodents.

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- Hygienic quality: food safety corresponds to a threshold quality and
the zero-defect standard must be reached for certain food-
microorganism systems when the presence of the microorganism in the
food can cause a problem for the consumer health.

What is the importance of microorganism in food?

Good (desirable) Bad (undesirable)

Food bioprecessing Food disease
Food biopreservation Food spoilage
Probiotics
•Food processing: food produce by using biological process. In this process, microorganisms are
used as starter culture to produce different types of fermented foods and beverages.

• Food biopreservation: is a food biological preservative by using antimicrobial metabolites (from

certain microorganisms) in order to control pathogenic and spoilage microorganisms in food.

•Probiotics are microorganisms that are believed to provide health benefits by promoting our
body's natural immunity and improving digestion system.
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I.2. PRIMARY SOURCES OF MICROORGANISMS

Microbial flora normally associated with plants and animals is therefore potentially
present in food. Moreover, an exogenous microbial intake is often inevitable
(environment, contact, manipulations, etc ...).

Animals and
derived products

Air

Faeces

Soil Water
Plants and derived
products

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Soil and water
Bacteria: Achromabacter, Enterobacter,Alcaligenes, Bacillus, Clostridium, Corynebacterium,
Micrococcus, Proteus, Pseudomonas, Serratia, Sarcina, Streptomyces, etc.
Mold: Aspergillus; Rhizopus, Penicillium, Trichothecium, Bothrytis, Fusarium etc ...
Yeasts: Saccharomyces, Rhodotorula, Torula etc.
Plants and derived products
Bacteria: Acetobacter, Enterobacter, Erwinia, Flavobacterium, Lactobacillus,Leuconostoc,
Streptococcus, Paracolobactrum, etc.
Molds: genera responsible for degradation of fruits and vegetables
Yeasts: Saccharomyces, Rhodotorula, Torula, etc. There is a relationship between mi

Animals and derived products

Bacteria: Bifidobacterium (Lactobacillus bifidus), Bacteroids, Escherichia, Proteus, Salmonella,

Shigella, Staphylococcus, Streptococcus, Clostridium, Paracolobactrum, Pseudomonas.
Yeasts: Candida, etc ...

Note that the total microbial load of a healthy man is close to 1017 and 1019 .

Air

Most bacteria and moulds and many yeasts are present.

Bacteria: Bacillus, Sarcina, Micrococcus are frequent because resistant to low HR
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 I.3. FOOD SPOILAGE
I.3.1. What is spoilage?
According to the Oxford English Dictionary to spoil is to ‘deprive of good or effective
qualities’. When a food is spoiled its characteristics are changed so that it is no longer
acceptable. Food spoilage may not always be microbiological in origin; a product may
become unacceptable as a result of insect damage, drying out, discolouration, staling or
rancidity for instance, but by and large most food spoilage is a result of microbial
activity. Food spoilage may be defined as any changes in the visual, smell and texture of
food that makes it unacceptable for consumption.

spoiled milk with release of bad Perfect milk (acceptable milk)

odour (unacceptable milk) 11
Microbiological food spoilage can manifest itself in several different ways,
some of which often occur in combination. Visible microbial growth may be
apparent in the form of surface slime or colonies, degradation of structural
components of the food can cause a loss of texture, but the most common
manifestation will be chemical products of microbial metabolism, gas,
pigments, polysaccharides, off-odours and flavours.

However, spoilage is also a subjective quality; what is spoiled for one

person may be perfectly acceptable to another. The perception of spoilage is
subject to a number of influences, particularly social; foods acceptable in
some cultures are unacceptable in others. Some products such as well
matured cheeses and game birds that have been hung for extended periods
are esteemed by some people and highly objectionable to others. Affluence
is another contributory factor – many are not in the position to be able to
discard food due to some slight sensory defect. In the 18th and 19th century
navy, sailors often preferred to eat in dark corners so that they could not see
the weevils and maggots in their food.

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 I.3.2. Types of food spoilage

a) Physical spoilage
• Deshydration of vegatable

A dry lemon
b) Chemical spoilage
• Browning of fruits and vegetable

Browning of fruits A: Apple B: Banana

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c) Microbial spoilage

Microbiological food spoilage can manifest itself in several different ways, some of
which often occur in combination. Visible microbial growth may be apparent in the
form of surface slime or colonies, degradation of structural components of the food can
cause a loss of texture, but the most common manifestation will be chemical products of
microbial metabolism, gas, pigments, polysaccharides, off-odours and flavours.

Therefore, the proliferation of microorganisms, like fungi (moulds, yeasts) and bacteria,
in a food product results in changes in the organoleptic qualities that are generally
detectable when the number of microorganisms exceeds 106 per g of food product.

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The main criteria associated with food spoilage: Odour or smell ; Texture;
Shape; Slime formation; Colour; Gas production.

1˚) Relationship microorganism and composition of food

• From the carbohydrate foods (and derived products)

- Polymers (starch, cellulose): hydrolysis: modified texture

- Dimers and monomers (sucrose, maltose, lactose, glucose, fructose, etc.):
fermentations: formation of acids and carbonyl compounds. For example:
incidence on taste and aroma.

• From protein foods (and derivatives)

- Polymers (proteins): hydrolysis: modified texture
- Amino acids: decarboxylation, deamination, desulfurization, etc. :
changes in taste, odour, formation of toxic catabolites

• From food lipids (and derivatives): oxidation and lipolysis: change in taste.

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2˚) Modification of odour

The development of microorganisms

in a product is first detected by odour
changes due to the sensitivity of our
olfactory system.

The detection of threshold of organic

volatile compounds is on average 10-6
- 10-9 g (10-12 for pirazine
derivatives).

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Generally, these modifications are biphasic:

-A large part of the food is transformed into a dominant product

(acetic acid - ethanol): it can be an alteration (sour ...) or a desired
transformation.
If the product is rich in carbohydrates and has a pH> 6 there is a
tendency for lactic fermentation. If the product has a pH <6 and
without oxygen, tendency to alcoholic fermentation.
This primary spoilage is detectable from a threshold of about 108
germs / g.

-Production of characteristic odors linked to volatile organic

compounds (smell - taste) or not (taste). The detection threshold of
these odorous compounds varies from 10-6 to 10-12 (derivatives of
pirazine).

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 Some examples of perception thresholds

Volatil compounds Odor µg/l

2-isobutyl, 3-methoxypirazine Pepper 2.10-3
Methylmercaptan Coffee 2.10-2
2,4-decadienal Chiken 7.10-2
Ethyl-2-methyl butyrate Apple 10-1
3-Hexenal Tomato 3.10-1
g-decalactone Peach 7.10-1
Acetate d’amyle Banana 5
Ethanol Alcohol 105

These compounds contribute to the quality of certain fermented

products (wines, cheeses) or depreciation when these odors are
unpleasant (odor of ammonia, mercaptans, amines, etc ...).
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•Analyzed by gas chromatography, these organic volatile compounds
sometimes make it possible to identify the microorganisms that
produced them.
• The odor compounds produced by the microorganisms are mostly
detected when the microbial charge reaches 106 to 107 germs / g.

•So, it is generally not possible to attribute to each microorganism the

genesis of a particular odor. This production depends on the food
composition, the temperature, the strain microorganism, etc.

• However, molds often generate a musty (complex) or rancid smell,

while the bacteria generate pleasant, fruity or unpleasant odors.
Pseudomonas : linden odor (media low in organic matter)
Achromobacter or Flavobacterium : smell of apple or turnip (beer)
Bacillus subtilis: rotten melon odor
Streptomyces: musty smell.
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*Meat: a microbial development on the surface results in a
relenting odor from 107 germs / g when stored at 10° C and an
ammoniacal smell and H2S when stored at temperature. This is
known as putrefaction, which is a phenomenon sometimes
sought after (pitching).

*Fish: putrefaction generates ammoniacal odors (formation of

trimethylamine, mercaptan, dimethylsulfide, H2S etc ...). In
mackerel there are, for example, 2 phases: the first corresponds
to the production of lactic acid (sour) and the second to the
genesis of ammoniacal odors.

*Cheeses: Cl. Butyricum synthesizes butyric acid with a

characteristic unpleasant odor. An ammoniacal odor occurs
after proteolysis.

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3˚) Modification of taste

• They are linked to the presence of volatile or non-volatile compounds.

•The most frequent is an acidification linked to the production of lactic

acid. Various qualifications are used to describe this transformation:
stinging, embittering, ... This modification is favourable with certain
products (cheeses, sauerkraut, sausage) In the case of vinegar it is acetic
acid that is produced.
•The thresholds for perception of the taste of certain compounds are
given in the table below:

NaCl 0,25 %

Saccharose 0,5 %

HCl 0,007 %

quinine 5.10-5 % 21
• Some of the tastes related to the presence of some microorganisms are
described below:

Nutty taste : Leuconostoc citrovorum : diacetyl : butter. This same

microorganism induces a taste of margarine in citrus
juices
Rancidity : Pseudomonas
Malt taste : Yeasts in milk
Caramelized taste : yeasts or Streptococcus lactis var. Maltigenes in milk
Alcoholic taste : Yeasts
Sweet taste : yeasts (production of mannitol) : wine
Chalky taste : yeast (Endomycopsis or Trichosporum): bread
:Pediococcus, Lactobacillus, Leuconostoc transform
glycerol into Acrolein which combines with
Bitter taste: polyphenols: beer

Pungent taste : Production of CO2

Fruity tastes (biotransformations).

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 4˚) Changes in appearance and colour
•These changes are chronologically detectable visually well after the appearance of
odours. In a first phase, these are small areas that have variable shape characteristics
(round, flat, domed, irregular ...).

•Their appearance (opaque, matt, shiny, rough ...) and / or their colour (white, black,
yellow, red ...) are multiple. These areas are composed of bacteria, yeasts and
mucous secretions that extend to the surface of the food and form a coating often
sticky, viscous and sticky.

Spoilage of red bread

by Serratia marcescens
Meat spoilage (discoloration, bad smell..)

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•The proliferation of molds is characterized by the formation of colored zones with
centrifugal evolution. These areas may have varied aspects (felting, rough spots, ...).

Carrot spoiled by moulds

• Changes in color result from one or more phenomena:

- synthesis of one or more pigments by the microorganism. All colors are possible
(white - black - blue - green - yellow - red ..). The producer genres of pigment most
encountered are:
Micrococcus, Pseudomonas, Chromobacterium, Serratia, Bacillus, Flavobacterium,
Rhodotorula. 24
-transformation of an endogenous pigment into the food. Oxidation of
carotene (loss of the orange color of many plant products), changes in
myoglobin (numerous brown to green derivatives).

- Cellular destructions involving enzyme and substrate (PPO-quinone).

This phenomenon is common in plant products.
•production of a reactive and chromogenic component (H2S generating
various black sulphides).

5˚) Modification of the structure and texture

•The structure of a food product is linked to the presence of

macromolecules such as pectins, celluloses, hemicelluloses (starch
and proteins) in plant products and proteins in animal products.

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•If contaminating microorganisms synthesize and excrete hydrolases
(pectinases, proteases, etc.), softening occurs. For a given
microorganism, this softening is all the greater as the microbial load is
high:
-Favourable Phenomenon (Tenderising by proteolysis, clarification
of fruit juices by pectinases)
-Unfavourable phenomenon: loss of shape etc ...

For example, some enzymes in a tomato help it to

ripen, but other enzymes cause it to decay. Once
enzymic spoilage is under way, it produces damage to
the tomato skin, so moulds can begin to can attack it
as well, speeding the process of decay.

• The synthesis of polymers (dextrans from sucrose with Leuconostoc)

increases the viscosity of certain syrups o juices. 26
•The production of gas (CO2 most often) induces the formation of cracks or bubbles and alters
the packaging (e.g. spoilage of Canned Foods)

Spoilage of Canned Foods often manifests itself through microbial gas production which causes
the ends to distend and a number of different terms are used to describe the extent to which this
has occurred. The spore-forming anaerobes Clostridium can be either predominantly proteolytic
or saccharolytic but both activities are normally accompanied by gas production causing the can
to swell. Cans may sometimes swell as a result of chemical action. Defects in the protective
lacquer on the inside of the can may allow the contents to attack the metal releasing hydrogen.
These hydrogen swells can often be distinguished from microbiological spoilage since the
appearance of swelling occurs after long periods of storage and the rate at which the can swells is
usually very slow.
In cases where microbial growth occurs without gas production, spoilage will only be apparent
once the pack has been opened. Bacillus species, with the exceptions of B. macerans and B.
polymyxa, usually break down carbohydrates to produce acid but no gas giving a type of spoilage
known as a ‘flat sour’, which describes the characteristics of both the can and the food.

Spoilage of Musselman's Peach

(presence of gas produced by certain
microorganisms
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6˚) Modification of nutritional value

In the case of products obtained by fermentation, the structure, the hygienic,

organoleptic and nutritional qualities are currently well controlled.

The microorganisms involved in these processes consume high-energy molecules and

the caloric value of the fermented products is therefore generally lower than that of the
initial product.

These same microorganisms have a favourable role in synthesizing molecules with

biological activity such as vitamins or catabolizing toxic or antinutritional products
(non-fermentable carbohydrates C1-C6: stachyose, toxic proteins).

For most of our foods, the development of a superficial microbial flora is made from
simple carbohydrates and non-protein nitrogen. Thus, in the case of meat and fish,
surface alteration resulting in the formation of silt and characteristic odours is virtually
accompanied by proteolysis, and therefore a significant change in nutritional value up to
109 germs/cm2.

When proteolysis phenomena appear, they are followed by the formation of amino acid
derivatives which give the products odour, taste and texture such that they become
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inconsolable.
 I.4. FACTORS AFECTING THE GROWTH AND SURVIVAL OFMICROORGANISMS

I.4.1. Intrinsic factors (Food characteristics)

1˚) Biological structures

•The presence of envelopes, shells, skins etc. Gives certain foods
excellent protection against microbial growth (seed testas, fruit husks,
shells of nuts, eggs, skin of animals, etc.).

•The alteration of these natural protections often results in contamination

/ proliferation. The primary purpose of packaging is to protect the
stabilized or non-stabilized food from contamination. It should be noted
that there are edible packaging.

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2˚) Antimicrobial agents naturally present
•All foods were at some stage part of living organisms and, as such, have been equipped
through the course of evolution with ways in which potentially damaging microbial
infections might be prevented or at least limited.
• Examples:
- Fresh milk contains lactenins and anti-coliform factors with limited activity over time.
- The egg contains active lysozyme on Gram positive germs.
-The cranberries contain active benzoic acid on yeasts and molds; Compounds such as
thymol (thym), eugenol (clove) or cinnamic aldehyde (cinnamon) have antimicrobial
activities

Some essential oil components with antimicrobial activity 30

3˚) Biochemical composition of Food

•To proliferate, microorganisms must find nutrients in the food. Note that
the most dangerous microorganisms are heterotrophic chemo-
organotrophs and must therefore find their energy in the components of
the food. They must also find water, a source of nitrogen, minerals and
some of the vitamins and growth factors.

•The greater the diversity of composition of a food (animal products

such as meats and derivatives, milk, etc.), the greater its susceptibility to
be culture medium for growth of microorganisms.
4˚) pH

• The acidity or alkalinity of food has a profound effect on the growth

and metabolism of micro-organisms.

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•Plotting microbial growth rate against pH produces an approximately
symmetrical bell-shaped curve spanning 2–5 pH units, with a maximum
rate exhibited over a range of 1–2 units.
Growth rate
Escherichia coli

Acetobacter
Brucella melitensis

pH
1 4 7 10

• In general, bacteria grow fastest in the pH range 6.0–8.0, yeasts 4.5–

6.0 and filamentous fungi 3.5–4.0. As with all generalizations there are
exceptions, particularly among those bacteria that produce quantities of
acids as a result of their energy-yielding metabolism. Examples
important in food microbiology are the lactobacilli and acetic acid
bacteria with optima usually between pH 5.0 and 6.0. 32
•Thus, it is indirectly the conformation that the proteins take in a medium at a
given pH which is responsible for the expression of the activity of these molecules.
If at a given pH the conformation is such that the macromolecule no longer has any
activity, growth will stop if the activity of this macromolecule is essential to the life
of the microorganism.
•According to pH, it is usual to consider two groups of foods: those whose pH is
less than 4.5 and those whose pH is greater than 4.5 In the first category, the
dangerous microorganisms for human consumption do not generally multiply and
Clostridium botulinum does not produce its toxin.

•Acidophilic microorganisms (pH: 1-4.5) yeasts, moulds and certain bacteria

which produce the acids (acetic, lactic, propionic ...).

• Basophilic microorganisms (pH: 4.5 – 9.5): most pathogenic bacteria.

• pH of the food promotes all the better the proliferation that it will be near the optimum
pH of growth.

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 Table I.1. Approximate pH values permitting the growth of selected pathogens in food.
Microorganism Minimum Optimum Maximum

Clostridium perfringens 5.5 - 5.8 7.2 8.0 - 9.0

Vibrio vulnificus 5.0 7.8 10.2

Bacillus cereus 4.9 6.0 -7.0 8.8

Campylobacter spp. 4.9 6.5 - 7.5 9.0

Shigella spp. 4.9 9.3

Vibrio parahaemolyticus 4.8 7.8 - 8.6 11.0

Clostridium botulinum toxin 4.6 8.5

Clostridium botulinum growth 4.6 8.5

Staphylococcus aureus growth 4.0 6.0 - 7.0 10.0

Staphylococcus aureus toxin 4.5 7.0 - 8.0 9.6

Enterohemorrhagic Escherichia coli 4.4 6.0 - 7.0 9.0

Listeria monocytogenes 4.39 7.0 9.4

Salmonella spp. 4.21 7.0 - 7.5 9.5

Yersinia enterocolitica 4.2 7.2 9.6 34
 Table I.2. pH ranges of some common foods

Food pH Range

Dairy products Butter 6.1 - 6.4

Buttermilk 4.5

Milk 6.3 - 6.5

Cream 6.5

Cheese (American mild and 4.9; 5.9

cheddar)

Yogurt 3.8 - 4.2

Meat and poultry Beef (ground) 5.1 - 6.2

Ham 5.9 - 6.1

Veal 6.0

Chicken 6.2 - 6.4

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 Fish and Shellfish Fish (most species) 6.6 - 6.8

Clams 6.5

Crabs 7.0

Oysters 4.8 - 6.3

Tuna Fish 5.2 - 6.1

Shrimp 6.8 - 7.0

Salmon 6.1 - 6.3

White Fish 5.5

Fruits and Vegetables Apples 2.9 - 3.3

Apple Cider 3.6 - 3.8

Bananas 4.5 - 4.7

Figs 4.6

Grapefruit (juice) 3.0

Limes 1.8 - 2.0

Honeydew melons 6.3 - 6.7

Oranges (juice) 3.6 - 4.3

Celery 5.7 - 6.0

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Plums 2.8 - 4.6

Watermelons 5.2 - 5.6

Grapes 3.4 - 4.5

Asparagus (buds and stalks) 5.7 - 6.1

Beans (string and lima) 4.6 - 6.5

Beets (sugar) 4.2 - 4.4

Broccoli 6.5

Brussels Sprouts 6.3

Cabbage (green) 5.4 - 6.0

Carrots 4.9 - 5.2; 6.0

Cauliflower 5.6

Corn (sweet) 7.3

Cucumbers 3.8

Eggplant 4.5

Eggs yolks (white) 6.0 - 6.3 (7.6- 9.5)

Lettuce 6.0

Olives (green) 3.6 - 3.8

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 Onions (red) 5.3 - 5.8

Parsley 5.7 - 6.0

Parsnip 5.3

Potatoes (tubers and sweet) 5.3 - 5.6

Pumpkin 4.8 - 5.2

Rhubarb 3.1 - 3.4

Spinach 5.5 - 6.0

Squash 5.0 - 5.4

Tomatoes (whole) 4.2 - 4.3

Turnips 5.2 - 5.5

The pH of the food is sometimes evolutionary (transformation of

glycogen into lactic acid during rigor mortis, level of maturity of
vegetables and fruits ...) and can thus vary from several units

38
 5˚) Water activity
•Microorganisms need water in an available form to grow in food products. The
control of the moisture content in foods is one of the oldest exploited preservation
strategies. Food microbiologists generally describe the water requirements of
microorganisms in terms of the water activity (aw) of the food or environment.

•Water activity is defined as the ratio of water vapor pressure of the food substrate to
the vapor pressure of pure water at the same temperature :
aw = p/po where p = vapor pressure of the solution and po = vapor pressure of the
solvent (usually water).

•The aw of pure water is 1.00 and the aw of a completely dehydrated food is 0.00. The
aw of a food on this scale from 0.00 - 1.00 is related to the equilibrium relative
humidity above the food on a scale of 0 - 100%. Thus, % Equilibrium Relative
Humidity (ERH) = aw x 100. The aw of a food describes the degree to which water is
"bound" in the food, its availability to participate in chemical/biochemical reactions,
and its availability to facilitate growth of microorganisms.

39
•Microorganisms capable of developing in the products with low water
activity are called Xerophiles, those in highly sweetened or salted media
respectively of Osmophiles and Halophiles.

• Ways to lower the water activity in foods are numerous:

- physical (freezing, dehydration)
- additives (salting, sweetening ...)

They lead respectively to frozen, dried, salted and brined foods, jams and
sweets (cf course of food technology).

• For aw <0.65 no microorganism can grow (they can survive).

•For aw <0.85 no pathogenic microorganism can grow except for certain

moulds which excrete mycotoxins.

40
Water activity of some
microorganisms
1 0.9 0.8 0.7 0.6

Clostridium botulinum

E. coli, Salmonella sp, VBibrio, Pseudomonas, Achromobacter, Enterobacter, etc.

Most bacteria, some yeasts, Basidiomycetes

Bacillus, Lactic bacteria, yeasts, Mucorales, Fusarium

Staphylococcocus, Debaromyces, Cladosporium

Saccharomyces, Penicillium

Halobacterium, Halococcus, Aspergillus

Eurotium
Saccharomyces
rouxii, Xeromyces

41
Table I.3. Approximate aw values of selected foodcategories.

Animal Products aw

fresh meat, poultry, fish 0.99 - 1.00

natural cheeses 0.95 - 1.00

pudding 0.97 - 0.99

eggs 0.97

cured meat 0.87 - 0.95

sweetened condensed milk 0.83

Parmesan cheese 0.68 - 0.76

honey 0.75

dried whole egg 0.40

dried whole milk 0.20

42
 Plant Products aw

fresh fruits, vegetables 0.97 - 1.00

bread ~0.96

bread, white 0.94 - 0.97 Note that The

bread, crust 0.30 principle of design
baked cake 0.90 - 0.94 of foods with
maple syrup 0.85 intermediate
jam 0.75 - 0.80
humidity (AHI) is
based on the
jellies 0.82 - 0.94
simultaneous use
uncooked rice 0.80 - 0.87
of several agents
fruit juice concentrates 0.79 - 0.84
depressants of
fruit cake 0.73 - 0.83 water activity.
cake icing 0.76 - 0.84

flour 0.67 - 0.87

dried fruit 0.55 - 0.80

cereal 0.10 - 0.20 43

 I.4.2. Extrinsic factors (Environmental limitations)
1˚) Temperature
In microorganisms, the temperature increases the speed of all reactions which it is the
seat (anabolism and catabolism); We observe an increase in the growth rate with the
increase in temperature following the Arrhenius law.
Arrhenius:
µ acceleration of reactions

Proteins denaturation

Temperature
min T Optimal T max T
Effect of temperature on growth rate

However, as the temperature increases, the rate of denaturation of bacterial proteins

(enzymes in particular) increases. When all protein molecules with metabolic activity
are denatured, the germ has a zero growth rate and often, if enzymes involved in the
expression of its genome are inactivated, it dies because these phenomena are very
often irreversible. 44
The growth curves (changes in the number of germs as a function of
time) are variable according to the microorganisms, which makes them
classified into three main categories:

Table I.4. Classification of microorganisms according to the temperature

Temperature °C (°F)

Group Minimum Optimum Maximum

Thermophiles 40 - 45 (104 - 113) 55 - 75(131 - 167) 60 - 90(140 - 194)

Mesophiles 5 - 15 (41 - 59) 30 - 45(86 - 113) 35 - 47 (95 - 117)

Psychrophiles (obligate
psychrophiles) -5 - +5 (23 - 41) 12 - 15(54 - 59) 15 - 20 (59 - 68)

Psychrotrophs
(facultative
psychrophiles) -5 - +5 (23 - 41) 25 - 30(77 - 86) 30 - 35 (86 - 95)

45
 Table I.5. Approximate minimum, maximum and optimum temperature values in °C
permitting growth of selected pathogens relevant to food

Microorganism Minimum Optimum Maximum

Bacillus cereus 5 28 55
Campylobacter spp. 32 42 45
Clostridium botulinum types A& B* 10 30 50
Clostridium botulinum type E** 3 25 45
Clostridium perfringens 12 43 50
Enterotoxigenic Escherichia coli 7 35 46
Listeria monocytogenes 0 30 45
Salmonella spp. 5 35 45
Staphylococcus aureus growth 7 35 48
Staphylococcus aureus toxin 10 40 46
Shigella spp. 7 37 45

Vibrio cholera 10 37 43
Vibrio parahaemolyticus 5 37 43
Vibrio vulnificus 8 37 43

Yersinia enterocolitica -1 28 42

46
•Cold is a widely method used for controlling the growth rate of microorganisms. In the
refrigerator, the shelf life is approximately 3 to 5 days, a time corresponding to an
unfavorable proliferation of the cryophilic (psychophilic) microorganisms. Freezing at -
18 ° C completely stabilizes the food in which no growth of microorganism can occur.

2˚) Types of packaging/atmospheres

•Many scientific studies have demonstrated the antimicrobial activity of gases at

ambient and sub-ambient pressures on microorganisms important in foods.

• Gases inhibit microorganisms by two mechanisms:

- First, they can have a direct toxic effect that can inhibit growth and proliferation. E.g.
Carbon dioxide (CO2), ozone (O3), and oxygen (O2) are gases that are directly toxic to
certain microorganisms. This inhibitory mechanism is dependent upon the chemical and
physical properties of the gas and its interaction with the aqueous and lipid phases of
the food.

47
 Oxidizing radicals generated by O3 and O2 are highly toxic to anaerobic bacteria and
can have an inhibitory effect on aerobes depending on their concentration. Carbon
dioxide is effective against obligate aerobes and at high levels can deter other
microorganisms.

- second inhibitory mechanism is achieved by modifying the gas composition, which

has indirect inhibitory effects by altering the ecology of the microbial environment.
When the atmosphere is altered, the competitive environment is also altered.
Atmospheres that have a negative effect on the growth of one particular microorganism
may promote the growth of another. This effect may have positive or negative
consequences depending upon the native pathogenic microflora and their substrate.
Nitrogen replacement of oxygen is an example of this indirect antimicrobial activity.

•A variety of common technologies are used to inhibit the growth of microorganisms,

and a majority of these methods rely upon temperature to augment the inhibitory
effects. Technologies include modified atmosphere packaging (MAP), controlled
atmosphere packaging (CAP), controlled atmosphere storage (CAS), direct addition of
carbon dioxide (DAC), and hypobaric storage.

•E.g. A nitrogen atmosphere or vacuum packaging preventscontamination by aerobic

microorganisms.
48
 Cheeses packaged under vacuum

Food vacuum packaging machine

Potato Chips packaged under gas nitrogen

Nitrogen Gas Flushing Sealing Machine 49
 Table I.6. Examples of gas mixtures used for various MAP products.

Product % CO2 % O2 %N2

Fresh meat 30 30 40

15 - 40 60 - 85 0

Cured meat 20 - 50 0 50 - 80

Sliced cooked roast beef 75 10 15

Eggs 20 0 80

0 0 100

Poultry 25 - 30 0 70 - 75

60 - 75 5 – 10 20

100 0 0

20-40 60-80 0

Pork 20 80 0

Processed Meats 0 0 100

Fish (White) 40 30 30

Fish (Oily) 40 0 60

60 0 40 50
 Product % CO2 % O2 %N2
Hard cheese 0 - 70 30 - 100
Cheese 0 0 100
Cheese; grated/sliced 30 0 70
Sandwiches 20 - 100 0 – 10 0 - 100
Pasta 0 0 100
70 - 80 0 20 - 30
Baked goods 20 - 70 0 20 - 80
0 0 100
100 0 0

3˚) Storage/holding conditions

•In this module, the storage conditions will be limited to the storage/holding
temperature, and the time/temperature involved in cooling of cooked items, and the
relative humidity to which the food or packaging material may be exposed.

•Note that other factors that may be included as important considerations for storage,
such as the effectiveness of the packaging material at conserving certain characteristics
(see the course of food technology)

51
•When considering growth rate of microbial pathogens, time and temperature are
integral and must be considered together. As has been stated previously, increases in
storage and/or display temperature will decrease the shelf life of refrigerated foods
since the higher the temperature, the more permissive conditions are for growth.

•At the same time, those foods that have been cooked or re-heated and are served or
held hot may require appropriate time/temperature control for safety. For example, the
primary organism of concern for cooked meat and meat-containing products is C.
perfringens. Illness symptoms are caused by ingestion of large numbers (greater than
108) of vegetative cells. The organism has an optimal growth range of 43 - 47 °C and a
growth range of 12-50 °C . Generation times as short as 8 min have been reported in
certain foods under optimal conditions. Thus, time/temperature management is essential
for product safety.
•We have many examples in literature of foodborne illness that have resulted from
cooling food too slowly, a practice that may permit growth of pathogenic bacteria. E.g.
the spore-forming pathogens that have relatively short lag times and the ability to grow
rapidly and/or that may normally be present in large numbers. Microorganisms that
possess such characteristics include C. perfringens, and Bacillus cereus. As with C.
perfringens, foodborne illness caused by B. cereus is typically associated with
consumption of food that has supported growth of the organism to relatively high
numbers. 52
•The relative humidity of the storage location affects both the water activity of
the food (dynamic equilibrium) and the growth of microorganisms on the
surface of the food. For example, when a food has a water activity of 0.6, it is
necessary to avoid that the relative humidity conditions of the surrounding
atmosphere lead to an increase in surface water activity to a compatible value
with microbial growth.

4˚) Antimicrobials used during the food manufacture

The addition of antimicrobial compounds to food products (additives) or the

use of various antimicrobial agents in the food production environment
(disinfecting, cleaning agents, etc.) is regulated and will not discussed in this
module .

53
Table I.7. Preservatives frequently used in conjunction with main groups of foods in the U.S.

Foodstuff Nitrate, Sulfur Acetic Propionic Sorbic Benzoic BHA Smoke Nisin Parabens
Nitrite Dioxide Acid Acid Acid Acid and
BHT

Fat - - + - ++ + + - - +
Emulsions

Cheese - - - + ++ (+) - - + -

Meat ++ - - - + - - ++ - -
Products

Seafood + + ++ - + + - ++ - (+)
Products

Vegetable - + ++ - ++ ++ + - - -
Products

Fruit - ++ + - ++ ++ (+) - - +
Products

Beverages - (+) - - ++ ++ + - - +

Wine - ++ - - ++ - - - - -

Baked goods - - + ++ ++ - - - - (+)

Confectionery - - - - ++ (+) (+) - - -

54
 I.5 FOOD SHELF LIFE DETERMINATION AND PREDICTION
The establishment of validated methodologies for the determination of food shelf-life is currently
demanded by both food industries and Health Authorities at national and international scale. It is
well known that most foods are perishable, since they are subjected to modifications in their
structure, composition and properties during storage before consumption. These changes are of
physico-chemical origin attributed to food composition together with the action of intrinsic and
extrinsic environmental factors, and also microbiological, where spoilage flora play an important
role. These modifications are “translated into“ sensorial deterioration at a specific time point.

Food shelf-life can be defined as “the term or period a product may be stored before a specific
element of the product makes it unsuitable for use or consumption”. This element could be of
biological or physicochemical nature.

Understanding how different properties of a food, its environment and its history can influence
the microflora that develops on storage is an important first step towards being able to make
predictions concerning shelf-life, spoilage and safety.

It is therefore essential to know the evolution of the microbial flora throughout the life of the
product and in particular the most probable evolution concerning the nature of the floras
(qualitative) and their numbers (quantitative) of the food product from the factory outlet until
consumption.

55
In the last years, different procedures have been reported for the establishment of shelf-
life, mainly based on the detection of microbial alteration, as well as physico-chemical
and sensorial changes. The traditional approach consists of setting a cut-off point along
thestorage period at the time when any of the measured attributes exceeds a pre-
established limit
.
Experimental work usually includes the storage of food product at different
temperatures, performance of microbial analysis and the assessment of spoilage by
sensorial testing. In the case of foods whose shelf-lives might be conditioned by the
presence and proliferation of pathogenic microorganisms, experiments also involve
challenge testing with the target organism prior to storage.

The cut-off point has been traditionally referred as quality limit (if deterioration of food
is known to be produced by physico-chemical changes), or safety limit (if deterioration
of food is due to the presence of nocive chemical substances and/or pathogenic
microorganisms, parasites or virus at levels of concern). This method is usually labour-
intensive and expensive.

56
 Table I.8. Example of determination of shelf life of juice heated to 75 ° C for 5 minutes
( traditional method)
Storage location Room temperature Refrigetor (4°C)
(27°C )
Shelf life (days)
0d 10 d 20 d 30 d 10 d 20 d 30 j
Parameters
Organo -
leptic Color Orange Orange Orange Orange Orange Orange Orange
Taste Sweet Sweet Sweet Sweet Sweet Sweet Sweet
Aroma orange orange orange orange orange orange orange
deposit volume 0 3 ,46 3,80 3,86 3,90 3,96 3,96
at the bottle (± 0,09) (± 0,08) (± 0,07) (± 0,07) (± 0,07) (± 0,07)
bottom (%)

Stability pH 4,03 4,08 4,08 4,08 4,04 4,04 4,04

(± 0,09) (± 0,04) (± 0,04) (± 0,04) (± 0,06) (± 0,07) (± 0,07)
Brix 10,8 10,8 10,8 10,72 10,8 10,8 10,8
(± 0,45) (± 0,45) (± 0,44) (± 0,54) (± 0,45) (± 0,45) (± 0,45)
Total aerobic 0 0 0 0 0 0 0
microbial flora

Total anaerobic 0 0 0 0 0 0 0
microbial flora

Nutritional Vitamin C 35 ,97 35,91 34,84 34,49 35,97 35,9 35,79

value content (mg/l) (± 3,68) (± 3,63) (± 3,78) (± 3,7) (± 3,68) (± 3,67) (± 3,67)

57
Regarding microbiological proliferation of spoilage and/or pathogenic
microorganisms, predictive microbiology is recognized as a reliable tool
for providing an estimation of the course of the bacteria in the foods, and
indirectly, provide an estimation of shelf-life of the product in the cases
when the cause of food spoilage or unacceptability is known to be
microbiological. Indeed, mathematical modelling is a science-based
discipline which aims to explain a reality with a few variables, and
whose applications have been extended beyond research as a real added-
value industrial application.

The main concept behind the application of predictive microbiology for

the determination of shelf-life based on spoilage is the specific spoilage
organisms (SSO), which are associated to sensorial changes and
spoilage. As such, the end of shelf-life can be defined as the time
needed for SSO to multiply from an initial contamination level to a
spoilage level, or the time invested by SSO to produce a certain
metabolite causing sensorial rejection.
58
In the case of pathogens, challenge test protocols are available for the
determination of kinetic parameters (named maximum growth rate
[µmax] and lag time [lag]) of pathogen organisms in ready-to-eat foods..
In addition, European Regulation No. 2073/2005, recommends the
performance of predictive microbiology studies in order to investigate
compliance with the criteria throughout the shelf-life.

In general, proliferation of pathogens for which absence is required

although might be present in foods, growth/no growth models are
generally accepted as useful tools for the determination of
the probability of growth, whereas those pathogens for which hazardous
levels (i.e. Bacillus cereus) or toxin-producing organisms (i.e.
Staphylococcus aureus) have been set, growth kinetic models are more
appropriate to estimate the time until reaching such levels.

59
•Roberts and Buchanan proposed mathematical models to predict the
shelf life of food products. This duration is a polynomial function taking
into account the factors influencing microbial survival and growth
(structure, composition, pH, aw, temperature, etc.).
Modeling
External Factors Determination
of:
-Initial
Food composition, pH, aw, redox potential,
microbial load
temperature, gas, HR, PO2, P CO2, presence of other No
microorganisms (antagonism, synergy, ...)
-Charges
maximum
tolerable at
the deadline of
shelf life (DC)
Nc Log Nc

-Lag time t1
µmax
Log No
-Maximun
growth rate
Internal Factors (µmax)
Physiological condition, age, genotype, stress, ... t1 DC

60
E.g. Predicted growth of spoilage organisms during two chill distribution chains

61
There are many factors that may affect the shelf life of a product. Some
factors relate to the food itself (intrinsic factors), such as moisture and
pH, while others are external to the product (extrinsic factors), e g. the
packaging conditions, materials and storage conditions. By
understanding which are the most important factors impacting on the
shelf life of a food, it may be possible to manipulate these factors to
extend the shelf life. Information on the factors that influence and cause
food deterioration and spoilage and thus the shelf life of a food can be
found in section I.3 and I.4.

Altering the composition, formulation, processing or packaging may

inadvertently lead to a decrease in the shelf life or make the food more
susceptible to the growth of spoilage or even pathogenic
microorganisms. So it is important to assess any changes proposed for
their potential to have an adverse effect on shelf life. This
will be especially important if the safety of a food relies on a number of
interacting factors or hurdles to inhibit the growth of pathogenic bacteria.
62
 I.6. Fermented and Microbial Foods
Microbes can however play a positive role in food. They can be consumed as foods in
themselves as in the edible fungi, mycoprotein and algae. They can also effect desirable
transformations in a food, changing its properties in a way that is beneficial.

Almost without exception, fermented foods were discovered before mankind had any
knowledge of micro-organisms other than as witness to the effects of their activity. It
was simply an empirical observation that certain ways of storing food effected desirable
changes in its characteristics (Table I.9). Originally the most important of these changes
must have been an improvement in the shelf-life and safety of a product, although these
became less important in the industrialized world with the advent of alternative
preservation methods such as canning, chilling and freezing. Modern technologies have
in no way diminished the sensory appeal of fermented products however.

In food fermentation, conditions of treatment and storage produce an environment in

which certain types of organism can flourish and these have a benign effect on the food
rather than spoiling it. The overwhelming majority of fermented foods is produced by
the activity of lactic acid bacteria and fungi, principally yeasts but also, to a lesser
extent, moulds. Both groups of organisms share a common ecological niche, being able
to grow under conditions of low pH and reduced aw, although only lactic acid bacteria
63
and facultative yeasts will prosper under anaerobic conditions.
Table I.9. Some fermented foods

Food Ingredients Geographical Distribution

Busa Rice, millet, sugar Turkey

Beer Barley, sorghum Widespread
Cheese Milk Widespread
Chicha Maize and others S. America
Dawadawa Locust beans W. Africa
Gari Cassava Nigeria
Idli/dosa Rice and black gram India
Injera Tef Ethiopia
I-sushi Fish Japan
Kefir Milk Eastern Europe
Kenkey Maize, sorghum Ghana
Kimchi Vegetables Korea
Koko Maize, sorghum Ghana
Leavened bread Wheat Europe, N. America
Lambic beer Barley Belgium
Mahewu Maize S. Africa
Nam Meat Thailand
Ogi Maize, sorghum, millet Nigeria
Olives Mediterranean Area
Palm wine Palm sap Widespread
Poi Taro Hawaii
Puto Rice Philippines
Salami Meat Widespread
Salt stock, cucumbers Cucumbers Europe, N. America
Sauerkraut Cabbage Europe, N. America
Sorghum beer Sorghum S. Africa
Sourdough bread Wheat, rye Europe, N. America
Soy sauce, miso Soy beans S.E. Asia
Tempeh Soy beans Indonesia
Tibi Fruit Mexico
Yoghurt Milk Widespread
64
As a consequence, they frequently occur together in fermented foods;
in some cases members of both groups act in concert to produce a
product while in others, one group plays the role of spoilage organisms.
Some examples of these are presented in below Table .

Table I.10 Yeasts and lactic acid bacteria in fermented foods

Yeasts1 Lactic acid bacteria2 Yeasts and lactic acid bacteria

Modern European beers Yoghurt Sourdough bread
Bread Sauerkraut Kefir
Wine Salami Soy sauce
Cider Cheese African beers
Lambic beer

1 The presence of lactic acid bacteria in these foods is often associated with spoilage
2 The presence of yeasts in these foods is often associated with spoilage

65
 I.6.1. Yeast
A major taxonomic study of the yeasts by Kreger–van Rij (1984) describes about 500 species
divided into 60 genera of which 33 are considered to be Ascomycetes, 10 Basidiomycetes and 17
Deuteromycetes. A number of yeasts, though certainly not all, are able to grow anaerobically
using a fermentative metabolism to generate energy. The majority, if not all, of these fermentative
yeasts grow more effectively aerobically and anaerobic growth usually imposes more fastidious
nutritional requirements on them.

Although there is a large diversity of yeasts and yeast-like fungi, only a relatively small number
are commonly associated with the production of fermented and microbial foods. They are all
either ascomycetous yeasts or members of the imperfect genus Candida. Saccharomyces
cerevisiae is the most frequently encountered yeast in fermented beverages and foods based on
fruits and vegetables, an observation which is reflected in the existence of more than eighty
synonyms and varieties for the species.

Because of its ability to grow at low water activities in the presence of high concentrations of
sugar or salt, Zygosaccharomyces rouxii is espe-cially associated with the fermentation of plant
products in which the addition of salt is an integral part of the process. Many strains of Hansenula
anomala and Debaryomyces hansenii can also grow in fairly concentrated salt solutions and the
latter is frequently isolated from brined meat products and fermented sausages.

66
One of the most important yeasts associated with spoilage is Zygo-saccharomyces
bailii. It has the ability to grow at relatively low water activities and low pH, as well as
being remarkably resistant to preservatives, such as sorbic, benzoic and ethanoic acids,
sulfur dioxide and ethanol, commonly used to prevent microbial spoilage of fruit juices,
fruit juice concentrates, fermented beverages, pickles and sauces. Z. bailii is strongly
fermentative and spoilage of products stored in plastic packs and glass bottles can lead
to explosion of the containers. The survival of a single cell in a product containing an
appropriate nitrogen source and fermentable carbohydrate can result in spoilage, so
pasteurization or membrane filtration before filling, followed by stringent hygiene to
prevent post-treatment contamination, are essential.

I.6.2. Lactic acid bacteria

The term lactic acid bacteria (LAB) has no strict taxonomic significance, although the
LAB have been shown by serological techniques and 16S ribosomal RNA cataloguing
to be phylogenetically related. They share a number of common features: they are
Gram-positive, non-sporeforming rods or cocci; most are aerotolerant anaerobes which
lack cytochromes and porphyrins and are therefore catalase- and oxidase-negative.
Some do take up oxygen through the mediation of flavoprotein oxidases and this is used
to produce hydrogen peroxide and/or to re-oxidize NADH produced during the
dehydrogenation of sugars.

67
Table I.11. Principal genera of the lactic acid bacteria

Genus Cell Morphology Fermentation Lactate isomer DNA (mole %GC)

Lactococcus cocci in chains homo L 33–37

Leuconostoc cocci hetero D 38–41

Pediococcus cocci homo DL 34–42

Lactobacillus rods homo/hetero DL, D, L 32–53

Streptococcus cocci in chains homo L 40a

a S. thermophilus
(Other genera that are currently included in the lactic acid bacteria, Carnobacterium,
Enterococcus, Oenococcus, Vagococcus, Aerococcus, Tetragenococcus, Alloiococcus,
Weissella)

68
Lactic acid bacteria are often inhibitory to other micro-organisms and this is the basis of their
ability to improve the keeping quality and safety of many food products.

Bacteriocins are bactericidal peptides or proteins which are usually active against species closely
related to the producing organism. They production by lactic acid bacteria has been extensively
studied in recent years and a number have been described. Interest in them stems from the fact
that they are produced by food-grade organ-isms and could therefore be regarded as ‘natural’ and
hence more acceptable as food preservatives. A few promising candidates have been found but
many others have a spectrum of activity which is too limited to be of any practical utility.

Since then a number of claims have been Table I.12. Beneficial effects claimed for
made for lactic acid bacteria, particularly lactic acid bacteria
in association with fermented milks (Table Nutritional improvement of foods
Inhibition of enteric pathogens
9.6). So much so, that live cultures of
Alleviation of diarrhoea/Constipation
lactic acid bacteria (and some others such Hypocholesterolaemic action
as Bifidobacterium spp.) consumed in Anticancer activity
foods are frequently termed ‘probio-tics’ Simulation of the immune system
(Greek: for life). Much of the evidence
available on these putative benefits is
however inadequate or contradictory at
present, and many remain rather ill
defined.
69
Several studies have shown improved nutritional value in grains as a result of lactic
fermentation, principally through increasing the content of essential amino acids. Such
improvements however may be of only marginal importance to populations with a
varied and well balanced diet. It has also been reported that fermentation of plant
products reduces levels of antinutritional factors which they may contain such as cyano-
genic glycosides and phytic acid, although this effect is often the result of other aspects
of the process such as soaking or crushing rather than microbial action. Some have
claimed that fermentation of milks increases the bioavailability of minerals, although
this is disputed.

Lactic acid bacteria have been reported to stimulate the immune system and various
studies have described their ability to activate macro-phages and lymphocytes, improve
levels of immunoglobulin A (IgA) and the production of gamma interferon. These
effects may contribute to a host’s resistance to pathogens and to the antitumour activity
noted for LAB, mainly Lactobacillus acidophilus, in some animal models. An additional
or alternative possible mechanism proposed for the antitu-mour effect is the observed
reduction in activity of enzymes such as b-glucuronidase, azoreductase and
nitroreductase in faecal material when LAB are ingested. These enzymes, produced by
components of the intestinal flora, can convert procarcinogens to carcinogens in the gut
and their decreased activity is probably due to inhibition of the produc-ing organisms by
LAB.
70
NOTE: In this section we have listed a limited selection of fermented
foods which we believe illustrates their diversity and importance as well
as some of their general microbiological features. There is a large and
growing literature on this topic and details of others among the plethora
of fermented foods produced can be found in some of the references
recommended as further reading in this course. In addition, the modules
of Microbial physiology and Enzymatic Engineering, and Industrial
Biotechnology will also discuss the different fermentation types.

71