FOOD

BIOTECHNOLOGY
Eight environmental sources of micro-organism found in
food are given below:
1. Soil and water
2. Plants and plant products
3. Food utensils
4. Intestinal tract of human and animals
5. Food handlers
6. Animal feeds
7. Animal hides
8. Air and dust
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Microbial spoilage of foods. Cause of spoilage classification of foods by ease of spoilage.

Factors affecting kinds and numbers of microorganisms in food.

Spoilage of food by micro organisms

Cause of spoilage:
Spoilage may be due to one or more of the following
1. Growth and activity of micro organisms
2. Insects
3. Action of the enzymes of the plant or animal food.
4. Purely chemical reactions i.e., those not catalysed by enzymes of the tissues or of micro
organisms

5. Physical changes such as those caused by freezing, burning, drying, pressure etc.
Classification of foods by ease of spoilage:
1. Stable or non perishable foods:
These foods which do not spoil unless handled carelessly include such products as sugar,
flour and dry beans.

2. Semi perishable foods:

If these foods are properly handled and stored, they will remain unspoiled for a fairly long
period.
Ex: Potatoes, apples etc.
3. Perishable foods:
It includes most important daily foods that spoil readily
Ex: Meats, fish, poultry, milk, eggs, fruits and vegetables.
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Contamination may increase number of micro organisms in the food. Wash water may
add surface bacteria in butter, plant equipment may add spoilage organisms to foods during
processing. Increased “ bio burden” of micro organisms, especially those which cause spoilage,
makes preservation more difficult.
Growth of micro organisms in food obviously will increase number of micro organisms.
Pretreatment of foods may remove or destroy some kinds of micro organisms and
inactivate part or all of the food enzymes.

● Washing may remove organisms from the surface or may add some from the wash water.
●
If washing is by means of an antiseptic or germicidal solution, numbers of organisms may be
greatly reduced.
● High temperatures will kill more organisms treatment with rays, ozone, SO2,
germicidal vapors will reduce numbers.

Factors affecting the growth of micro organisms in food:

Associative growth:
Associations of micro organisms with each other are involved in spoilage or fermentation
of most foods.

● Competition between the different kinds of bacteria, yeasts and molds in a food ordinarily
determines which one will outgrow the others and cause its characteristic type of spoilage.

● If conditions are favourable for all, bacteria usually grow faster than yeasts and yeasts
faster than molds.

Micro organisms are not always antagonistic or antibiotic to each other, however and
may sometimes be symbiotic i.e., mutually helpful.
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Some times they are synergistic i.e., when growing together they may be able to bring
about changes such as fermentations.

Ex: Pseudomonas syncyanea growing alone in milk produce light brownish tinge and
streptococcus lactis no color change in milk. When two organisms grow together, a bright blue
color develops resulting from PH effect on the brown pigment produced by P. syncyanea.

Metabiosis in which when one organism makes conditions favourable for growth of the
second. Both organisms may be growing at the same time, but more commonly one succeed the
other.

Ex: Raw milk at room temperature normally first supports an acid fermentation by streptococcus
lactis and coli form bacteria until the bacteria are inhibited by the acid they have produced. Next
the acid folerant lactobacilli increase the acidity further until they are stopped. Then film yeasts

and molds grow over the top, finally reducing the acidity so that proteolytic bacteria can become
active.

Some bacteria are:

• Acidophilic bacteria, e.g. Lactic acid bacteria (pH 3.3–7.2) and acetic
acid bacteria (pH 2.8–4.3).
• Basophilic bacteria e.g. Vibrio parahaemolyticus (pH 4.8–11.0) and
Enterococcus spp. (pH 4.8– 10.6).
– Increasing the acidity of foods either through fermentation or the
addition of weak acids could be used as a preservative method.
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Factors affecting growth and survival of microorganisms in foods. Intrinsic factors -

Nutrient content, pH, buffering capacity, Redox potential (Eh), Inhibitory substances and
biological structures (Antimicrobial barriers and constituents) water activity.
Factors affecting the growth and survival of micro-organisms in foods.
Intrinsic parameters:

The parameters of plant and animal tissues that are inherent part of the tissues are referred
to as intrinsic parameter. These parameters are as follows:

1. pH:
PH: It is the negative logarithm of the hydrogen ion activity.
1
PH = - log (aH) = log
(a H)

1
= log [H ]
−1

pH= Hydrogen ion activity

[ H]= Hydrogen ion concentration.
+

Every micro organism has a minimal, a maximal and an optimal pH for growth.
Bacteria grow fastest in the pH range 6.0 - 8.0, yeasts 4.5 - 6.0 and filamentous fungi 3.5 - 4.0.
Usually between pH 5.0 & 6.0.

Inherent acidity:
Some foods have a low pH because of inherent property of the food. Ex: Fruits &
vegetables.
Biological acidity:
Some foods develop acidity from the accumulation of acid daring fermentation. Ex: curd,
sauerkraut, pickles etc.
Molds can grow over a wide range of pHvales than the yeast and bacteria. Film yeasts
grow well on acid foods such as sauerkraut and pickles. Most yeasts do not grow well in alkaline
substrates.

Bacteria which are acid formers are favoured by moderate acidity. Active proteolytic
bacteria, can grow in media with a high pH (alkaline.) Ex: Egg white.

The compounds that resist changes in pH are important not only for their buffering capacity
but also for their ability to be especially effective within a certain pH range.
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Vegetable juices have low buffering power, permitting an appreciable decrease in pH with
the production of small amount of acid by lactic acid bacteria during the early part of sauerkraut
and pickle fermentations. This enables the lactics to suppress the undesirable pectin-
hydrolyzing and proteolytic competing organisms.
Ex: Milk - High in protein content, act as good buffer. Lactic acid converted to pyruvic acid by
glycolytic pathway.
Acid again converts to lactic acid by lactic dehydrogenase enzyme. After 5-

10 minutes, there will be decreased in pH. Hence the lactic acid bacteria survives and activity
slows down. Once the acidity increase, yeasts and molds will take upper hand and all the
products used by these organisms. The quantity of acid decreases and pHincreases to neutral.
Proteolytic bacteria acts on caesin and these proteins are broken down and gives bad smell
accompanied by removal of NH3. pH increases and neutral due to deamination. Then lipolytic
organisms which utilise the fat present and utilises the short chain fatty acids through hydrolysis
which gives still bad smell.

Egg white where the pH increases to around 9.2 as CO2 is lost from the egg after laying.
Fish spoil more rapidly than meat under chill conditions.
The pH of post- rigor mammalian muscle, round 5.6 and it is lower than that of fish (6.2 - 6.5)
and this contributes to the longer storage life of meat.

The ability of low pH to restrict microbial growth has been employed since the earliest
times in the presentation of foods with acetic and lactic acids.
Fruits are acidic than vegetables
pHof milk - neutral.
Fruits generally undergo mold and yeast spoilage than vegetables.
Redox potential (Eh): - Oxidation - reduction potential:
Oxygen tension or partial pressure of oxygen about a food and the O R potential or
reducing and oxidising power of the food itself, influence the type of organisms which will grow
and hence the changes produced in the food. The O R potential of the food is determined by

1. Characteristic O R potential of the original food.

2. The poising capacity i.e., the resistance to change in potential of the food.
3. The oxygen tension of the atmospHere about the food.
4. The access which the atmospHere has to the food.
Head space in an “evacuated” can of food contain low oxygen tension compared to air.
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Yeasts - Aerobic and facultative.

Bacteria - Aerobic, anaerobic and facultative.
High O - R potential favours aerobes and facultative organisms.
Low O-R potential favours anaerobic and facultative organisms.
However some aerobes grow at low O-R potential O-R potential of a system is usually written
Eh and measured and expressed in terms of millivolts (mv).

Highly oxidised substrate would have a positive Eh and a reduced substrate have a negative Eh.
Aerobic microorganisms require positive Eh. Ex: Bacillus, micrococcus, pseudomonads
acinetobacters.
Anaerobic micro organisms required negative Eh. Ex: Clostridium

Most fresh plant and animal foods have a low and well poised O - R potential in their
interior because plants contain reducing substances like ascorbic acid and reducing sugars where
as animal tissues contain -SH (Sulf hydryl) and other reducing groups

Heating and processing may alter the reducing and oxidising substances of food.

Ex: Fruit juices lost reducing substances by their removal during extraction and filtration by
their removal during extraction and filtration and therefore have become more favourable for the
growth of yeasts.

3. Nutrient content:
Food is required for energy and growth of micro organisms.
Carbohydrates especially the sugars are commonly used as an energy source. Complex
carbohydrates such as cellulose can be utilized by few organisms and starch can be hydrolysed
by any a limited number of organisms. Many organisms cannot use the disaccharide lactose
(Milk sugar) and therefore do not grow well in milk.

Maltose is not attacked by some yeasts. Some micro organisms hydrolyze pectin of the
fruits and vegetables.
Limited number of micro organisms can obtain their energy from fats by producing lipases.
Aerobic their energy from fats by producing lipases. Fats are hydrolysed to glycerol and fatty
acids. Aerobic micro organisms are more commonly involved in the decomposition of fats than
are anaerobic ones and the lipolytic organisms usually are also proteolytic.
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Hydrolysis products of proteins, peptides and amino acids serve as an energy source for
many proteolytic organisms when a better energy source is lacking. Meats are decomposed by
proteolytic sps Ex: Pseudomonas sps:

Ex: Some lactic acid bacteria grow best with polypeptides as nitrogen foods, cannot attack
casein.

Some microorganisms use fermentable carbohydrates and results in acid production which
suppresses the proteolytic bacteria and hence it is called sparing action on the nitrogen
compounds.

Many kinds of molds are proteolytic but very few yeasts are actively proteolytic.

Accessory food substances or vitamins needed by the organisms. Some micro organisms are
unable to manufacture some vitamins.

Meats are high in B vitamins and fruits are low, but fruits are high in ascorbic acid.
Egg white contains biotin but also contains avidin. This avidin ties up biotin mating it
unavailable to microorganisms and eliminating possible spoilage organisms.
Thiamine, pantothenic acid, folic acid group and ascorbic acid are heat labile and drying
causes loss in these compounds.

Storage of foods for long periods may result in decrease in level of the accessory growth
factors.
Each kind of bacterium has a definite range of food requirements. Some micro organisms
can use other carbon compounds such as organic acids and their salts, alcohols and esters.
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Pseudomonas spp may be satisfied by simple compounds such as ammonia or nitrates or more
complex compounds such as amino acids, peptides or proteins.
Antimicrobial barriers and constituents (or) Inhibitory substances and biological
structure:
Inhibitory substances: These originally present in the food or added purposely to prevent
growth of micro organisms.
Freshly drawn milk - Lactenins, anticoliform factors.
Egg white - Lysozyme

Cran berries - Benzoic acid

Allicin - Garlic, onion, leeks.

Phytoalexins are produced by many plants in response to microbial invasion.

Antifungal compound phaseolin produced in green beans
Eugenol - Allspice (pimento), cloves, cinnamon
Thymol - thyme and oregano

Cinnamic aldehyde - cinnamon and Cassia

Inclusion of cinnamon in raisin bread retards mould spoilage.

Humulones contained in the hop resin and isomers produced during processing, impart the
characteristic bitterness of beer.
Oleuropein - The bitter principle of green olives have antimicrobial properties.
Lysozyme present in milk, egg is most active against gram positive bacteria.
Egg - Ovotransferrin, avidin ovolflaroprotein.

Milk - Lactoferrin
Ovoflavo protein and avidin in egg white which sequester biotin and riboflavin restricting
the growth of those bacteria.
Milk has capacity to generate antimicrobials in the presence of hydrogen peroxide. The
milk enzyme lactoperoxidase will catalyse the oxidation of thiocyanate by H2O2 to produce inter
alia Hypo-thiocyanate. This can kill gram negative bacteria and inhibit gram positives.

Propionic acid produced by the propionibacteria in a swiss cheese is inhibitory to molds.

Nisin produced by certain strains of Streptococcus lactis may be useful in inhibiting lactate
fermenting, gas forming clostridia in curing cheese. Heating foods may result in the formation of
inhibitory substances. Ex: Heating lipids may hasten auto oxidation and make them inhibitory.
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Browning concentrated sugar syrups may result in the production of furfural and hydroxyl
methyl furfural which are inhibitory to fermenting organisms.
Biological structures of food on the protection of foods against spoilage has been
observed.
Ex: 1) Inner parts of healthy tissues of living plants and animals are sterile or low in microbial
content.
2) Protective covering on the food like shell on egg, skin on poultry, shell on nuts, rind or skin on
fruits and vegetables, artificial coating like plastic or wax.
3) Layers of fat over meat may protect the part of the flesh or scales may protect the outer part of
the fish.
Water activity:
Micro organisms have an absolute demand for water. Without water, no growth can occur.
The exact amant of water needed for growth of micro organisms varies. This water requirement
is best expressed in terms of available water or water activity (a W).

aw = Vapourpressureofthesolution
Vapour pressure of the solvent
aw for pure water is 1.00
For 1.0 m solution of the ideal solute, the aw would be 0.9823.
Water activity also defined as the ratio of the partial pressure of water in the atmosphere in
equilibrium with the substrate, P, compared with the partial pressure of the atmosphere in
equilibrium with pure water at the same temperature, P0.
P 1
Aw = = ERH
Po 100

ERH = Equilibrium relative humidity.

AW Values
0.98 and above Fresh meat, fish, fresh fruits and vegetables, milk,
canned vegetables, in brine, canned fruits in light
syrup.

0.93 - 0.98 Evaporated milk, tomato paste, processed cheese,

bread, canned cured meats, permented sausage,
gouda cheese.
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0.85 - 0.93 Dried beef, raw ham, aged cheddar cheese,

sweetened condensed milk, dry or fermented
sausage.
0.60 - 0.85 Dried fruit, flour, cereals, jams & jellies, nuts.
Below 0.60 Chocolate, confectionary, Honey, Biscuits,
Crackers, Potato chips, Dried eggs, milk and
vegetables.

Water is made unavailable in various ways:

1. Solutes and ions tie up water in solution. Therefore an increase in the concentration of
dissolved substances such as sugars and salts effectively dry the material. Water tends to leave
the microbial cell by osmosis.

2. Hydrophilic colloids (gels) make water unavailable.

3. Water of crystallization or hydration is usually unavailable to micro organisms.
Each micro organisms has a maximal, optimal and minimal a w for growth.
Low aw - decrease in the rate of growth of organisms.
Factors that may affect water activity (aw). Requirements of micro organisms include the
following.
1. The kind of solute employed to reduce aw. Potassium chloride usually less toxic than NaCl.
And less inhibitory than sodium sulphate.
2. The nutritive valve of the culture medium. The better the medium for growth, the lower the
limiting aw.
3. Temperature: Most organisms have the greatest tolerance to low a w at about optimal
temperatures.
4. Oxygen supply: Growth of aerobes takes place at lower aw in the presence of air than in its
absence.
5. pH Most organisms are more tolerant of low aw at pH valves near neutrality than in acid or
alkaline media.
6. Inhibitors: The presence of inhibitors narrows the range of aw for growth of micro organisms.
Methods for the control of aw are
1. Equilibrium with controlling solutions
2. Determination of the water - sorption isotherm for the food.
3. Addition of solutes.
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Methods for measuring or establishing aw valves of food:

1. Freezing point determinations by Clausius - Clayperson equation.
2. Manometric techniques
3. Electrical devices.
Favourable aw for bacteria to grow in foods - 0.995 to 0.998. They grow best in low
concentration of sugar or salt. 3-4% sugar and 1-2% salt may inhibit some bacteria.
Some general conclusions related to water requirement of micro organisms are

1. Each organism has its own characteristic optimal a w.

2. Bacteria require more moisture than yeasts and yeasts more than molds.
Minimum aw required for bacteria - 0.91

Minimum aw required for yeasts - 0.88

Minimum aw required for molds - 0.80
Minimum aw required for Halophilic bacteria - 0.75
Minimum aw required for Xerophilic fungi - 0.65
Minimum aw required for Osmophilic yests - 0.60
3. Micro organisms that can grow in high concentrations of solutes e.g. sugar and salt have low
water activity (aw). Osmophilic yeasts grow best in high concentrations of sugar.
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Extrinsic factors - Relative Humidity, Temperature, Gaseous Atmosphere. Chemical

changes caused by microorganisms - changes in nitrogenenous organic compounds, non-
nitrogenous organic compounds, organic acids, other compounds, Lipids, Pectic substances
Extrinsic parameters (Environmental limitations)

1) Relative humidity: (RH)

Relative humidity and water activity are interrelated. When food commodities having low
water activity are stored in an atmosphere of high RH water will transfer from the gas phase to
the food

Ex: Grain silos or in tanks in which concentrates and syrups are stored.
Storage of fresh fruits and vegetables requires very careful control of relative humidity. It
RH is too low, many vegetables will lose water and become flaccid. It is too high then
condensation may occur and microbial spoilage may be initiated.

2. Temperature:
Microbial growth can occur over a temperature range from about -8°C up to 100°C. at
atmospheric pressure.
Thermophiles have optimum - 55-75°C
Mesophile have optimum - 30 -40°C
Psychrophiles (Obligate psychrophiles) - 12 - 15
Psychotroph (facultative) - 25-30
Micro organisms can be classified into several physiological groups based on their cardinal
temperatures. Low temperature affects the uptake and supply of nutrients to enzyme systems
within the cell. Many microgranisms responds to growth at lower temperature by increasing the
amount of unsaturated fatty acids in their membrane lipids and that psychrotrophs generally have
higher level of unsaturation in a fatty acid decreases its melting point so that membranes
containing higher levels of unsaturated fatly acid will remain fluid and hence functional at lower
temperatures.

As the temperature increases above the optimum, the growth rate declines as a result of
denaturation of proteins.
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Gaseous atmosphere:
Oxygen comprises 21% of the earth’s atmosphere and is the most important gas in contact
with food under normal circumstances.
Inhibitory effect of CO2

Moulds and bacteria are sensitive to CO2 condensation. Some yeasts such as Bettanomyces
spp how tolerance to high CO2 levels.
Growth inhibition is usually greater under aerobic conditions than anaerobic and the
inhibitory effect increases with decrease of temperature, presumably due to the increased
solubility of CO2 at lower temperatures. CO2 dissolves in water to produce carbonic acid which
decreases PH and partially dissociates into bicarbonate anions and protons. CO2 also affects
solute transport, inhibition of key enzymes involving carboxylation, decarboxylation reactions in
which CO2 is a reactant and reaction with protein amino groups causing change in their
properties and activity.

Chemical change caused by micro organisms:

Different chemical changes are possible because great variety of organic compounds are
present in foods and numerous kinds of micro organisms that can decompose them may grow in
the food.

Following changes are observed in foods.

1. Changes in Nitrogenous organic compounds:
Most of the nitrogen in foods is in the form of proteins. Proteins are hydrolysed to
polypeptides, simpler peptides or amino acids before they can serve as nitrogenous food for most
organisms.

Proteinases catalyze the hydrolysis of proteins to peptides gives bitter taste to foods.
Peptidases catalyze the hydrolysis of polypeptides to simpler peptides and finally to
amino acids.

Proteinases Peptidases
Proteins Peptides Polypeptides
Peptidases
Polypeptides amino acids
Anaerobic decomposition of proteins, peptides or aminoacids result in the production of
obnoxious odors called putrefaction.
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Putrefaction results in foul smelling, sulphur containing products such as hydrogen,

methyl and ethyl sulfides and mercaptans, plus ammonia, amines (Ex: histamine, tyramine,
piperidine, putrescine and cadaverine), indole, skatole and fatty acids.
When micro organisms act on amino acids, they may deaminate them, de-carboxylate
them or both.

Ex: Escherichia coli produces glyoxylic acid, acetic acid, and ammonia from glycine.
Pseudomonas produces methylamine and CO2 clostridia gives acetic acid, ammonia,
methane from alanine these three organisms produces

1) α - Keto acid, ammonia and CO2

2) Acetic acid, ammonia and CO2
3) Propionic acid, acetic acid ammonia and CO2 respectively.
Desulfatomaculum nigrificans an obligate anaerobe, can reduce sulphate to sulphide and
produces H2S from cystine.

Other nitrogenous compounds include

1. Amides, imides and urea from which ammonia is the principal product.
2. Guanidine and creatine which yield urea and ammonia.
3. Amines, purines and pyrimidines which may yield ammonia, CO2 and organic acids.
Changes in Non nitrogenous organic compounds:
Main non nitrogenous foods for micro organisms, mostly used to obtain energy but
possibly serving as source of carbon, include carbohydrates, organic acids, aldehydes and
ketones, alcohols, glycosides, cyclic compounds and lipids.

Carbohydrates:
Carbohydrates act as energy source by micro organisms. Complex, di, tri or
polysaccharides usually are hydrolyzed to simple sugars before utilization.
A monosaccharide (glucose) aerobically would be oxidised to carbon-dioxide and water.
Glucose anaerobically decompose to

a) An alcoholic fermentation by yeasts with ethanol and CO2 as the principal products.
b) A simple lactic fermentation as by homo-fermentative lactic acid bacteria.
c) A mixed lactic fermentation by hetero-fermentative lactic acid bacteria with lactic and acetic
acids, ethanol, glycerol and CO2 as the chief products.
d) The coli type of fermentation as by coliform bacteria with lactic, acetic formic acids, ethanol,
CO2, H2 etc.

e) The propionic acid fermentation by propionic bacterium

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f) Butyric - butyl - isopropyl fermentations yields butyric and acetic acids, CO2 & H2.
Organic acids:
Organic acids usually occurring in foods as salts are oxidized by organisms to carbonates,
causing medium to become alkaline. Aerobically the organic acids may be oxidized completely
to CO2 and water. Saturated fatty acids or ketonic derivatives are degraded to acetic acid.
Other compounds:

Alcohols usually oxidised to the corresponding organic acids.

Ethanol to acetic acid; Acetaldehyde to acetic acid.

Lipids:
Fats are hydrolysed to glycerol and fatty acids by lipase.
Phospholipids may be degraded to their constituent phosphate, glycerol, fatty acids and
nitrogenous base.

Ex: choline
Pectic substances:
Protopectin in plants converted to pectin.
Pectin is a water soluble polymer of galacturonic acids. Pectinesterase causes hydrolysis
of the methyl ester linkage of pectin to yield pectin acid and methanol.