Milk Microbiology

• The science of micro-organisms is called

microbiology.
• Most living things are classed into two
kingdoms, animal and plant, but as micro-
organisms do not fit into either of these
kingdoms, they are classified together with
algae, protozoa and viruses in a third kingdom
called “Protista”.
• Biotechnology in the modern sense is largely
associated with industrial utilization of the
properties of living cells or components of
cells to obtain
– production of various products, specifically
effective medicaments such as hormones and
certain vaccines.
Bacteria
• Bacteria are single-celled organisms which
normally multiply by binary fission, i.e. splitting
in two.
• The most widely used method of staining
bacteria was introduced by the Danish
bacteriologist Gram and is called Gram staining.
• Bacteria, according to their Gram stain
characteristics:
– red, Gram negative and blue, Gram positive.
 Morphology of bacteria
Shape of bacteria
• Spherical or round
• Rod
• Spiral
Size of bacteria
• Cocci vary in size between 0.4 and 1.5 mm (1 mm =
0.001 mm).
• The length of bacilli can vary between 1 and 10 mm,
though a few species are larger or smaller.
 Conditions for growth of bacteria
1. Nutrients
• Protein, fat, carbohydrates, vitamins and minerals
2. Water activity
• The aw can be calculated according to the formula
aw = p/po
– p = vapour pressure of the food at t°C,
– po = vapour pressure of pure water at t°C
• Bacteria cannot normally develop at aw < 0.9.
• For yeasts the aw should be < 0.88
• For moulds it should be < 0.8 to stop growth.
• A low aw value does not however stop the activity of
enzymes.
• Symbiosis = permanent union between
organisms, each of which depends for its
existence on the other
• Antibiosis = coexistence where one organism
produces substances which inhibit the growth
of another organism
3. Classification by temperature preference

• Psychrotrophic (cold-tolerant) bacteria are psychrophilic or mesophilic

strains which can reproduce at a temperature of 7°C or below, regardless
of the optimum temperature.
– Psychrophilic (cold-loving) bacteria have an optimum growth
temperature below 20°C.
– Mesophilic bacteria (loving the happy medium) have optimum growth
temperatures between 20 and 44°C.
• Thermophilic (heat-loving) bacteria have their optimum growth
temperatures between 45 and 60°C.
• Thermoduric (heat-enduring) bacteria endure high temperatures – above
70°C. They do not grow and reproduce at high temperatures, but can
resist them without being killed.
• Psychrotrophic bacteria are of particular interest to the dairy industry,
because microbiological activity in farm milk and market milk usually takes
place at a temperature of 7°C or below.
4. Moisture
• Organisms grow very well at an available moisture content of
20%.
• Reduction to 10% limits growth, and at an available water
content of less than 5% there is no growth (with the exception
of moulds).
5. Oxygen
• Aerobic bacteria need oxygen from the atmosphere for
growth.
6. Light
• Light is not essential to most bacteria because they do not
contain chlorophyll and do not synthesis food in the same way
as plants.
• Instead light tends to kill bacteria if it contains ultraviolet light,
which causes chemical changes in the DNA and cell protein.
7. Osmotic pressure
• Bacteria cannot tolerate strong solutions of sugar or
salt, i.e. high osmotic pressures.
8. pH – acidity/alkalinity
• Micro-organisms cannot tolerate strong acidity or
alkalinity.
• Bacteria prefer a pH close to neutral, i.e. 6.8 – 7.4.
• Moulds prefer a low pH, 4.5 or lower.
• Fresh milk has a pH normally falling between 6.5
and 6.7. Sour milk has a pH of 4.6 and lower.
The most important biochemical and enzymatic systems in
milk products are those responsible for the following effects:

• Breakdown of carbohydrates
• Breakdown of protein
• Breakdown of fat
• Breakdown of lecithin
• Production of colour
• Production of mucus or slime
• Production of odours
• Reduction of oxygen
• Diseases
 Breakdown of carbohydrates
Breakdown of carbohydrates by:
• hydrolysis
• alcoholic fermentation
• lactic acid fermentation
• coliform type fermentation
• butyric acid fermentation
Fermentation usually results in various products such as
• organic acids (lactic acid, butyric acid, etc.)
• alcohols (ethyl alcohol, butyl alcohol, etc.)
• gases (hydrogen, carbon hydroxide, etc.).

The most important forms of fermentation in milk are:

Alcoholic fermentation of lactose to alcohol and gas.
• lactose is broken down to ethyl alcohol and carbon dioxide.
• usually takes place under anaerobic conditions and mainly
by yeasts and moulds.
Lactic acid fermentation of lactose to lactic acid.
This reaction is used in the manufacture of cheese, yoghurt
and other acidified products.
Coliform (mixed acid and butanediol) fermentation of lactose,
• resulting in a wide variety of end products, for example lactic
acid, acetic acid, succinic acid, formic acid, butanediol, ethyl
alcohol, carbon dioxide and hydrogen.
Butyric acid fermentation under strict anaerobic conditions by
the Clostridium bacteria.
• In butyric fermentation lactose is broken down to butyric
acid, carbon dioxide, hydrogen and, in some cases, butyl
alcohol.
As a general rule carbohydrate fermentation in milk results in
the production of acid (souring) and sometimes gas
(depending on the organisms).
 Breakdown of protein
Breakdown of protein in liquid milk takes place in two major stages
called peptonisation and consists of:

Curdling (sweet as opposed to sour) or clotting of the milk by

rennin-like enzymes.
• This fault in milk is called sweet curdling, a defect which is
common in pasteurised milk which is stored warm.
Proteolysis of the protein, resulting in production of ammonia,
which is alkaline.
• The degree of amino free acids and ammonia in cheese gives an
indication of its age and maturity as proteolysis progresses.
• Blue, or mould ripened, cheese has rapid proteolysis, resulting in
production of large amounts of ammonia.
 Breakdown of Fat
• The process where fat is broken down by enzymes is called lipolysis,
from the Greek roots lipo meaning fat and lysis meaning
breakdown.
• The major enzyme concerned is lipase. During lipolysis the fat is
hydrolysed to glycerol and three separate fatty acids. Some of the
fatty acids are volatile and give off strong smells. One example is
butyric acid, which gives the characteristic, rancid taste.
• Pure fat is relatively resistant to microbiological breakdown. Milk
fat, in the form of butter and cream, contains protein, carbohydrate,
minerals, etc. for growth and is therefore more susceptible.
• Many bacteria and moulds which break down proteins also break
down fat oxidatively.
 Breakdown of lecithin
• Lecithin, the phospholipid included in the membranes round
the fat globules, is a chemical combination of glycerol, two
fatty acids, phosphoric acid and choline, an organic alkali.
• Strains of Bacillus cereus produce enzymes, lecithinases,
which hydrolyse the lecithin into diglyceride and phosphoryl
choline.
• The membranes of the fat globules are split, resulting in an
unstable fat emulsion often appearing in the form of flocks or
lumps floating on the surface of the milk or cream. This fault
in milk or cream is called “bitty cream” or “broken cream”.
• Further breakdown of the choline into trimethyl amine will
result in a fishy smell and taste.
 Pigment and colour production
• The process of colour production is called chromogenesis and the
organism causing the production is referred to as chromogenic after the
Greek roots chromo meaning colour and genesis meaning birth or origin.
• takes place at lower temperatures.
• Aerobic conditions are also necessary for chromogenesis.
There are two types of pigment:
– endo-pigment, which stays in the cell
– exo-pigment, which diffuses out of the cell into the surrounding food
There are three basic colour groups:
– Carotenoids, which are yellow, green, cream or golden
– Anthrocynins, which are red
– Melanins, which are brown or black
• The name of an organism often refers to the colour it produces. Example:
Staphylococcus aureus = “the golden Staphylococcus”.
Mucus production
A number of bacteria produce mucus or slime, which is utilised in certain
cultured products such as yoghurt and långfil, a Swedish ropy milk.
Odour production
A number of organisms produce strong odours or smells. Examples are:
• Moulds, which produce a musty smell
• Actinomyces, which produce an earthy smell
• Yeasts, which produce a fruity smell
• Pseudomonas, which produce a fruity/fishy smell
• Coliforms, which produce a cowlike and dirty smell
• Lactococcus lactis var. maltigenes, which produces a malty smell
Reducing power
All micro-organisms have some degree of reducing power, i.e. the power to
remove oxygen.
In milk the most powerful reducers are Lactococcus, coliforms and Bacillus.
These are largely responsible for the reduction of oxygen in dye-reduction
tests such as “Resazurin” and “Methylene blue”, indicating the degree of
microbiological content and keeping quality .
Disease production (Toxins) Organisms which produce
diseases are called pathogenic from the Greek roots
pathos, suffering and genesis, origin.
Organisms bring about disease in human beings, animals
and plants by attacking and breaking down living cells
and producing poisonous substances called toxins. The
organisms responsible may die, but the toxin can remain
and cause the disease.
Examples are Staphylococcus, Salmonella and Clostridium,
which cause food poisoning, Salmonella typhosa (causing
typhoid fever), Clostridium letani (causing tetanus) and
Corynebacterium diphtheriae (causing diphtheria).
 Bacteria in milk
• When milk is secreted in the udder it is virtually sterile. But even
before it leaves the udder it is infected by bacteria which enter
through the teat channel.
• In cases of bacterial udder inflammation (mastitis), the milk is
heavily contaminated with bacteria and may even be unfit for
consumption
• In the course of handling at the farm, milk is liable to be infected by
various micro-organisms, mainly bacteria.
• Bacterial count in milk: For milk to be classed as top quality, the
bacteria count, the CFU (Colony Forming Units), should be less than
100 000 per ml.
• Rapid chilling to below 4°C contributes greatly to the quality of the
milk at the farm.
– slows down the growth of the bacteria in the milk
Bacteria groups in milk:
• Lactic acid bacteria
• Coliform bacteria
• Butyric acid bacteria
• Propionic acid bacteria
• Putrefaction bacteria
 Lactic acid bacteria
• Lactic acid bacteria are facultatively anaerobic.
Most of them are killed by heating to 70°C, though
the lethal temperature for some is as high as 80°C.
• Lactic acid bacteria prefer lactose as a source of
carbon. i.e. the end product may be almost
exclusively lactic acid (homofermentative
fermentation), or other substances may also be
produced, such as acetic acid, carbon dioxide and
ethanol (heterofermentative fermentation).
 Coliform bacteria

• Coliform bacteria are facultatively anaerobic with an optimum

temperature of 30 – 37°C.
• They are found in intestines, in manure, in soil, in contaminated
water and on plants.
• They ferment lactose to lactic acid and other organic acids,
carbon dioxide and hydrogen
• They break down milk protein, resulting in an off flavour and
smell. Some coli bacteria also cause mastitis.
• Coliform bacteria can cause serious trouble in cheesemaking.
Besides causing off flavour, the relatively strong gas formation will
result in an unwanted texture at an early stage (early blowing).
• Coliform bacteria are killed by HTST pasteurisation.
 Butyric acid bacteria
“The Cheese Destroyers”:
• Clostridium tyrobutyricum
• Clostridium butyricum c acid.

• Butyric acid bacteria are anaerobic spore-forming micro-organisms with an optimum

temperature of 37°C
• They do not grow well in milk, which contains oxygen, but thrive in cheese where anaerobic
conditions prevail.
• Clostridium butyricum, a group containing both lactose and lactate fermenters, The bacterium
can cause both early and late butyric acid fermentation in cheese and Clostridium
tyrobutyricum, which ferments lactates (lactic acid salts) and can cause late butyric acid
fermentation.
• These fermentation processes produce large quantities of carbon dioxide, hydrogen and
butyric acid.
• The cheese acquires a fermented, ragged texture and a rancid, sweetish taste of butyric acid.
• Over the years large quantities of cheese have been spoiled by butyric acid fermentation.
• These bacteria cannot be killed by pasteurisation when they occur in the heat-resistant spore
form. It is therefore necessary in practice to resort to special production engineering
techniques to prevent butyric acid fermentation.
Propionic acid bacteria
• Propionic acid bacteria comprises a number of species of varying appearance.
• Optimum temperature is about 30°C, and several species survive HTST
pasteurisation.
• They ferment lactate to propionic acid, carbon dioxide and other products.
• Pure cultures of propionic acid bacteria are used (together with certain lactobacilli
and lactococci) in the manufacture of Emmenthal, Gruyère, Jarlsberg, Grevé and
Maasdam cheese, where they are responsible for the formation of eyes and
contribute to the characteristic flavour.

Putrefaction bacteria
• Putrefaction bacteria produce protein-splitting enzymes.
• They can therefore break down proteins all the way to ammonia. This type of
breakdown is known as putrefaction.
• Some of them are used in dairy processing, but most of them cause trouble.
Putrefaction bacteria:
• Brevibacterium linens (useful)
• Pseudomonas fluorescens (harmful)
• Clostridium sporogenes (harmful)
Fungi are divided into:
• yeasts
• moulds
Important factors for yeast
growth
• nutrients
• moisture
• acidity
• temperature
• oxygen
 Yeast
• Yeast can grow in media with pH values ranging from 3 to 7.5. The
optimum pH is usually 4.5 – 5.0.
• Yeast cells do not usually grow at temperatures below the freezing point
of water or above about 47°C. The optimum temperature is normally
between 20 and 30°C.
• Growing cells are normally killed within 5 to 10 minutes at temperatures
of 52 to 58°C. Spores (ascospores) are more resistant but are killed when
exposed to 60 – 62°C for a few minutes.
• Yeast has the ability to grow both in the presence and in the absence of
atmospheric oxygen, i.e. yeast cells are facultatively anaerobic.
• Yeasts are generally undesirable organisms from the dairy point of view,
with one exception. Kefir, a Russian cultured product, is fermented with a
mixed culture of yeasts and lactic acid bacteria in a grain-shaped
aggregate.
• Yeast organisms are otherwise unwelcome in the dairy because they cause
serious faults in cultured products including cheese and butter.
 Moulds
Moisture
• Moulds can grow on materials with a very low water content
• Some can tolerate concentrations of sugar and salt with high osmotic
pressure.
• Example: Fruit preserves and sweetened condensed milk.
Oxygen
• Moulds normally grow in aerobic conditions. Oxygen is necessary for the
formation of conidia, and for the growth of mycelia.
Temperature
• The optimum growth temperature for most moulds is between 20 and 30°C.
Acidity
• Moulds can grow in media with pH values from 3 to 8.5.
• Many species, however, prefer an acid environment. Example: cheese,
yoghurt, citrus fruit and fruit juices.
 Importance of moulds in the dairy
• As with yeasts, moulds do not survive ordinary pasteurisation temperatures, 72 – 74°C for some 10 to 15
seconds. The unwanted presence of these organisms is therefore a sign of reinfection.
• There are many different families of moulds. Some groups which are of
• importance in the dairy industry are Penicillium and milk mould, Geotrichum candidum.
Penicillium
• Some species of penicillia play an important part in dairy processes.
• Their powerful protein and fat splitting properties make them the chief agents in the ripening of Blue
cheese, Camembert, etc. The Blue-cheese mould is called Penicillium roqueforti and the Camembert
mould Penicillium camemberti.
Milk mould
• The milk mould Geotrichum candidum is on the borderline between yeast and mould. The mould occurs
on the surface of cultured milk as a fine, white velvety coating.
• This mould contributes to the ripening of semisoft and soft cheeses.
• It may cause rancidity in butter.
• Moulds on the surfaces of cheese and butter can cause discoloration and also give the product an off
flavour.
• Strict hygiene is necessary in the dairy in order to prevent products from being affected by moulds during
• processing.
• Walls and ceilings, for example, must be kept scrupulously clean in order to prevent moulds from settling
there.