Lesson 1

INTRODUCTION AND SIGNIFICANCE OF DAIRY MICROBIOLOGY

1.1 Introduction
As per the definition, a branch of biology that deals with the study of microorganisms and their
different activity is termed as microbiology. Since ages, these microorganisms are playing a
potential role in human welfare both as useful and harmful biological agents. This leads to an
extensive study of these micro-organisms with the aim of understanding their growth and
nutritional requirements for their production or destruction both.
The different branches of microbiology are:
• Agricultural and soil microbiology
• Aquatic and marine microbiology
• Dairy and food microbiology
• Environmental microbiology
• Industrial microbiology
• Medical and pharmaceutical microbiology
• Rumen microbiology
• Space microbiology
1.2 Definitions
Milk and milk products occupy a more significant role in the human food profiles. The study
of microorganisms that are associated with milk and milk products in all aspects is defined as
“Dairy Microbiology”.
1.2.1 Milk
Milk is described as a whole, fresh, clean, lacteal secretion obtained from the complete milking
of healthy milch animal (excluding this milk is obtained within 15 days before or five days
after calving to render the milk practically colostrum-free) containing the minimum prescribed
levels of fat and solids non-fat.
1.2.2 Milk hygiene
Milk hygiene is concerned with the production of clean, wholesome milk that is free from
bacteria or other disease causing micro-organisms and maintenance of this condition from farm
to the consumers. In general milk is considered as the most nutritious and complete food for
neonates and adult human beings both.
Dairy industry provides an excellent example where bacteria, yeasts, moulds and viruses are
very important in determining the quality of final product. The control and destruction of
undesirable microorganisms, as well as their intentional introduction and utilization are
problems that need special attention. The nutritional qualities of milk make it a desirable food
for humans and other young animals. However, these nutritional values also permit growth of
many microorganisms, some of which cause undesirable changes in milk and its products.
Milk’s sanitary qualities are influenced by many factors in the course of production, processing,
and delivery to the consumers. An example of dairy industry which is dependent to a larger
extent on the desirable enzymatic changes caused by microorganisms is cheese making. The
flavours and the texture of cheese are largely ascribed to the conversion of milk constituents
by various species. Few bacterial and mould species are added intentionally to cheese during
manufacturing and much of the microbial activity in that takes place during ripening are due to
microbial species that enter the milk by chance at different stages. The taste and aroma are
competitive assets of butter. Starter cultures, that are mixed bacterial cultures selected for acid-
and flavor-producing ability, are important in the manufacture of flavored butter. The
conditions in which optimum flavor develops and the relationships with microbes are well
known, and certainly the changes brought about by the starters are desirable. Yogurt, sour
cream, and buttermilk are some of the examples of fermented milks made by different
intervention of microorganisms to milk, cream, and skim milk, respectively. Each of those
products has a characteristic microflora that is partly responsible, for its characteristic flavor
and texture. It is also possible to ferment milks having desired properties (i.e. flavor and acid-
producing) by using starter cultures.
Microbial conversion of certain constituents of milk into economically valuable products like
vitamins, solvents, and food adjuncts can be done commercially these days. On the other hand,
milk constituents that have no economic significance, or that are usually wasted, must be
converted to stably oxidized and non-obnoxious substances prior to their discharge into the
environment. Here also microbial activity is responsible for the desired changes in the organic
constituents of dairy wastes dung of sewage treatment.
Microbes are undesirable in milk or its products, if these deteriorate flavor or texture, and
produce diseases. It is important that dairy microbiologists understand the facts that, influence
the deterioration of milk and the methods through which this type of deterioration can be
prevented. Souring of milk or cream is undesirable under majority of situations, but souring
(i.e. production of lactic acid) is essential also for the manufacture of cheese or cultured milk
products. Discolorations, sliminess, ropiness, putrefaction, rancidity, gassiness, and many other
defects are caused by different microbes that grow in dairy products. Milk occasionally has
been the carrier of microbes that can cause disease. However, these agents of disease can be
effectively controlled. As the new processes and products are developed by the dairy scientists,
these must be checked to ensure that these are safe for consumers.
Many ordinances and other regulations under which milk is produced and handled specify
quantitative, qualitative, and microbial standards. These standards have shown that under
desirable conditions, the numbers of microbes gaining entrance to the product will not exceed
certain levels and, also, that proper handling of these products will not permit growth beyond
certain levels. To a certain degree, the numbers of microbes permitted on these standards
usually are far much below the numbers necessary to cause spoilage. Practically this results
into a gradual up-gradation of microbial standards for different dairy products. Because of this
type of control, the packaged milk produced by dairy industries is of high microbiological
quality and hence, safe to the consumers.
1.3 Significance of Microbes in Milk
• Information on microbial load can be used to assess its sanitary quality and the conditions of
production
• Bacteria, if permitted to multiply, cause spoilage of milk
• Milk is potentially susceptible to contamination with pathogens, however precautions must
be taken to destroy them
• Certain microbes produce chemical changes that are desirable in the production of cheese,
yogurt and fermented milk products.
There is a need for knowledge of the microbiology of different dairy products as shown by the
three distinct considerations placed below:
1.3.1 Spreading of diseases via milk
Different studies have proved that diseases in both man and animals are sometimes caused by
the pathogens spread by milk. Although, the presence of such microorganisms is not specific
only to foods from dairy but from a variety of other sources also. However, it is especially
serious with dairy products because these are mostly consumed without being heated and thus
without destruction of harmful micro-organisms. Accordingly, the sources of pathogens in
dairy products, conditions under which they grow, and methods of prevention or destroying
them is essential. Such a knowledge is important to the manufacturer of dairy products, because
of the responsibility of producing only safe foods, and it is of extreme importance for the
supervisor of dairy plants, as the inspection is the final protection efforts to the consumer.
Although, the manufacturer and supervisor should ensure consumer's safety, but it is also
desirable that the consumers are well aware to decide whether a particular dairy product is
satisfactory or not? Similarly, the farmer using milk or milk products for animals feeding
should know the probability of introducing diseases into his herds through such products.
The hygienic milk production practices, proper handling and storage of milk and pasteurization
have decreased the threat of milk-borne diseases, such as tuberculosis, brucellosis, and typhoid
fever. However, there have been
a number of milk-borne illnesses resulting from the ingestion of raw milk, or dairy products
made with milk that was not properly pasteurized or was poorly handled leading to post-
processing contamination. The following bacterial pathogens are of major concern to in raw
milk and other dairy products:
• Bacillus cereus
• Campylobacter jejuni
• Escherichia coli
• Listeria monocytogenes
• Salmonella spp.
• Yersinia enterocolitica
It should be noted that moulds, mainly the species of Aspergillus, Fusarium, and Penicillium
can also grow in milk and dairy products. If conditions permit, these moulds may produce
mycotoxins that can be a health hazard to the consumers.
1.3.2 Spoilage of milk
Micro-organisms, as a result of their growth or biochemical activities, cause undesirable
changes in milk and, are responsible for spoilage. The producer of milk should be aware of the
sources of micro-organisms causing rapid changes, conditions favoring their growth, and
methods of preventing their activity. The manufacturer of milk products must contend with
problems similar to those of producer of milk and additional ones also, as butter, cheeses, etc.
are frequently stored for longer periods, during which may further decrease quality. The
problem of spoilage is especially important with the cheeses they require ripening, since
conditions must be favorable for growth of certain desirable microorganisms and may also
allow the growth development of undesirable ones. The initial microbial quality of raw milk is
quite crucial for the production of good quality dairy foods. Spoilage is a term used to describe
the deterioration of a food’s texture, color, odor or flavor to the point, where it becomes
unsuitable for human consumption. Microbial spoilage of food often involves the degradation
of protein, carbohydrates, and fats by the microorganisms or their enzymes. In milk, the
microorganisms that are mainly involved in spoilage are psychrotrophs. Most psychrotrophs
are destroyed by pasteurization, however, some like Pseudomonas fluorescens; Pseudomonas
fragi can produce proteolytic and lipolytic extracellular enzymes that are heat stable and
capable of causing spoilage. Some species and strains of Bacillus, Clostridium,
Corynebacterium, Arthrobacter, Lactobacillus, Microbacterium, Micrococcus, and
Streptococcus can survive pasteurization and grow at refrigeration temperatures that can cause
spoilage problems in milk and milk products.
1.3.3 Developing desirable microorganisms in some milk products
The pleasing flavor of dairy products as butter, cheeses, and fermented milks, and the desired
texture of many dairy products are largely due to the development of certain microorganisms
during manufacture and ripening. An understanding of the action of these organisms,
conditions favoring their growth, etc., is important and necessary if products having a
uniformly high quality are to be produced and marketed. The manufacturing methods used for
certain dairy products were developed empirically even before the role of microorganisms was
understood, but various valuable improvements have resulted from knowledge of the action of
the important micro-organisms.

Lesson 2
SOURCES OF CONTAMINATION IN MILK
2.1 Introduction
Milk when secreted into an uninfected animal’s udder is sterile and invariably, it becomes
contaminated during milking, cooling and/or storage. It is an excellent medium for the growth
of bacteria, yeasts and moulds that are the common contaminants of any food material. Their
rapid growth, particularly at high ambient temperatures can spoil the milk for liquid
consumption and for manufacturing dairy products. This can be avoided to a greater
extent by adopting the basic rules of clean milk production.
2.2 Sources of Microbial Contamination of Milk
Microbial contamination of milk can be from the internal and/ or external sources (Fig 2.1) that
are described in the following section.
Fig. 2.1 Various sources of contamination during milk production
2.2.1 Interior of udder
Varying numbers of bacteria are found in aseptically drawn milk with the reported counts of
<100-10,000 CFU/ml from normal udder, but an anticipated average is 500-1000 CFU/ml in
advanced countries. Microorganisms enter the udder through the duct at the teat tip that varies
in length (from 5-14 mm) and its surface is heavily keratinized. This keratin layer retains the
milk residues and exhibit antimicrobial activity.
Fig. 2.2Fig. 2.2 Secretion of milk in udder
During progress of a milking, bacteria are present in the largest numbers at the beginning and
then gradually decrease. This is mainly due to the mechanical dislodging of bacteria,
particularly in teat canal, where the numbers are probably highest. Because of this discarding
of first few streams of milk helps in lowering the counts of microbes in milk. Milk from
different quarters also vary in [Link] species of bacteria that are found in milk, as
it comes from udder are very limited as given in Table 2.1.
Table 2.1 Presence of different microbial groups in raw milk
Group of microbes Percent range
Micrococci 30-99
Streptococci 0-50
Asporogenous Gram positive rods <10
Gram negative rods <10
Bacillus spores <10
Other groups of microbes <10
Though micrococci are slow growing, but if allowed to grow, they cause acid formation and
proteolysis. These are mostly non-pathogenic. Streptococci are less frequent than micrococci.
Streptococcus agalactiae may be present even in non-clinical mastitis and thus it appears to be
a natural inhabitant of udder. Among Gram positive rods, Corynebacterium bovis has been
found in large numbers. It is non-pathogenic, but if grown causes rancidity. If an animal is
infected from mastitis, microbial contamination from within the udder of animal contributes
notably to the total numbers of microbes in the bulk milk, when compared with the milk
originated from a healthy animal. The influence of mastitis on the total bacterial count of milk
depends on the type of the infecting microbe. Most common microbial agents of mastitis in
milch animals are given in (Fig 2.3) are Staphylococcus aureus, Streptococcus agalactiae,
Streptococcus dysgalactiae, Streptococcus uberis, Escherichia coli and Corynebacterium
pyogenes.
Fig. 2.3 Most common microbial agents of mastitis
2.2.2 Exterior of udder
In addition, to the udder infections, unclean udder and teats of animal also contribute
significantly to the total bacterial counts of milk. The microbes that are naturally associated
with the skin of the animals as well as those derived from the environment, where the cow is
housed and milked are predominant in the milk. The environmental conditions such as soil,
manure, mud, feed or bedding; determines what kind of microbes will dominate in milk.
Udder and teat become soiled with dung, mud, bedding material such as saw dust, straw etc.
With heavily soiled udder teats the counts may be 1,00,000 cfu/ml. The bedding material in
winter has high number of bacteria, mainly psychrotrophs, coliforms and Bacillus spp. Udder
microflora is not affected much by simple washing. Economy washing with sodium
hypochlorite accompanied by drying, helps in reducing the number of microbes. Different
category of microbes that occurs in the exterior of udder are
• Predominantly micrococci and coagulase negative staphylococci exist.
• Next, on the teat surface are faecal streptococci, but Gram negative bacteria including
coliforms are less. Coliforms do not survive well on teat surface.
Aerobic thermoduric organisms are entirely Bacillus spp. The more frequent are B.
licheniformis, B. subtilis,B. pumilis and less frequent ones are B. cereus, B. circulans and B.
firmus.
• Teat surface may also contain clostridial spores that are usually found in cows fodder, bedding
and faeces
Fig. 2.4 Common psychrotrophic bacteria found on the teat surfaces
Psychrotrophic and thermoduric bacteria predominate on the teat surfaces. The psychrotrophs
that can grow at 7ºC and below are mostly Gram negative rods, and the major ones are
Pseudomonas fluorescens, followed by Alcaligenes, Flavobacterium and coliforms (Fig 2.4).
On the other hand, thermodurics on teat surfaces are often bacterial spores (a dormant and non-
reproductive structure; highly resistant to radiations, desiccation, lysozymes, high temperature,
starvation and disinfectants) that are typically found in the soil (Fig 2.5). When these spores
enter the bulk milk, they may survive during pasteurization and cause a number of post-
pasteurization problems.

Fig. 2.5 The structure of bacillus and clostridial spores in milk

2.2.3 Coat of cow

The coat serves as a vehicle to contribute bacteria directly to milk. The hairs around udder,
flanks and tail contribute to the higher bacterial count in milk. The coat may indirectly
contribute microbes into air, especially Bacillus spp. The coat may carry bacteria from the
stagnant water pools, especially ropiness causing milk microbes.
2.2.4 Animal shed and surroundings
Milk produced on farms with poor hygiene practices may undergo significant spoilage and
have a shorter shelflife, when compared to milk produced under hygienic conditions. Microbes
associated with the bedding materials include:
• Coliforms
• Spore-formers
• Staphylococci
• Streptococci
• Other Gram negative bacteria
2.2.5 Milking staff
The staffs involved at different stages of milk production plays a pivotal role in maintaining
hygiene and preventing milk contamination. The hand contacts or dislodging of dust and dirt
particles by milker may add varieties of microbes to milk. Risks of contamination from milker
are definitely higher, when cows are hand milked in comparison to when they are machine-
milked. Soiled clothes and hands increase the risk of contamination of milk and milking
equipments many folds. Milker with infected wounds on hands contributes pathogenic
Streptococcus spp. and micrococci. If wet hand milking is practiced, the microorganisms
present in lubricants like fore-milk, water or saliva of the milker and bacteria from hands and
teats will enter the milk. The common microbial pathogens from humans causing diseases such
as typhoid, paratyphoid and dysentery may contaminate the milk. Microbial pathogens causing
scarlet fever, septic sore throat, diptheria, cholera etc. contaminate the milk.
2.2.6 Milking equipment (storage containers and transportation systems)
Improperly cleaned milking and cooling equipments are one of the main sources of milk
contamination. Milk residues left on the equipment contact surfaces supports the growth of a
variety of microbes. Although natural inhabitants of the teat canal, apex and skin;
microorganisms associated with contagious mastitis do not grow well on these equipments, it
is possible that certain strains associated with environmental mastitis may grow to a significant
level. Since, it is very difficult to remove all milk residues and deposits from the milk contact
surfaces of milking equipments; hence equipment with smooth surfaces and minimal joints
should be used. The tanker and collecting pipes are also the potential sources of contamination,
if not adequately cleaned. In addition, biofilms can easily build up on the enclosed, hard to
clean surfaces (Fig. 2.6).
Unclean or improperly cleaned milk cans and lids if they are still moist, results in multiplication
of thermophilic bacteria like Bacillus cereus. Improperly sterilized milking machines contain
thermoduric micrococci, Bacillusspp. and Microbacterium spp. predominantly compared to
coliforms and streptococci. Rubber hoses predominantly contribute to pseudomonas rather than
thermodurics.
2.2.7 Water supplies
At dairy-farms, the water can be a predominant source of microbial contamination. Water used
in production should be of good bacteriological quality. Inadequately or uncleaned, storage
tanks, untreated water supplies from natural sources like bore wells, tanks and rivers, may also
be contaminated with the faecal microbes (e.g. Coliforms, Streptococci and Clostridia). In
addition, a wide variety of saprophytic bacteria ([Link], Coliforms, other Gram
negative rods, Bacillus spores, Coryneform bacteria and lactic acid bacteria) may also be
present in water and may contaminate the milk potentially. The warm water used for udder
washing is potent source of Pseudomonas and Coliforms which may even cause mastitis.
2.2.8 Airborne contamination
Aerial contamination of milk by bacteria is insignificant, in comparison to microbes with those
that are derived from the teat surfaces. The microbial counts of air in sheds rarely exceed 200
cfu/l. Micrococci account for >50% of the aerial microflora. Air contains dust, moisture and
bacteria; hence its entry should be minimized in milk.
Micrococci, Coryneforms, Bacillus spores, streptococci, and Gram negative rods are the major
genera present in air. In general, more air incorporated into milk leads to the faster growth of
bacteria. Following are some of the practices that increase aerial counts in milk:
• Sweeping of floors just before milking process
• Handling hay and feed shortly before milking process
• Brushing of animals prior to milking process
• Having the dusty bedding materials for animals
• Allowing dust and dirt to accumulate on the walls or ceiling of sheds
Lesson 3
HYGIENIC MILK PRODUCTION
3.1 Introduction
In order to get high quality milk, certain hygienic practices such as appropriate sanitation and
disinfections of the teats, dairy utensils and equipment, properly good quality water and
mastitis control measures; are essentially required at dairy farm. The ultimate quality of dairy
products offered to the consumer, is determined by the complete process (from animals
production till consumers utilization). The practices for milking ensure that milk is produced
and stored under hygienic conditions and utensils/ equipment used in the whole processes of
milk production are well maintained as per the recommendation.
3.2 Principles of Clean Milk Production
Clean milk means that it comes from the udder of healthy animals, has good flavour, free from
dirt, contains relatively fewer bacteria and none of those are harmful to human health. High
quality milk should have:
• Longer keeping quality
• Proper nutritive value
• Normal taste, colour, odour and
• Free from extraneous matter
3.2.1 Animal management
[Link] Clean and healthy cows
The animals apparently with good health should only be purchased and quarantined, aside from
testing for contagious diseases, if any annual testing for tuberculosis and brucellosis invariably
should be done. Also examine periodically for udder and other infections. Infected animals
should be treated by a qualified veterinarian and should be isolated from the normal herd so as
to avoid the further spread of infection.
Clip long hair around flanks, udder and teats regularly. Animal should also be washed and
groomed daily probablybefore milking so that the dirt particles in the air do not fall into milk.
Time to time, hair from hind legs, udder and tail of the animal should be shaved-off. This is
important in the case of buffaloes, as they usually wallow in dirty ponds and carry mud and
filth on their body.
The udder and teats should be definitely washed gently with antiseptic solutions prior to
milking in such a manner that no damage is done to the orifices and clefts between the quarters
of the udder. Keep two separate soft cloths for wiping after washing with plain water and
disinfecting solution. A third washing with a mild detergent solution and a separate cloth is
recommended for wiping the teats after milking. Addition of hypochlorite (500 ppm) helps in
disinfecting the udder. Quaternary ammonium compounds (200 to 400 ppm) are better
substitutes due to their less harmful effect on tissues. Under Indian subcontinent conditions,
the easily available Dettol or Savlon may be diluted suitably and used to disinfect the udder
and teats. Disposable paper towels may be used instead of cloth, if affordable. However, under
the Indian conditions, these may be impractical. Milk of the infected animal should never be
pooled with bulk milk, until the animal recovers from the illness fully.
3.2.2 Animal housing management
A neat and clean housing is quite important to have healthy animal that will produce hygienic
milk at farm. At many instances, the animal sheds are the breeding places for flies and
mosquitoes that attack the animal, causing various kinds of physical discomforts and infectious
diseases. If the design of animal shed is not appropriate, the metabolic gases like methane,
moisture and carbon dioxide produced by the animals and ammonia gas produced
by the microbes acting on the dung will not find an easy exit that will not only adversely affect
the health of theanimals residing there but also the human workers (Fig. 3.1).

Fig. 3.1 Fundamentals of clean milk production

In many cases, animals are kept inside where the people live, which may be dangerous to both
the animal as well as humans. Similarly, the flies and the mosquitoes also will find their way
to the milk, directly from the environment or when the animal flips during milking. Hence, the
sheds needed to be designed along the following recommendations:
• Animal stables have to be located on high ground with a natural sloppy drainage
• Have pucca (concrete) floor, water proof, hard and easy for cleaning
• Drains constructed have a decent width and depth, and slope
• Ensure proper drainage of dung and urine directly to the sewer or frequent removal
• Mangers should be smooth without sharp angles.
• Ensure proper aeration in shed
• Ensure maximum comfort to the animal by providing air space of 500 cft per animal
• Have provision for regular supply of clean and fresh water
• Periodical lime washing has to done.
The existing sheds should be maintained properly by:
• Keeping the shed clean, wash regularly and dry as far as possible
• Milk houses should be free from dust and stable odours
• Removing the dung, urine frequently, away from the shed
• Spraying a recommended chemical in dung pit to stop the breeding of mosquitoes
• Use fly repellents ( i.e. phenyl) inside the shed and/ or at the farm premises
3.2.3 Feeding management
• Always feed the animal with a high nutritional valued diet, as a healthy animal will yield the
cleaner milk
• Feeding the animals with healthier diets will reduce the chances of occurrence of diseases
• Clean the water tubs and the feeding manger regularly to avoid microbial growth
• Never feed dusty feed concentrates. Feed either pellets or slightly moistened feed
• Avoid feeding silage and hay during milking
• Never feed the animal with leftover feed that may be spoiled with moulds or other microbes
• Never allow the animal to drink dirty water as it may lead to waterborne infections
3.2.4 Personal hygiene
[Link] Disease control
It is really important that the milker should never have the symptoms of any communicable
disease. They should not also have open cuts including sores, boils or infected wounds that will
definitely be the source of microbial contamination to milk. In case of stomach upset, the milker
should never be allowed for milking the animal. This will prevent the communication of udder
disease and also contamination of milk. Periodic medical check up for milkers should be
carried. Milker should be healthy, free from infectious diseases like Cholera, Typhoid, Scarlet
Fever, Tuberculosis etc.
[Link] Cleanliness
• Milker should always wear neat and clean clothing and caps.
• Milker should also maintain adequate personal cleanliness ( i.e. haircut, trimming of beard
and cutting the
nail regularly) to avoid any microbial contaminations
• Cuts, blisters or boils, if any must be covered with clean dressing
• Washing of hands with detergents and drying with clean towel is must prior to hand milking
• Never smoke, eat drink or spit while milking
• Avoid coughing and sneezing while milking
3.2.5 Milking management
• Better, if the animal let out milk without the calf
• Clean the shed before milking and dispose the dung away from the shed
• Clean the animals and wash the udder with clean water
• Properly wipe and dry the udder after washing using clean dry napkin
• Washing of hands with detergent and drying of hands with towel is must
• Use utensils preferably made up of stainless steel without any crevices that are easy to clean
• Correct milking practices should be adopted to prevent any damage/ injury to teats
• Carry gentle, fast and complete milking
• Use milkman’s rope for restraining the animal
• Practice dry, full hand milking
• Discard fore-milk
• Use strip cup for detecting mastitis
• Separate utensils are recommended for washing of udder and for milking
• Never pour the milk of animals having contagious diseases
• Milking should be as fast as possible because the excitation by the animal lasts approximately
only for 7 minutes
• Always ensure complete milking as the microbes will grow on the left over milk in udder
resulting in mastitis
• Always keep the milk covered to avoid airborne contamination
• Disinfect the teats by teat dip (i.e. disinfectants, iodophor, etc) after milking, to restrict entry
of microbes to the teat canal
• Never allow milk to flow from animals affected by any disease
• Always discard the milk from the animal treated with antibiotics for the prescribed days, as
the milk may have antibiotic residues that may affect the quality of the milk, starter failure or
health of a consumer
• Never use paper, cloth etc to air tight the lid of the milk can
• If the calf is used for excitation, wash the udder after suckling also
3.2.6 Management during collection
• The place of milk collection should be maintained absolutely clean to prevent any microbial
contamination.
• Adequate provisions should be made for cleaning and sanitizing the utensils/ vessels used for
milk collection and transport.
• Sanitizers should be kept away from the milk collection place.
• Lids of cans should not be kept on the ground and should be tightly fitted on cans to prevent
spillage.
• Do not use hay, grass etc. to give a proper fitting to the lid. Milk should always be kept
covered.
• Ensure that milk cans are kept in shade before loading in the truck. Trucks without covers or
hood should not be used, as they will expose the milk to sunlight and hence, accelerate the
growth of microbes.
• Transportation of other materials along with the milk cans should be avoided, as this may
lead to contamination.
• Careless handling of cans lead to dents, which also act as points for the growth of microbes,
and it also becomes very difficult to clean such cans. Most of the microbial growth takes place
during the time lag
between milk collection and its receipt at the dock. This time has to be reduced to the possible
extent to minimize the microbial growth.
• One that produces and handles milk should attempt to prevent microbial contamination at
each stage.
3.2.7 Hygiene of milking utensils
The milking utensils should be of uniform size. Having small mouths to avoid external
contamination. These should be preferably made up of non-rusting and non-absorbent materials
(i.e. aluminium or galvanized iron). Stainless steel is ideal, but costly. All the utensils should
be free from dents, cracks and crevices. The utensils should be scrubbed and cleaned before
and after each milking. The detergents and chemicals used should be noninjurious, and non-
abrasive in nature. At farm, use of washing soda coupled with exposure to sunlight or use of
detergents-cum-disinfectants (iodophors) is recommended. Properly cleaned vessels should be
placed in inverted positions for the complete drainage of water after milking, so as to avoid
contamination from air, insects, rodents, reptiles etc. In villages, where milk collection is done
by co-operative societies, the use of community milking parlours with facility to clean and
disinfect udders teats as well as milking equipments is needed.
[Link] Cleaning and sterilisation of milking equipments
While producing clean milk, the cleanliness and sterility of equipments are of prime
importance. Cleaning and sterilization are complementary to each other as none alone will
achieve the desired results. These can either be separate or combined processes as in the case
of chemical sterilisation.
The term ‘cleansing’ is frequently used to indicate cleaning combined with sterilization and
satisfies the condition that all equipment surfaces are free of milk residues and microbes. The
cleaning procedure be comprised of:
• Pre-rinse with water to remove all extraneous soil and to wet the surface
• Removal of soil from the surface by solution, saponification or a combination thereof
• Dispersion of un-dissolved soiling matte
Removal of detergent solution along with suspended and dissolved soil, and
• Final rinse to remove trace of detergent.
[Link] Detergent cleaning
The detergents help to free the surface of the milking equipments from fat and other milk
residues. The type and strength of detergents used depend on the method of washing.
Detergents in general are not disinfectants, however, strong alkaline detergents, if used in hot
are bactericidal in nature. When alkaline detergents are used for hand washing, the
concentration should not be more than the equivalent of 0.25% sodium carbonate. This is
not applicable in cleaning in place.
[Link] Cold water rinse
The outside dirt and residual milk from the surfaces should be removed by a lukewarm water
rinse. This should be done immediately after the vessels are emptied because, if the milk solids
are allowed to dry on the surface, it will be extremely difficult to remove these by rinsing.
[Link] Hot detergent wash
Hot detergent wash is best done in a wash trough; however the temperature of the solution
should not exceed 46°C, so as to be comfortable. The amount of detergent used should be
nearly equal to 115 g of soda ash or 230 g of washing soda per 45 l of water. If the concentration
of alkali is stronger than this, it may lead to the gradual defatting of the skin. The milking
equipments should be brushed in hot detergent with suitable brushes to remove
the surface residues effectively.
[Link] Final clean rinse
The final traces of detergent should be removed by a rinse of clean water. In case the water is
hard, hot water should be used, as a cold rinse will leave a deposit on the utensils.
[Link] Steam sterilisation
The efficiency of steam in destroying micro-organisms is greater than that of scalding hot
water. However, under Indian conditions, the latter is more practical. In large organized farms,
where milking machines are used, the use of steam is more convenient. Moist heat a few
degrees below boiling point (~96°C) for about 10 min are sufficient to kill all microorganisms
other than the resistant spores.
[Link] Hot water sterilisation
The boiling or scalding water may be used in small farms, where the number of equipments to
be sterilized is too small to warrant the installation of a boiler. As with steam, scalding water
is used after the cleaning process Temperature should be as near the boiling point as possible
and never below 85°C. The utensils should be immersed for 1 min, but where it is not possible,
boiling water should be poured over the milk-contact surfaces till they are too hot to touch.
[Link] Sterilisation by chemicals
Sterilisation of farm equipments with the use of chemicals is preferred overuse of steam, as it
does not involve high capital expenditure. Whereas, steam sterilisation is controlled by time
and temperature, chemical sterilization is dependent on several factors:
• Strength of the disinfectant
• Effective contact time
• Temperature
• Speed of action of the disinfectant and its specificity against different microbes
• Ability of the disinfectant to wet and also to penetrate any deposit on the surface, and
• Type of surface.
The common groups of chemical sanitizers used are hypochlorites (calcium and sodium),
organic chlorinecontaining chemicals (chloramines, dichlorodimethylhydrantoin, and
trichloroisocyanuric acid), quaternary ammonium compounds and iodine compounds
(iodophor).
3.2.8 Hygiene of milking environment
The places, where housing, feeding and milking are done, need special attention to minimize
the contamination of milk. The animals are housed and milked in the same building and this
system has been practiced for years, with variation in different countries according to climatic
conditions. The animal house should be carefully designed and constructed so as to provide
comfortable and healthy housing for the animal and enable them to be milked in clean
conditions. The major points such as siting, planning and layout, walls and floors of the housing
and ease to clean them, stall divisions, adequate water supply, lighting and proper ventilation,
drainage facilities, dung disposal and isolation chambers for sick animals should be taken into
consideration for clean milk production.
3.3 Straining of Milk
A clean muslin cloth should be tied on the mouth of milk-collecting vessel to strain off all the
extraneous matter. Although, the straining would not remove but reduce the number of
microorganisms, by expelling all the particulate matter thus, improving the aesthetic appeal of
milk for consumer.
3.4 Cooling of Milk
The strained milk should preferably be chilled immediately to 4°C to prevent the proliferation
of microorganisms. In places, where milk is stored in cans before transportation, bulk can
coolers are the best options. Some of the other cooling options practiced may be air-cooling,
water-cooling, and ice and mechanical cooling. The cooling aids usually used are household
refrigeration, direct expansion surface cooler, expansion bulk tank, ice bank and chilled water.
An effective cooling will prevent growth of mesophilic and thermophilic microbes.
3.5 Transportation of Milk
The basic system of milk transportation in India comprises the transport from farm to the
collection centre (either in small vessels or cans), from collection centre to the chilling centre
(in small tankers) and from chilling centre to the processing plant (in insulated road tankers).
The quality of milk will deteriorate during transit, if the surfaces that are in contact with milk
are not sufficiently clean and milk is at high temperature supporting the growth of microbes.
All the measures recommended for clean milk production at the farm should be strictly adhered
to, so that the initial quality of milk is good. Milk should be held for minimum time at farm at
ambient temperatures. In general, transport of uncooled milk can be justified only if great care
has been taken in its production (micro-organisms <100,000/ml) and if the milk is processed
or chilled to a low temperature not more than 3 h after its production. The collection centre
should be equipped with a basic cooling system. For larger quantities of milk, a surface cooler
or plate chiller with a tank storage system is recommended, especially if the holding period
between reception and transport to the dairy is long. The milk should be chilled to 4°C at the
chilling centre. At dairy, the temperature of the milk should not exceed 4°C, when it is received
in road tankers. Cleaning and sterilization of all equipment used, whether small containers,
cans or road tankers, should be carried out immediately after emptying. The tankers are usually
cleaned in processing dairies using manual method or cleaning in place.

Lesson 4
MORPHOLOGY AND CLASSIFICATION OF DAIRY BACTERIA
4.1 Introduction
Micro-organisms found in milk can be studied as groups possessing one or more major
characteristics in common. These may be divided on the basis of their action on milk
constituents, for example acid-former, proteolytic, saccharolytic etc. Another approach of
classification is based on the morphology of these micro-organisms. These types of
classification of dairy industrial microbes have been discussed in the following sections in this
lesson.
4.2 Classification Based on Shape, Size and Arrangement of Cells
The bacteria have been classified as cocci, bacilli, spirilla based on the shape of the cells.
However, some bacterial cells appear in different shapes or may be lacking uniform shape and
are known as pleomorphic ([Link], Bifidobacterium) (Fig. 4.1). Further based on the
arrangement of cells bacteria are also classified into different groups. The arrangement of cells
linked side by side similar to that like matchsticks is known as palisade arrangement.
4.2.1 Cocci
Bacterial cells with spherical or ellipsoidal shape are called as cocci and may have the
following type of arrangement of cells:
a) Diplococci: cells divide in one plane and remain attached in pairs (e.g. Neisseria)
b) Streptococci: cells divide in one plane and remain attached after some divisions, in form of
chains (e.g. Streptococcus)
c) Tetrads: cells divide in two planes and form 4 cells (e.g. Pediococci)
d) Sarcinae: cells divide in 3 planes and form a cuboidal arrangement
e) Staphylococci: cells divide in 3 planes in an irregular pattern producing bunches of cocci.
4.2.2 Bacilli
Bacteria with cylindrical or rod like cells are called as bacilli and these may have the following
type of arrangement of cells.
a) Diplobacilli: cells divide in one plane and remain attached in pairs.
b) Streptobacilli: cells divide in one plane and remain attached after some divisions, in form of
[Link] may be rounded as in Lactobacillus bulgaricus or squamosed as in Bacillus
anthracis.
4.2.4 Spirilla
Bacteria with spiral or helical shaped cells are called as spirilla and may have the following
characteristic shape.
Vibrio: rod shaped cells having single curves
Spirochetes: rod shaped cells having many curves
Few curves: true spirilla
Fig. 4.
1 Some of the Morphological features
4.3 Classification Based on Temperature
4.3.1 Mesophillic
Microorganisms capable of growing between 20 and 40°C with the optimum growth
temperature of 37°C are termed as ‘Mesophiles’. All pathogenic organisms are mesophillic in
nature (e.g. S. aureus, E. coli).
4.3.2 Psychrotrophic
Microorganisms that are capable of growing at or below 7°C (refrigerated) but the optimum
growth temperature are between 15 and 20°C are termed as psychrotrophs. These are the
significant spoilage organisms of refrigerated milk and milk products (e.g. Pseudomonas spp.,
Alkaligenes spp.).
4.3.3 Thermophilic
Organisms capable of growing over 50°C with optimum growth temperature of 55°C are
termed as thermophiles. They are the important organisms causing outbreaks in heat processed
milk and milk products. Some are capable of growing between 40-85°C. Organisms produce
enzymes at rapid rate, so that enzymes are replaced quickly
(e.g. Bacillus stearothermophilus, Streptococcus thermophilus).
4.3.4 Thermodurics
Organisms capable of withstanding pasteurizing temperatures of 63°C/30 min. with optimum
growth temperature of 35-37°C are termed as thermodurics. They form important flora of
pasteurized or heat processed foods (e.g. Micrococcus varians).
4.4 Classification based on Oxygen Requirement
4.4.1 Aerobic
Organisms capable of growing in the presence of oxygen are termed as aerobic organisms.
They can grow in a standard air atmosphere of 21% oxygen. They are more efficient in
utilization of available nutrientsm([Link] spp.).
4.4.2 Anaerobic
Organisms which cannot grow in the presence of oxygen but can grow in the presence of CO2
are termed as anaerobic organisms. They do not use O2 for energy yielding reactions. They are
however poisoned by O2. Some tolerate low concentrations of O2. They produce catalase and
peroxidase enzymes.
High tolerance – Clostridium perfrigens
Moderate tolerance – Clostridium tetani
4.4.3 Facultative
Organisms which can grow either in the presence or absence of oxygen are termed as
facultative organisms (e.g. E. coli, Lactococcus lactis ssp. lactis).
4.4.4 Microaerophillic
Organisms which grow best at 1-15% of O2 levels are known as microaerophilic. They can use
O2 for energy yielding reactions but cannot withstand more than 21% of O2
([Link] jejuni).
4.5 Physiological Grouping
4.5.1 Acid producers
The organisms capable of fermenting lactose to form lactic acid are known as acid producers.
Lactic acid coagulates milk by producing precipitation of Casein at 4.6 pH.
Homofermenters: Lactococcus, few Lactobacilli
Heterofermenters: Few Lactobacilli, Lueconostoc spp.,
4.5.2 Gas producers
Gas producers are the organisms capable of producing CO2 or \ and H2 from lactose
fermentations (e.g. E. coli, yeasts, and Clostridium spp.).
4.5.3 Proteolytic
Proteolytic organisms degrade milk proteins into soluble components by enzymes known as
proteinases or proteases (e.g. Bacillus spp, Pseudomonas spp.).
4.5.4 Lipolytic
Lipolytic organisms are capable of attacking milk fat by enzymes such as lipases liberating
glycerides and fattyacids (e.g. Pseudomonas spp, Achromobacter lipolyticum; Moulds:
Geotrichum candidum, Penicillium roqueforti).
4.5.5 Sweet curdling
Organisms capable of causing curdling of milk by rennin like enzyme before the development
of sufficient acidity are known as sweet curdling organisms (e.g. B. subtilis, B. Cereus and
Enterococcus liquefaciens).
4.5.6 Ropiness
Ropiness organism produce change in the viscosity of milk or forming threads when the milk
is poured from one container to other, due to production of gums, mucins etc. (e.g. Alcaligenes
viscosus).
4.5.7 Flavour producing
Many organisms are capable of producing different flavours in milk during their growth. Some
of the flavours and associated organisms are listed below:
Fruity – Pseudomonas fragi
Malty – Lactococcus lactis subsp. lactis biovar maltigenes
Fishy – Proteus icthyosmius
Unclean – E. coli
4.5.8 Colour fermentations
Many organisms are capable of producing different colours in milk during their growth by
producing chromogenic
compounds. Some of the colours and associated organisms are listed below:
Yellow: Pseudomonas synxantha
Blue: Pseudomonas cyanogenes
Green: Penicillium

[Link] OF MARKET MILK

[Link] and standards of market milk

Market milk may usually refer to the fluid milk that is sold for direct consumption and
thus excludes milk consumed on the farm and that used for the manufacture of dairy products.
According to the international commission on Microbiological Specifications for
Food(ICMSF), market milks are heated,fresh milk products consumed primarily in fluid form,
e.g., pasteurized, sterilized or ultra high temperature(UTH) treated milk as given under PFA
rules 1955 are defined as follows.

[Link] milk

Milk which has been brought to boil

2 .Pasteurized Milk

The term pasteurization, when used in association with milk and milk products means
heating milk of different classes by a heat treatment as indicated below and cooling to a suitable
temperature before distribution. Pasteurized milk of different classes shall show a negative
phosphatase test. The terms “pasteurization”, ”pasteurized “ and similar terms shall be taken
to refer to the process of heating every particle of milk of different classes to at least 63c and
holding at such temperature continuously for atleast 30 min, or heating it to at least 75.5c, and
holding at such temperature continuously for at least 15 sec or an approved temperature time
combination that will serve to give negative phosphatase test. The pasteurized milk of different
classes shall be cooled immediately to a temperature of 10c or less.
[Link] milk
The term sterilization, when used in association with milk means heating continuously
to a temperature of 115c for 15min or 145c for 3 sec or equivalent temperature time
combination to ensure preservation at room temperature for a period of not less than 15days
from the date of manufacture Sterilized milk shall show absence of albumin by a negative
turbidity test. Sterilized milk shall sold only in the container in which the milk was sterilized.

4. Standardized milk

Cow milk or buffalo milk or sheep milk or goat milk or a combination of any of these
milks that has been standardized to a fat and solids not fat percentage to the adjustment of milk
solids . Standardized milk shall be pasteurized and shall show a negative Phosphate test.

5 .Flavoured milk

By whatever name this type of milk is called it may contain nuts, chocolate, coffee or any
other edible flavour, edible food colour and cane sugars. Flavoured milk shall be pasteurized
, sterilized or boiled. The type of milk shall be mentioned on the label.

[Link] milk
 This is a homogenized product prepared from milk fat, non-fat milk solids and water.
Recombined milk shall be pasteurized and shall show a negative phosphatase test.

[Link]

Mixed milk is a combination of milk of cow,buffalo, sheep , goat or any other milch
animal and may be a combination of any of these milks which has been made and conforms to
the standards.

[Link] milk

It is the product made from milk from which almost all the milk fat has been removed
mechanically.

9 .Toned milk
Toned milk is the product prepared by admixture of cow or buffalo milk or both with
fresh skimmed milk, or by admixture of cow or buffalo milk or both, has been standardized to
fat and solids by adjustment of milk [Link] shall be pasteurized and shall show a negative
Phosphatase [Link] fat or non-fat milk solids are used, it shall be ensured that the products
remain homogeneous and no deposition of solids takes place on standing.

10. Double toned milk

The definition of this product is exactly the same as given for this toned milk except that
the standardized percentage for fat and solids not fat is different.

[Link] quality of market milk

Raw milk, as produced at the farm level, is expected to contain a variety of
microorganisms, including pathogens and thus is not suitable for direct consumption. In
addition to potential health risks, raw milk cannot keep for long, especially if stored above
refrigeration temperatures, due to the presence of various spoilage producing microorganisms.
Hence for increasing its shelf like and safety for human consumption, the raw milk is heat
processed before selling as market milk. Pasteurization is the most common method of market
milk.

MICROFLORA OF MARKET MILK

Microflora of pasteurized milk

The microorganisms encountered in market milk (pasteurized) may either be those

surviving the pasteurization temperature or those entering the product as post- pasteurization
contaminants. To the former category belong the thermoduric and thermophilic
microorganisms whereas in the latter are included psychrotophic, coliforms and other heat-
sensitive bacteria.

The heat resistance of microorganisms during heating depends on several factors as

given below:

1 pH: As the pH of the medium decreases, the number of survivors also decrease.
 2 Age of cells: Young cells are more susceptible to thermal destruction than the older
cells.
3 Growth temperature: commonly the organisms are more heat-resistant when grown at
their optimum than when grown at lower temperatures.
4 Composition of medium: Bacterial cells are killed faster by heating in aqueous media
as containing additives like proteins, starch, fats or other organic molecules.
5 Initial microbial number: The number killed due to heat-treatment in any interval of
time is a function of the number initially present.
6 Presence of free water: In general , desiccation increases heat resistance of organisms
as free water assists penetration of heat.

A. THERMODURIC MICROFLORA
a) Definition: In dairy industry, thermoduric micro-organisms are those that
survive pasteurization temperatures but do not multiply at these temperatures. The chances of
their survival are relatively more in HTST as compared to holder method of pasteurization. These
can be detected by laboratory pasteurization of sampleat 62.8c for 30 min followed by planting
out and incubation at 32c.
b) Most of the thermoduric microorganisms in pasteurized milk are mesophilic but few
are psychrotrophic in nature. The commonly occurring thermoduric bacteria in pasteurized milk
include micrococci, microbacteria, aerobic sporefomers streptococci and Arthrobacter. Certain
others like Lactobacillus spp, Clostridia may also be encountered infrequently.

B. THERMOPHILIC MICROFLORA
a) Definition: In dairy industry, the term applies to those bacteria which can not
only survive but also grow at pasteurizationtemperature. Since their upper limit for growth is 70c,
they can grow well during holder method of pasteurization. The standard procedure for their
detection in laboratory is to incubate agar plates at 55c as this is the optimum temperature for
most of the thermophilic species.
b) Types: In pasteurized milk, generally aerobic or facultative anaerobic spore
forming rods constitute majority of the thermophilicmicroflora.
Examples Lactobacillus thermophillus
Bacillus spp
B stearothermophilus,
B thermoacidurans
B calidolactis
B coagulans
C PSYCHROTROPHIC MICROFLORA
a) Definition: In dairy industry, the term is used to define those organism which
can grow appreciably at commercial refrigeration temperatures (2-7c) irrespective of their
optimum temperature of growth. Majority of the psychrotrophs in pasteurized milk are
mesophilic while only few are psychrophilic.
b) Types: These are two categories of psychrotrophic bacteria in pasteurized milk.
1. The first category comprising psychrotrophic thermoduric bacteria which are
Gram positive rods and cocci, include the genera Bacillus, Clostridum and Streptococcus.

2. The second category comprises psychrotrohic bacteria which are Gram negative
rods belonging to the genera Pseudomonas, Flavobacterium, Alcaligenes,
Achromobacter,Acinetobacter etc.,

5.3 MICROBIOLOGY OF CREAM

 Cream is the fat-rich portion of milk obtained on keeping it undisturbed for
sometimes or on centrifuging. According to the definition given in PFA Rules 1955 cream
excluding sterilized cream is the product of cow or buffalo milk or a combination there of
which contains not less than 25% of milk fat.

Types of cream

A. On the basis of use

1. Market cream : cream which is directly consumed.
2. Manufacturing cream: cream, which is diverted to manufacture of milk products like butter,
ghee, ice-cream, various dairy desserts etc. In India, cream is commonly used for butter
and ghee manufacture.
3. Bakers cream: the cream used by the bakers and other in the food industry , is commonly
called bakers cream. It usually contains 32 -40 % fat. The microbiological problems with
this product are more since it is subjected to more unhygienic practices.
B. On the basis of fat content.
1. Table cream
2. Light cream containing 20-25% milk fat
3. Coffee cream
4. Whipping cream
5. Heavy cream containing 30-40% milk fat
6. Plastic cream containing 65-85% milk fat
In UK eight types of cream defined only in respect of their fat content, are
produced. Half cream sterilized cream whipping cream whipped cream double cream clotted
cream

c. On the basis of processing

i. untreated cream: cream which is not processed or cream which is not obtained from unprocessed
milk.
ii. pasteurized cream : cream which is pasteurized or cream which is obtained from pasteurized milk.
iii. Sterilized or canned cream: cream which is sterilized
iv. UHT cream: cream which has been subjected to UHT processing
v. Frozen cream: cream which is frozen to below 20°C
vi. Sour cream: cream obtained ripening the pasteurized sweet to high acidity (0.6% lactic acid).
vii. Clotted cream: cream obtained by skimming off the surface layer of cream heated (77°-80°C) and
cooled in shallow pans.
viii. Cream powders : made y dehydrating fresh cream of mixture of an hydrous milk fat and skim milk
powder.
ix. Artificial or reconstituted cream: made from butter milk
x. Imitation or synthetic cream: made by emulsifying edible fats or oils in water other permitted
additives.

Chemical composition
 In cream, milk fat may vary from 18 to 85% and the SNF constituents are present
in lower proportion than in milk ; the higher the fat content in ceam, the lower SNF content.

Chemical composition of cream

Percentage
Constituent
I II

25 50
Fat
2.54 1.69
Protein

3.71 2.47
Lactose
0.56 0.37
Ash
Solids-not-fat(SNF) 6.80 4.55

Total solids 31.80 54.55

Water 68.20 45.45

Microbiological standards

Indian standardized institution (ISI) has given the following standards for
raw and pasteurized cream

i)Raw cream

Plate count/ml grade

Below 4,00,00 very good

4,00,000-2,000,000 good

2,000,000-10,000,000 fair

Over 10,000,000 poor

Coliform count/ml

Notmore than 100 satisfactory

ii) pasteurized cream

the plate count/ml should not exceed 60,000 and coliform count /ml should
not be more than 10.

The microbiological standars in other countries have been laid down as

follows:

Country

Canada - counts less than 50,000 per g No coliforms in 1g

phosphatase negative

Northern Ireland - untreated cream –counts less than 50,000 per g

pasteurized cream – no coliforms in 1 g

Sweeden - counts less than 100,000 per g coliforms less

than 10 per g aerobic spores less than 100/g

Microbiological quality of cream

The micro environments of cram and milk are he same except the difference
in the proportion of various constituents like fat,proteinlactose etc. as a result,
the initial microbiological quality of cream is almost parallel to that of milk
, but in later stages, cream presents more problems than milk because of
longer storage and erratic distribution pattern.

[Link] of butter

Butter is commonly defined as a fat concentrate which is obtained by

churning cream, gathering the fat into a compact mas and then working
[Link] to the definition given under PFA rules (1955), table (creamery)
butter means the product obtained from milk, cream or curd of cow or buffalo
or a combination of ,with or without the addition of common salt and no
colouring matter except annatto or carotene may be added.

Types of butter

Based on the manufacturing process and the type of raw material used, the
following types of butter are available in the market in different parts of the
world.

A. creamery butter : butter made in creamery or dairy plant.

B. Dairy butter (USA) : butter made on a dairy farm. Generally it
has a sour flavor since it is made from sour cream. Creamery butter has a
more uniform quality as compared to dairy butter.

C. desi butter : butter made in the indian household usuall from

curd by churning

pasteurized cream butter :butter made from pasteurized sweet cream has a
milder flavour compared to that from unpasteurizied cream

unpasteurized cream butter : made from unpasteurized cream

sweet cream butter : butter made from sweet cream i.e, cream with acidity
not exceeding 0.20%

sour cream butter : butter made from sweet cream i.e, cream with more than
0.20% acidity.

neutralized sour cream butter: butter made after neutralizing the sour cream
with chemical neutralizers.

ripened cream butter – butter made from cream that has been ripened which
means the inoculation and incubation of cream with a desirable flavour
producing butter culture under optimum conditions so that typical butter
flavoured is developed ,. Such butter is normally unsalted and if salted , it
usually should not contain more than 0.5% salt since the combined effect of
low pH and high salt will [Link] higher salt addition,
neutralization is recommended.

unripened cream : butter obtained from unripened [Link] has a mild flavour
unlike ripened cream butter.

salted butter: butter obtained by mixing of salt at recommended

concentration.

unsalted butter : butter which has no added salt

fresh butter : butter that has not been kept in cold storage and usually it is not
kept for more than three weeks.

cold storage butter: butter that has been kept in cold storage usually for one
to six months.

Chemical composition

The composition of a typical salted indian butter is as follows :

Ingredient amount (%)

Milk fat 80.2

Moisture 16.3

Common salt 2.5

Curd 1.0

According to PFA rules (1995), the creamery butter should meet the
following composition

Milk fat - not less than 80% by weight

Moisture - not more than 3% by weight

Curd - not more than 1.5% by weight

The almost universally accepted standards for butter are prescribed either as
16% moisture maximum or 80% fat minimum ( except a limit of 78% in UK).
Some countries also specify for solids- not-fat (usually 2% maximum).

Microbiological standards

The standards and recommendstion of indian standards institution for butter

are as follows:

SPC: no standards have been suggested

Coliforms: presence of more than 10 coliform per ml butter is an index of

insufficient pasteurization or contamination of butter from external sources
like wash water, equipment and other sources during man8facture and
packaging.

Yeast and molds

Y and M count/ml quality

Less than 20 good

21- 50 fair

51- 100 poor

More than 100 very poor

Keeping quality: if an off- flavour develops on keeping butter in a glass jar
either at 37°c for 48 hours or 21.1°c for 7 days, a bad keeping quality is
predicted.

[Link] of ice cream

Ice cream should represent less of bacteriological problems than most other
dairy [Link] ingredients used in ice cream may include cream with or
without added milk solids, sugars, gelatin, stabilizer,and eggs or egg solids.
The ingredients are combined to form the mix. Certain flavours such as
chocolate may be added . The mix is pasteurized , cooled, aged and frozen
into final products. The sanitary problems in ice cream manufacture
includes, selection of ingredients of proper quality, proper pasteurization of
the mix and the cleen handling of the mix after pasteurization . once the
ingredients are frozen microorganisms cannot multiply in it.

Factors affecting numbers of bacteria in ice cream:

Bacterial content of ice cream is dependent on the quality of the ingredients

used. Cream employed in ice cream varies widely in bacteriological
condition. Pasteurization of the cream normally destroys a high percentage
of the bacteria originally in the cream. Thermoduric bacteria, such as
micrococci , streptococci and sporeformers may be present in the cream .
cans used to hold milk products or mix should be thououghly sterilized before
using. In spite of the high pasteurizing temperature used, if considerable
number of thermoduric bacteria are present , it is likely that many will survive
the pasteurization.

Egg products and gelatin may contribute large number of bacteria , including
coliform types to ice cream . sugar in general contains only small number of
bacteria and is of little importance as source of bacteria in ice cream.
Flavouring materials should be recognized as source of organisms in ice
[Link] and nut meats may constitute important sources of bacteria since
they often are added in the raw state just as the mix enters the freezer. These
nuts carry a wide variety of organisms including aerobic
sporeformers,micrococci and [Link]. colouring materials also are agents
introducing microorganisms is icecream. Colouring materials are some times
added after pasteurization and therefore contamination from them assumes
considerable importance.

Types of bacteria in ice cream:

A wide variety of bacterial species have been isolated from ice cream. These
include micrococci, streptococci,aerobic and anaerobicsporeformers,E-coli,
[Link],Psedomonas species and many other pathogenic and non-
pathogenic bacteria. Acid forming species make up a considerable
percentage of the flora .the general microfolra of icecream of icecream
resembles that of pasteurized milk or cream. If favourable conditions for
growth of bacteria are provided a variety of organisms grow in icecream and
these include yeasts and molds.

[Link] of concentrated milk products:

Here we shall confine ourselves to the indianindigenous milk products which

are generally prepared by evaporating the milk in iron pans. The two most
common such products found in many parts of indian are “ khoa and rabri”.
Khoa is a type of concentrated milk made by boiling milk in an open pan to
evaporate the moisture until only a solid dough –like mass remains. Almost
95 % of water in the milk is removed in the process. Generally whole milk is
used to make “khoa”.usually nearly 4 pounds of milk is used at a time.
Boiling must be continuous and vigorous so that evaporation occurs as
quickly as possible. The keeping quality of khoa depends entirely upon the
degree of contamination and the extent to which it is exposed to conditions
favourable to the growth of micro organisms subsequent to its manufacture
. As during the process of manufacture of “khoa “ a continued boiling process
is followed , almost all the bacteria present in milk are killed, still a few
sporeformers may survive. Therefore, the bacteria present in khoa are mostly
due to the faulty methods of its handling after preparation. Khoa is usually
wrapped with green leaves of plants and so a great number of yeasts and
molds get entry into it from these sources. As khoa is rich as food material
and at the same time it contains sufficient moisture and no sugar is added in
its preparation it is very easily [Link] may also be contaminated with
pathogenic bacteria and these may cause outbreak of disease.

Rabry is another product of milk that is prepared by heating in a wide top

open pan. In this case the milk is not boiled but is heated to a temperature a
which a skin may form on the surface. Skin formation is encouraged by
fanning the surface of the milk during heating. The skin which forms during
heating is carefully reoved from time to time by use of a special dipper,
usually made of [Link] successive skins are removed they are placed on
the upper exposed inside edge of the pan, one upon the other. The process is
continued until the remaing milk Is about 1/8th the volume of that with which
the process [Link] is added at this stage at the rate of 2 ounces per 2
pounds of the original milk. The skins are then removed from the sides of the
pan and immersed in the sweetened concentrated milk and is further
evaporated for a [Link] in the preparation of rabri milkis not boiled
though it is heated for a consideration of the [Link] the process of
making, equipments used areare not sterilized and the bamboo dipper may
act as a source of contamination. Besides these sources handling of it after
preparation also may result in contamination of the [Link] most
common organisms found in rabri are sporeformingrods,micrococci,molds
and [Link] producing bacteria may also get entrance to it.

Both khoa and rabri are very extensively used in india,but their methods of
preparation are very primitive and no care is taken as regards the hygienic
quality of the products. It is suggested, therefore that proper mechanical
devices,as in the preparation of other evaporated milk products,should be
introduces in the manufacture of theses two important products. If the
technology of its production could be improved much of the problems of its
contamination and the period of preservation might be improved.

[Link] of dried milk:

Dried milk products represent the most stable products from the standpoint of microbial [Link] long
as they kept in a dry state no mcroorganisms of any type can develop in them. In fact the microorganism
gradually decrease in numbers in dried foods during their storage. At present, milk is dried by the roller
process and also in the various spray systems. Both whole milk and skim milk are dried. milk powder in
tight containers sometimes is packed in an inert gas to delay deterioration.

Since the low content of moisture (less than 5 %) absolutely prevents any bacterial growth any storage
temperature , the bacterial content of dried milks reflect the original conditions of milk and the conditions
under which they are determined by plate count method. It is necessary to use warm water to reconstitute
the [Link] water should be at 43 to 49°C. the second important requirement is to use a low incubating
temperature , 30 to 32°C instead of 37°C. the lower temperature is needed for incubation of
microbacteria,which require low temperature for their growth. The rest of the procedure is the same as that
used for milk. Numbers of bacteria in dried milk may also be determined by direct microscopic method.
In that case the milk powder may be dispersed advantageously in 1.25% sodium citrate solution.

The number of bacteria in dried milk after different periods of storage

Amount of Number of bacteria in one gram of dried milk

moisture %
after2 after4 after 12
months months months
fresh after6
months

22508 7818 2487 655 261

 9.14

Though the microorganisms prevent in dried milk powder cannot multiply

they cause serious defects in the manufacture of different products.
Bacillusmesentericus if present in the milk powder may cause a very serious
defect known as ropy bread originating from the use of such milk powder.
When starter culture are prepared from dried milk, sporeformers present in it
may give rise to serious troubles. Microorganismsfound in spray-dried milk
include thermoduric streptococci such as S. thermophiles, micrococci,
lactobacilli, achromobacter,aerobic and anaerobic sporeforming types,
molds,yeast,coliform bacteria and species of tiny rods known as
microbacteria.

Lesson 6
[Link] ASSOCIATED WITH RAW MILK AND THEIR
SIGNIFICANCE
11.1 Introduction
Milk, like many other foods and environment around us, contains bacteria that can be classified
into three
categories:
• Beneficial
• Spoilage
• Harmful or pathogenic
Beneficial bacteria help in making food products, for example, cheese, yogurt, and buttermilk.
Spoilage causing bacteria leads to bad smell/ taste and poor look of the product. Most bacteria
in fresh milk from healthy animals are usually harmless. However, rapid changes in the health
of a milk animal or of the handler, or contaminants from polluted water, dirt, manure, air, cuts
and wounds can make raw milk potentially dangerous, if these factors introduce harmful
bacteria to milk, as it provides an excellent medium for the growth
of bacteria. Milk is sterile, when it is secreted in udder but gets contaminated before it leaves
udder. However, contaminating bacteria are usually harmless except in case of mastitis, but
can lead to different spoilage problems. Microorganisms causing spoilage may be categorized
as psychrotrophs, mesophiles or thermophiles. These microbes are of great significance in milk
as:
• Information on microbial counts of milk can be a diagnostic criteria for the judgement of its
overall quality
• If multiply, bacteria can cause spoilage of milk much faster
• Milk is highly susceptible to contamination with pathogens and precautions must be taken to
minimize it and to destroy pathogens, if these gain entry
• Certain types of microbes produce chemical changes that are desirable in the production of
different dairy products, for example cheese, yogurt etc.
6.1.1 Harmful or pathogenic bacteria

The consumption of raw milk was prevalent in the society even prior to the industrial revolution
and discovery of pasteurization. Before that nobody knew about the infectious microbes and
their association with water, milk and milk based products and the various diseases they cause.
Initially, no testing procedure was available to determine the quality of raw milk and also to
discriminate between healthy and infectious milk. With the advent of modern scientific
techniques, now it is possible that the milk is free from harmful microbes but many problem
may still persist like eradication of their toxic metabolites. Significant work has been done to
combat all these issues like enforcement of sanitation and testing procedures at farm level and
implantation of various practices at commercial level. However, pasteurization continues to be
an effective method in control of these unseen
problems. These days with innovative techniques one can identify microorganisms at genomic
level to establish
preventive measures effectively.
6.2 Microflora of Raw Milk
Milk provides a wealth of nutritional benefits, but raw milk can harbour dangerous
microorganisms that can pose serious health risks to the consumers. Milk from cows, sheep, or
goats that has not been pasteurized to kill harmful bacteria is raw milk. This raw, unpasteurized
milk can carry dangerous bacteria such as Salmonella, E. coli, and Listeria that are responsible
for causing numerous food-borne illnesses.
Milk has been considered as the most significant part of our food, as it contains all essential
nutrients, suitable pH, and water activity for the growth of microbes. Hence, there is a high risk
for these products to get contaminated with spoilage causing microbes. Hence, it is worth to
know more about its chemistry, spoilage and different preservation methods, so that microbes
responsible for negative effects could be identified. Milk is a good source for the growth of
microorganisms therefore, it is essential to understand more about the the types of
microbes associated with milk, their control measures and beneficial uses.
Milk when secreted into the udder of an animal is considered to be as sterile; and it gets
contaminated later from the microflora residing in the udder tissues and most of the times the
contaminants are non-infectious except in case of mastitis. Milk becomes more contaminated,
when it comes in contact with an external environment during milking, transportation and
storage. Other factors such as utensils, machines, handlers, water, air and soil, also contributes
greatly to the micro flora of milk. This way bacteria, yeast and moulds get entry into milk
constituting the normal microflora. The number of contaminants added from various sources
depends on the level of precaution taken to avoid contamination.
The presence of these non-pathogenic microbes in milk is not very serious, but if these multiply
quickly, these may cause souring of milk, putrefaction and development of different taints,
undesirable flavours and colours of milk and milk products (Table 11.1). Thereby, it is essential
to develop the suitable preventative measures to check spoilage of such highly nutritious food
commodities that are perishable in nature
6.3 Microbial Contaminants from the Surroundings

A. Animal and its udder: The animal and its teats contribute greatly to the microflora of milk
that includes those that are resided on skin and also that are present in the niche of animal,
where it is reared and/ or milked.

B. Utensils and sanitary practices: The hygienic conditions inside milking system influence
total microbial load of raw milk. Milk residues left behind on equipments may support the
growth of different microbes that on subsequent milking may lead to spoilage milk. These may
account for 100,000 to a billion microbes per millilitre in the cracks, scratches of utensils etc.
The microbial load of raw milk finally will be concluded on the basis of different contaminants
in the surroundings. Milk microflora contains a mixed population of lactic acid bacteria,
mesophiles and thermophiles, thermoduric, psychotropic and pathogenic microbes. The milk
microflora will be influenced by a number of environmental parameters that affects the growth
microbes.

6.4 Psychrotrophs

Psychrotrophs are the microorganisms that have the ability to grow near freezing temperature
in the range of 0 to 7°C. The typical representatives of this class are both Gram negative and
positive microorganisms.

Gram negative includes: Pseudomonas, Achromobacter, Aeromonas, Serraita,

Alcaligenes,Chromobacterium and Flavobacterium spp.

Gram positive includes: Bacillus, Clostridium, Corynebacterium, Streptococcus,Lactobacillus

andMicrocobacterium spp.

The quality of raw milk has been considerably maintained by refrigeration on farms, and in
processing plants. However, the current practices in India for the collection and storage of the
raw milk favor the growth of psychrotrophs that are, able to grow below 7°C, regardless of
their optimal growth temperature. Besides, their rapid growth ability in refrigerated milk,
psychrotrophs produces heat stable extracellular proteases, lipases, phospholipases etc.
Psychrotrophs are those bacteria that can grow at 7°C, although their optimal growth
temperature is higher. During cold storage after milk collection these dominate the micro-flora,
and their extracellular enzymes, mainly proteases and lipases, contribute to the spoilage of
dairy products. The extracellular enzymes can resist pasteurization (72°C for 15 s) and even
ultra-high temperature (138°C for 2 s) processing. The lipases, by hydrolyzing triglycerides,
cause flavour defects associated with fat breakdown in cream, butter and cheese. Proteases are
associated with bitterness in milk, gelation of sterilized milk, and reduced yields of soft cheese.
Most proteases can degrade caseins and are highly heat stable. Overall, psychrotrophs play a
greater role in spoilage of refrigerated milk. The numbers of psychrotrophs that develop after
milk collection depend on the storage temperature and time. Under sanitary conditions, <10%
of the total microflora are psychrotrophs incontrast to >75% under unsanitary conditions.
Pseudomonas is the most prevalent psychrotroph in raw milk.

6.4.1 Significance of psychrotrophs in milk

Psychrotrophs are not only important from the spoilage point of view but also emerged as
psychrotrophic pathogens like Listeria monocytogenes, Yersinia enterocolitica etc. that made
the situation more severe. Over the last few decades, listeriosis has become a major food-borne
disease, as L. monocytogenes can cause meningitis, septicemia, and abortion. Nearly, 20 to 30
% of people, who get listeriosis die from it and there is a longtime between food consumed and
symptoms developed. Therefore, the control of psychrotrophs is important to stop spoilage, as
well as health hazards to the consumers.

The cold storage of milk favours the growth of psychrotrophs. Following pasteurization or
other heat treatment of raw milk to destroy or remove the microbes, spore germination or
recontamination can still cause quality deterioration; furthermore, heat-resistant extracellular
proteinases and lipases produced by psychrotrophs before processing are a major spoilage
factors during storage of milk. The detection and control of psychrotrophs and their heat
resistant enzymes are major concerns of quality control in dairy industry. Thermo-resistant
psychrotrophs: These are the psychrotrophs that survive pasteurization. For example -
Arthrobacter, Microbacterium, Streptococcus, Corynebacterium, Clostridium; are spore
formers and anaerobic. Among these thermo-resistant psychrotrophs; Bacillus spp.
(sporeformer) dominates and secretes extracellular heat resistant proteinases, lipases and
phospholipases (lecithinases). Control of psychrotrophs and their enzymes

• Following hygienic practices in different aspects of milk handling

• Minimizing storage time of milk
• Following effective HACCP system
• Strict maintenance of refrigeration at 4°C
• Use of non thermal techniques like use of high pressure for preservation
• Combination of heat treatment with aseptic filling or packaging
• Use of natural preservation by bacteriocins
• Treatment of milk with carbon dioxide or nitrogen
• Thermization of milk before chilling and storage
The suggested standards for psychrotrophs in raw milk are:
• Less than 104 cfu per ml - Satisfactory
• Above 104 cfu per ml – Unsatisfactory

Lesson 7

[Link] OF PROCESSING ON MICROORGANISMS IN MILK

7.1 Introduction

In Indian subcontinent milk production is regularly increasing during the last few decades and
acquires the position of nutritious food. Different plans have been introduced in past to improve
the production and quality omilk from farm to consumers. However, in India dairy sector is
still dominated by the rural people, especiallfarmers and at farm the advanced facilities to
protect the milk from spoilage are scanty but upcoming. Becausof this, a major portion of total
milk produced is spoiled by different factors during milking, transportation, handling and
storage of milk. The final quality of milk or its products is directly related to the hygienic
quality of raw milk produced. Therefore, milking production practices plays a major role in
determining the hygienic quality of raw milk or milk products as discussed earlier.

Table 7.1 Hygienic parameters pH and microbial counts (cfu/ml)] of freshly drawn milk
7.2 Effect of Cooling on Milk
Cooling of milk is a process of bringing the temperature of the milk below ambient temperature
using a cooling medium. The purpose of the cooling is to reduce the spoilage of milk by
preventing or retarding the growth of different group of microorganisms. Milk contains various
microbes derived from different sources. The most important spoilage and pathogenic micro-
organisms in milk are mesophiles with a growth temperature ranging
between 20°C to 40°C. Cooling reduces the metabolic activity of micro-organisms by reducing
the activity of the enzymes necessary for metabolism, thereby reducing the growth and
reproduction of the bacteria, especially mesophiles.

From the above data it is evident that the critical temperature is 10°C as beyond this the growth
increased further. Hence in practice, milk is cooled to below 5°C to increase the shelf life of
raw milk.
7.3 Bactofugation
Centrifugal force provides an efficient means of improving the bacteriological quality of milk.
The principle is bacterial super centrifugation but when it is carried out under pasteurization,
it is called ‘bactofugation’. However, there is a tendency to use the term irrespective of
processing temperature. Bactofugation removes bacteria, both living and dead, from treated
substances, whereas traditional heat treatment kills bacteria and leaves them in food.
Bactofugation is important in food-stuffs infected with bacteria, especially those containing
thermo-stable endotoxins.
The purposes of bactofugation are:
• To improve the hygienic quality
• To avoid heat resistant bacteria without resorting to excessive heating
• Where exceptionally high degree of bacteriological purity is sought
Bactofugation is not a substitute for pasteurization or sterilization but used in conjunction with
them to improve their efficiency.

7.3.1 Advantages of bactofugation

• In cheese:
Prevents swelling in certain cheeses by butyric acid bacteria which are heat resistant
Removal of bacteria without pasteurization which enables raw milk cheese production with
more typical cheese flavor.
• In milk Powder: To reduce the count of microbes and significant removal of heat resistant
bacteria.
• Sterilized milk: The severity of heat treatment can be reduced.
• Cream: Bittiness caused by heat resistant Bacillus cereus is avoided.
7.4 Thermization
Thermization is the least heat treatment followed in dairy industry. Heat treatment used is in
the range of 63- 65°C/15-20 s and cooling to below 6°C, optimum being 66-70°C/15 s.
Purpose of Thermization is:
• To destroy enzyme ‘lipase’ because milk produced on farm may not be taken daily to dairy
• To extend storage of raw milk during refrigeration
• To maintain daily production of products, when supply is reduced
It is designed to lower the number of psychrotrophs of raw milk to take care of lipolysis and
proteolysis.
7.4.1 Factors affecting thermization process
• Raw milk - Initial load should be less than 5,00,000 cfu/ml. As load increases, the
corresponding count after thermization in 3 days storage at 6°C also increases because of
elimination of only of psychrotrophs
• Heat treatment above 65°C is effective, but increasing the temperature at constant holding
time promoted of shelf life than increasing holding time at constant temperature. As
temperature increases more number of flora of enterobacteriaceae are destroyed
• Some bacteria may increase in number e.g. S. thermophilus (at 68°C/10 s)
7.5 Destruction of Microbes by Heat
Microbes are destroyed by heat, when microbial proteins are coagulated and enzymes required
for their metabolism are inactivated. The death of microbes is also due the thermal denaturation
of the secondary and tertiary structure of macromolecular cellular organizations
7.6 Pasteurization of Milk
Louis Pasteur (1860-64), a French scientist, heated wine to 50-55°C to increase the keeping
quality and this method known as pasteurization has been frequently used in industry with
some modification. As per the definition, pasteurization is: LTLT= 63°C/30 min or HTST=
72°C/15 s and cooled to 5°C or below.
7.6.1 Objectives of pasteurization
• Destroy all the pathogenic organisms present in milk.
• Reduce the load of non-pathogenic organisms and increase shelf life.
The most important heat resistant pathogen is Mycobacterium tuberculosis. Thus, the minimum
heat treatment was established to guarantee the inactivation of M. tuberculosis. The
pasteurization deals effectively with almost all pathogens satisfactorily except for Bacillus
cereus Milk contains alkaline phosphatase enzyme that is inactivated at a temperature-time
combination similar to that of pasteurization. Alkaline phosphatase can be measured by a
simple chemical test known as phosphatase test.
Some of the conditions of pasteurization are placed below:

7.6.2 Effect of pasteurization on Raw Milk Microflora

Raw milk contains a mixed microflora arising from several sources. Holding temperature prior
to pasteurization is quite important. Above 10°C, all the micro-organisms in raw milk multiply
actively. This includes heat resistant non-spore formers and spore formers. If this number of
heat resistant micro-organisms increases the total count of milk after Pasteurization will also
increase.
<10°C – Growth of contaminating strains rapidly falls.
<5°C – Gram negative rods grow significantly like Pseudomonas that are capable of producing
heat resistant enzymes. B. cereus may grow and produce toxins.
Theoretically it is not possible to destroy all the microbes in milk by heat treatment but the
purpose of Pasteurization is to reduce probability of pathogens surviving the process to such a
level that the public health risk from drinking such milk is negligible.

7.6.3 Effect of pasteurization on thermodurics in milk

A. Source of thermodurics from milk:
Gram-positive organisms: Only Alcaligenes tolerans survives pasteurization
• Gram-positive cocci
a) Micrococci – large number in raw milk but outgrown by others at 7°C. These are unable to
grow due to lactenin, a natural inhibitor in milk. These also occur on dairy equipments.
b) S. thermophilus, E. faecalis and S. uberis: A number of strains of these are thermoduric but
grow slowly at refrigeration temperature between pasteurization and consumption. Milk
containing high thermodurics also contains appreciable number of haemolytic streptococci.
c) Anaerobic spore formers – unable to grow because of high redox potential of milk but
sometimes isolated from milk.
d) Aerobic spore formers – survive pasteurization
Mesophilic – B. licheniformis most important followed by B. pumilus, B. subtilis
Thermophilic – B. stearothermophilus
Psychrotrophic – B. coagulans, B. circulans, B. mycoides
e) Coryneform groups – Survive and form substantial portion of microflora of pasteurized milk
but do not grow at low temperature.
B. Inadequately cleaned utensils.
C. Accumulated in pasteurization plant due to improper cleaning methods.
D. Re-pasteurization of returned milk: because of growth of thermodurics on storage after first
pasteurization.
E. Use of pasteurized skim milk or cream for standardization.
F. Summer months due to poor cooling.
7.6.4 Effect of pasteurization on psychrotrophs in milk
• Pseudomonas, Flavobacterium, Acaligenes, Achromobacter are destroyed by pasteurization.
• Part of normal flora of milk and grow fast as temperature increases up to 25°C.
• Pasteurization destroys all psychrotrophs to the extent that survivors do not deteriorate flavor
deterioration even over extended storage. But post pasteurization contamination is a major
problem.
• Psychrotrophs can cause the problems in pasteurized milk like unclean flavor, putrid, fruity,
rancid, sour, ropy, greenish yellow discolorations.
7.6.5 Effect of pasteurization on thermophilic organisms in market milk
These are non-pathogenic and usually associated with high acidity and off-flavors. Facultative
thermophiles grow at 37°C and at 55°C however obligate thermophiles do not grow at 37°C
but grow at 55°C and even up to 70°C.
There are some factors that affect the thermophile count:
i) Raw milk – contains few thermophiles but these may increase on storage at higher
temperature and gain access via soil beddings, feeds etc.
ii) Pasteurizer – inside surface and holder tubes, pre heaters, filter cloths etc. HTST – no
problem of thermophiles because of too short residence time of milk (total 70-80 s), but filter
cloths used are problematic.
iii) Re-pasteurization and returned milk:
a) addition of returned milk
b) dripping from bottle fillers to raw milk contain very high thermophiles
c) skim milk or cream from returned milk for standardization.
iv) milk foam left in equipment is an excellent source
v) dead ends where hot milk is allowed to stand for any appreciable time.
7.6.6 Effect of pasteurization on coliforms
Coliforms are undesirable in pasteurized milk. Placed below there are some factors that effects
the counts:
a) Raw milk – improper sanitation of production i.e. fecal contamination through water,
exterior of animal and
improperly cleaned utensils.
b) Pasteurizing plants:
i. Improperly cleaned pipelines, pumps, fillers, bottles etc.
ii. Condensate drippings at various places
iii. Personnel unsanitary practices
iv. Defective or worn-out equipment having pits and pockets that favor accumulation of milk
solids.
v. Defects - gassiness, ropiness, unclean flavor, medicinal, bitter flavor
7.6.7 Effect of pasteurization on pathogens
Pathogen presence is due to improper pasteurization and, post processing contamination both.
No recorded cases of pathogens, if pasteurization is done as per IDF standards. Incidents
of Staphylococcus,Salmonella, Campylobacter, and Yersinia are reported but B. cereus was not
reported though it can survive pasteurization.
The possible sources of post-processing contamination:
(a) Human carriers
(b) Thermophiles in final regeneration are shed into pasteurized milk
(c) Holes in plate heat exchanger of regeneration section allow raw milk mixes with pasteurized
milk that can be avoided using high pressure on processed milk
7.6.8 Effect of pasteurization on keeping quality
Keeping quality of pasteurized milk is 5-7 days at refrigeration temperature. Spoilage depends
on type and number of micro-organisms and also on temperature of storage. Unclean and bitter
flavors are due to Gram-negative rods and bitter off-flavors are due to B. cereus at 106 cfu/ml.
Acid production, coagulation, protein destabilization are brought by acid producers like
Lactococcus lactis ssp lactis.
During Pasteurization, Psychrotrophs are destroyed but enzymes produced by these are
‘thermostable’. Proteases, lipases and phospholipase have less time to react at low temperature
storage because of keeping quality of 5-7 days.
7.7 UHT Processing of Milk
The purpose of UHT treatment of milk is to produce sterilized milk that is meant to:
• Keep without deterioration i.e. remain stable and of good commercial value for a sufficient
period
• Be free of microorganisms and toxins harmful to the health of consumers
• Be free of microorganisms liable to proliferate during storage
IDF recommended a temperature 135-150°C/1-4s for UHT treatment of milk.
7.7.1 Microbes in milk and their response to UHT
Class I: Microbes killed by conventional pasteurization temperature of 71-72°C for 15-30 s.
This eliminates most vegetative cells of bacteria like S. aureus, haemotylic streptococci, Gram-
negative enterococci (E. coli, Salmonella sp.), Pseudomonas, B. abortus, M. tuberculosis, all
yeasts and moulds.
Class II: Resistant to HTST, but sensitive to UHT i.e. 135-150°C/1-4s HTST is tolerated by
some thermoduric vegetative cocci like enterococci, some micrococci, microbacteria,
thermophilic bacilli (L. bulgaricus, Lactobacillus lactis), S. thermophilus, thermoduric aerobic
and anaerobic spores.
Class III: Obligate thermophilic soil bacterium B. stearothermophilus are known to withstand
UHT treatment of milk. Some spores of mesophilic bacilli and clostridia may survive, if milk
is heavily contaminated.
B. stearothermophilus is most resistant and constitute greatest hazard in spoilage of sterilized
milk product.
7.7.2 Spoilage of UHT milk
It is caused due to
(a) Enzymes those are heat resistant
(b) Post processing contamination either from packaging operation or improper cleaning of
system.
Spoilage is characterized by bitter off flavor, gelation and coagulation of milk proteins.
Psychrotrophs and aerobic spore formers do produce proteases and lipases at a storage
temperature of as low as 4°C. Some of these enzymes are most heat resistant. Enough enzymes
are produced at 5°C by as few as 103-104pseudomonas/ml to cause significant loss of native
milk proteins and bitter flavor. Most of UHT spoilage can be traced to packaging failures.
Aseptic filling of UHT milk is of utmost importance. Since contamination with one viable
bacterium is able to reproduce in milk will inevitably spoil the product during storage within
few days. Contamination risk with sterility of packaging material and contamination is mainly
by class I and II micro-organisms, is mainly due to
• During change of paper rolls Worn out gaskets in sterile portions of equipment that harbor
microbes
• Condensed water at the filling pipes
• Faulty sealing of packages.
If contamination by insufficient sterility of packaging material is due to faulty sealing or
corroded plates of plate heat exchanger, then the contamination micro-flora is most variable
and varies between package to package. If contamination is due to gaskets or condensed water,
then all contaminated packages contain uniform microflora. Microbes that will enter the
product more probably are those found in water stagnant on dairy floors –
Pseudomonas, Micrococcus, Enterococcus Bacillus and some yeasts. But these do modify milk
visibly (i.e. coagulation, proteolysis, staining and flocculation) or produce off flavors or a
measurable property (i.e. pH, acidity, redox potential) but UHT milk cannot attain cell
concentration of >105/ml so these must be detected by direct counts on culture medium.
7.7.3 Quality control of UHT milk
Satisfactory process shows spoilage level not higher than one per 1000 containers.
Packs test, 50 to 100 units’ daily/ line, if the line gives 4000 units/ hour. This amounts to 0.1 –
0.2% of the whole production.
• Pre incubation of packs for sufficient period is done. The temperature should be between 25-
30°C/7-9 days and incubated at 55°C for thermophilic spores. The appearance, pH, taste and
blowing of packages are recorded.
• The defective packages are tested by streaking on agar plate and incubated at 30°C/2 days or
55°C/4days. Streaking is done to know the nature of micro-organism by going morphological,
oxidase and catalase tests.
• Interpretation should be:
Thermophilic spores – insufficient heating of the product
Mesophilic spores - survived heat treatment or recontamination.
7.7.4 Methods to improve the quality of UHT milk
• Use raw milk of low SPC and psychrotrophs
• Milk collected from hygienic milk shed areas
• Immediately after collection, milk is subjected to pasteurization (or) thermization
• Test sample of milk in sealed ampoule in oil bath at 130 – 140°C
• Better use recombined or reconstituted milk with very low spore count
• LP system preservation of farm milk
• Bactofugation with 1000 l/h flow rate
• Nisin – active against spore formers (aerobic/anaerobic) but that is not approved by PFA.
7.8 Sterilization of Milk
Sterilization means exposing the milk to 118 - 120°C for 15 to 20 min. Sterilized milk should
contain neither bacteria nor bacterial spores.
7.8.1 Bacterial spores of raw milk
Raw milk contains a considerable number of bacterial spores although this number may be low
in comparison with total bacterial count.
Bacterial spores gain entry into milk mainly by contaminate with:
- Dust particles
- Soil and
- Manures
Thus, the density of spores in the milk is higher, when the animal is milked in stable than in
open air. More than half of spores in raw milk belong to the species of Bacillus licheniformis.
Other spores of considerable importance are B. pumillus, B. subtilis. But lesser number of
spores of B. cereus, B. circulance, B. megatherium, B. stearothermophiulus (thermophilic) and
Clostridium spp. are also encountered. Among the mesophilic spores present in raw milk those
of B. subtilis are most thermoresistant.
7.8.2 Survival of bacteria in sterilized milks
Because of deleterious effect of sterilization upon certain physic-chemical properties of milk,
there is often tendency to use the minimum amount of heat treatment, with the result
occasionally bacteria are developed in sterilized product. When B. subtilis is present, it gives
extremely ‘bitter taste’ and ‘proteolysis’ occurs.B. circulans presence in sterilized milk
produces ‘carbolic taint’. When sterilized milk is incubated at higher temperature, it is possible
that more species may be found. The presence of B. coagulans and of facultative
thermophilic bacilli can be demonstrated by tests at 37°C. Test at 55°C can demonstrate the
‘presence of bacteria of B. coagulans and groups of obligate and facultative thermophilic
bacilli. Presence of B. cereus and B. subtilis indicates the lower intensity of heat treatment. The
spores of B. cereus are less thermo-resistant than those of B. subtitis. Hence the presence of B.
cereus in greater in number than B. subtitis indicates insufficient degree of heat treatment. The
presence of B. circulans that produces carbolic taint is often associated with improperly washed
bottles. These bacteria grow even at rather low temperature quickly and abundantly form spores
in milk from returned bottles contaminate the bottle washing machine. When the temperature
and alkalinity of detergent solutions are not sufficiently efficient, large number of B. circulans
spores may survive the treatment and infect clean bottles.
The in-bottle-treatment fails to kill the spores; hence, more infected bottles are returned and
contaminate the bottle washing machine. Thus, a vicious circle is set up which is fatal to the
product unless measures are taken to improve the standards for bottle sterilization.
The presence of B. coagulans and thermophilic bacilli is usually associated with the filler. For
reasons of economy and quality milk is mostly bottled at higher temperature. These bacteria
develop in filler, forms spores and contaminate milk during bottling, often surviving later in
bottle treatment. Development of B. coagulans group can be easily controlled by using filling
temperature and their growth is retarded at 60°C and prevented at 65°C.
The development of thermophilic bacilli can be controlled only thorough frequent cleaning and
disinfection of filler. At a maximum bottling temperature of 80°C, thermophiles develop much
lesser than at frequently used bottling temperature of 65-70°C.
Anaerobic spore formers are practically never present unless milk has been strongly under
sterilized, as anaerobic spore forms are less resistant then aerobic spores.

Lesson 8
MASTITIS MILK – SUITABILITY FOR PROCESSING AND PUBLIC HEALTH
SIGNIFICANCE
8.1 Introduction
Milk obtained from animals suffering from the infected udder is termed as ‘mastitis’. IDF
defines mastitis as ‘an inflammation of udder, almost always of microbial origin. The mastitic
milk has higher microbial count and somatic cell count and has altered composition
accompanied by reduced yield.
Mastitis is a parenchymal inflammation of the mammary gland that is caused by microbes that
invade the udder, multiply and produce toxins, which are harmful to the mammary gland. It is
characterized by physical, chemical and usually bacteriological changes in milk and
pathological changes in glandular tissues of the host animal. Mastitis is one of the most
important deadly diseases of milch animals, responsible for heavy economic losses due to
reduced milk yield, milk discard after treatment (9%) cost of veterinary services (7%) and
premature culling. Mastitis is a global problem that adversely affects animal health, quantity
and economics of milk production and huge financial losses. Unlike clinical mastitis, in sub
clinical mastitis there are neither visual abnormalities in milk like blood clots, flecks etc. nor
in mammary gland like swelling, hotness etc.
8.2 Different Forms of Mastitis
Mastitis can be classified based either on symptoms or on causative micro-organisms.
8.2.1 Classification based on symptoms
Swollen, hot, red and painful udders.
• Acute or clinical: Macroscopic changes to udder or milk, readily detectable by milker.
• Chronic: Little compositional changes with almost complete absence of pain in udder.
• Sub-acute/ sub-clinical: Most common form, udder and milk appear normal. Diagnosed by
detecting pathogens and somatic cells and change in milk composition
8.3 Causative Microorganisms
Mastitis is caused as a result of udder infection with one or more of the causative micro-
organisms. These microbes enter through the teat tip into the teat duct, where these get
colonized due to the presence of left over milk and subsequently, spread throughout the udder
causing infection.
8.3.1 Microorganisms associated with mastitis
• Most common causatives are Staphylococcus aureus and Streptococcus agalactiae
Coliforms : Escherichia coli , Enterobacter, Klebsiella, Citrobacter.
• Other Streptococci: Streptococcus uberis, Streptococcus dysgalactiae, Streptococcus
faecalisand Streptococcus pyogenes.
• Other Staphylococci : Staphylococcus epidermidis and Staphylococcus albus.
• Corynebacteria : Corynebacterium bovis, Corynebacterium pyogenes.
• Ricketsia: Coxiella burnetii.
• Yeast: Cryptococcus neoformans, Candida pseudotropicalis.
• Molds: Asperigillus spp.
8.3.2 Classification based on causative microorganism
• Contagious mastitis: Streptococcus agalactiae (as natural inhabitant of udder)
• Common mastitis: Species of Streptococcus, Staphylococcus and Escherichia coli
• Summer mastitis: Corynebacterium pyogenes
• Environmental mastitis: Streptococcus uberis
8.4 Compositional Changes in Mastitis Milk
The colonization of mammary glands by mastitis causing microorganisms trigger a series of
events that in turn causes major compositional alterations.
• Initial increased level of pathogenic bacteria occurs, which is closely followed by
considerable increase in somatic cell count.
• Subsequently, there is a wide range of related effects like impaired synthetic ability of the
secretary tissue causing lower milk yield and altered levels of major and minor milk
constituents and increased infiltration of blood constituents i.e. serum proteins into milk.
Overall milk from the infected quarters in different cases of mastitis may have the following
altered constituents:
8.4.1 Increased constituents
Total whey proteins (i.e. bovine serum albumin, immunoglobulins), sodium, chloride and other
ions like Cu, Fe, Zn, various enzymes and certain glycoproteins increased significantly in
mastitic milk. The pH of milk also increases.
8.4.2 Decreased constituents
Lactose, fat, total casein (i.e. alpha and beta fractions) decrease but gamma fraction increase,
some whey proteins (i.e. alpha-lactalbumin and beta globulin), potassium and other minerals
like calcium, magnesium and phosphorus decreased. On the whole mastitic milk in general has
a lower SNF, fat, casein and lactose and higher serum proteins, chloride ions and pH
8.5 Significance of Mastitis Milk
Mastitis in lactating animals affects the yield, quality and public health aspects of milk. With
severe clinicalmastitis, abnormalities of milk are easily observed and milk is discarded. Such
milk normally would not enter the milk chain. But when milk of cows with sub-clinical
mastitis, i.e. with no visible changes, is accidentally mixed into bulk milk, it enters food chain
and can be dangerous to consumer. Although, pasteurization destroys all human pathogens,
there is concern, when raw milk is consumed or when pasteurization is incomplete or faulty.
Milk and other dairy products are frequently infected with S. [Link] of infected animals
is the main source of enterotoxigenic S. aureus of animal origin. For example, certain S. aureus
strains produce heat-resistant enterotoxins that cause nausea, vomiting and abdominal cramps,
when ingested by humans and are responsible for staphylococcal food poisoning. Toxins are
produced due to improper cooling of milk, during cheese making from raw milk and also due
to post-processing contamination. These toxins cannot be destroyed by heating. The bovine
mammary gland can be a significant reservoir of enterotoxigenic strains of S. aureus. S.
agalactiae is an important bovine pathogen, especially as a cause of both clinical and sub-
clinicalmastitis in dairy animals. Mastitis constitutes a source of economic loss for the dairy
industry due to its effects on milk quality. It not only lowers the quality of cheese and other
milk products and decreases milk yield. It also reduces nutritive value of milk due
to the changes in its composition, increases processing problems and off-flavours. It also
decreases the shelf life of fluid milk products, due to the growth of spoilage causing bacteria.
Moreover, the impact of mastitis involves the additional cost of therapeutic strategies and
veterinary services. S. agalactiae is considered a major cause of increased somatic cell
counts as related to standards in bulk tank milk. SCC increases in milk because of mastitis,
hence, milk quality, and lactose and casein contents decreases. Milk yield of a cow with an
infected quarter may to the extent of 40%, while animal does not show any apparent clinical
signs of mastitis. A reduction in milk quality ultimately leads to loss of income to the dairy
farmers.
Another public health concern regarding mastitis is antibiotic residues in milk due to extensive
use of antibiotics in the treatment and control of disease. Antibiotic residues in foods can lead
to severe reactions in people allergic to antibiotics at low levels, cause sensitization of normal
individuals and development of antibiotic-resistant strains of bacteria.
Compliance with recommended withholding time helps minimizing the risk of antibiotic
residues to occur in milk and meat which is the producers’ responsibility.
8.5.1 Milk yield
The milk yield and also the productive life of milch animal are adversely affected due to
mastitis.
8.5.2 Milk quality
The quality of milk as said above is lowered due to a number of compositional changes.
8.5.3 Suitability for fermented dairy products
Mastitic milk is not suitable for making fermented milk products. In cheese making the
following effects are noticed in product on while using mastitic milk.
[Link] Lower product yield
This might be due to increased fat losses in whey and reduced starter activity.
[Link] Poorer product quality
Rennet clotting time is increased causing decreased curd firmness and a loose final texture of
cheese. Also results in lack of adequate flavor development due to retarded starter activity.
8.5.4 Other fermented products
Mastitic milk is not suitable for preparing fermented milks like dahi, yoghurt, cultured butter
milk, kefir etc. where product quality is directly dependent on starter cultures. The starters are
affected in terms of their growth and activity (acid and flavor production) in sub clinical
mastitic milk. For example, both lactobacilli and lactic streptococci are inhibited in milk from
Streptococcal and Staphylococcal cases of clinical mastitis. Both the rate of acid production
and final acidity attained in mastitic milk are lower than that in normal milk. Even at less than
5% level of mastitic milk in the pooled milk, the aroma, taste and consistency of end product
are adversely affected. Diacetyl production by Lactococcus lactis ssp. Lactis
biovardiacetylactis is lower in mastitis milk.
8.5.5 Public health significance
Some of the mastitis causing microbes which are excreted in milk is also pathogenic to humans.
For example, certain strains of Staphylococci associated with mastitic milk cause food
poisoning due to enterotoxin production. The storage of mastitic milk under favorable
temperatures leads to elaboration of enterotoxins that are not inactivated even during
pasteurization and spray drying of milk. Other pathogens and food poisoning causing
bacteria like Salmonella spp and enteropathogenic strains of certain coliforms(E. coli and
Klebsiella spp) might also be associated with public health hazards through mastitic milk.
Lesson 9
FOOD INFECTION, INTOXICATION AND TOXI INFECTION
9.1 Introduction
A variety of pathogens may gain access in to milk from different sources and cause different
types of milk borne illness. Milk and its products may carry microbes or their ‘toxins’ to the
consumers. Based on the type of illness produced these are categorized as food infection,
food intoxication and food toxi-infection.
9.2 Types of Food Borne Illness
9.2.1 Food intoxication
Ingestion of pre-formed toxins, already synthesized by microbes in food brings about
poisoning syndrome in the consumers and hence, is called food intoxication. Toxins affecting
gastro intestinal tract are called as enterotoxins
Examples includes: Staphylococcal food poisoning, botulism, diarrohea caused by
Escherichia coli, Cholera mycotoxicosis etc.
9.2.2 Food infection
Ingestion of viable pathogens along with food leads to their establishment in consumers and
hence is termed as food infection.
Examples includes: Typhoid, shigellosis, septic sore throat, scarlet fever etc.
9.2.3 Food toxi-infection
Some micro-organisms can produce toxins ‘in situ’ after getting ingested with food and infect
intestine. Such type of illness is referred as food toxi-infection.
Examples includes: Bacillus cereus food poisoning and Clostridium perfringens gangrene
9.3 Milk Borne Infections
9.3.1 Salmonella poisoning
Salmonellosis is a disease resulting from the ingestion of salmonella along with contaminated
food. This is mainly a food-borne illness and milk and milk products appear to be commonly
involved. There are about 2000 closely related biotypes of salmonella that can cause food
poisoning in humans. It is during the mishandling of milk that the causative microorganism
may enter these foods and their subsequent growth during holding may lead to salmonellosis.
[Link] Causative microorganism
• Salmonella typhi– Typhoid
• Salmonella paratyphi A, B, C – Paratyphoid
• Salmonella enteritidis – Food poisoning
[Link] Sources
• Water is one of the most important sources of salmonella, as it frequently gets contaminated
through faecal matter. If polluted water is used for washing of utensils and equipment it can
also lead to contamination of milk. Silage is another source of salmonella.
• Handlers are the carriers in active cases.
• External agents like flies also acts as a source of contamination.
• Animals suffering from salmonellosis, under certain conditions, excrete viable micro-
organisms in their milk.
[Link] Symptoms
a) Typhoid fever: Characterized by continuous fever, inflammation of intestines, formation
of intestinal ulcers, enlargement of spleen, characteristic raised spot eruptions on the
abdomen and toxemia.
b) Paratyphoid fever: Resembling typhoid fever but it is milder than typhoid.
Approximately 105 – 107microorganisms per ml are required to cause infection.
c) Salmonellosis: Nausea, vomiting, abdominal pain, diarrhoea, chills, headache, prostration,
muscular weakness, drowsiness, moderate fever, restlessness etc.
[Link] Incubation period
7 – 14 days for typhoid and 1 – 7 days for paratyphoid
[Link] Diagnosis
Microorganism may be present in feces; urine and blood are identified by microscopic,
cultural, fermentative and serological tests. Widal test is used for typhoid.
[Link] Prevention and control
• Adequate treatment of water
• Infected individuals, who had infection should not handle milk
• Follow hygienic conditions during production, processing and storage
• Educating the people about food and personal hygiene
• Periodic examination of individuals who handle foods for public consumption
• Pasteurization and other adequate heat treatment of milk and milk products
• Control of flies
• Treatment of affected individuals
9.3.2 Bacillary dysentery (Shigellosis)
This is one of the common food-borne infection and outbreaks are associated with
consumption of unpasteurized milk.
[Link] Causative micro-organism
• Shigella dysenteriae
• Shigella sonnei
• Shigella flexneri
[Link] Source
• Milk contaminated with water, flies and utensils
• Milk handlers
[Link] Symptoms
Shigellosis is an acute intestinal disease characterized by diarrhoea with blood, pus or
mucous, fever, vomiting, abdominal cramps and tenesmus.
[Link] Diagnosis
Isolation of microorganism by taking rectal swabs and plating on desoxycholate citrate agar,
incubation period is 1 – 4 days.
[Link] Prevention and control
• Strict sanitary condition should be enforced in dairy workers particularly in pasteurization
plants and retail outlets dispensing milk in bulk
• Attendants looking after the patients should be prohibited from contact with milk or utensils
• Take precautions to exclude flies
9.3.3 Streptococcal infection
Streptococcal infections like septic sore throat, scarlet fever and food poisoning are treated to
the consumption of milk and its products. Humans and animals both can contaminate milk
with streptococci. Although streptococcal food poisoning through milk and milk products is
not well established, yet a few strains of Group D Streptococci or enterococci have produced
toxic metabolites in milk and their toxigenic potentials is ascertained in animal models.
[Link] Causative microorganism
Streptococcus pyogenes – Scarlet fever, septic sore throat, tonsillitis and septicaemia
Streptococcus agalactiae – Mastitis in animals
[Link] Sources
• Animals infected with Streptococcus agalactiae may harbor microorganisms in udder and
acts as a source of pathogens
• Personnel directly connected with the care and milking of animals in a dairy herd may act as
vectors of mastitis and cause mastitis in other animals as these carry the pathogens on skin of
hands etc
• Enterococci are chiefly of fecal origin and fecal contamination of milk and milk products
may introduce lot of enterococci in these products
• Milking machines may help in the transfer of pathogens to milk and also to other animals
with the help of infected teat cups
• Human carriers of Streptococcus pyogenes contaminate milk during handling and
processing
• Human carriers disseminating pathogenic Streptococcus pyogenes may also infect animals
that develop clinical or sub-clinical udder infections, and in turn pass out these
microorganism in to milk
• Milk products may get contaminated from raw milk infected with streptococci
[Link] Symptoms
a) Septic sore throat: Characterized by high and irregular fever, sudden onset of fever,
inflammation and swelling of lymph glands of throat and sometimes involving the formation
of abscess around tonsils and in cervical lymph glands.
b) Scarlet fever: It is an acute febrile disease of throat, accompanied by a scarlet rash.
Invasion of other parts of body may occur by microorganisms resulting in infection of middle
ear, kidney. Scarlet rash is due to elaboration of a toxin.
c) Food poisoning: The symptoms resembles staphylococcal food poisoning, however, these
are of mild type. Incubation period is 1 – 3 days.
d) Diagnosis: For septic sore throat, throat swabs should be taken and observed for the
presence of hemolytic streptococci of lancefield group A. Dick test is used to detect scarlet
fever. Food poisoning can be diagnosed by the isolation of causative microorganisms their
enterotoxin typing and toxigenicity.
[Link] Prevention and control
• Adequate heat treatment of milk
• Holding milk at lower temperature
• Rejection of milk from the suspected quarters
• Milk showing abnormal changes should not be pooled
• Regular checking of the health of dairy workers
• Faucal contamination of milk should be avoided
9.4 Milk Borne Intoxications
9.4.1 Staphylococcal poisoning
Infection of milk by some strains of Staphylococcus aureus leads to the elaboration of
enterotoxins by these that cause gastro-enteritis in humans.
[Link] Toxins
Staphylococci produce different metabolic end products such as:
• Haemolysin / Staphylolysin (α, β, γ and δ)
• Leukocidin
• Necrotizing factor
• Lethal factor
• Enterotoxin
• Coagulase
Among different toxins, enterotoxin is responsible for major food poisoning out breaks.
Staphylococcus aureus is capable of growing and producing enterotoxins in raw milk at a
concentration of 106microorganisms per ml.
However, not all the strains of microorganisms are capable of producing enterotoxins. The
toxin production is faster in milk with low count. In poor quality milk, competition with other
microorganisms and changes brought about by these e.g. acidity, depletion of nutrients inhibit
toxin production
[Link] Sources
Human handlers are the main source as the microorganisms and present in nose, skin,
wounds, pimples and boils. Animals affected with mastitis are the other sources.
[Link] Symptoms
Nausea, vomiting, abdominal cramps, diarrhoea, sweating, headache, prostration etc.
Mortality is low, incubation period is 1 – 16 h., and symptoms last for 24 – 48 h.
[Link] Diagnosis
Biological methods, serological methods, coagulase test and thermo nuclease test.
[Link] Prevention and control
• Adequate heating destroys staphylococci but not the enterotoxin. So, heating within few
hours of production would inhibit the multiplication of staphylococci and hence, no toxin
production
• Adequate cooling immediately after producing milk also inhibits multiplication of
staphylococci.
• Post pasteurization contamination should be avoided
• Infected handlers should not be allowed to handle milk
• Animals having mastitis should be isolated
[Link] Diagnosis
Microscopic examination, animal inoculation and cultural identification.
[Link] Prevention and control
• Adequate heating of product destroys toxin (1000C per 10 – 20 min.)
• Hygienic conditions is adopted during production of milk
• Chilling after production should be essential
• Acidity inhibits the causative microorganism
• Reject bulged cans and spoiled foods
9.4.3 E. coli poisoning
E. coli is known to be associated with enteritis in infants and adults as well as with traveler’s
diarrhoea and food poisoning. Milk and milk products like ice cream, kulfi, dried milks,
cheese etc can be contaminated with toxigenic E. coli that can produce the enterotoxins under
favorable conditions. A minimum of 105 to 107 cells per gram are required to produce
enterotoxins sufficient to cause food poisoning.
[Link] Toxins
E. coli can produce two types of enterotoxins named as heat labile and heat stable toxins. The
labile toxin can be inactivated at 65˚C per30min.
[Link] Sources
• Water supplies contaminated with fecal matter
• Unhygienic practices by handlers may introduce the microorganism in to milk chain
• Infected animal may also excrete the microorganisms.
[Link] Symptoms
Children are more susceptible. Symptoms due to heat labile toxins are almost similar to
symptoms of cholera. Massive watery diarrhoea occurs due to accumulation of fluid in the
intestines. In case of heat stable toxin, the symptoms include diarrhoea with or without vomit
that is not bloody and without inflammatory exudates in stool.
Fever may be present in children but not in adults. Incubation period is 8 – 24 h.
[Link] Prevention and control
• Fecal contamination of water supply should be avoided
• Handlers should follow strict hygiene practices
• Cross contamination from infected animals should be avoided
9.4.4 Cholera
Cholera is one of the acute diarrheal diseases caused by Vibreo cholerae. It frequently occurs
in the form of massive epidemics. Unhygienic practices are mainly responsible for outbreak.
Although, cholera is mainly water borne disease, the involvement of milk may also transmit
the disease. The causative microorganism adheres to the epithelial lining of mucosa in the
small intestines, where it produces enterotoxin that causes loss of fluid and electrolytes from
the body followed by dehydration.
[Link] Source
• Milk infected by soiled hands of a patient
• Use of infected water for dairy purpose
• Adulteration of milk with contaminated water.
[Link] Symptoms
In mild cases of cholera, diarrhoea appears to be the only symptom. In severe cases,
diarrhoea, vomiting, rice water stools, abdominal pain, thirst, dehydration are major
symptoms. The disease runs a short course terminating in death, sometimes within 12 h after
the appearance of first symptoms. Incubation period is few hours to 5 days, usually 3 days.
[Link] Diagnosis
Intra-peritoneal inoculation of Guinea pigs with pure culture results in the death of the animal
within 24 h.
[Link] Prevention and control
• Proper pasteurization of milk and its products.
• Sanitary disposal of human excreta
• Protection of water by boiling
• Sanitary preparation and handling of products
• Control or destruction of house flies
• Public health education
• Isolation of patients and carriers
• Disinfection of stools and vomit and articles soiled by the patients
• Food left by the patients should be burnt
• Room of patient should be thoroughly cleaned and disinfected
• Use of cholera vaccine in exposed population group
9.4.5 Fungal intoxications
The common types of fungal intoxications that result through milk are those caused by
Asperigillus and other molds like Pencillium, Fusarium etc.
[Link] Aflatoxicosis
Aflatoxicosis is a common type of fungal intoxication caused by the common molds,
Aspergillus flavusand Aspergillus parasiticus by virtue of their ability to produce aflatoxin.
Aflatoxin can be present in milk as preformed toxin or it can be secreted in milk by the milch
animal that has been fed with aflatoxin containing feed. Therefore, even with satisfactory
sanitation and hygiene during production and processing of milk, the mycotoxins can be
transferred to consumer. Upon ingestion, the aspergillus toxins are metabolized by milch
animals and are secreted into milk in form of M1 and M2 that are also toxic to the consumers.
a) Types of aflatoxins: Aspergillus flavus produces B1, B2, B2a. B1 is the most abundant
and most toxic of the aflatoxin. G1, G2 and G2a toxins (produced by Aspergillus flavus only)
are extremely heat stable, potent and exhibit very strong toxicity apart from being highly
carcinogenic. The recommended upper permissible limit of aflatoxin in foods is 0.5μg/kg.
b) Source: Aerial contamination and soil contaminated foods introduce mold spores in milk
and milk products.
c) Symptoms: Liver hyperplasia, tissue hemorrhage, anorexia, hepatitis and finally death in
animals. The vital organs like spleen, pancreas and kidney may also be involved in
aflatoxicosis.
d) Prevention and control:
• Prevent contamination of milk and milk products
• Prevent fungal growth by storing the product under proper conditions and by use of fungi-
static agents
• Detoxification of aflatoxins by physical, chemical or biological agents. However, this is
relatively impractical in dairy products.
9.5 Milk borne Toxi – Infections
9.5.1 Bacillus cereus poisoning
It occurs due to ingestion of contaminated food with Bacillus cereus or its spores and
produces 3 types of toxins:
a) Haemolysin
b) Lecithinase
c) Enterotoxin
[Link] Sources
• From mastitis udder
• Spores from animal’s teat and skin equipments
• Soil – directly or indirectly
[Link] Symptoms
Two types
• Diarrohea: Abdominal pain and cramps, profuse watery diarrhea, rectal tenesmus. Moderate
nausea with rare vomiting. Lecithinase releases phosphoryl choline, a toxic substance from
lecithin
• Vomiting: Acute attack of nausea and vomiting diarrhea is not common
[Link] Prevention and control
• Proper cooling of milk
• Maintenance of environmental hygiene and air quality
• Hygiene during production, processing etc.
9.5.2 Clostridium perfringens poisoning
Clostridium perfringens- It causes gas gangrene. The anaerobic conditions inside canned
foods are favourable for growth of C. perfringens. Five different enterotoxins - A, B, C, D
and E are produced, where A and C causes food poisoning.
[Link] Sources
• From soil and faeces – dust, fodder, milk
• Faecal contamination of water
[Link] Symptoms
Diarrhoea, vomiting, abdominal pain; incubation period is 8-22 h. Sometimes it is fatal.
[Link] Prevention and control
• Proper cooling of milk
• Maintenance of environmental hygiene and air quality
• Hygiene during production, processing etc.
Lesson 10
MILK BORNE DISEASES
10.1 Introduction
The spoilage and pathogenic micro-organisms causing illnesses could come from the animal,
handler, environment, water, equipment, air, and raw materials and due to poor sanitation
practices. The knowledge about sources of contamination will be helpful in preventing or
reducing such spoilage causing and pathogenic microbes in milk.
Milk is, an efficient carrier for a variety of disease producing microbial agents. With mass
collection and distribution of milk in industrial countries, the potential of milk for disease
transmission became a widespread problem. The disease control, however, can be maintained
only by constant supervision of the health of dairy animal and by adequate controls
at all points from the time the milk leaves the udder until it reaches the consumer. While the
problems of ensuring a safe milk supply are of different orders of magnitude in economically
advanced and in developing countries, yet there are essential similarities. In both cases, where
a highly mechanized system with extensive distribution services from a centralized milk plant
is employed, the slightest relaxation of attention at any crucial links in the milk chain from
the farm to consumer invites problems.
A second factor that is common to advanced and developing countries, is the disease causing
microbes.
Such microbial agents can be conveniently classified as :
a) Communicable disease causing microbes - viruses, rickettsiae, bacteria, protozoa, and
other parasites-and/ or their toxins;
b) Specific and non - specific sensitizing agents; and
c) Toxic chemicals - pesticides, preservatives, drugs, radionuclides, and other substances.
10.2 Milk as Vehicle of Microbes
Milk, by virtue of possessing all sorts of nutritional factors, can serve as an excellent media
for microbes, especially including pathogens. Bacteria have the ability to utilize various milk
constituents to grow and multiply. While growing at the expenses of milk constituents these
microbes release certain metabolites like lactic and other organic acids, gases, enzymes,
flavouring compounds, pigments, toxins etc in the system which may be useful and/or
harmful, and thus, effects the quality of milk. Generally, these metabolites lead to different
spoilage conditions in milk products and make these unfit for consumption. For this reason,
raw milk is inherently dangerous and should not be consumed by anyone, at any time, for any
reason. However, this has been a practice in past in northern parts of India, might be due to
the unawareness of consumers.
Food safety experts say that pasteurization saves lives. Pasteurization has reduced food-borne
illnesses from milk to one-fourth in comparison to before the technique was widely adopted
in early 1900s to about 1 percent now. Although all are vulnerable to illness from E. coli or
Salmonella poisoning, children and people with weak immune systems are at higher risk from
drinking raw milk. The number of infections is growing, as raw milk is gaining popularity
and people are taking a risk.
Milk serves as a potential vehicle for transmission of diseases under certain circumstances.
Pathogens grow and multiply to produce certain toxic metabolites and make itself an
extremely vulnerable commodity from public health point of view. The microbiological
health hazards arising from the consumption of contaminated milk has grown in recent past
and has resulted in intensification of food hygiene programme world over. Although, the
occurrence of incidences of food borne illness has been considerably reduced in developed
countries, mainly due to adoption of strict microbiological quality control and sanitary
practices during production, processing and distribution of milk yet, the situation is continues
to be grim in developing countries like Indian subcontinent where, such practices are
impracticable even at present. Milk spoilage is manifested by a reduction in aroma, flavor,
texture and nutritional value of foods. In extreme cases the dairy products become totally
unpalatable. In addition, some microbes are known to release toxins that may cause damage
to health of consumers.
10.3 Different Sources of Pathogens
A variety of pathogens may gain access to milk from a number of sources and cause different
types of food borne illness. Milk and its products may carry microbes as such or their toxic
metabolites called toxins to the consumers.
10.3.1 Animals
The health of dairy animals is a very important parameter because a number of diseases
including brucellosis, Qfever, salmonellosis, staphylococcal and streptococcal infections and
foot and mouth disease virus may be transmitted to man through milk. The microbes causing
these diseases may be transmitted to milk either directly from the udder or indirectly through
the infected body discharges that may drop, splash or be blown in to milk.
10.3.2 Handlers
The diseased persons may transmit diseases like typhoid fever, scarlet fever, diphtheria,
septic sore throat, infantile diarrhea by contaminated hands or by coughing, sneezing and
talking during milking or subsequent handling of milk at farm level.
10.3.3 Environment
Dairy farm environment may also introduce pathogens in to milk products at different stages
of production and processing. Some common air borne pathogens are like Group A
Streptococci, Corynebacterium diptheriae, Mycobacterium tuberculosis, Coxiella burnetii
and some viruses of respiratory origin. Contaminated water, fodder and unclean vessels and
containers used for handling milk and other unhygienic conditions at farm and plant may
significantly contribute to pathogens and spoilage causing micro-organisms in milk.
10.4 Different Terms Used in Milk Borne Infections
10.4.1 Outbreak
According to Communicable Disease Centre, an outbreak of food-borne disease is defined as
an incident in which two or more persons experience a similar illness usually gastro-intestinal
after ingestion of a common food.
10.4.2 Etiology
Etiology of a food-borne disease is the confirmation or identification of the causative agent of
the disease through laboratory diagnosis.
10.4.3 Epidemiology
Epidemiology of a food-borne disease is a systematic approach to locate the cause and mode
of transmission of the disease, so that corrective measures can be applied.
10.5 Investigation of Milk Borne Diseases
There exists a systematic monitoring and surveillance system to investigate the causes of
food-borne illness in developed countries but, there is lack of adequate investigating system
in developing countries and as a result, no follow-up action is taken to avoid reoccurrence.
Generally gastro intestinal disorders are perhaps the greatest single cause of morbidity in
developing countries.
For investigation of an outbreak the following steps are to be followed:
• A detailed description of gastro-intestinal cases should be made.
• A record of food eaten and a common source of infection should be identified, if large
number of individuals is involved.
• History of previous illness of personnel handling milk should be traced.
• Evidence of enteric disorders, scratches, wounds, sores, pyogenic infections or other
evidence of sepsis should be looked for and swabs should be taken.
• Sanitary facilities and practices used in plant should be recorded.
• A detailed veterinary record of animals should be obtained with particular attention to
recent cases of mastitis.
• Pooled milk samples from one or several animals should be taken aseptically, immediately
cooled and held cool until delivered for examination.
After identification of the suspected animals carrying the causative microorganism, the
individual samples should be obtained.
10.6 Diseases From Animal to Man And Milk to Man
10.6.1 Tuberculosis
The causative microorganism is Mycobacterium tuberculosis. German physician Robert Koch
(1843-1910) revealed the micro-organism, Mycobacterium tuberculosis from contaminated
raw milk, and its role in infant mortality.
Koch also reported that another strain, M. bovis, was responsible for tuberculosis in cows,
and that it was species specific and believed that cow strain would not infect humans. Milk
borne tuberculosis is directly or indirectly related to consumption of raw milk from infected
dairy herds. The tuberculosis traceable to raw milk was the result of external contamination
or lesions in the udders of cows racked with bovine tuberculosis. The milk buckets, too, were
easily contaminated by workers. There are two types of tuberculosis, pulmonary and non
pulmonary type. Pulmonary is caused by human type of microorganisms that affects mainly
respiratory tract. Bovine type bacillus cause non pulmonary tuberculosis.
Tuberculosis of cattle is produced by Mycobacterium bovis. Avian type of the microorganism
may cause both types of tuberculosis.
• Mycobacterium tuberculosis – Human
• Mycobacterium bovis – Cattle and human
• Mycobacterium avium – Birds, swine but rarely human
Human type tuberculosis bacilli may gain access to milk from milkers and other handlers. It
causes human type tuberculosis in cattle. This cannot be immediately noticed and may give
tuberculin negative test but after 2 – 3 months, this test will be positive. Such suspected
animals are usually held under observation and rested periodically. If the reaction disappears,
these are restored to their normal status in herd. Such cattle may excrete bacilli in their milk
from apparently normal udders.
Milch animals other than cattle are also affected with tuberculosis mainly by bovine type.
Buffaloes and goats are less frequently affected by tuberculosis. Bovine type infection in man
appears to be practically nonexistent, in spite of a considerable proportion of cows being
infected. It may mainly be attributed to the habit of boiling milk before consumption. Sour
milk may kill human and bovine tuberculosis bacilli within 18 – 24 h Avian type tuberculosis
bacilli also cause natural infections in cattle. Human infection with avian type bacilli is quite
rare.
[Link] Symptoms
Tuberculosis is characterized by the onset of paranchymal pulmonary infiltration
recognizable by X-ray examination, pleurisy, followed by advanced stage that is
accompanied by cough, fever, and fatigue and weight loss. Incubation period is 4 – 6 weeks
from infection to demonstrable primary lesion.
[Link] Prevention and control
• Animals should be subjected to tuberculin test.
• Animal suffering with tuberculosis should be isolated.
• Proper heat treatment of milk. The traditional habit of boiling every lot of milk before
consumption in India is good, in combating the incidence of tuberculosis.
• Overcrowding of animals must be avoided and living conditions must be improved
• Tuberculosis patients should be prohibited from handling cattle as well as milk.
• Proper disinfection should be followed.
10.6.2 Brucellosis
It is one of the most common milk-borne diseases. Brucellosis, also called Bang's disease,
Crimean fever, Gibraltar fever, Malta fever, Maltese fever, Mediterranean fever, rock
fever, or undulant fever, is a highly contagious zoonosis caused by the ingestion of
unsterilized milk or meat from infected animals or close contact with their secretions.
Brucella spp. are small, Gram-negative, non-motile, non-spore-forming, rod shaped
(coccobacilli) bacteria. This function as facultative intracellular parasites causing chronic
disease that usually persists for life. Symptoms include profuse sweating and joint and muscle
[Link] fever, the disease now called brucellosis first came to the attention in 1850s in
Malta during the Crimean War. In cattle, this disease is also known as contagious abortion
and infectious abortion.
The popular name undulant fever originates from the characteristic undulance (or ‘wave-like’
nature) of fever, that rises and falls over weeks in untreated patients. In 20th century, this
name, along withbrucellosis, gradually replaced the 19th century names Mediterranean
fever and Malta [Link] in humans is usually associated with the consumption of
unpasteurized milk and soft cheeses made from milk of infected animals, primarily with
Brucella melitensis and with occupational exposure of workers, veterinarians and slaughter
house workers. Some vaccines used in livestock, B. abortus, also cause disease in humans, if
accidentally injected. Brucellosis induces fevers, sweating, weakness, anaemia, headaches,
depression and muscular and body pain. The main symptoms are like muscular pain and
sweating and the duration of disease can vary from a few weeks to months or years. In the
first stage of disease, septicaemia occurs and leads to undulant fevers, sweating and
migratory arthralgia. Blood tests characteristically reveal leukopenia and anemia, and
demonstrate positive Bengal Rose and Huddleston reactions. During episodes of Malta fever,
melitococcemia (presence of brucellae in blood) can usually be demonstrated by means of
blood culture in tryptose medium. If untreated, the disease can become chronic. The
focalizations of brucellosis occur usually in bones and joints and spondylodiscitis of
lumbar spine accompanied by sacroiliitis is very characteristic of this disease.
[Link] Diagnosis of brucellosis
• Blood cultures in tryptose broth, bone marrow cultures. The growth of brucellae is
extremely slow and the culture poses a risk to lab personnel due to high infectivity of
brucellae.
• Demonstration of antibodies against the agent either with, ELISA or 2-mercaptoethanol
assay for IgM antibodies associated with chronic disease
• Histologic evidence of granulomatous hepatitis
• The preferential erosion of antero-superior corner of lumbar vertebrae and marked
osteophytosis are suspicious of brucellic spondylitis
[Link] Prevention and control
• Segregation of infected herd to avoid cross infection and if possible the infected animals
should be slaughtered.
• Herds should be properly vaccinated.
• Adequate heat treatment should be given to milk for the destruction of causative microbial
agents.
10.6.3 Diphtheria
Diphtheria is caused by only toxigenic strains of Corynebacterium diphtheriae. Rarely, a
diphtheria-like illness is caused by a toxigenic strain of C. ulcerans or C. pseudotuberculosis.
C. diphtheriae has three biotypes: gravis, intermedius, and mitis. The gravis biotype is
associated with the most severe disease, but any strain may be toxigenic. All clinical isolates
of C. diphtheriae should be tested for toxigenicity. Nontoxigenic strains can cause sore throat
and other invasive infections, and are associated with endocarditis.
[Link] Illness
Classic diphtheria is an upper-respiratory tract infections characterized by sore throat, low-
grade fever, and an adherent pseudomembrane of the tonsil(s), pharynx, and/or nose. The
disease can involve almost any mucous membrane. For clinical purposes, diphtheria can be
classified according to the site of the infection:
[Link] Anterior nasal diphtheria
Anterior nasal diphtheria usually presents with mucopurulent discharge from nose that may
be bloody and a white pseudomembrane on nasal septum.
[Link] Pharyngeal and tonsillar diphtheria
Pharyngeal and tonsillar diphtheria, the most common type of infection, initially presents
with malaise, sore throat, anorexia, and low-grade fever. Within a few days, a bluish-white
pseudomembrane forms on one or both tonsils that can extend to the tonsillar pillars, uvula,
soft palate, pharynx and nasopharynx. Over time, the pseudomembrane evolves, assuming a
dirty gray color with areas of green or black necrosis surrounded by a minimal amount of
erythema. Attempts to remove the pseudomembrane cause bleeding. With severe disease
patients can develop edema of the anterior neck.
If a significant amount of toxin is absorbed into the blood stream, patients may develop
pallor, rapid pulse, coma and death. The differential diagnosis of diphtheria includes
streptococcal pharyngitis, viral pharyngitis, Vincent's angina, infectious mononucleosis, oral
syphilis and candidiasis.
[Link] Laryngeal diphtheria
If the infection involves larynx, it may occur either as an extension of pharyngeal form, or as
laryngeal involvement alone. Patients can present with fever, hoarseness and a barking
cough. The pseudomembrane can cause potentially fatal airway obstruction.
[Link] Cutaneous diphtheria
Cutaneous diphtheria, caused by either toxigenic or nontoxigenic strains of C. diphtheriae, is
usually mild, typically consisting of non-distinctive sores or shallow ulcers, and rarely causes
toxic complications. The disease may present as a scaling rash or as clearly demarcated
ulcers. A chronic skin lesion may harbor C. diphtheria along with other microorganisms.
Skin infections with C. diphtheria are common in tropical climates, and this is likely the
reason for high levels of natural immunity among local populations in these regions.
[Link] Reservoir
Infected humans are the reservoir.
[Link] Modes of transmission
Diphtheria is transmitted from person to person through respiratory droplets or less
commonly, through contact with discharge from skin lesions. Historically, raw milk and
fomites were known to have served as vehicles.
[Link] Incubation period
The incubation period is usually 2–5 days (range 1–10 days).
[Link] Communicability
Persons are communicable for up to 4 days after treatment with effective antibiotics has been
initiated. Untreated persons generally shed bacteria from the respiratory tract or from skin
lesions for 2–4 weeks after infection. A chronic carrier state is rare, but known to exist, and
such a carrier may shed micro-organisms for 6 months or more.
[Link] Prevention and control
• Adequate heat treatment of milk.
• Infected person should not be allowed to handle milk and milk products.
• Unhygienic practices like sneezing and coughing by the dairy persons should be avoided.
• Proper vaccination of individuals against disease is an efficient prophylactic measure.
10.6.4 Q – Fever
Q-fever is caused by Coxiella burnetti. Raw milk is commonly implicated as a vehicle for
transmission of disease. Coxiella burnetti is more heat resistant than Mycobacterium
tuberculosis. It can survive pasteurization, if the specified temperature is not maintained and
also freezing temperatures. In view of the considerable heat resistance of this microbe the
time-temperature combination used in pasteurization is selected on the basis of heat
inactivation of this microorganism. It has been found to be viable for 2 years at –20˚C and
resist 0.5 per cent formalin and 1 per cent phenol. It has also been observed to remain viable
for 25 days in rennet cheese, 42 days in cottage cheese, whereas in yoghurt it is killed within
24 hours due to acidity. All these features make Coxilla burnetti a micro-organism of great
public health significance for its pathogenic potentials. Individuals, who have frequent direct
contact with animals, including veterinarians, meat workers, and sheep and dairy farmers, are
at higher risk. Q fever is rarely fatal. Most patients get Q fever by coming in contact with
animals infected with the Q fever bacteria, their tissues, or fluids. Transmission may occur
through breathing contaminated air or dust from an area with a large concentration of
animals. Tissues from animals giving birth pose a particular risk. People can also become
infected indirectly from animals through contaminated materials like wool, straw, and
fertilizer. There is a risk of Q fever from consumption of contaminated raw milk. Sheep,
cattle, goats, cats, dogs, some wild animals like bobcats and rodents, birds, and ticks carry the
bacteria. Most infected animals do not show signs of illness, but Q fever may sometimes
cause abortion.
Only about one-half of all people infected with C. burnetii show signs of illness. For patients
who become ill, the first symptoms of Q fever resemble flu and may include fever, chills,
sweats, headache, and weakness. Q fever may rarely progress to affect liver, nervous system,
or heart valve. Q fever is diagnosed by identifying the bacteria in tissues or through a blood
test that detects antibody to the micro-organism. Patients with mild transient illness usually
do not require treatment. Placenta, other birth products, and aborted fetuses should be
disposed of immediately. Seek veterinary assistance, if animals have reproductive or other
health problems.
[Link] Sources
Mostly human infection is by inhalation of infected dust of the fecal matter. Infected cattle
continue to excrete
the microorganisms in milk for a long time.
[Link] Symptoms
High fever, headache, weakness, malaise, severe sweating and virus like pneumonia.
[Link] Prevention and control
• Adequate heating of milk and cream
• Calving sheds should be away from the milking sheds and dairy
• Animals should be properly vaccinated
• Survey for determining the prevalence of infection in an area should be carried out
10.6.5 Viral infections
The rapid growth of the frozen and convenience foods has given rise to increased concern
about the possible role of such foods in the dissemination of viral infections. Although, food
is rarely reported a vehicle for viral distribution. A few breakouts of poliomyelitis have been
traced to raw milk. Personal contact and mechanical distribution by flies are believed
to be the usual routes of infection with enteric viruses. Outbreaks of infectious hepatitis have
occurred in which foods appear to be the most probable means of transmission.
Epidemiological evidence suggests that ingestion of raw clams or oysters taken from sewage
polluted waters could be a mean by which infectious hepatitis is spread. A number of factors
can influence the importance of any food as a vehicle for the spread of a virus. Among these
are the opportunities for contamination with infectious virus, the ability of a virus to survive
and remain infectious under conditions of handling and storage, the extent of adsorption to
food, the effect of gredients like flavourings and preservatives, and competition from other
contaminants. However, unlike bacteria, viruses will not multiply in the foods. They will
either survive at a constant level or die out. Since some frozen foods are subject to a great
deal of handling in manufacture, are kept frozen, and require little or no cooking in the home,
they would appear to be likely sources of viral distribution, should they become contaminated
during preparation.
10.6.6 Enteroviruses
These are a group of viruses that can cause severe epidemics of diarrhea in infants and
children on ingestion of contaminated milk. Among these most common human pathogens
are polio and coxsackie viruses. Milk and its products are commonly contaminated with
enteroviruses mainly through fecal contamination. Unheated milk contaminated after
pasteurization play a significant role in the transmission of disease, especially in developing
countries. However, polio virus can be inactivated in milk by pasteurization. Compared to
polio virus, coxsackie viruses are more resistant to heat treatment
10.6.7 Poliomyelitis
Like many other diarrheal diseases, poliomyelitis occurs commonly.
10.6.8 Sources
• Person to person contact is the main mode of spread of viruses
• Flies may also serve as a vector for the spread of the disease
• Fecal contamination of water and milk
[Link] Symptoms
First, there is a minor disease associated with viremia characterized by gastro intestinal
disturbance. Then headache, fever, muscle stiffness and paralysis associated with cell
destruction in central nervous system.
[Link] Prevention and control
• Proper pasteurization of milk
• Hygienic measures should be strictly followed to avoid fecal contamination
• Infected persons should not be allowed to handle milk.
• Immunization is also effective in preventing disease.
• Proper vaccination of the children confers protection against disease.
10.6.9 Infectious hepatitis
Among various viral diseases, infectious hepatitis is considered as one of the most serious
viral disease for which milk may be important vehicle for transmission. The illness is also
known as hepatitis A and is caused by Hepatitis A virus, whereas hepatitis B is not
transmitted through milk.
[Link] Sources
• Since the disease spreads through person to person contact, hence handlers can be an
important source Defective water supply in a milk plant may also introduce the virus to milk
• Polluted environment in milk plant may also contaminate milk
[Link] Symptoms
Nausea, vomiting, lethargy, abdominal pain, diarrhea, fever, chills, anorexia, lassitude, sore
throat, bile in urine and jaundice.
[Link] Prevention and control
• Proper sanitary conditions should be maintained during production and processing of milk
• Infected persons should not be allowed to handle milk
• Fecal contamination of water should be avoided
• Adequate heat treatment of milk is likely to inactivate the virus
Lesson 11
ANTIMICROBIAL SUBSTANCES IN MILK
11.1 Introduction
The germicidal properties of milk have been known for years but only recently the progress is
made in characterizing some of the antimicrobial substances in milks of different species (i.e.
cow and [Link] raw milk contains different levels of inhibitory substances that
restrict the normal development of certain bacteria and sometimes may even kill these. The
degree of inhibitory activity varies considerably in milks of different species viz., cow,
buffalo, human (as the maximum activity present in human milk), in milks of same animal at
different milking. The activity may even vary between milk samples collected from different
quarters of udder of same [Link] are two groups of antimicrobial substances in milk.
1. Inherent or naturally occurring substances in milk
2. Non inherent compounds secreted in to milk from other sources
11.2 Naturally Occurring Anti Microbial Substances
These compounds may be specific and non-specific in conferring protection to the new born.
In other words the specific factors are produced in response to specific antigen, whereas non
specific may have less specific antimicrobial
activity.
11.2.1 Specific factors
A. Immunoglobulins
B. Complement
C. Macrophages
D. Bifidus factor
11.2.2 Non specific factors
A) Iron binding protein like lactoferrin
B) Lysozyme
C) Lactanins
D) Lactoperoxidase
E) Vitamin binding proteins and fatty acid
11.3 Significanceof Anti Microbial Substances in Milk
• Health benefits: The primary significance of the antimicrobial substances detected in milk
can be related to their possible role in conferring protection to mammary gland and neonates
in mammals. The new borne therefore should essentially be given the colostrums, which is
very rich in most of these antimicrobial factors.
‘Breast feeding is the best for infants’ derives its substance from the abundance of
antimicrobial factors in mother’s milk, as against animal milk based infant formulae.
• Preservation of milk: Although, the presence of antimicrobial factors cannot solely be
dependent upon to prevent or even to retard significantly the development of mixed
population of microbes in raw milk, some microbes get markedly affected. However, an
artificial exploitation of milks natural antimicrobial system called lactopeoxidase system, for
developing a cold sterilization like process, can prove effective in preservation of raw milk.
Such a process holds great promise under tropical farm conditions in countries like India.
• Processing problems: Where raw milk is used for product making, e.g. in cheese making,
the acid development in cheese vat may be retarded by inhibition of starter activity due to the
presence of antimicrobial substances. In starter culture propagation, the heating of milk
(80˚C) is essential as it inactivates factors that may interfere with the normal starter growth

Lesson-12
[Link] TESTS FOR MILK
Methylene Blue Reduction Test (MBRT)
The methylene blue reduction test is based on the fact that the color imparted to milk by the
addition of a dye like methylene blue will disappear more or less quickly. The removal of the
oxygen from milk and the formation of reducing substances during bacterial metabolism
cause the colour to disappear. The agents responsible for the oxygen consumption are the
bacteria. The test is useful in assessing the bacteriological quality of milk by determination of
the time taken for the reduction of methylene blue in milk indicated by its colour change.
Principle
Oxidation reduction potential of a substrate may be defined generally as the chemical process
in which the substrate either loses or gains electrons. When an element or compound loses
electrons the substrate is said to be oxidized, while a substrate that gains electrons becomes
reduced.
Milk, as it exists in the udder has a sufficiently low redox potential to reduce the methylene
blue immediately. The processes like milking, cooling, dumping etc. raise the oxidation
reduction potential of milk to +0.3V, because of the incorporation of atmospheric oxygen. At
this particular O-R potential, methylene blue is in oxidized state. When bacterial cells
multiply in milk these, consume dissolved oxygen and as more and more oxygen is used and
gets depleted, the dye starts acting as electron acceptor instead of oxygen. As the oxidation
reduction potential decreases from + 0.06 to 0.01 V, methylene blue gets reduced. One atom
of hydrogen is taken up by the double bonded nitrogen of the dye that converts it into
colourless state. The greater is the number of microorganisms in milk, the greater is the
metabolic activity and the faster is the reduction of methylene blue.
MBRT is a rapid, sensitive and low cost, yet a simple quantification method to evaluate
viable count during a growth experiment. It is widely used in dairy industry to determine the
microbial load in the milk. This test involves the addition of methylene blue into a milk
sample and measuring the time required for decolouration. The disappearance of colour in a
short time indicates a high microbial load. The disappearance of colour is due to the removal
of oxygen from milk and formation of reducing substances during bacterial metabolism
Conversion of methylene blue to leucomethylene blue
Standard solution of methylene blue
One tablet of methylene blue thiocyante or chloride is dissolved in 200 ml of cold sterile
glass distilled water by gentle heating to facilitate dissolving and then add another 600 ml
distilled water.
Procedure
The samples of milk are mixed thoroughly. If the milk is in a bottle/ sachet, it shall be
inverted at least 25 times to mix the fat uniformly with the milk. Take 10 ml of milk into a
test tube and add 1 ml of standard methylene blue solution. Invert the test tube to mix the
milk and methylene blue solution. Place the test tube in a thermostatically maintained water
bath at 37.5C and note down the time of incubation. Observe the test tubes after 30 min for
decolourization reduction of dye.
Grading of milk
The quality of raw milk is adjusted by making the following observations
Grading of milk based on MBRT as per BIS standard
Factors affecting the MBRT
a. Cold milk holds more oxygen than warm milk
b. Pouring milk back and forth from one container to another increases the oxygen, and
c. During milking time much oxygen may be absorbed.
d. The rate of reduction of dye depends on the type of microorganism
e. Coli forms appear to be the most rapidly reducing microorganisms,
f. Closely followed by Lactococcus lactis spp. lactis, some of the faecal Streptococci, and
certain micrococci.

RESAZURIN REDUCTION TEST (RRT)

Resazurin reduction t is another method of dye reduction test and the principle of this test is
nearly similar to methylene blue reduction test. In MBRT the time for reduction of the dye is
measured, while in RRT, at a fixed period time, specific shade of colour and its intensity is
measured.
Principle
Unlike methylene blue the resazurin undergoes reduction through a series of colour shades
viz., blue, purple, and lavender, pink before completely getting reduced to colourless.
Resazurin dye which is blue in colour at the oxidation-reduction potential the colour of dye
changes to colourless (dihydroresorufin), which is a reversible reaction. Usually, the degree
of reduction of the dye is measured after a fixed time of incubation of milk sample in the
presence of dye.
Procedure
Take 10 ml of milk into a test tube and add 1 ml of working solution of Resazurin solution.
➢ Put air tight closure to prevent oxygen entry
➢ Invert the test tubes to mix the milk and Resazurin solution.
➢ Place the test tubes in a thermostatically maintained water bath at 37 - 0.5 C and note
down the time of incubation (10 min or 1 h).
➢ At the end of incubation match the colour of the milk with one of the colour standards of
Resazurin disc.
Grading of milk
The quality of raw milk is adjusted by using the following parameters.
Grading of milk by resazurin test

Advantages of dye reduction test

➢ Used for estimating the suitability of milk for liquid consumption.
➢ These tests are cheaper and less time is required.

Disadvantages
➢ Rate of reduction of dye varies considerably and is related to species and the rate at which
different micro-organisms grow at a particular temperature.
➢ Inhibitory substances like penicillin and other antibiotics prevent the growth of bacteria
and thus increase the reduction time.
PHOSPHATASE TEST
Pasteurisation is an essential process in the production of milk which is safe and free from
pathogens. Alkaline Phosphatase is an enzyme which is naturally present in milk, but is
destroyed at a temperature just near to the pasteurization temperature. Alkaline Phosphatase
test is used to indicate whether milk has been adequately pasteurised or whether it has been
contaminated with raw milk after pasteurisation. This test is based on the principle that the
alkaline phosphatase enzyme in raw milk liberates phenol from a disodium para-nitro phenyl
phosphate and forms a yellow coloured complex at alkaline pH (Scharer, 1943). The intensity
of yellow colour produced is proportional to the activity of the enzyme. The colour intensity
is measured by direct comparison with standard colour discs in a Lovibond comparator. The
test is not applicable to sour milk and milk preserved with chemical preservatives.
Apparatus required
1. Water-Bath -maintained at 37±l⁰C, thermostatically controlled.
2. Comparator - with special discs of standard colour glasses calibrated in μg p-nitrophenol
per ml milk, and 2 x 25 mm cells.
3. Test Tubes - of size 16 x 1.50 mm and rubber stoppers to fit.
4. Pipettes - 1, 5, and 10 ml.
5. Filter Paper - Whatman No. 2 or equivalent.
6. Litmus Paper

Reagants
1. Sodium Carbonate-Bicarbonate Buffer - Dissolve 3.5 g of anhydrous sodium carbonate and
1.5 g of sodium bicarbonate in one litre of distilled water.
2. Buffer Substrate - Dissolve 1.5 g of disodium p-nitrophenyl phosphate in one litre of
sodium carbonate-bicarbonate buffer. This solution is stable if stored in a refrigerator at 4°C
or less for one month but a colour control test should be carried out on such stored solutions.
Procedure
1. Pipette 5 ml of buffer substrate into a clean, dry test tube followed by 1 ml of the milk to
be tested. Stopper the tube, mix by inversion and place in the water-bath
2. At the same time place in the water-bath a control tube containing 5 ml of the buffer
substrate and 1 ml of boiled milk of the same kind as that under test that is pasteurized
homogenized, low fat.
3. After 2 hours, remove the tubes from the bath, invert each and read the colour developed
using the comparator and special disc, the tube containing the boiled milk control being
placed on the left of the stand and the tube containing the sample under test on the right.
Record readings which lie between two standard colour discs by adding a plus (+) or minus (-
) sign to the figure of the nearest standard.
NOTE - If artificial light is needed when taking these readings, an approved ‘day light’
source of illumination must be used.
STANDARD PLATE COUNT
Introduction
The standard plate count (SPC) is suitable for estimating bacterial populations in most types
of dairy products, and it is a reference method specified in the Grade A Pasteurized Milk
Ordinance to be used to examine raw and pasteurized milk. This procedure is also
recommended for application in detecting sources of contamination by testing line-samples
taken at successive stages in the processing.
Principle
The test employs aserial dilution technique for easy quantification of the micro-organisms.
The appropriate dilutions of the milk sample are mixed with a sterile nutrient medium that
can support the growth of the micro-organisms, when incubated at a suitable temperature.
Each bacterial colony that develops on the plate is presumed to have grown from one
bacterium or clump of bacteria in the inoculums. The total number of colonies counted on the
plates multiplied by the dilution factor to represent the number of viable micro-organisms
present in the sample tested.
Procedure
Sample preparation
➢ Mark each plate with sample number, dilution, and other desired information before
making dilutions.
➢ Before opening a sample container, remove from the closure all obvious materials that
may contaminate the sample. If desired, wipe the tops of unopened sample containers with a
sterile cloth or paper towel saturated with 70% ethyl alcohol.

Dilution of samples
➢ For SPCs, select dilution(s) so that the total number of colonies on a plate is between 30
and 300. For example, where an SPC is expected to reach a number 5000, prepare plates
containing 10-2 dilutions.
➢ Use a sterile pipette for initial and subsequent transfers from the same container, if the
pipette is not contaminated. If the pipette becomes contaminated before transfers are
completed, replace it with another sterile pipette. Do not flame to decontaminate. Use a
separate sterile pipette for transfers from each different dilution.

Plating
Melt the required amount of medium quickly in boiling water, in a microwave oven, or by
exposing it to flowing steam in a partially closed container, but avoid prolonged exposure to
unnecessarily high temperatures during and after melting. Discard melted nutrient agar or
tryptone dextrose agar that develops a precipitate. Do not melt more medium than will be used
in 3 hours. Do not re-sterilize the [Link] the melted medium.
Incubating
Incubate plates at 32 C or 37 C for 48 .3 h for SPC. Plates must reach the temperature of
incubation within 2 h. Avoid excessive humidity in incubator to reduce the tendency toward
spreader formation, but prevent excessive drying of the medium by controlling ventilation
and air circulation. Agar in plates should not lose more than 15% of its weight during 48 h of
incubation.
Counting of colonies on agar plates
Count the plates after the desired incubation period. Record the dilutions used and number of
colonies counted on each plate. If it is impossible to count at once, after the required
incubation store the plates at 0 to 4.4C for not more than 24 h. For each lot of samples, record
the results of sterility tests on materials used when pouring plates and the incubation
temperature used.
Table Grading of milk based on standard plate count test (BIS Standards)
REFERENCES
Britz, T.J. and Robinson, R.K. 2008. Advanced Dairy Science and Technology. 1st ed.
Blackwell Publ. Ltd.,
UK.
Fernandes, R. 2009. Microbiology Handbook: Dairy Products. Royal Society of Chemistry,
Revised
ed., London.
Marth, E.H. and Steele, J. 2001. Applied Dairy Microbiology. 2nd ed. CRC Press, Boca
Raton, USA.
Robinson, R.K. 2002. Dairy Microbiology Handbook - The Microbiology of Milk and Milk
Products. 3rd ed.
Wiley-Interscience, New York.
Walstra, P., Wouters, J.T.M. and Geurts, T.J. 2006. Dairy Science and Technology. CRC Press,
New York.