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Unit – I Dairy Microbiology
Definition of and composition of milk
Milk is a whitish liquid containing proteins, fats, lactose, and various vitamins and minerals that is produced by the mammary glands of all mature female mammals after they have given birth and serves as nourishment for their young. Milk is a white liquid produced by the mammary glands of mammals. It is the primary source of nutrition for young mammals before they are able to digest other types of food. Early-lactation milk contains colostrum, which carries the mother's antibodies to the baby and can reduce the risk of many diseases in the baby.
Milk (cow or buffalo or goat milk) may be defined as the whole, fresh, clean, lacteal secretion obtained by complete milking of one or more healthy animals, practically colostrum free and containing the minimum prescribed percentages of milk fat and milk solids not fat (SNF).
Colostrum ( first milk ) is a form of milk produced by the mammary glands of mammals just prior to giving birth. Colostrum contains antibodies to protect the newborn against disease, as well as being lower in fat and higher in protein than ordinary milk. Colostrum is very rich in proteins, vitamin A, and sodium chloride, but contains lower amounts of carbohydrates, lipids, and potassium than normal milk. The most pertinent bioactive components in colostrum are growth factors and antimicrobial factors. The antibodies in colostrum provide passive immunity, while growth factors stimulate the development of the gut. They are passed to the neonate and provide the first protection against pathogens.
The milk of cows, goats, buffalos or other animals is used as food by humans.
Composition of different species of milk
Species
Percentage of Composition Water Fat Protein Lactose Ash Elephant 67.8 19.6 3.1 8.8 0. Buffalo 84.2 6.6 3.9 5.2 0. Camel 86.5 3.1 4.0 5.6 0. Goat 86.5 4.5 3.5 4.7 0. Cow 86.6 4.6 3.4 4.9 0. Human 87.7 3.6 1.8 6.8 0.
Milk is a white or yellow-white, opaque liquid. The color is influenced by scattering and absorption of light by milk globules and protein micelles. A yellowish, color is derived from carotene present in that fat phase and from riboflavin present in the aqueous phase. Milk tastes mildly sweet, while its odor and flavor are normally quite faint. Milk occurs in the form of droplets or globules, surrounded by a membrane and emulsified in milk serum (also called whey). The fat globules (called cream) separate after prolonged storage or after centrifugation. The fat globules float on the skim milk. Homogenization of milk so finely divides and emulsifies the fat globules that cream separation does not occur even after prolonged standing. Proteins of various sizes are dispersed in milk serum. They are called micelles and consist mostly of calcium salts of casein molecules. Furthermore, milk contains lipoprotein particles, also called milk microsomes, which consist of the residue of cell membranes, microvilli, etc, as well as somatic cells which are mainly leucocytes.
Chemical composition of Milk
Various carbohydrates, fats, proteins, minerals and other ingredients are solubilized in milk serum.
Carbohydrates
Lactose is the major carbohydrate in the milk of most species. Lactose is a disaccharide composed of the monosaccharides D-glucose and D- galactose, joined in a β-1,4-glycosidic linkage. Lactose is cleaved to glucose and galactose in the intestine of the neonate by an enzyme activity called lactase (or β-galactosidase). The galactose is then converted to glucose by a different enzyme. Lactose is a major, readily digestible source of glucose which
Minerals
Calcium and phosphorous are the major minerals found in milk. These minerals are required in large quantities by the rapidly growing neonate for bone growth and development of soft tissues. Calcium and phosphorous mostly are associated with the casein micelle structure. Milk also contains most other minerals found in the body.
Some minerals, such as Zn, Mg, Fe, Cu, Mn, and Mo, are required by enzymes as cofactors. Minerals contribute to the buffering capacity of milk, the maintenance of milk pH, the ionic strength of milk, and milk's osmotic pressure.
Trace Elements
The milk concentration of some elements can be increased by increasing the amount in the diet. These particularly include I, B, Br, Co, Mn, Mo, Se, and Zn.
Vitamins
Milk contains all the vitamins except Vit C required by mammals. The fat soluble vitamins , A, D, E, and K, are found primarily in the milk fat; milk has limited amounts of vitamin K. The B vitamins are found in the aqueous phase of milk.
Vitamins are essential organic compounds required in the diet. The fat soluble vitamins (A, D, E, K) are associated with the milk fat globule.
Somatic cells
Milk also contains a range of other components. Milk contains leukocyte cells, also known as somatic cells in cow milk
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Sources of Microorganisms in Milk –
There are several principal sources of contamination of milk. From the time the milk leaves the udder, until it is dispensed into containers, everything with which it comes into contact is a potential source of more microorganisms. Milking performed under hygienic conditions, with strict attention to sanitary practices, will reduce the entry of microorganisms into the milk. Naturally the fewer the organisms that can get into the milk, the fewer have a chance to grow.
**1. Producing Animal
- Milking Area
- Utensils and Equipments
- Personnel (Milking men)
- Water
- Producing Animal**
Unless the producing animal is clean, and her flanks, udder and teats given special sanitary care just before milking, her body can be a source of considerable contamination. The first few streams of milk from each teat should, be collected, separated and discarded. This flushes out the organisms that have entered the teat through the teat opening.
Milk from a cow with an infected udder is likely to contain a large number of organisms. The probability of diseases of the udder contaminating the milk is very high. Mastitis, which is a disease causing inflammation of the udder, contributes considerable number of organisms, sometimes even blood cells, into the milk. Washing and massaging the cow's udder with a warm detergent sanitizer solution before milking serves to clean the area. The hair of all animals harbors organisms. The hair, dirt and dust often fall from the body into the milking pots or the teat cups of milking machines. The modern practice is to keep the flanks clipped to minimize contamination.
2. Milking Area –
The microbial content of air is greatly affected by many conditions and practices. Dried dirt and waste is picked up by all movements and carried about as dust in the atmosphere. For this reason, dust may be the source of almost every kind of contamination. The sprays which are sometimes used to cut down air contamination are not very useful. However, the main point of keeping the conditions clean and sanitary is not to raise dust.
3. Utensils and Equipments -
Utensils and equipments are known to be the greatest sources of contamination. They may account for as much as 100, 000 to a billion organisms per milliliter. Pails (containers, buckets), strainers (sieve), milking machines, cans, pipes, bottles, and other equipments used for the handling of milk are sometimes not properly washed and sanitized.
flavour. Cream is starting substrate. Butter is normally made by churning cream that has been soured by lactic acid bacteria.
- Yogurt.
It is made by fermenting milk with a mixture of Lactobacillus bulgaricus and Streptococcus thermophilus at 40°C. Flavour is due to accumulation of lactic acid and acetaldehyde.
- Cheese.
Cheese consists of milk curds that have been separated from the liquid portion of the milk (whey). The curdling of milk is done by enzyme rennin (casein coagulase or chymosin) and lactic acid bacterial starter cultures. Cheeses are classified as soft (high, 50-80% water content), semi hard (about 45% water) and hard (a low water content, less than 40%).
Undesirable changes carried out by microorganism in milk
Spoilage occurs when microorganisms degrade the carbohydrates, proteins, fats of milk and produce noxious, end products. Milk products as follows;
**1. Gas Production
- Ropiness / sliminess**
- Proteolysis
- **Lipolysis
- Sweet curdling
- Bitty Cream (or) Broken cream
- Development of abnormal Flavours
- Development of abnormal colour fermentations
- Gas Production:**
The production of gas mainly CO 2 by certain organisms in dairy products is responsible for a defect called “gassiness”. In some cases it is associated with acid production. In high fat milk there is foaming and the gas escapes the partially coagulated mass and the defect is called “Frothiness”. This is due to associative action of acid producing bacteria with yeasts. The production of gas
in canned dairy products causes bulging of cans and the defect is called „blowing ’ of cans.
Causative organisms:
i. Coliforms: E. coli, Enterobacter aerogenes ferment lactose of milk or cream into gas and acid. These are called ‘early gas producer’ and produce early blowing condition.
ii. Anaerobic spore forming bacteria: ex. Cl. butyricum, Cl. sporogenes — produce gas only in anaerobic conditions, mostly in canned dairy products like processed cheese, concentrated milk. They are called ―Late gas producers’ and produce late blowing condition.
iii. Lactose fermenting yeasts: ex. Candida psedutropicalis , yeasts produce CO 2 and small amounts of ethyl alcohol in milk and cream, whey at or below 37°C.
2. Ropiness / sliminess:
Ropy fermentation is brought about by the growth of bacteria leading to change in consistency of the produce that forms threads of viscous masses when poured. Ropiness develops only on storage and milk is drawn out as fine threads and may appear gel like consistency. Sometimes the change is so much pronounced that the milk can be drawn into long thread.
Causative organisms:
i. Gram – ve rods : Alcaligenes viscosus ii. Coli-aerogenes group : This group consists of ropy strains belonging to enterobacter, citrobacter, Serratia marcescens and related genera, iii. Aerobic spore formers : B. cereus, B. subtilis, B. circulans iv. S. lactics var hollandicus, L. casei, Lactobacillus delbruckeii ssp bulgaricus show ropiness before detectable acid development. Ropiness decreases as acidity increases.
caproic are responsible for the lipolytic off flavours, also referred to as rancidity (Hydrolytic rancidity).
Lipolytic microbes or enzymes: a) Psychrotrophes: Pseudomonas sp. mainly Ps. fragi, Ps. fluoresens, Achromobacter lipolyticum
b) Other types Micrococcus frendenreichii, Bacillus cereus, B. subtilis, B.coagulans
c) Yeasts & molds C. lipolytica, Geotricum candidum, Penicillium spp. and Aspergillus spp.
5. Sweet curdling
Curdling without pronounced acid production is called the sweet curdling. The defect is due to the production of an extracellular enzyme similar to rennin by bacteria which causes casein to precipitate in the term of small specks of curd before the development of sufficient acidity i.e between 6.2 and 6.6 pH.
Causative organisms :
i. Cocci: S. liquifaciens ii. Aerobic spore formers – B. cereus, B. subtilis iii. Psychrophilic spore forms - B. cereus, B. licheniformis, and certain Microbacterium spp. iv. Non spore forming rods: Proteus and Escherichia
6. Bitty Cream (or) Broken cream:
It is characterized by the appearance of flakes in the cream which do not mix again when milk is shaken. If such milk is used in tea, the flakes float on the surface making it unaccepted to mainly people. Flaking normally occurs before the changes in flavour or heat stability. Bacteria origin – Produced partly by the lecithinase enzyme of B. cereus. Bitty cream is the chief spoilage problem of pasteurized milk. Failure of refrigeration, Seasonal variation prolonged storage etc., are the main reasons.
7. Development of abnormal Flavours
a. Fruity Flavours : These are due to ethyl ester formation usually catalyzed by esterases from psychrotroph or lactic acid bacteria. Ester formation by Ps. fragi involves liberation of butyric & caproic acids from one and three positions of milk triglycerides and are esterified with ethanol. Predominate esters are Ethyl butyrate, Ethyl hexanoate.
b. Malty flavour : Caused by Malty strains Lactococcus lactis sp
c. Bitty flavour: Caused by proteolytic organisms especially Bacillus sp., and Pseudomonas sp. d. Fishy flavour: Caused by Ps. icthyosmius e. Potato flavor: Caused by Ps. mucidolens and Ps. graveolens f. Phenolic flavour: Caused by Bacillus circulans g. Soapy flavour: Caused by Ps. sapoticum h. Bitty/Musty flavour: Caused by Actinomyces and certain yeast i. Burnt of caramel flavour: Caused by Malty strains of Lactococcus lactis j. Barny flavour: Caused by Aerobacter oxytocum
8. Development of abnormal colour fermentations:
a. Yellow coloration : Pseudomonas synxantha b. Blue coloration: Pseudomonas cyanogens c. Green coloration: Penicillium roqueforte d. Black coloration: Pseudomonas nigrifaciens e. Red coloration: Serratia marcescens/Micrococcus resen f. Brown coloration: Pseudomonas fluorescens g. Greenish coloration: Pseudomonas fluorescens
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Microorganisms found in milk can also be described on the basis of the following characteristics.
- Biochemical activities.
- Temperature response.
- Ability to cause infection and disease (bovine and human origin) 1. Biochemical activities
If allowed to stand under condition that permits bacterial growth, raw milk of a good sanitary quality will rapidly undergo a series of chemical changes. The principal change is lactose fermentation to lactic acid.
i) Acid producing: - This change is brought about by aciduric lactic organisms, especially Strepotococcus lactis and certain lactobacilli. These include two distinct biochemical types, homo-and heterofermentative. In homofermentation lactic acid is the major product of lactose fermentation. Heterofermentative organisms, however, produce lactic, acetic, propionic, and some other acids, and some alcohols and gases such as CO 2 and H 2. Organisms continue to form lactic acid until the concentration of acid is itself too great for the organisms to remain live.
Microbacteria, micrococci, coliforms, etc. also ferment lactose to lactic acid and other products. Many Clostridium species and, some yeasts such as Torula lactic , and Torula cremoris ferment lactose with acid and gas production. As the acidity continues to increase and reaches a pH of 4.7, it eventually causes a precipitation of casein. Organisms capable of metabolizing lactic and other acids develop especially aciduric, yeasts and molds.
The acidity of milk is diminished and the alkaline products of protein decomposition such as amines, ammonia and the like are produced. This is accomplished by many species of the genera Bacillus, Clostridium, Pseudomonas, Proteus and numerous other forms.
ii) Proteolytic:
These microorganisms degrade casein or some insoluble casein derivatives to water soluble compounds through the action of organisms or their enzymes. E. g. Pseudomonas fluorescens, Ps. fragi, Alteromonas putrefaciens. Thermoduric bacteria especially Micrococcus caseolyticus , B. stearothermophilus B. cereus, B. subtilis.
iii) Lipolytic: - The action of microorganisms does not involve fat as readily as it does lactose and protein. Lipolysis results from the action of lipase produced by bacteria such as Pseudomonas, Achromobacter and by some yeasts and molds. Fat is hydrolyzed to glycerol and fatty acids. Some of the fatty acids, for example, butyric and caproic acid give milk products, distinctive and usually rancid, odours and flavours.
iv) Ropiness producing: - Several microorganisms also bring about certain objectionable changes in the milk which may not be deleterious to health. Ropines in milk are sometimes encountered. The milk become ropy or slimy and may be pulled out into long threads. It is produced by several organisms but the most important species is Alcaligenes viscolactis. A rapid fermentation of lactose in milk is sometimes observed and is known as stormy fermentation. This is brought about by Clostridium perfringens.
v) Gas producing: - The curd becomes torn to shreds by the vigorous fermentation and gas production.
vi) Colour producing: - Several organisms have been isolated from milk which imparts brilliant colours. Pseudomonas syncyanea imparts blue colour, pseudomonas synxantha yellow colour and Serratia marcescens red colour to the milk.
2. Temperature response
Microorganisms found in milk can also be described according their optimum temperature for growth and heat resistance. This is a very practical consideration since milk is preserved by employing low temperatures to prevent changes due to microbial activity and by high temperatures to reduce microbial population and destroy pathogens. All the four types of microorganisms i.e. psychrophilic, mesophilic, thermophilic and thermoduric are found in milk.
may be transmitted to man. The organisms causing these diseases may get into the milk either directly from the udder, or indirectly from infected body discharges, which may drop, splash, or be blown into the milk.
Some of the important diseases of human origin that have been transmitted by milk are
(1) Typhoid fever Salmonella typhi
(2) Diphtheria, Corynebacterium diptheriae
(3) Scarlet fever, Streptococcus pyogenes
(4) Dysentery Shigella dysenteriae
(5) Septic sore throat Hemolytic streptococci
(6) Poliomyelitis. PolioVirus
(7) Tuberculosis, Mycobacterium bovis
(8) Brucellosis Gram-negative rod Brucella abortus
(9) Q fever. Coxiella burnetii (Rickettsia)
(10) Pneumonia, Diplococcus pneumonia and Viruses
(11) Toxoplasmosis, Toxoplasma gondii (Protozoan)
(12) Anthrax, Bacillus anthracis
(13) Foot and Mouth disease F & M virus
(14) Hepatitis Hepatitis virus
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Changes in the flora of raw milk stored at room temp
Raw milk contains many types of microorganisms coming from different sources. Microbial spoilage of raw milk can potentially occur from the metabolism of lactose, proteinaceous compound, fatty acids (unsaturated), and the hydrolysis of triglycerides.
If the milk is refrigerated immediately following milking and stored for days, the spoilage will be predominantly caused by the Gram-negative psychrotrophic rods, such as Pseudomonas, Alcaligenes, Flavobacterium spp ., and some coliforms. Pseudomonas and related species, being lactose-negative, will metabolize proteinaceous compounds to change the normal flavor of milk to bitter, fruity, or unclean. The growth of lactose-positive coliforms will produce lactic, acetic, and formic acids, C0 2 , and H 2 leading to curdling and
souring of milk.
Some Alcaligenes spp and coliforms can also cause ropiness (sliminess) due to production of viscous polysaccharides. However, if the raw milk is not refrigerated soon, growth of mesophiles predominates e.g, Lactococcus, Lactobacillus, Enterococcus, Bacillus, and coliforms, along with Pseudomonas, Proteus , and others causing changes like souring and curdling of milk. Yeast and mold growth, under normal conditions, is generally not expected.
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Microbiological examination of milk
There are different approaches for checking the bacteriological quality of raw milk and they are broadly classified into
1) Direct tests A) Direct Microscopic Count (DMC) B) Standard Plate Count (SPC) / Pour Plate Technique or Colony Count Test 2) Indirect tests such as dye reduction tests A) Methylene blue reduction time test (MBRT test) B) Resazurin Reduction test (RR test) 3) Enumeration of Coliforms in Milk
1. DIRECT METHODS
A. Direct Microscopic Count test (DMC) B. Standard plate count test (SPC)
Disadvantages of DMC: -
This method may be the source of considerable error unless the food contains a high count. This is because a large factor is used for converting the number of organisms per field to the number per ml of food.
This method does not differentiate dead from living organisms.
Requirements: -
- Milk sample
- Slide
- Microscope
- Newman’s Stain (Tetrachloroethane, Ehyl alcohol, Methylene Blue) OR
- Methylene blue
- Xylene
Procedure: -
1 sq. cm area is marked on a clean grease free slide with glass marking pencil.
0.01 ml milk sample is spread on opposite side of the marked area.
Sample is air-dried and heat fixed.
The smear is flooded with Newman's stain for 1 minute, gently washed with tap water, air dried.
OR
- The smear is flooded with xylene for 1 minute to remove fat from the milk sample. Smear is stained with methylene blue solution for one minute and gently washed with tap water, air dried
- 10 fields are observed under oil immersion lens.
Direct microscopic counts per ml Bacteriological quality of milk
Less than 5, 00,000 Good 5, 00,000 to 40, 00,000 Fair 40, 00,000 to 2, 00, 00,000 Poor Over 2, 00, 00,000 Very poor / bad
B. Standard plate count test (SPC) / Pour Plate Technique or Colony Count Test :
The standard plate count method is also called as pour plate technique or colony count test. It is useful in the estimation of number of viable microorganisms in the given sample of milk. The test employs the serial dilution technique for easy quantification of the organisms in view of a wide range of bacterial population that may occur in milk. The appropriate dilutions of the milk sample are mixed with a sterile nutrient medium that can support the growth of the organisms when incubated at a suitable temperature. Each bacterial colony that develops on the plate is presumed to have grown from one bacterium in the inoculum. The total number of colonies counted on the plates multiplied by the dilution factor is taken to represent number of viable organisms present in the sample.
Preparation of dilutions of the milk sample: Transfer 1ml of the milk sample with a sterile pipette to 9ml of sterile water (1st dilution) which will make 1 in 10 dilution of the milk sample. Take 1ml from 1st dilution and transfer to second 9 ml sterile water to get 1 in 100 dilutions. Mix thoroughly and transfer 1ml from second dilution to third 9 ml sterile water to make 1 in 1000 dilution and so on till a series of required dilutions of the sample is ready. Use a fresh sterile pipette for each successive dilution.
Plating the sample and preparation of plates:
Transfer 1ml of each required dilution into sterile petri dish. To each petri dish add 15 to 20 ml of sterilized Nutrient Agar which was previously sterilized, melted and cooled to 45°C. Mix well, allow the agar to cool and set. Invert the plates and incubate at 37^0 C for 24 - 48 hours. After the incubation determine the average of the counts in the two plates and multiply this by the dilution factor. Raw milk is graded based on the following specifications SPC/ml Grade Not exceeding 2,00,000 Very good 2,000,00 to10,00,000 Good 10,00,000 to 50,00,000 Fair Over 50,00,000 Poor
