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Chapter: Microbiology and Immunology: Applied Microbiology: Bacteriology of Water, Milk, and Air

Bacteriology of Milk

Milk is an opaque white liquid, which provides the primary source of nutrition for newborns before they are able to digest other types of food. It

Bacteriology of Milk

Milk is an opaque white liquid, which provides the primary source of nutrition for newborns before they are able to digest other types of food. It is an emulsion of butterfat globules within a water-based fluid. Each fat globule is surrounded by a membrane, consisting of phospholipids and proteins. These emulsifiers keep the individual globules from joining together into noticeable grains of butterfat and also protect the globules from the fat-digesting activity of enzymes found in the fluid portion of the milk.

Bacterial Flora of Milk

Lactic acid bacteria: This group of bacteria is able to fermentlactose to lactic acid. They are normally present in the milk and are also used as starter cultures in the production of cultured dairy products, such as yogurt. Some examples of lactobacilli found in milk are:

a)        Lactococci, such as Lactic delbrueckii subsp. lactis (Streptococcuslactis) and Lactococcus lactis subsp. cremoris (Streptococcus cremoris).

b)         Lactobacilli, such as Lactobacillus caseiLactobacillus del-brueckii subsp. lactis (L. lactis), Lactobacillus delbrueckii subsp. bulgaricus(Lactobacillus bulgaricus), and Leuconostoc.

Coliforms: Coliforms are facultative anaerobes with an opti-mum growth at 37°C. Coliforms are indicator organisms; they are closely associated with the presence of pathogens but are not necessarily pathogenic themselves. They also can cause rapid spoilage of milk because they are able to ferment lactose with the production of acid and gas, and are able to degrade milk proteins. They are killed by pasteurization; therefore, their presence after treatment is indicative of contamination. E. coli is an example belonging to this group.

Significance of Presence of Microorganisms in Milk

Information on the microbial content of milk can be used to judge its sanitary quality and the conditions of production. If permitted to multiply, bacteria in milk can cause spoilage of the product. Milk is potentially susceptible to contamination with pathogenic microorganisms. Precautions must be taken, therefore, to minimize this possibility and to destroy patho-gens that may gain entrance. Certain microorganisms produce chemical changes that are desirable in the production of dairy products, such as cheese and yogurt.

Spoilage Microorganisms in Milk

The microbial quality of raw milk is crucial for the produc-tion of quality dairy foods. Spoilage is a term used to describe the deterioration of a foods’ texture, color, odor, or flavor to the point where it is unappetizing or unsuitable for human consumption. Microbial spoilage of food often involves the degradation of protein, carbohydrates, and fats by the micro-organisms or their enzymes.

In milk, the microorganisms that are principally involved in spoilage are psychrotrophic organisms. Most psychrotrophs are destroyed by pasteurization temperatures; however, some like Pseudomonas fluorescensPseudomonas fragi can produce pro-teolytic and lipolytic extracellular enzymes, which are heat sta-ble and capable of causing spoilage. Some species and strains of Bacillus, Clostridium, Corynebacterium, Arthrobacter,Lactobacillus, Microbacterium, Micrococcus, and Streptococcus species can survivepasteurization and grow at refrigeration temperatures, which can cause spoilage problems.

Pathogenic Microorganisms in Milk

Hygienic milk production practices, proper handling and storage of milk, and mandatory pasteurization have decreased the threat of milk-borne diseases, such as tuberculosis, brucel-losis, and typhoid fever. There have been a number of food-borne illnesses resulting from the ingestion of raw milk or dairy products made with milk that was not properly pasteurized or was poorly handled, causing postprocessing contamination. Milk-borne diseases are of three types:

·           Infections primarily of humans but transmitted through milk.

·           Infections primarily of animals that can be transmitted to humans.

·           Infections transmitted by milk contaminated with excreta of ticks and rats (Table 76-4).

It should also be noted that molds, mainly of the species of  Aspergillus, Fusarium, and Penicillium can grow in milk and dairyproducts. If the conditions permit, these molds may produce mycotoxins, which can be a health hazard.

Bacteriological Examination of Milk

Bacteriological examination of milk can be carried out by fol-lowing groups of tests:

a)        Colony counts

b)       Coliform counts

c)        Chemical tests, such as methylene blue reduction test, phosphatase test, and turbidity test

d)       Detection of specific pathogens

 Colony counts

This test is carried out by plate dilution methods. Raw milk may contain 500 to several million bacteria/mL of milk.

 Coliform counts

The presence of coliforms in milk indicates improper pasteuri-zation of milk, or postpasteurization contamination of milk. This is because all coliforms are destroyed during the process of pasteurization of milk. This test is carried out by inoculating varying dilutions of milk into MacConkey medium and noting the production of acid and gas after 48 hours of incubation at 37°C. The presence of acid and gas indicates the presence of coliforms in milk.

 Chemical tests

Methylene blue test: This is a test used since long to demon-strate bacterial contamination of milk. It is an indicator of the number of viable bacteria present in the milk. It is a rapid and inexpensive way of indicating poor-quality milk that had been  unrefrigerated. The basis of the test is that the presence of via-ble bacteria in milk reduces methylene blue and decolorizes the milk when kept in a dark place. The test is performed by using a 1:300,000 solution of methylene blue. One milliliter of the methylene blue solution is added to 10 mL of the milk sample in a test tube. Both the milk and methylene blue solution are mixed by shaking and then placing the mixture in a water bath at 37°C for 30 minutes in dark. Untreated milk can be considered as sat-isfactory if it fails to decolorize the dye within 30 minutes.

Phosphatase test: Alkaline phosphatase is normally presentin milk and is inactivated if pasteurization has been carried out effectively. Successful pasteurization, which kills nonspor-ing pathogens, also inactivates alkaline phosphatase. The test depends on the ability of the enzyme to liberate p-nitrophenyl phosphate after breaking down disodium p-nitrophenyl phos-phate. The test determines the amount of alkaline phospha-tase present after pasteurization by measuring the amount of p-nitrophenyl phosphate it liberates, which is known by devel-opment of a yellow color that is quantitated by a colorimeter.

Turbidity test: This is the definitive test for checking the ster-ilization of milk, thereby distinguishing it from the untreated milk and milk that has been merely pasteurized. The degree of heating necessary for sterilization causes all the heat-coagulable proteins in milk to become precipitable by ammonium sulfate. If the amount of heat applied to milk is insufficient for steril-ization, some of its protein will not be precipitated by ammo-nium sulfate and will be detected by its coagulation, resulting in turbidity when a filtrate of ammonium sulfate treated-milk is boiled. The absence of turbidity indicates that the milk has been boiled or heated to at least 100°C for at least 5 min.

 Detection of specific pathogens

Mycobacterium tuberculosis and Brucella species are the specificpathogens that can be detected in milk to know transmission of these bacteria through milk. Specific selective media for these bacteria are employed to detect these in infected milk. For example, M. tuberculosis can be isolated from centrifuged deposit of milk by inoculation in Lowenstein–Jensen (LJ) medium or by inoculation in guinea pigs. Similarly, Brucella spp. may be isolated from milk samples by inoculating in serum dextrose agar or by intramuscular inoculation in guinea pigs. Subsequently, these bacteria are identified by their cultural and biochemical properties. Brucella spp. in infected animals can also be demonstrated by milk ring test, whey agglutination test, and demonstration of brucella antibodies in serum.

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