Microbiological Examination of Dairy Products

GS/M.Sc./FOOD/3608/08

B.K.K.K.Jinadasa

Microbiological Examination of Dairy Products

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Introduction Milk contributes a greater number of the essential nutrients for human nutrition than any other single food, some in relatively large amounts. It is an outstanding source of calcium, which is needed all through life for healthy bones and teeth. It also supplies many other vitamins including riboflavin and, when fortified, vitamin-D which helps structural and tissue development. The protein in milk is one of the best quality proteins that any food offers. And milk is not only a beverage. It can be used in or on cooked food such as gravies, puddings and cereals. It can be consumed in the form of cheese, butter, ice cream or milk drinks, during meals or snacks. Due this highly nutritious nature of the milk and milk foods, it serves as an excellent growth medium for a wide range of microorganisms. The microbiological quality of milk and dairy products is influenced by the initial flora of raw milk, the processing conditions, and post-heat treatment contamination. The most significant source of contamination is dairy utensils and milk contact surface including milk pall or milking machines. It can also be subject to contamination during transport, storage and manufacturing processes. Milk is an excellent culture medium for many kind of microorganism. Its high water activity, moderate pH and available nutrients are the principal factors which contribute to microbial growth. Therefore, it is necessary to maintain high standards of hygiene during its production and related manufacturing processes. The predominant microorganisms living in milk under improper handling and storage conditions are Gram positive. Spoilage occurs when microorganisms degrade the carbohydrates, proteins, fats of milk and produce noxious, end products. It may be seen that Lactobacillus or Streptococcus species ferment the lactose to lactic acid and acetic acids turning the mi1k sour. The importance of microbiology to the dairy industry has been demonstrated by foodborne illness associated with consumption of milk and dairy products that had been contaminated with pathogenic organisms or toxins. Undesirable microorganisms constitute the primary hazard to safety, quality, and wholesomeness of milk and dairy foods. Consequently, increased emphasis has been placed on the microbiological analysis of milk and dairy products designed to evaluate quality and to ensure safety and regulatory compliance.

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The focus of dairy microbiology, there are three microbiological analysis methods that can be carried out to standardize milk. 1. 2. 3. Total colony count E.coli type count Methylene blue dye reduction test

3.1Dye reduction test for milk Introduction Dye reduction methods depend on the ability of microorganisms to alter the oxidation-reduction potential of the medium. They are in consequence a measure of the activity of microorganisms in the test system rather than of the numbers in the sample. Suitable indicator dyes include methylene blue and resazurin. The length of time taken to reduce the dye depends upon the mass and activity of bacteria present in the sample. The greater the number present shorter the time required for reduction. The methylene blue reduction test is based on the fact that the colour imparted to milk by the addition of the dye. Methylene blue will disappear more quickly. The removal of oxygen from milk and the formation of reducing substances during bacterial metabolism cause the colour to disappear. Methylene blue Materials Glass tubes with rubber stoppers Two milk samples Methylene blue Water bath Leuco methylene blue

Microbiological Examination of Dairy Products

Procedure
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10 ml from each milk sample were added to sterilized glass tubes and 1 ml of methylene blue was added. Sterile stoppers were fitted to those test tubes and gently mixed (do not shake). Then the tubes were placed in the water bath maintained at 36ºC in an inverted position for incubation. Tubes were covered to keep out of light. A blank tube was placed without methylene blue to compare the colour. Samples were checked for discoloration after 30 minutes of incubation. Readings were taken at hourly intervals. After each reading, tubes were inverted before placing in the water bath. Time taken for methylene blue discoloration was recorded. Results Milk Sample Raw Milk More than 8 hours Sterile milk Time taken for Decolourization <20 minutes

Discussion Fresh milk sample contained considerable amount of microorganisms whereas sterilized milk sample didn¶t contain such amount of microorganisms to reduce the colour. According to the data table we can say that there were 4 × 106 - 2× 107 microorganisms present in the fresh milk sample.

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3.2. Enumeration of bacterial numbers in milk sample by the plate counting Materials Sample Milk, Sterile pipettes (1.0ml) Sterile Petri dishes Sterile molten nutrient agar Procedure A dilution series of the sample to be tested was made by transferring aseptically 1.0ml of the homogenized sample to the tubes containing 9.0ml of sterilized Ringer¶s solution. Tubes were mixed well by rolling tubes between the palms of the hands to ensure even dispersal of cells in the mixture. 1.0ml of 10-3, 10-4, 10-5 dilution series was transferred to separate three Petri dishes and 15ml of molten agar was poured to each of them. Petri dishes were gently rotated on the table top clockwise and anticlockwise to ensure to ensure uniform distribution of the cells in the medium. Petri dishes were kept to solidify 15 to 20miniutes and they were incubated at 37 0C for 48 hrs. After the incubation period colonies were counted. Results and Readings Sterilized milk No of colonies:Not found in plates Raw milk Dilution which had 30 - 300 colonies Number of colonies per plate = = 10-5 107

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Calculation Dilution factor Number of colonies / 1ml of sample = = 10-5 107 × 105 1.07 x 107

Average Number of colonies/1ml of sample = Conclusion.

Given Sterilized milk sample is considerably lower number of Microorganisms or no viable microorganism found due to sterilization process. Estimated number of microorganisms in the 1ml of the given raw milk sample is 1.07 × 107 Discussion The microbiological diagnostic procedures used for assessing the overall quality and safety of the Dairy foods products and the standard plate count is one of most common technique use all over the world. By this method only viable cells are counted and it allows isolation of discrete colonies that can be subculture in to pure culture, which may be then easily studied and identified. In conclusion, all methods have limitations. One of the major limitations to the plate count method is the relatively narrow countable range (generally considered to be 25-250 CFU bacteria on a standard petri dish). The currently prevailing confusion between the Limit of Detection (1 CFU) and Limit of Quantification (25 CFU) for the plate count method creates a larger degree of variability in microbiology data than is necessary. In other way the major disadvantages of this method are; time consuming, more experience labour concern, relatively expensive when analyze a large number of samples. Also it is having overnight incubation and is not suitable for rapid assessment of bacterial count. The most of milk products are bearing short shelf life and the long testing procedure is not suitable for these products. If food must be held until testing is complete, distribution is delayed and the storage cost can add considerably to the cost of processing. It is necessary to use more glassware in this procedure. It needs for greater manipulation Mayresult in erroneous counts due to personal errors in dilution or plating. Some pathogens do

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not grow under conditions provided. (Ex: Mycobacterium tuberculosis) and this method not suitable to detect pathogenic bacteria count. The actual number of bacteria obtained by the plate method does not represent because of various biological factors. The air supply and the temperature are not suitable for all types of bacteria. Therefore most of the time value obtained from the plate count is lower than actual number of viable bacteria presence in the product.

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