COMPANY PROFILEDairy was stared in 1977 with a Handling capacity of 1.0 LLPD Under BSDC in operation FLOOD-1 Management of the Dairy handed over to NDDB on 01.10.1981 Management handed over to VPDUSS Ltd, Patna by NDDB on 01.07.1988. Plant capacity expanded from 1.0 LLPD to 1.5 LLPD in operation Flood-II in 1994. BACKGROUND Patna was one of the milk shed identified under operation Floo1 for Implementation of programme. A100, 000 It/day capacity Feeder Balancing Dairy (FBD) and 100MT / day capacity Feed plant (CFP) were set up under this programme. In order to implement the programme properly and also organizing the milk procurement activity and for the management of above two plants. The Bihar State Dairy Corporation (BSDC) was found in the year 1972.The Dairy Corporation was to develop the dairy corporation both at the village level and milk shed level on “Anand Pattern” and it was expected that the milk shed level co-operation would take over the entire infrastructure created in due course. The co- operation after recruitment and training of necessary staff positioned the procurement and input wing from 1975. A spear head team (SHT) was deputed from National Dairy Development Board (NDDB) from the same year for helping the co-operation in organizing and developing the co-operation. Through the progress could not achieve the goals for which it was established. Subsequently, the stable government felt it necessary to request the NDDB for taking over the infrastructure created on management basis. The NDDB took over the management of the infrastructure with effect from first October 1981 under the banner of Patna Dairy Project (PDP). Progress of Patna Dairy Project The National Dairy Development Board (NDDB) immediately after Taking over the project positioned an integrated Spear Head Team (SHT) to restructure the milk procurement activities and also for steam lining the Working of the Feeder Balancing dairy (FBD) and Capacity Feed plan(CFP) under the management of national Dairy Development Board (NDDB) the project has not only made excellent progress but had been able to establish the fact that the co-operative could function equally well in Bihar to and what is essential in proper atmosphere and guidance. Along with the organization of milk procurement activities and Management of both t he plants on commercial lines, the National Dairy Development Board (NDDB) took special care to develop the Vaishal Patliputra Dugha Utpadak Sahkari Sangh Limited (VPDUSS), the milk shedLevel co-operative for taking over the project once the dairy board withdrawsIt management. National Dairy Develoment Board (NDDB) handed overThe Patna Dairy project (PDP) to vaishal patliputra Dugha UtpadakSahkari Sangh Limited (VPDUSS) with effect from first July 1988. Feeder Balancing Dairy The Feeder Balancing Dairy (FBD) with a capacity to handle 1.5Lakhs litre per day has facilities for manufacture of milk powder, butter, ghee, ice cream, peda, paneer and plain / misti dahi and lassi. The production and marketing of table butter the brand name “SUDHA” was introduced from 1st October 1993 and the response has been encouraging. The marketing of “SUDHA” brand of ice cream in Patna after test marketing in August- September 1994 was formally launched from April 1995. The initial response has been more than satisfactory. The marketing of ‘SUDHA’ brand plain / misti dahi in Patna was started in October – November 2001 and was formally launched from 2001.The initial response for this product too has been overwhelming. Cattle Feed Plant The role of balanced feed is not only increasing milk production but also sustaining the same by ensuring regular conception need not be cover Emphasized. Realizing the same union has been making consistent efforts for popularizing the consumption of balance feed by the milk producers. In addition to catering the needs of the dairy co- operation societies Cattle feed is sold through dealers in rest of the state for beter capacity Utilization of the plant. Further realizing the importance of introduction of Latest technologies in the field, the production and sale of by pass protein Feed was started from the year 1989-90. The response for this feed is encouraging. MILK MARKETING The marketing of liquid milk in sachet was introduced from the year1981 it’s off. However initially the thrust was for organizing the milk Procurement activities to stabilize the same at reasonable level. Nevertheless there was some natural growth in the milk marketing over the years. However for various reasons there was some stagnation for few years in the quantity of milk marketed with certain modifications in the policy decisions and because of the concerted efforts, the quantum of milk being marketed steadily growing. QUALITY AND PRODUCTIVITY ACTIVITIES The Dairy plant Management programme (DPMP) was introduced in the year 1992 followed by Quality Assurance Programme (QAP) in the year 1993 with the help of National Dairy Development Board (NDDB). This Resulted in bringing about a positive change leading to viability of the project coupled with lowering of operational costs on one hand and improved quality of production on the other. Consequent to the liberalization and globalization of Indian economy in early 90’s it was felt that the Organization should strive to make its total outlook approach and system of highest standards. Accordingly it was decided in the year 2001 that the organization should go in for ISO certification both in quality management System and food safety. This process was successfully completed leading to ISO 9001-2000 and HACCP (15-15000_ certification by Bureau of Indian Standard, in March 2002. THRUST AREAS 1. To make SUDHA Board, a market leader by making Sudha milk and Milk product consumer delight and ensuring that the esteemed customers get value for money. 2. Consolidation of DCSs already organized leading to increase milk Prodocurement. 3. Further improvement in the involvement / participation of members in their co- operatitives. 4. Bridging the flush / clean gap further. 5. Popularization all the input programme. 6. Increasing the through puts and sale of milk and milk products as Well as cattle feed, by pass protein feed and UMB. 7. Reducing further the handling losses and increasing the utilization of plant capacities. 8. Optimizing the utilization of all consumables. 9. Human resource Development through training, orientation etc to the Employees at all level for ensuring better motivation for involvement leading to all round progress of the organization. OBJECTIVE OF THE PLANT The VPDUDD Ltd. is committed to continually and proactively Seeking to live up to the expectations and need of its esteemed milk produces and customers and delight them with quality products and services. It shall strive to delight its customers by making available high quality and safe food with innovative features. Its endeavor shall be to bring qualitative charge in the socioeconomic Status for its member producers by ensuring remunerative price for their Produce and offering quality products at competitive price to consumers through professional excellence. The milk union based on ANAND pattern shall achieve this novel goal through- a) Selecting appropriate, cost–effective and eco-friendly process and technology. b) Improving the sanitation in the city area by forcing the cattle out of The city area to rural area. c) Developing the dairying in Bihar to meet the nutritional requirement of the people either by supply fluid milk or by the product. d) Empowering its employees to excel in every sphere through continual skills, process and system. e) Providing vibrant work environment that result in excellence. f) Applying HACCP principles & Q.A activities in quality management system that results in production of sale food. Pathogens in Milk & Milk products 1. Introduction Milk is an excellent food and protective medium for pathogens, whose Growth depends mainly on temperature and competing microorganism and their metabolites. Several of them produce toxins, and many are spore formers. Their disease producing capacity depends upon the initial load of infection in the milk and on the subsequent dilution, processing, time lapse before the milk is consumed and other factors. Pathogenic microorganisms in milk are derived from the dairy animal itself, the human handler or the environment. One of the most important extraneous, sources of infection is a contaminated water supply. Insects, dirt, manure and poor sanitation are other pathogen in milk and its products. With due care in milk production and handling the modern processing facilities and good hygienic practices, pathogens can be controlled. There have, however, been reported cases of outbreak of Disease attributed to pathogens in milk products. It is therefore necessary to take maximum care to insure that the product is safe for consumption. This needs through understanding of products, process, equipment, environment and pathogens, among others. This issue of the techniques and next one, catalogue the important pathogens in milk with brief description of their Characteristics. THOGENS IN MILK & MILK PRODUCTS AND DISEASES CAUSED BY THEM. S.NO 1. NAME OF PATHOGEN Bacillus anthrasis DISEASE Skin infection 2. 3. 4 5. 6. 7. 8. 9. 10 11 12. 13 . Bacillus cereus Brucella abortus Camphylobacter jejuni Clostridium botulinum Clostridium Perfringes Coxiella burnetti Escherichia coli Listeria monocytogens Salmonella spp Shigella spp Staphylococcus aureus Mycobacterium tuberculosis Diarrhoeal illness Brucellosis Bacterial diarrhea Respiratory failure Gastroenteritis Q. fever Gastroenteritis, Colitis Meningitis & Abortion Enteric fever & Typhoid Dysentery Gastroenteritis Tuberculosis 1.Bacillus anthracis Characteristies (i)General Description Useally large motile,spore rod shaped, non- Formation capsulated. Gram positive bacteria (ii) Source (iii) Pathogenicity Human Cutaneous (Skin infection) Inhalation (affecting lungs) gastrointestinal forms of infection (Animals) (iv) Growth parameter # temperature (v) Shedding in milk 7°c to 49°c No any animal with Anthrax. Either ceases tolactate or gives Milk that is body. Yellowish.visibly abnormal Growth in Milk Associated Dairy foods (vi) Inactivation parameter No May be raw material Vigorous boiling for 2 to 3 minutes Elimination of infected additives from the food chin. Products from diseased and dying animals should be rejected animals should be rejected for human consumption .through cooking of animal products offers protection from vegetative cells of B. anthracis. 2. Bacillus cereus Characteristics Description Anthrax and Diseased animal, soil air Anthrax (a fatal disease) (i) General Source Rod shaped aerobic spore froming gram +ve bacteria Air, water, fodder. Food (ii) .soil.udder,milking equipment etc (iii) # humans Pathogenicity Food born illness (Infection) intoxication Diarroheal illness (resembles C. perfringens food poisoning # Infection Dose Food poisoning due to enterotzin Production at high population level (more than 106/g ) particularly in starchy food #Toxin Type Heat labile enterotoxin (produce in small intestine) Heat stable (120°c for 90°c miniutes) Enterotoxin (iv) Growth parameter 7°c to 49°c( mesophillic organism capble of growing at 7°c to 12°c ) 0.93 minimum 4.3 to 9.3 Shedding in Milk No Yes These organism are common contaminants Of raw milk and invariably present in pasteurized milk and product. Rapid growth Of vegetative cells during periods of temp. abuse is presumably responsible for high incidence if this organism in milk during summer months. Presence of B.cereus in powered milk probably Possess the greatest puplic health concern because both pasteurization and spray-drying induce germination and out growth od spores in the reconstituted products. B.Cereus can give rise to “bitty” cream and sweet curdling in pasteurized milk. Due to the spores # Temperate #Water Activity #PH (v) #Growth in Milk # Associated Dairy Foods uriving the pasteurization treatment and the vegetative cells. Spores of B. cereus can spoil UHT milk (vi)Inactivation parameters Heat sterilization done in an autoclave or by ultra high treatment, is enough to reduce. B. cereus spore population to a level to ensure no public health risk and microbiogical stability Of the product Increase in pasteurization temp. above 78°c reduces the shelf life of milk due to Activation of spores of B cereus (vi) Control measures Widespread occurrence of B.cereus in the nature environment ensure its continuous recovery from milk and other dairy products during all stages of production .However dairy related outbreak of B.cereus poisoning are readily prevented by sanitary handling. Prevented by sanitary handling. Minimizing contamination of raw milk at the BACTERIOLOGICAL STANDARDS OF MILK & MILK PRODUCTS. (as per PFA, effective from Dec.2005) PASTEURIZED MILK PARTICULARS SPC Coliforms E.Coil STANDARDS Not more than 30000/gm Absent in 0.1 ml. Absent in 1 gm Salmonella Shigella Staphylococci aureus Yeast & Molds Anaerobic spore formers Listeria monocytogenes Absent in 25gm Absent in 25gm Absent in 1 gm Absent in 1gm Absent in 1gm Absent in 1 gm PASTEURIZED FLAVOURED MILK PARTICULARS SPC Coliforms E.Coil Salmonella Shigella Staphylococci aureus STANDARDS Not more than 30000/gm Absent in 0.1 ml. Absent in 1 gm Absent in 25gm Absent in 25gm Absent in 1gm YEAST & Molds Anaerobic spore formers Listeria monocytogenes Absent in 1 gm Absent in 1gm Absent in 1gm Milk power PARTICULARS SPC Coliforms E.Coil Salmonella Shigella STANDARDS Not more than 50000/gm Absent in 0.1 ml. Absent in 1 gm Absent in 25gm Absent in 25gm Staphylococci aureus Yeast & Molds Anaerobic spore formers Listeria monocytogenes Absent in 0.1 gm Absent in 1gm Absent in 1gm Absent in 1 gm DAHI PARTICULARS SPC Coliforms E.Coil Salmonella Shigella Staphylococci aureus Yeast & Molds Anaerobic spore formers Listeria monocytogenes STANDARDS Not available Absent in 10 gm Absent in 1 gm Absent in 25gm Absent in 25gm Not more than 100/gm Not more than 100/gm Absent in 1gm Absent in 1 gm GHEE PARTICULARS SPC Coliforms E.Coil Salmonella Shigella Staphylococci aureus STANDARDS Not more than 5000/gm Absent in 0.1 ml. Absent in 1 gm Absent in 25gm Absent in 25gm Absent in 1 gm Yeast & Molds Anaerobic spore formers Listeria monocytogenes Absent in 1gm Absent in 1gm Absent in 1 gm ICE-CREAM PARTICULARS SPC Coliforms E.Coil Salmonella Shigella Staphylococci aureus Yeast & Molds Anaerobic spore formers Listeria monocytogenes STANDARDS Not more than 250000/gm Not more than10/gm Absent in 1 gm Absent in 25gm Absent in 25gm Absent in 1 gm Absent in 1gm Absent in 1gm Absent in 1 gm PANEER PARTICULARS SPC Coliforms E.Coil Salmonella Shigella Staphylococci aureus Yeast & Molds Anaerobic spore formers Listeria monocytogenes STANDARDS Not more than 5000/gm Not more than 90/gm Absent in 1 gm Absent in 25gm Absent in 25gm Not more than 100/gm Not more than 250/gm Absent in 1gm Absent in 1 gm ICE-CREAM MIX PARTICULARS SPC Coliforms E.Coil Salmonella Shigella Staphylococci aureus Yeast & Molds Anaerobic spore formers Listeria monocytogenes STANDARDS Not more than 25000/gm Not more than 10/gm Absent in 1 gm Absent in 25gm Absent in 25gm Absent in 1 gm Absent in 1gm Absent in 1gm Absent in 1 gm ICE-CANDY PARTICULARS SPC Coliforms E.Coil Salmonella Shigella Staphylococci aureus Yeast & Molds STANDARDS Not more than 25000/gm Not more than 10/gm Absent in 1 gm Absent in 25gm Absent in 25gm Absent in 1 gm Absent in 1gm Anaerobic spore formers Listeria monocytogenes Absent in 1gm Absent in 1 gm DRINKING WATER STANDARD AS PER IS:10500 (Mg/Lit) PARAMETERS Aluminum Arsenic Cadmium Chloride Chlorine Chlorine(total) Cobalt Coliforms (total/ml) Colour Copper Cyanide BIS LIMIT (Mg/ lit) 0.03 0.05 1.00 75.0 250 0.20 0.05 0.00 10.0m 5.00 0.05 Hardness Hydrogen peroxide Lead (Pb) Magnesium (Mg) Manganese(Mn) Mercury(Hg) pH phenols Selenium TDS Sulphate Surfactants Turbidity(non-microbial) 1.00 300 0.30 30.0 0.10 0.001 6.5- 8.5 0.001 0.05 500 200 0.2 5 NTU LACTOMETER PRINCIPLE: A lactometer is used to find out the amount of water in the milk. It works on the principal of specific gravity of milk. It consists of a test tube and a meter bulb. Toned milk Double toned milk Standardized milk Sudha Gold At 27c lactometer reading 29c lactometer reading CLR = = = = = = = = 8.5% 9.0% 8.5% 9.0% 30 28 Lactometer reading CLR / 4 x.2 x fat +.66 = SNF Total Solid Moisture content = 28/4x.2x54.6x0.66 7x1.78 = 8.78% = Fat +SNF = 5.6 + 8.78 = 14.38 = 100-14.38 = 85.62% Gram Staining of Plain Dahi &Misti Dahi Procedure:Prepare Smear on a glass slide ↓ Heat fix using a spirit lamp Bacterial cell fixed on slide ↓ put a drop of crystal violet and wait For 1 miniute Remove excess stain with tap water ↓ Bacterial cell are stained ↓ Stain with gram’s iodine and wait For 1 minute Remove excess stain with tap water ↓ Decolourize the stain with 95% ethanol ↓ put 1 drop of saffraning and wait For 1miniute Put 1 drop of saffranine and wait for 1 mimiute ↓ Wash with tap water. Result:Plain Dahi- Gram +ve coccous Misti Dahi- Gram + ve coccous and some are diplococcus. Aim: Methylene blue reduction test for microbial contamination of milk. Requirement: test tubes, Test tube stand, pipette, Milk sample (Raw milk, pasteurized sudha milk), Methylene blue solution. Principal: Methylene blue reduction test is done to judge the shelf life of milk, to now the probable quality of milk, to know the sanitary condition of lant.herincipal behind is that a milk sample that contains a large population of actively metabolizing microorganisms will contain a markedly decreased concentration of dissolved oxygen because of growth of organisms. In other words, the oxidation-reduction potential of the sample is greatly lowered. The dye MB a redox indictor, loses its color in an anaerobic environment & is said to be reduced. The Methylene blue reductase said to be reduced. The ethylene blue test is designed to screen the quality of milk, which may contain large population of enteric organisms & streptococcus lactis, which are potent reducer of dye. The speed at which Reduction occurs following addition of ethylene blue to a sample of milk indicates quality of milk. This determination id made as follows; 1. 2. 3. 4. Reduction within 30 minutes-very poor quality. Reduction occurring within 30 mins to 2 hrs-poor quality of milk Reduction occurring between 2& 6hrs- fair quality of milk Reduction occurring between 6to 8 hrs- good quality of milk. Procedure: Methylene blue solution was prepared. The temperature of water bath was fixed at 37°c 10ml of each 2 sample of milk (Raw milk, pasteurized milk) were poured in test tube. Then 1 ml of Methylene blue solution was added into the test tubes. The test tubes were incubated at 37°c in water bath & observation was taken Observation: Raw milk: decolorized (blue to white) within 30 mins. Pasteurized milk: decolorized after 6 hrs. Result Methylene blue Reduction time 0 to 30 mins Milk sample Classification of Approximate Milk numberof bacterial/ml Raw milk Verypoor quality >20,000 361 to 480 mins Pasteurized milk Good quality <500,000 Conclusion: from above observation it can be conclude raw milk was poor quality of milk because microbial load in that milk was more, as it was not boiled as it decolorized after 6hrs Microbial load was least in this milk. Precaution: • Methylene blue was prepared carefully. • Proper aseptic condition were made (sterilized tubes, sterilized rubber, pipette should be used.) • Temperature of water bath should be maintained properly as required & test tube should be appropriately labeled. Aim: To detect the antibiotic residue in milk. Requirement: Bromocresol purple agar embedded with Bacillus Stereothermophilic var. calid ollactis, raw milk, 1 nutrient table, and pipette. Pricipal: The Delvo test is one of the tests used to detect the presence drugresidue in milk. The principle of the method involves germination growth of spores of specific bacteria (Bacillus Stereothermophilic var. calidollactis,) embedded in agar upon the addition of nutrients & following incubation at a specified temperature. If milk is free of inhibitory substances the growth of these spores produces acid. Which changes the colour of the pH indicator (bromocresol purple) in agar from purple to yellow? However, if milk contains certain inhibitory subsequently the acid production by the bacteria the medium remains purple in colour. Procedure: Ml of milk is added to bromocresol purple agar embedded with B Stereothermophilic. • 1 nutrient tablet is added with forceps. • Leaved foe 4hrs at 64.5°c in water bath as it is a growth temperature essential for these bacteria. • Then after seeing the colours of tubs further observation is made. • Observation: As raw milk samples from 5 different places Islampur, Bidouli, Sukhmal, Sarojnagar, Bhadura were used. It was observed that bromocresol purple agar turned to yellow colour. Result : All samples from 5 different places give antibiotic –ve test. Conclusion: Thus, by seeing above observation we can say that, all samples are antibiotic free & it shows that as antibiotic free & it shows that as antibiotic is absent in milk samples it mean bacteria is surviving in it & able to convert lactose of media to lactic acid & colour turn ti yellow. Precaution: • Proper aseptic conditions were made • Water bath temperature is set accurately so that proper growth environment is given to bacteria. Aim : To check the cleaning efficiency of the tank / tanker / pipelines. Principal: proper sanitary condition of the plant is the most important step in a food processing industry. There must be proper cleaning of the tank, tankers & pipelines. Micro- orgaisms are very important in proper fermentation processes. Apart from a few microorganisms which are hazardous for human health. Therefore proper conditions should be maintained to inhibit or check the growth of microorganism. Raw material from different sources is collected in large tanks & tankers. Pasteurized are passed from one to another through various pipelines. SO, proper care should be taken to prevent post-pasteurization contamination of milk. Requirement: Cotton swab, dilution, nutrient agar, violet red agar media, sterilized pipettes, sterilized petriplates, colony counter etc. PROEEDURE:Preparation of diluents Stock solution- 34 gm potassium dehydrogen phosphate is dissolved in 1000 ml distilled water. 1.25 ml of the above solution was then diluted to make 1000 ml. The solution was sterilized at 121°c & 151b / inch2 pressure for 15-20 minutes. Preparation of decimal dilution 5 ml of dilution was poured on swab. It was sterilized at 121°c 151b/inch² pressure for 15 to 20 minutes. With the help of swab, microbial sample were obtained from different tanks, tankers pipelines. Inoculation & incubation 1ml of diluents was transferred to sterilized petriplates. 10ml of the melted cooled nutrient agar & violet red media was added to each petriplates. Each pert plate was rotated gently, immediately after addition of the media, for inform distribution of the organisms & allowed to solidify the agar. Second layer of violet red agar medium was poured after solidification for anaerobic conditions. All the plants were incubated in inverted position for 24-48 hrs at 37°c. Silo Cream tank Vat Tank Uncountable No growth Uncountable Uncountable 36× 45=1620 No growth No growth Uncountable 2× 45=90 No growth No growth Silo pipe Blank No growth Calculation & result: It has concluded that containers containing raw milk have uncountable bacterial colonies whereas containers in which pasteurized milk is kept free from any type of contamination. Therefore, we can say that sanitaryCondition of the plant is good. Precaution: • Aseptic condition should be maintained. • Media should be properly prepared. • All plates should be sterilized. EXPERIMENT Aim: To count the coliform of pasteurized milk Principle: The coliform group of bacteria comprises all aerobic, facultative anaerobic gram negative non sore forming rods able to ferment lactose with the production of acid & gas at 30°c, 35°c or 37°c within 48hrs. Common source of these organism‘s intestinal tract of warm blooded animals. Certain bacteria of non-feacal origin are also members of this group. Typically, three bacteria are classified in the genera Escherichia, Enterobacteria & Klebsiella. The presence of these coliforms in dairy products is suggestive of unsanitary conditions or practices during production, processing or storage of milk & milk products. The coliform estimates are performed on raw milk to determine the degree of contamination during milk production. While the tests on pasteurized milk are referred to detect post pasteurization contamination. Requirement: milk sample, sterile pipettes, sterile test tubes, sterile petri plates, autoclave, colony counter. Procedure: Preparation of diluents Stock solution -34 gm potassium dihyrogen phosphate is dissolved in 1000ml distilled water. 1.25ml of the above solution was then diluted to make 1000 ml .the solution was sterilized at 121°c & 151b/ inch² pressure for 15-20 minutes. Preparation of decimal dilution 1ml of the test sample was pipette & added to 9ml diluents. This primary dilution was shaked using mechanical shaker for 5 to 10 seconds. 1ml of this primary dilution was transferred into another tube containing 9 ml sterile diluents, contact between pipette & the diluents & mixed to obtain 10-1 diluents. Inoculation & incubation 1ml of diluents was transferred to sterilized petriplates. 10ml of the melted cooled nutrient agar & violet red agar media was added to each petriplates .Each petriplates were rotated gently, immediately after addition of the media, for inform distribution of the organism & allowed to solidify the agar. Second layer of violet red medium was poured after solidification for anaerobic conditions. All the plates were incubated in inverted position for 24-48 hrs at 37°c Observation: Bacterial colonies were observed on the plates, which were counted using colony counter of both the milk samples. 67,66 & 63 colonies on respective three plates, which give an average of 65. Therefore, CFU colony forming unit = no of colonies × 1 0 =65 ×10=650/ml Result: As the number of bacterial cells per ml is less than 30,000 therefore, milk is property pasteurized. Precaution: • Septic condition should be maintained. • Media should be properly prepared. • All plates should be sterilized. EXPERIMENT Aim: Alkaline phosphates test to check the efficiency of passsteurized milk. Requirement: Constant temperature, water bath maintained at 37.5 ± 0.5oc Pipettes, volumetric flask, measuring cylinder, test tubes with s toppers, milk samples (raw milk, pasteurized milk) Chemical reagents Buffer solution: 1.5 gm of sodium bicarbonate & 3.5 gm of sodium carbonate in distilled water & volume is made up to 1000ml. Buffer substrate solution: 0.15 gm of substrate (disodium p nitro phenyl phosphate) into a 100ml volumetric flask & volume is made up with buffer solution. Principal: Phosphates enzyme, at pH 9.5 & temperature 37°c spits the substrate, p nitro phenyl phosphate to give p nitro phenol, which is yellow colored in alkaline solution. Phosphatase present in milk is destroyed during pasteurization, Therefore, Phosphatase test is performed to determine the efficiency of milk in dairy plant. Procedure: 1ml of each 2 sample of milk (raw milk & pasteurized milk poured in test tubes. • Then 5 ml of buffer substrate solution is added into the test tubes. • • The test tube was incubated at 37°c in water bath & observation was made. Observation: Seeing the colour of milk made observation. If, colour changes to yellow then considered as positive test. If milk is pasteurized properly then colour of milk not change it remains white. First reading is made at 10mins & next at 30 mins. Raw milk: Raw milk became in a faction of second. Pasteurized milk: Remains white. Result: As raw milk’s colour changes to yellow colour it means phosphatase +ve test. Pasteurized milk gives –ve test. Conclusion: Thus, by observing the colour of milk sample, we can say that (pasteurized milk sample provided to us is properly processed, as Alkaline phosphates enzyme present in milk denatured at pasteurization temperature. Precaution: Aseptic condition solution should be maintained. Buffer substrate should not be exposed to direct sunlight. Standard plate count (SPC) of pasteurized milk (Smart, Shakti) Principal: The standard plant count (SPC0 is one of the oldest and most widely Used method for enumerating bacteria. Classically SPC procedures are used to determine the total plate count (TPC) or Aerobic plate count (APC) or total viable count (TVC).SPC is the standard, method to which other screening tests are compared. The methods involves preparation of the decimal dilution of the milk sample, transferring a know volume (generally 1ml) of the appropriate dilution of the sample into petri dishes adding a prescribed nutrient agar into each dish and incubating the dishes at a specified incubation period. After the incubation period, the colonies developed in each plate are counted and the total viable count is calculated using the dilution factor employed in the tests. Requirements: Milk sample (Smart, Shakti), sterile pipettes, sterile test tubes, sterile petridishes, autoclaves, colony counter. Procedure: Preparation of diluents Stock solution: 1. 1000 ml distill water in flask 2. Add 34gm potassium dihydrogen phosphate 3. 1.25 ml of the above solution was then diluted to make 1000ml 4. The solution was sterilized at 121°c and 151b/ inch2 pressure for 15-20 minutes. Preparation of decimal dilution 1ml of the test sample was pipette and added to 9ml diluents. This primary dilution was shaked using mechanical shaker for 5-10 seconds. 1ml of this primary dilution was transferred into another tube containing diluents, avoiding contact between pipette and the diluents, and mixed to obtain 10-2 dilution. Similarly 10-³ dilution was made adding 1ml of 10-² dilution to 9ml sterile diluents. Inoculation and incubation 1ml of 10-3 dilution was transferred to sterile labeled petriplates. 10ml of the melted, cooled agar medium was added to each pertriplates. Each petriplates was rotated gently, immediately after addition of the medium, for uniform distribution of the organism and allowed to solidify the agar. All the plants were incubated in inverted position for 48hours at 37°c Observation: Bacterial colonies were observed on the plates which were counted colony counter of both milk samples. 10, 15, and 18 colonies on plantes incubated with shakti 12, 16, and 20 colonies on plantes incubated with smart Calculation and result The number of bacteria per ml of the milk sample was calculated. Viable cell per ml=mean plant count x10³ Direct microscopie somatic cell count (DMSCC) Principle: The direct microscopic estimation of somatic cell count in milk is referred to as the DMSCC. The number of somatic cells in raw milk provides a measure of the presence and the extent of mastitis or certain other abnormal milk secretion. Direct microscope somatic cell count is the officially recognized procedure for confirming the somatic cell count in milk previously estimated by one of several screening tests. Test results are reported in actual counts of somatic cells per milliliter of milk. Requirement: milk sample, slides, Newman’s strain, distill water Procedure: 1. A square of 1cm2 areas was drawn on the slide ↓ 2. 0.1ml of milk sample was spread on that area, and was heat fixed ↓ 3. The slide was dipped in Newman’s stain for 20 minutes. ↓ 4. The slide was rinsed in water until all the surplus dye was washed off. 5. The slide was air-dried before examine the film under microscope. Calculation and expression of result: Compute somatic cell count as follows and express the result as direct microscopic somatic cell count (DMSCC) per ml of milk. DMSCC per ml= no. of somatic cells in a single strip x5333 Observation: S.N Sample collected No of cells observed DMSCC per ml 1 Bhagwanpur Nil 0 2 Bairichak 5 27775 3 Gangachak Nil 0 4 Daulatpur 6 33330 5 Bhadaura 3 16665 Fluorescence image of somatic cells before image processing Result: Milk collected from sudha dairy does not come from diseased animals. Precaution: 1. Milk sample should be fresh 2. Raw milk should be used 3. Excess of stain should be properly washed off. Air exposure of media on different section of the plant Principal: The atmosphere contains all the major group of microbes ranging from the algae to the viruses. The microbial flora of air is transient and variable. Air is not a medium in which microorganisms can grow but is a carries particulate matter, dust and droplets, which may be laden with microbes. The no, and kinds of microorganisms containing the air are determined by the source of contamination in the environment. Medium used for air exposure was made acidic for the proper growth of yeast and moulds and also to restrict the growth of bacteria. Each plate was exposed for a given period of time in different section of the plants such as paneer section, dahi section, ice –cream section, peda section. Requirements: PDA media, sterile Petri plates, tartaric acid etc. Procedure: 1. 10ml of the melted cooked PDA media was added to each Petri plate. ↓ 2. Plates were allowed to solidify ↓ 3. Plates were exposed to air for 5 minutes ↓ 4. All the plates were incubated in inverted position for 72 hours at room temp. Observation: Fungal colony observed on the plates. Numbers of yeast and molds colonies were counted. S.N 1 2 3 4 Sites Ice cream section Paneer room packing No of colonies Time of No of mold/ yeasts / molds exposure min m3 16 13 13 8 5mins 5mins 5mins 5mins 16x5=80 13x5=65 13x5=65 8x5=40 Peda packing room Dahi packing room Result: A large number of different molds and fungi were obtained at different section of plant. Precaution: Aseptic condition should be maintained. Media should be properly prepared. DETERMINATION OF PROTEN IN MILK BY FORMAL TITRATION (PYNE’S METHOD) PRINCIPLEWhen formaldehyde is added in milk which was previously titrated against Standard alkali to the end point of an indicator like phenolphthalein, it bind with the amino group of the milk protein and releases equivalent amount of protein and releases equivalent amount of protein which could be titrated against the alkali to the same end point. The amount of alkali used in second titration is the measure of the amino group originally present in the protein. APPARATUSPipette-10ml, 2ml,1ml Granduated burette. Erlenmeyer flask-100ml REAGENTNeutral formalin, saturated potassium oxalate solution, N/10 sodium hydrocide, phenolphthalein indicator PROCEDURE1. Take 10gm of milk into 100ml flask. 2. Add 5 drops of phenolphthalein indicator. 3. Add 0.4ml of saturated potassium oxalate and keep this solution undisturbed for 4-5 minutes. 4. Titrate the milk against the standard alkali solution to its end point. 5. Add 2 ml of neutral formalin and mix well. 6. Titrate against the standard alkali to the same end point as before. 7. Record the volume of alkali in second titration. ResultPercentage of protein=R*1.7 COMPOSITION OF MARKET MILK PER 100 gm S.N PARTICULERS UNITS TONED MILK STD MILK 4.60 DOUVLE TONED MILK 1.60 SUDHA GOLD MILK 1 FAT Grams 3.10 6.10 2 SNF Grams 8.60 8.6 9.10 9.10 3 PROTEIN Grams 3.20 3.20 3.55 3.55 4 LACTOSE Grams 4.70 4.70 4.80 4.80 5 MINERAL Grams 0.70 0.70 0.75 0.75 6 CALCIUM Mg 118 118 128 128 7 PHOSPHORUS Mg 90.0 90.0 96.0 96.0 8 WATER Grams 88.3 86.8 89.3 84.8 9 VITAMIN A I.U 150 200 75.0 200 10 CALORIES K cal 59.0 73.0 48.0 90.0 POISONOUS METAL LIMITATION METALS LIMITS (Maxm) REMARKS Lead ppm.lollies) 0.5ppm All milk /milk pdts. Maxm 1.0 Arsenic 0.1ppm milk. Maxm 0.5ppm Icecream / lollies) Copper 30ppm All milk / milk pdts. Zinc 50ppm All milk / milk pdts. Cadmium 1.5 ppm All milk/milk pdts. Mercury 1ppm Milk. Methyl mercury 0.25ppm All milk / milk pdts. MILK ENZYMES Nomenclature, classification general properties and salient characteristics of some important milk enzymes. An enzyme is a biological catalyst elaborated by the living cells of mammary tissue gain entrance into milk accidentally or unavoidably during the secretory process. Enzymes are proteins they are denatured or inactivated by high temperature process, a pH of optimum activity and exhibit specificity for certain substance. Milk contains a number of enzyme about 21 enzymes have been purified, isolated or definitely identified in bovine milk, milk enzymes play a vital role in assessing milk quality and shelf-life. Enzymes in milk have extensively utilized as yard-stick. For evaluation a variety of biological events operative in milk, these specific enzyme catalyzed a variety of biological events operative in milk, these specific enzyme catalyzed operations in milk have been summarized below. 1. Correlation with existence of pathogens. The enzymes in milk are heated sensitive. The presence of certain enzymes in milk is of great importance in relation to heat treatment e.g. Alkaline phosphates activity in milk has time honoured cheek for the degree of pasteurization. The thermal death point of Mycobacterium tuberculosis and the enzyme alkaline phosphates coincide with each other hence the activity of latter serves as an index for the presence of former in milk. 2. Correlation with spoilage. The enzyme lipase acts on milk fat, produces hydrolytic rancidity and thereby causes the spoilage of butterfat and products containing fat. 3. Correlation with under diseases. Increase in level of A. esterase, aldolase, and catalase are the established consequences of Udder disease and some other physiological disorder. Leucocytes count of milk has a correlation with the level of catalase in milk. 4. Correlation with flavor defects. Xanthine oxidase has been implicated in the oxidative degradation of dairy products resulting in flavor defects. 5. Correlation with bacterial action Lysozyme has antibacterial or immunological significance and is associated with keeping quality of milk. 6. Correlation with stability of emulsion. Ribonuclease may influence the stability of fat emulsion since it is associated with the microtonal component of the fat globule membrane. The principal enzymes present in milk are- 1. Aldolase EC 4.1.2.13 2. α Amylase EC 3.2.1.1 3. β Amylase EC 3.2.1.2 4. Carbonic anhydrase EC 4.2.1.1 5. Catalase EC 1.11.1.6 6. Cytochrome –c-reductase EC 1.6.99.3 7. Diapheraes EC 1.6.4.3 8. A-esterase EC 1.6.4.3 9. B-estrase EC 3.1.1.8 10. C-estrase EC 3.1.1.8 11. Lactose synthetase EC 2.4.1.22 12. Lipase EC 3.1.1.3 13. Ly sozyme E C 3.2.1.17 14. Phosphoprotein phosphalase E C 3.1.3.16 15. Phosphtase alkaline EC 3.1.23.1 16. Peroxidase EC 3.4.4 17. Protease EC 3.4.4 18. Rhodonase EC 2.8.1.1 19. Ribonucease EC 2.7.7.16 20. Solase 21. Xanthine oxidize EC 1.2.3.2 Other enzymes present in milk include. Sulphydril restuetase, sulphydryl oxidase, Oleinase, Transaminase, Sorbitol anhydrase, phosphohexose isomerase, β Glueuronidase, Flavo kinase etc. These enzymes depending upon their mode of action on milk Constituent’s physiological components can be categorized as follows. 1. Hydrolytic group of enzymes e.g. lipase, esterase and protease 2. Enzymes having physiological signification e.g. eatalase, lysozyme etc. 3. Enzymes associated with the microsomal particles of milk e.g. alkaline phosphates, xanthine oxidease, cytochrome –c- reductase etc. 4. Enzyme whose roles are yet to be defined confirmed e.g. ribonuelease, rhodonase, carbonic. Milk enzyme can be classified broadly according to the substrate they act upon carbohydrates, proteases act upon protein, lipase act upon lipids and so forth. Their classification proposed by International union of biochemistry (IUB) However appears to be the one most widely used according to which enzyme are divided among six major classes. 1. Oxidoreduclases which catakyze oxidation or reduction reaction 2. Transterases which catalyze the transfer of specific chemical moiety. 3. Hydrolase Which hydrolyse substrate with concomitant uptake of water 4. Lyases which catalyze the addition of a group to double bonds or conversely removes groups from substrate leaving double bond. 5. Isomerases which cause isomerization. 1. Ligases [synthetase] Which catalyses the condensation [bonding together] of two Molecules coupled with the cleavage of pyrophosphate bond of ATP or similar triphosphate Each class is then divided into sub-class, each sub-class sub-subclass and finally each sub-subclass contain several enzymes. Or example the serial classification number the first digit 3 from the left represent the class of hydrolases. The second digit 1, represent sub-class of carboxylic ester hydrolyses and the final digit 3, represent the enzymes glycerol ester hydrolyses. It is believed that these enzymes are normal constituent of cell or tissue and during milking process concomitant with the cell rupture these enzymes are spilled into milk. It has been suggested that these enzymes are secreted in milk for the benefit of the young having rather underdeveloped or in compete digestive system. Milk is not a homogenous solution of enzymes. Enzymes occur in milk in four distinct phases. 1. 2. 3. 4. Water soluble Associated with cream or lipid Bound to easein Enzymes present in microsomal particles. A brief account of milk enzymes present in SUDHA DAIRY: 1. ALDOLASE- A glycolytic enzyme associated in the sequence of reaction involved in the metabolism of carbohydrates i.e. hydrolyses fruclose-1, 6-diphosphate into dihydroxyacetone and phodphoglyceric aldehyde milk exhibit significant aldolase activity serum. The enzymes are associated with the fat globules, on separation isconeentrated in cream in cream layer. Aldolase is rather unstable in milk, the activity decrease rapidly at 37° and 45°c. . 2. AMYLASE- This enzymes catalyses the hydrolysis of α-1,4-D glycosidic linkages in starch and glycogens α-amylase content of milk is very low but high in mastitis milk. Α-amylase is inactivated by the pasteurization temperature but β-amylase is fairly heat resistant, α-amylase is considered normal constituents of cows milk white β-amylase is only in some milk. 3. CARBONIC ANHYDRASE- This enzyme reversibly catalyzes the hydration of CO2 & dehydration of carbonic acid. Carbonic anhydrase CO2+H2O → Η 2CO2 This enzyme is a Zine containing protein 4 CATALASE- In catalyzes decomposition of H2O2 into water and oxygen. 2H2O2 catalase → 2H2O+O2 Its concentration in milk is directly proportional to lencoyle eoant catalases content of colostrums and mastitis milk is significantly higher then that in normal milk and its appearance in milk is dependent upon the physiological condition of the animal. For this reason the Catalase content of milk has been proposed as a means of detecting mastitis, catalases activity and particularly with feed. Catalase activity is present in both cream & shine and its coprecipitation with cascin have also been reported like from other sources. Catalase of milk also appears to be a haemato protein .Heating at 60-75 for 30 minuts destroys the catalase and its activity is greatly impaired if it is heated for the same length of time at some what lower temperature. 1. CYTOCHROME-C-REDUCTASE- The enzyme appears to be concentrated in milk microsomes its activity is measured spectrophotometically. The rate of reduction of Cytochrome C 2. DIAPHORASE- This enzyme catalyses the hydrogenation of lipoamide in the presence of a hydrogen acceptation. Its activity is measured by following the reduction of 2,6- dichlorophenol indophenols. In the enzyme appears to be associated with microsomes. 3. ESTERASES- the term esterase embraces a variety of enzymes which catslyse the hydrolysis of esters. Although lipase is also an esterase its activity. Generally is regarded to be confined to glycerol esters. Three esterase namely A, B and C have reported. A esterase (aryl esterase) is a typical esterase which hydrolyzes phenyl acetate at a higher rate than phenyl butyrate. Aliphatic esters normally are attacked. B esterase (carboxy or glycerol ester hydrolyser) hydrolyzes aliphatic and aromatic esters but not the choline esters. It is sensitive to organophosphates but not to serine. C esterase (Choline) splits esters more rapidly than aromatic and aliphatic esters. It is sensitive to organospates and serine. A & C esterases activity is high in colestrum and she formers. A esterase activity of milk appears to be related to mastitis. B esterase contains glycerol hydrolase also lactase synthetase.It eatalyzes the synthesis of lactose from UDP galaelose and α-D glucose. This enzyme originates from the microbes of the mammary gland cell. Brodbeek and Ebner (1966) while purifying this enzyme from bovine milk demonstrated it resolve into two fraction designated as A and B neither fraction was active as enzyme, but gained the activity when the two fraction combined together. Fraction A and B were identied as galactosy transferase and α facalbumin. The galtosy transferase is found in much body tissue but α laealbumin only in lactating mammary gland. This protein modifies he galactosy transferees in such a manner that it transfers galactose to glucose only and hence the synthesis of lactose in mammary gland. 4. LIPASEE- It catalyzes hydrolysis of glycerol esters (fat & oils) in emulsion several workers have isolated many lipases each differing from each other in many respects, in milk there are two forms of lipases Viz. a) Plasma lipase: associated with casein and requires activating treatment like homogenization, agitation or foaming before it produces raneidity. b) Membrane lipase: adsorbed on the fat globule membrane which is catalyzed by cooling of milk. Purified lipase possesses a single pH optimum and a temperature optimum of 37°c. It is unstable and highly sensitive towards light, heat and serval reagents. Various milk constituents have a profound effect upon its activity. Saltsand casein being inhibitory, the observed lipase activity in a complete milk system may be the net result of the inhibitory action of various milk constituents. Milk lipase has drawn particularly and preferential attention over other enzymes in milk because of its established role in the development of flavor in milk. Milk processing and storange invole lipase action with a resultant production of undesirable rancid, flavor. This enzyme is responsible for inducing altreration; however it is a notorious enzyme in regard to its hydrolytic rancidity production in milk and milk products. 1. LYSOZYME – Milk of large number of species contain lysozyme and human milk is the richest source. Bowing milk contain 13µg of lysozyme per 100 ml while human milk contain 36 µg/ ml. the enzyme from bowing and human milk differs considerably in their physiochemical properties. The isoeletric point of bovine and human milk lysozymes are pH 9.5 and 11.0 with optimum ph for activity at 7.9 and 6.85 respectively. This enzyme has specific behavior or lying certain bacteria such function is achieved by the hydrolysis of the β-(1-4) linkagae between acetyl glucosamine and n-acetyl muramic presenting polymer from in its probable role described as associated with a) Natural antibacterial factor in fresh milk. b) In relation to infant nutrition. c) The mechanism of natural immunity d) Keeping quality of milk It is remarkably stable to heat (human milk lysozyme 1 more heat sensitive particularly at acid pH . It is relatively a small protein with a milexular weight approximately 18,000 which is slightly higher than that of egg white lysozyme. 1. PEROXIDASE- It catalyzes decomposition of H.O in the presence of an oxidizable compound or hydrogen donor milk peroxidase often referred to as lactoperoxidase perhaps the first enzyme reported in milk. Milk is one of the best sources of peroxidase. It represents as much as 1% of the total serum protein of the milk It appears to be associated with albumin or when protein compound of milk. This enzyme is more heat stable when compared with other enzyme in milk. It exhibit regeneration properly when inactivated by metod. This enzyme can be used to detect 11.0 when added as a preservative in milk. It is a home protein with a content of about 0.07%. 11 PHOSPHATASE- This class of enzyme hydrolyzes phosphoric acid esters A large number of phosphor of phosphates exists in nature such as phosphorous esterase phosphoric – esterase’s, phosphorylases, pyrophosphatases, phosphoprotein phosphatases etc. Milk contains several phosphatases but most of the work done has been on two phosphatases. Alkaline Phosphatase: It is a phosphomonoesterase with a pH optimum of 9.6. Its activity in normal cow’s milk vaties considerably.The relative distribution of the enzyme in milk fraction is established. About 30-40 present of the enzyme is convent rated in cream where it is adsorbed on the fat globule as microtonal particles, the balance is (60-70) present) is distributed throughout. The skim milk probably in the liproteins particles. It is a native enzyme of milk and plays an important role in energy transfer mechanism of living brings. The complete loss of activity of this enzyme synchronizes with pasteurization temperature of milk. Therefore its activity is eidely used as yard- stick of pasteurization efficiency of a milk processing plant. The well known phosphatase test is based on this characteristic. The main substrates used for assessing its activity include disodium phenyl phosphate,p-nitrophenyl phosphate phenolphthalein phosphate etc. The enzyme exhibits the characteristic of reappearing or becoming activated in heated milk especially in HTST processes. Acid phosphates: Its concentration is low in milk its optimum pH is 4.0. It is unstable when exposed to sunlight or UV radiation but it is very heat resistance requiring 96°cFor 5 minute for its complete destruction. PROTEASE- It catalyzes hydrolysis of the peptide linkages of protein to small fragments. Milk protease acts in slightly alkaline medium; its action is retarded in acidic- medium. Its optimal activity is at pH 8.5. It is inactivated by heat at 75-80°C and in acid medium it is destroyed at 72° For 10 minutes. Its activity is not likely to have any serious effect in milk processing however while preparing evaporated milk by HTST method reactivation of protease is possible it may however not be a milk protease. RIBONUCLEASE- Bovine milk contain 25mg / 1 of this enzyme, which catalyzes the hydrolysis of ribonucleic acid like ribonuclese A from bovine pancrease, milk ribonuclease is also a basic low molecular weight protein. It exists primarily in whey. It is believed to be related with the immunological characteristic. SALOLASE- It catalyzes the hydrolysis of phenyl salicylate. It has also been detected in bovine mammary gland.. XANTHINE OXIDASE- It is rather non- specific, enzyme since it Catalyzes the oxidation of purines, phyrimidines aldehyde. Xanthine oxidase is a prominent enzyme in milk .It is present in the milk of mare and human. It is part of microsomal particles located on fat globule Membrane.It is a metallo –flavor protein containing molybdenurm and iron as the metals in the ratio of 1:2: (1.3+.5):8 as Protein: FAD: MO”Fe. The enzyme has been implicated in the oxidative deterioration of milk fat is another characteristic feature of this enzyme. It appears to be activated by variation in temperature treatment such as cooling, heating, agiation and various chemical agents such as detergents. Heat sensitivity of this enzyme increases on storage homogenization and treatment with proteolytic and lipolytic enzyme. CONTROLPOSTPROCESS CONTAMINATION OF MILK INTRODUCTION Raw milk received in dairy plant contain bacteria mostly from the contamination during its handling and bacterial growth. Milk is therefore pasteurized as soon as possible after milking in which processes not of other bacteria are killed .This makes milk safe for human consumption and Increases its shelf life. However proper processing of milk alone is not adequate to ensure the safety and quality of final products, all the bulk in the subsequent “milk chain” need to be property manage. In the absence of this, a dairy plant runs the risk of unsafe and a poor quality product with attendant disastrous results. Consider for example the following incidences:• There was an outbreak of staphylococcal entertain poisoning due to Contaminated processed milk of one dairy company in Japan in 2000 Over 11000 people were affected with 165 requiring hospitalization. Infection was traced to one plant where a single value in the production line had been inadequately cleared. The company suffered a loss of over 125500 crores. • In the U.S.A, 29 outbreak were recorded between 1995 and 1997 Related to dairy product of which 34% were caused by contaminated milk. A Salmonella spp. was isolated in outbreak and one each due to Campylobacter and Listeria. A market survey of processed milk of 10 brands in India in 1999 Reported high level of E. coli in milk of several brand. In an incident in U.S.A in 1985, 16000 persons suffered salmonella due to drinking of pasteurized low fat milk of 2 brands. The milk was found to be contaminated with Salmonella typhinurium. The contamination of milk occurred due to cross contamination with raw milk in the pasteurizer. • • There are only a few example many more have been reported and many have remained unreported. Inadequately processed milk or recontamination caused poor quality and unsafe milk, which cost the dairies heavy economic losses and dairies of reputation. Reason od low quality and unsafe processed milk include inadequate pasteurization process or post pasteurizations process contamination .In most of cases of unsafe pasteurized milk the reason found has been post Process contamination (PPC). Therefore while it is important to ensure proper pasteurization of milk, it is equally important to take all precaution to avoid PPC. Its major reason of PPC and their control measure. COMMON RECONTAMINATING MICRO-ORGANISM Pasteurized milk has been responded to be contaminated with several types of spoilage causing and / or pathogenic micro-organism. Some microbial hazards identified in pasteurized milk Campylobacter jejuni, Listeria monocytogenes, Salmonella spp Staphylococcal entertoxin,yresinia entercolitica, shiga toxin producting E. coli (STEC). Gram negative (psychrotroph) Pseudomonades Bacillus cereus Recontamination of pasteurized milk gram- negative psychotropic bacteria (GNP), responsible for spoilage of milk has been reported to occur in filluing step. Pseudomonades have been found to be the most frequently occurring bacteria in refrigerated pasteurized milk. Gram- positive spore (GPS), such as Bacillus cereus spores, also recontamination processed milk and are sometimes alone responsible for its spoilage. The contamination site could be dead ends, pockets and traps where bacteria in case the CTP system are ineffective. Enter pathogenic E.coli and salmonella have been reported to be present in pasteurized milk due to recontamination. Likewise campylobacter spp, staphylococcus aureus and Listeria monocytogenes have been found in recontaminated milk. POST PROCESS CONTAMINATION ROUTES Milk may be contaminated via a myriad of contact surface of processing and packaging equipment and plant environments. Milk residues on inadequately cleaned surface; tanks, pipes and valves can support the Survival and growth of microbial contaminants. Spores of Bacillus cereus are very hydrophobic and will attach to the equipment surfaces where they may germination and form biofilm at sites that are difficult to clean. Contamination of milk by Bacillus cereus has been demonstrated in silos, Pasteurizer, milk pipelines with bad welding and packaging machines. Very often recontamination has been found to occur in process by the rinsing water inside and allows milk to come to contact with the surrounding air and with its aerosois. Condensed water on the equipment may also find its way into the milk and packaging material might be contaminated. Recontamination of milk could take place at various sites in equipment and pipe work such as dead ends, pockets and traps and mixing processed milk receiving back from stores. Outer Environment ↓ Plant Environment Pasteurizer Packaging machine Pasteurised Personnel Packaging film Raw Milk Modern pasteurizers are often complex and although efficient these do present possibilities of cross contamination of milk, plant treated cooling milk is passed through regeneration and cooling water section where it is separated by relatively thin plates from raw milk and chilled water, Respectively. Should any of these is a signification risk of recontamination of milk with pathogen or spoilage micro- organism. Further, the cleaning in place (CIP) lines and other associated lines in the pasteurizer might, if incorrectly designed or installed, allow raw milk to by pass the pasteurizer completely. PRECAUTIONS AGAINT RECONTAMINATION Prevention of recontamination of pasteurized milk is of major importance in Production of pasteurized milk that is both safe and of satisfactory shelf- life. Some control measures against contamination of milk with specific pathogens are listed below. However pasteurizers and filling machines are usually designed and constructed to minimize the possibility of the pasteurized product being contaminated, however adequate precaution are required to ensure that post process contamination does not take place. Some important precautions are elaborated below:1)SOURCE: plant environment PATHWAY:- Usually indirect via contamination of equipment. Also possible via personnel and packaging. PRECAUTION:- Eliminate contamination of pasteurized milk side of regeneration by leakage etc. Correct environmental sanitation. 2)SOURCE:- Equipment ROUTE: - Direct as following:i) Contamination of equipment by raw milk, coolant leaked in refrigeration or cooling section, respectively, due to gasket failure, split or pin hones in pasteurizer plates improper pipelines / valves arrangement. ii) Contamination by stagnant milk deposits at dead ends, values gaskets etc. iii) Inadequate CIP and manual cleaning where necessary. PRECAUTIONS:Vent interspaces between seals to atmosphere to provide an ingredient visual in designs of gasket failure. • Maintain a positive pressure balance at least 0.5 bar (0.5kg /sq.cm), between pasteurized milk and raw milk in the regeneration section. • Ensure correct positioning of flow diverter and associated pipe work to avoid contamination of flow resumes after diversion. • Restrict operating period to 8 hrs. • Testing for corrosion cracks and pinholes by a lithium injection test twice a year. • Visual inspection every day for backpressure control. • Milk contract surface of pasteurizing plant should be fabricated from high grade stainless steel and polished by electro-polishing to avoid crevices and Consequent entrapment of soil, hands etc. should be insured to the highest possible standard. • Follow suitable cleaning and sanitizing programmers for pasteurizer, storage tanks, silos etc. • Extra cleaning in case of more than 3days between processing runs. • Dismantling and inspection every 5 years. SOURCR:- Raw milk PATHWAY: Direct or indirect via contamination of plant environment, passive transfer or hands of personnel etc. PRECAUTIONS I) Correct design of equipment and related pipe work II) III) IV) Correct operation and maintenance of pasteurizer Correct plant layout Control of personnel movement and avoidance of ‘hand on’ during operation involving milk or milk contact surfaces. 4) Source:- personnel ROUTE: i) Direct due to personnel suffering clinical illness or being chronic carrier of pathogens. ii)Indirect due to introduction to plant of contamination from outside environment. PRECAUTION:i) Follow appropriate medical and exclusion policies. ii) Ensure good personnel hygiene and correct use of protective clothing and footwear. iii) Prohibit raw products such as eggs being brought into the plant by farmer or worker for sale to fellow farmers. 5) SOURCE :-Packing ROUTE Failure to adequately sterilize the packaging film in the packing machine. II. Contamination of packaging from plant environment etc. I. PRECAUTIONS:I. Ensure correct functioning of required ultra-violet light system so that the packaging film is properly sterilized II. Protect packaging from contamination. III. Cleaning and disinfections of packaging machine and buffer storage tank according to conformed procedures. IV.Disinfection of specific machine part by alcohol spraying. V. Adherence to keep hygienic condition during filling. VI.Follow conformed procedure regarding start, stop and interruptions. VII.Remove first 20 packs. It is of particular importance to ensure that there are no cross contamination. It should be ensured that pipelines and valves cannot be arranged and / or fail in such a way that Pasteurized product or pasteurized products line could be contaminated. It is critical to ensure that the flow diversion value is correctly installed and operated so that under- processed Milk is not carried forward. The operation of the flow diversion should be checked each time plant is started up and the correct operation of recorded per assured. Recording thermometer should check daily against calibrated mercury in – glass thermometer and holding time validated annually. Unnecessary product handling steps between processing and filling should be avoided. In addition, the lines leading to the filler should be designed for efficient cleaning in place. Packaging machines the selves should be designed with emphasis on clean ability and potential for contaminations during filling. The location of the filler is also important .It is undesirable to place milk filler in the same room as an open cheese vat when the environment could contain significant bacterial load. It is a good practice if the filler is placed in a designated filler room that operates under specific white room guidelines i.e. filtered air –employed dress codes etc. The packaging materials should be stored in a clean and sanitary manner to avoid contamination. CONCLUSION It was really fun as well as a new experience for me to make this project. I felt much more informed and learned after completing this project. With my raid of variety of dairy products, Patna Dairy project (PDP) has Emerged as a unique name in the field of dairy industry. The quality of products, the plants are easily determined from the satisfaction of its loyal customers. For Patna Dairy plant, there is still a lot to be done and achieved. The way PDP has penetrated the lanes and by lanes of Patna and its suburbs reflects nothing but its success and commitment. My informants were very useful and it was really fun gathering information and doing experiments and various tests in bacteriolocal department.