Summary and Conclusion Milk and milk products are consumed daily by Indian citizens. This type of food is easily perishable and support growth of foodborne pathogens keeping this view in mind the investigation was focused on 1) Microbiological analysis 2) Chemical analysis 3) Effect of ph on pathogens 4) Effect of Nacl on pathogens 5) Effect of temperature on pathogens 6) Effect of seasonal vairiation on growth of microorganisms obtained from milk and milk products. Milk can also serve as potential vehicle for transmission of some diseases under certain circumstances. Moreover, by virtue of processing almost all the essential nutritional factors, milk can also serve as an excellent culture and protective medium for certain microorganism which may include potential pathogens capable of causing serious health problem to the consumers. In this way, milk may serve not only as vehicle of transmission of disease causing organisms but it can also allow there pathogen to grow multiply and produce certain toxic metabolites. Milk and milk products derive from dairy cow s milk can harbour a variety of microorganisms and can be important sources of food borne pathogens (Oliver et. al., 2005). Should the dairy industry be concerned about food safety? The answer is yes, and there are several good reasons why such as; bulk tank milk contains several food borne pathogens that cause human 296
diseases, outbreak of disease in humans have been traced to the consumption of raw unpasteurized milk and have also been traced back to pasteurized milk. Raw unpasteurized milk is consumed directly by dairy producers and their families, farm employees and their families, neighbors, etc. Raw unpasteurized milk is consumed directly by a much larger segment of the population via consumption of several types of cheeses including ethnic cheeses manufactured from unpasteurized raw milk, entry of food borne pathogens via contaminated raw milk into dairy food processing plants can lead to persistence of these pathogens in biofilms and subsequent contamination of processed food products, pasteurization may not destroy all foodborne pathogens in milk and faulty pasteurization will not destroy all foodborne Pathogens. A logical approach to control foodborne pathogens in dairy farms should be to define areas in the dairy farm that serve as foodborne pathogens, reservoirs and management practices that contribute to the persistence and spread of pathogens from these reservoirs. Milk and milk products cannot be preserved in its fresh condition for a long time and soon it gets fermented as a result of life activities of the different type of microorganisms that gain entrance in to milk from various sources. 297
Chemical and Microbial analysis of milk and milk products: Total solids: The concentration of total solid content in milk powder was maximum i.e. 97.30% whereas, total solid content of milk was minimum i,e. 14.66%. Microorganisms required moist condition for their growth and milk powder has least moisture, while milk has high water content so there is a least possibility of growth of milkborne pathogen in milk powder as compared to milk. Fat content: The concentration of fat was maximum in ghee i.e. 99.96% and minimum in curd i,e. 5.1%. All microorganisms do not have lipolytic activity. The fat content in the ghee was maximum indicating less possibility of growth of milkborne pathogens. Fat content donot serve the source of nutrition for microorganisms so the growth of microorganisms in ghee was found to be minimum and in curd was maximum. Protein content: The concentration of protein content of curd was maximum i.e. 6.88% where as protein content of chocobar sample was minimum 298
i.e. 4.04%. Microorganism s need nitrogen for their growth, protein is a good source of nitrogen for the growth of microorganisms. It is clear that the milk and milk products have enough protein content to promote the growth of milkborne pathogens. Therefore growth of microorganisms in curd was maximum and in chocobar was minimum. Titrable acidity: Titrable acidity of curd was maximum i.e. 3.2% and that of milk and chocobar sample was minimum i.e. 0.1%. The acidity is a important factor affecting the survival of organisms in milk and milk products shrikhand, curd and amrakhand have high titrable acidity so growth of milkborne pathogen in fermented product was maximum and in chocobar was minimum. Sugar content: Sugar content of amrakhand was maximum i.e. 42.2% and the sugar content of ice cream was minimum i.e.18.36%. Sugar is a best carbon and energy source for the growth of microorganisms and in many milk products like Ice cream, Chocobar, Shrikhand and Amrakhand external sugar is added in milk in high concentration as it prevents the growth of milkborne pathogens so milk product which contain sugar concentration less than 1% has a maximum chance of 299
microorganisms and milk products containing sugar concentration greater than 1% has a minimum chance of microorganisms. Total ash: Total ash content of milk powder sample was maximum i.e. 7.35% whereas total ash content of chocobar was minimum i. e.0.41%. Thus, Total ash content of solid product was maximum and total ash content of liquid product was minimum. As the microorganisms grow well in moist conditions, microorganisms grow well in chocobar rather than milk powder. Mineral content: Copper content of amrakhand was maximum i.e. 1.52(mg/100g) whereas copper content of milk, curd and ghee sample was minimum i.e. <0.00001(mg/100g). Zinc content of shrikhand sample was maximum i.e. 0.65 (mg/100g) whereas Zinc content of amrakhand, milk, ice cream, chocobar, curd and ghee sample was minimum i.e. <0.00001. Manganese content of shrikhand sample was maximum i.e. 2.43(mg/100g) where as manganese content of milk and curd was minimum i.e., <0.00001. The concentration of iron in milk powder was higher than milk. Iron content of shrikhand sample was maximum i.e. 14.62(mg/100g) 300
where as iron content of milk, curd and ice cream was minimum <0.0001. Calcium content of ice cream sample was maximum i.e., 0.79(mg/100g) whereas calcium content of chocobar was minimum i.e., 0.49(mg/100g). Magnesium content of milk powder sample was maximum i.e., 1.17(mg/100g) whereas magnesium content of amrakhand was minimum i.e., 0.12(mg/100g). Potassium content of milk powder sample was maximum i.e. 38.1(mg/100g) whereas potassium content of chocobar sample was minimum i.e., 34(mg/100g). The possible reason for this may be due to proximity to industrial and traffic areas which increases significantly the copper, zinc, manganese, iron, calcium, magnesium and potassium concentration in milk. The minerals like copper, zinc, manganese, iron, calcium, magnesium and potassium are essential for activity of many enzymes produced by microorganisms and these minerals are responsible for the growth of microorganisms in milk and the milk get contaminated. If proper pasteurization is not carried out there is a contamination in milk and milk products which cause food poisoning. 301
Sodium content: Sodium content of milk powder sample was maximum i.e., 275(mg/100g) whereas sodium content of chocobar sample was minimum i.e., 41.3(mg/100g). High sodium content in milk product inhibit the growth of microorganisms, sodium content of milk powder sample was maximum and chocobar was minimum so growth of microorganisms in milk powder was minimum and chocobar was maximum. Water activity: Water activity of milk, ice cream, chocobar and curd was maximum i.e. 1 whereas water activity of milk powder was minimum i.e. 0.2. Microorganisms require water for the growth of microbial population ultimately growth of pathogen. It is very clear that water activity of milk was highest and microbial population was minimum, water activity in milk powder was lowest and microbial population was maximum. Viscosity: Viscosity of ghee sample was minimum 58.65(cps/min) whereas viscosity of shrikhand, amrakhand, ice cream, chocobar sample was maximum >10000(cps/min). The viscosity of the milk and milk products indicate that the ghee sample is having lowest viscosity and lowest microbial population. Whereas viscosity of 302
shrikhand, amrakhand, ice cream, chocobar sample has highest viscosity and highest microbial population so from the above result it is clear that the viscosity is the essential factor for the growth of microorganisms. Salt content: Salt percent are in ghee sample was minimum i.e. 0.017% whereas salt percent of milk powder was maximum i.e. 1.70% salt percentage higher than 6% is inhibitory for the growth of pathogens. It is assumed that in many milk products like icecream, chocobar, shrikhand and amrakhand salt is added in low concentration so growth of microorganism was low. In milk powder has high salt concentration as it prevents the growth of milk borne pathogens. Microbial analysis of milk and milk product: Bacterial count: Microbial count of pasteurized milk was minimum i,e. 3 10 3 /ml whereas the microbial count of curd was maximum i,e. 23 10 7 /ml. Physicochemical conditions favour the growth of microorganisms so microbial count of milk was maximum and microbial count in curd was minimum. 303
Fungal count: Fungal count of pasteurized milk was minimum 3g/ml whereas the microbial count of amrakhand was maximum i.e. 70g/ml. Fungi prefer acidic ph so the growth of fungi in fermented products was maximum and in milk was minimum. Correlation coefficient between bacterial population and various parameters of milk and milk products: The low degree of negative correlation existed -0.22 (p<0.05) in between total solid and bacterial count,-0.20 (p<0.05) in between milk fat and bacterial count, the low degree of positive correlation existed 0.44 (p<0.05) in between protein and bacterial count, the high degree of positive correlation existed in between titrable acidity and bacterial count 0.86 (p>0.05),the low degree of negative correlation existed in between sugar and bacterial count, value of r being (- 0.33),the negative correlation existed between total ash and bacterial count population in all milk products samples, value of r being -0.14 (p> 0.05). Thus low degree of negative correlation existed in between total ash and bacterial count, the low degree of negative correlation existed in between copper and bacterial count, value of r being -0.23 (p> 0.05), the low degree of negative correlation existed in between zinc and bacterial count, value of r being -0.19 (p> 0.05), the low degree of negative correlation existed in between manganese and 304
bacterial count, value of r being -0.26 (p> 0.05), the low degree of negative correlation existed in between iron and bacterial count, value of r being -0.23 (p> 0.05), the low degree positive correlation existed in between calcium and bacterial count, value of r being 0.20 (p> 0.05), the low degree of negative correlation existed in between magnesium and bacterial count, value of r being -0.24 (p> 0.05), the low degree of negative correlation existed in between potassium and bacterial count, value of r being -0.15 (p> 0.05), the low degree of negative correlation existed in between sodium and bacterial count, value of r being -0.28 (p> 0.05), the low degree of positive correlation existed in between water activity and bacterial count, value of r being 0.35 (p> 0.05), the low degree of negative correlation existed in between viscosity and bacterial count, value of r being -0.38 (p> 0.05), the low degree of negative correlation existed in between salt and bacterial count, value of r being -0.14 (p> 0.05). Thus, it seems that microbial population depends on physiochemical condition. The above physiochemical condition favors the growth of bacteria and the physiochemical condition for fungal growth are different than bacterial growth hence the correlation coefficient values of fungal growth with various parameter were totally different from bacteria. 305
Correlation coefficient between fungal population and various parameters of milk and milk products: The low degree of positive correlation existed between total solid and fungal count, value of r being 0.44 (p> 0.05), the low degree of negative correlation existed between milk fat and fungal count, value of r being -0.38 (p> 0.05), the low degree of positive correlation existed in between protein and fungal count, value of r being 0.38 (p> 0.05), the high degree of positive correlation existed between titrable acidity and fungal count, value of r being 0.87 (p> 0.05), the low degree of positive correlation existed between sugar and fungal count, value of r being 0.40 (p> 0.05), the low degree of positive correlation existed in between total ash and fungal count, value of r being 0.13 (p> 0.05), the high degree of positive correlation existed between copper and fungal count, value of r being 0.61(p> 0.05),the low degree of positive correlation existed between zinc and fungal count, value of r being 0.36 (p> 0.05), the low degree of positive correlation existed between manganese and fungal count, value of r being 0.47 (p> 0.05), the high degree of positive correlation existed in between iron and fungal count, value of r being 0.61 (p> 0.05), the low degree of positive correlation existed between calcium and fungal count, value of r being 0.03 (p> 0.05), the low degree of 306
correlation existed between magnesium and fungal count, value of r being -0.10 (p> 0.05), the low degree of correlation existed in between potassium and fungal count, value of r being 0.18 (p> 0.05), the low degree of positive correlation existed between sodium and fungal count, value of r being 0.44 (p> 0.05), the low degree of negative correlation existed between water activity and fungal count, value of r being -0.20 (p> 0.05), the low degree of positive correlation existed between viscosity and fungal count, value of r being 0.08(p> 0.05), the low degree of positive correlation existed between salt content and fungal count, value of r being 0.23 (p> 0.05). High degree correlation existed in titrable acidity, protein, calcium, water activity and bacterial count it means that this physiochemical parameter highly affect the growth of microorganisms or is essential for the growth of microorganisms and low degree correlation existed in total solids, milk fat, sugar, total ash, copper, zinc, manganese, iron, magnesium, potassium, sodium, viscosity, salt and bacterial count it means that above physiochemical parameters does not affect the growth of microorganisms or not that much essential for the growth of microorganisms. High degree of correlation existed in total solid, protein, titrable acidity, sugar, total ash, copper, zinc, manganese, iron, potassium, sodium, viscosity, salt had marked effect on fungal count and low 307
degree correlation existed in milk fat,magnesium,water activity had not any marked effect on fungal count. Isolation and Identification of bacterial pathogens: Of the 32 Shrikhand samples, Staphylococcus aureus was present in 8 samples. Salmonella typhi was present in 7 samples, Escherichia coli was isolated from 4, Shigella dysentriae present in 5 samples. Of the 24 Milk Packet samples, Staphylococcus aureus was present in 6samples. Salmonella typhi was present in 9 samples Escherichia coli was isolated from 3, Shigella dysentriae present in 4 samples. Of the 9 Milk powder samples, Salmonella typhi was present in 1sample. Of the 21 Amrakhand samples, Staphylococcus aureus was present in 2 samples. Salmonella typhi was present in 1 sample Escherichia coli was present in 1 sample, Shigella dysentriae present in 1 sample. Of the 15 Ice cream samples, Staphylococcus aureus was present in 3 samples. Salmonella typhi was present in 1 sample, Shigella dysentriae present in 1 sample. 308
Of the 15 Curd samples, Staphylococcus aureus was present in 3 samples. Salmonella typhi was present in 2 samples, Shigella dysentriae present in 1sample. Of the 7 Chocobar samples, Staphylococcus aureus present in 1 sample. Escherichia coli present 1 sample, Shigella dysentriae present in 1 sample. Of the 7 Ghee samples, Staphylococcus aureus was present in 1sample. Salmonella typhi was presenting 1sample, Shigella dysentriae present in 1 sample. The milk and milk products get contaminated after the processing. Post contamination of the milk and milk products by various intestinal pathogen cause human health hazard. The presence of S. aureus, S. typhi and E. coli and S. dysentriae in various milk and milk products indicate that these milk products were contaminated either during packaging, during transport or during retail marketing. The presence of these pathogen in the milk and milk product is a serious problem. It is clear from the results that every food sample is contaminated by milkborne pathogens hence it is suggested that at most care should be taken during the packaging handling and storage of these boxes. Effect of environmental parameter on growth of microbial populations: Effect of ph on pathogens: 309
Of the 130 Staphylococcus aureus isolates 22 shows growth at ph optima 5 at 37 0 C, at ph optima 6 at 37 0 C and 42 0 C, at ph optima 7 at 37 0 C, 42 0 C and 47 0 C. Of the130 Salmonella typhi isolates 22 shows growth at ph optima 5 at 37 0 C, at ph optima 6 at 37 0 C and 42 0 C, at ph optima 7 at 37 0 C, 42 0 C, 47 0 C and 52 0 C. Of the 130 Escherichia coli isolates 9 shows growth at ph optima 5 at 37 0 C, at ph optima 6 at 37 0 C and 42 0 C, at ph optima 7 at 37 0 C, 42 0 C, 47 0 C. Of the 130 Shigella dysentriae isolates 14 shows growth at ph optima 5 at 37 0 C, at ph optima 6 at 37 0 C and 42 0 C at ph optima 7 at 37 0 C, 42 0 C and 47 0 C. S.aureus, S.typhi, E.coli and S.dysentriae were able to grow at ph 5 only at 37 0 C incubation temperature. They were also able to grow at ph 6 at 37 0 C and 42 0 C incubation temperature. It is further seen that all these organisms were able to grow at ph 7 at 37 0 C as well as at 27 0 C and 47 0 C.Only S.typhi was able to grow at ph 7 at 57 0 C. Thus it is clear from the table that the optimum ph for the growth of all milkborne pathogen seems ph 7. It is further observed that no growth of any pathogens was detected at ph 8 and above. Each micro organism has a specific ph range of growth parameter where it can grow and reproduce successfully. It is well known that 310
most of the faecal pathogens prefer ph 7 and 37 0 C temperature for their growth. Milk and milk product also have ph near neutrality. The pathogens present in milk are adapted to ph 7. All milkborne pathogens are also adapted to human body temperature (37 0 C). Our results have also shown that the pathogens isolated from milk have shown optimum ph 7 and optimum temperature 37 0 C. Effect of Nacl on growth pathogens: Staphylococcus aureus, Salmonella typhi, Escherichia coli and Shigella dysentriae have shown good growth at 2% Nacl concentration at 37 0 C incubation temperature. Staphylococcus aureus, Salmonella typhi, Escherichia coli have shown growth at 4% Nacl concentration at 37 0 C incubation temperature also at 4% Nacl concentration at 47 0 C incubation temperature. Further it was observed that Staphylococcus aureus and Escherichia coli have shown growth at 6% Nacl concentration and 47 0 C incubation temperature. It was also found that Salmonella typhi have shown growth at 2% Nacl concentration at 37 0 C incubation temperature and 47 0 C incubation temperature at 4% Nacl concentration and 37 0 C incubation temperature and at 4% Nacl at 47 0 C incubation temperature. Salmonella typhi and Shigella dysentriae doesn t show any growth at 6% Nacl. Nacl is inhibitory to the growth of bacteria above 1% concentration in our results we found that all pathogenic bacteria 311
can tolerate 2% Nacl concentration whereas only E.coli was able to tolerate 6% Nacl concentration. The chemical analysis of various milk and milk products have shown at the Nacl concentration was ranging from 2% to 6% thus it seems that the concentration of Nacl higher than 6 prevent the growth of most of the pathogens. Effect of temperature on growth of pathogens: S. aureus and E. coli showed growth at 37 0 C, 42 0 C and 47 0 C temperature and isolates of S. dysentriae showed growth at 37 0 C temperature. S. typhi showed growth at the temperature 37 0 C, 47 0 C, and 57 0 C.Temperature above 60 0 C kills all pathogens present in milk and in dairy industries. Temperature above 77 0 C is sufficient to kill the pathogens. This is 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. The contamination takes place after pasteurization from equipment, cans, bottles and water. Effect of seasonal variation on growth of microbial populations obtained from milk and milk products after 24 hrs incubation: The effect of seasonal vairiation on growth of microorganisms in shrikhand was 5.88 10 2 cfu/ml in autumn, 7.32 10 2 cfu/ml in summer. Growth of microorganisms in milk was 2.5 10 2 cfu/ml in 312
autumn, 9.4 10 2 cfu/ml in summer. Growth of microorganisms in milk powder was 1.12 10 2 cfu/ml in autumn, 6.0 10 2 cfu/ml in summer. Growth of microorganisms in amrakhand was 5.14 10 2 cfu/ml in autumn, 6.20 10 2 cfu/ml in summer. Growth of microorganisms in ice cream was 3.24 10 2 cfu/ml in autumn, 6.9 10 2 cfu/ml in summer. Growth of microorganisms was 4.1 10 2 cfu/ml in autumn, 8.0 10 2 cfu/ml in summer. Growth of microorganisms in chocobar was 0.26 10 2 cfu/ml in autumn, 3.9 10 2 cfu/ml in summer. Growth of microorganisms in ghee was 0.9 10 2 cfu/ml in autumn, 3.4 10 2 cfu/ml in summer. It is obvious that higher incubation temperature favour the growth of intenstinal pathogens hence it is clear that the chances of milkborne outbreaks are more in summer than in autumn. It is obvious that higher incubation temperature favour the growth of intenstinal pathogens hence it is clear that the chances of milkborne outbreaks are more in summer than in autumn. All together, these results show that urgent measures are needed to ensure lean and safe milk production at pastoral community level, by the promotion of good hygiene practices. These would preferably need to focus on efficient cleaning of vessels, milker hands and udder and improve the health condition of dairy animals. These measures should be applied massively because this criterion remains very poor in all of dairy households and also the high level of 313
counts found in the milk. One may suppose that such milk may pose a public health risk and suggests the need for more strict preventive measures. The results of the present study indicate that strict preventive measures should be adopted to ensure contamination free milk products for the good health of all consumers. For this, consciousness and care is required from the point of generation to the point of consumption of these widely consumed milk products. Generally, fresh raw milks collected from retailers were heavily contaminated. Possible reasons for the counts could be due to infected udders of the cows, unhygienic milking procedures or equipment and/or inferior microbiological quality of water used for cleaning utensils and animals, as well as the milk storage conditions. The milking process, especially the equipment associated with it, introduces the greatest proportion of microorganisms in to raw milk. In conclusion, initial high levels of contamination with pathogens in traditional fermented skimmed milk are critical for the safety of fermented foods. Minimizing contamination of the raw materials is therefore another way of controlling pathogen levels in the final product. Measures should be taken to prevent the transmission of pathogens in to fermented foods at both the household and commercial levels. At the commercial level, improvement of product quality and safety could be achieved by 314
applying Good Manufacturing Practices (GMP), Good Hygienic Practices (GHP) and the Hazard Analysis and Critical Control Point (HACCP) system, attempts to provide HACCP guidelines for some traditional fermented foods. However, educating food handlers, particularly mothers and food vendors, the food hygiene is a strategy that can be used in efforts aimed at to preventing food-borne diseases. The study concludes that heat treatment of milk did not significantly affect the number of total bacteria. However, during shelf life the count significantly increased. Different bacteria spoilage as well as pathogens were isolated following heat treatment of milk indicating unhygienic conditions during production, processing and storage. The quality of milk in our region especially in countries like India, Pakistan and Bangladesh is very poor. Quality deterioration of milk starts just after milking, when it is carried out under unhygienic conditions. The main contributory factors on the farm are animal mishandling, feeding, unhygienic milking, transportation and poor storage conditions. All these results can be explained by the fact that the method of production of the various traditional foods are usually primitive compared to modern way of food preparation and the major risk enhancing factors are the use of contaminated raw materials, lack of pasteurization, use of poorly controlled natural fermentation, inadequate improper storage and maturation conditions. 315