Milk is balanced food having high biological value in nature due to which it gains significance in diet of immense population on earth. At the same time it is an excellent medium for growth of an organism. Raw milk (RM) often contains microorganisms which may cause food borne diseases (Headrick et al., 1998). Fresh milk drawn from a healthy cow normally contain low number of microorganisms (less than 1000 ml^-1). But contamination of milk and milk products mostly occurs at milk collection places due to manure, water, soil, milkman hands, utensils and flies, which results in increase of 100 folds or more times microbial load at normal temperature, due to which raw milk may contain organisms over 2,000,000 cfu/ml before processing and may reach to poor quality also (Kameni et al., 2002). Presence of food-borne pathogens in milk results due to the direct contact of milk with contaminated sources in the dairy farm as well as excretion from the udder of an infected animal (Zubeir et al., 2006), which creates majority of  hygienic quality problems. Major reason of presence of higher bacterial counts is due to poor production hygiene or slackened pasteurization (Harding, 1999). Improper storage of milk, fluctuation in the temperature during storage/ transportation of milk from village to city for selling to fulfill daily need may encourage a growth of microorganisms and there is a tremendous increase in the microbial load (Sharpe and Bramley, 1977). Adulteration especially addition of contaminated water is a potent source for the bacterial contamination of the milk. Occurrence of pathogenic bacteria results in changes in flavor / appearance of raw milk due to their activities may even cause serious health problem (Younis, 2003). The presence of certain pathogenic bacteria such as Salmonella spp, Escherchia coli, Staphyllococcus aureus (Ryser, 1998) and Escherichia coli and Streptococcus spp (Lingathurai et al., 2009) in milk emerged major public health threats.

Standard plate count, coliform and enterobacteria represent microbiological quality of milk and milk products (Szita et al., 2008).  Almost all half of the total coliform is attributed to faecal coliforms including E. coli indicating great possibility of the occurrence of enteric pathogens in milk (Grimaud et al., 2007). However organisms E. coli and Streptococci are most common contaminants of fecal origin and could be an important factor of gastrointestinal infection including food poisoning and food borne illness (Kumar and Prasad, 2010).  E. coli is most frequently contaminating organism which is reliable indicator of fecal pollution. Clostridium organisms are typical colonizer of sediments i.e. fecal contamination of fresh water; hence it also has been suggested as an alternative indicator of fecal pollution. Streptococcus and Staphylococcus organisms mostly derived from udder of unhealthy animal i.e. mastitis. Moreover presence of food borne pathogens creates major threat to human health. However higher bacterial counts always indicates poor hygiene during production (Harding 1995), which may cause food borne diseases (Ammara et al., 2009).

In view of growing public awareness about food safety and its quality at the same time to avoid the health and economic losses of consumers due to food borne pathogens present study was aimed to determine the microbial quality of milk sold in Parbhani city, Maharashtra with an objective to investigate the occurrence and load of microorganisms in milk with special reference to food borne pathogens.

Materials and Methods

Raw Material

A total of hundred milk samples were collected from vendors of local market in Parbhani city during Jan- Mar, 2015. Two hundred and fifty mililitre (250ml) milk of each sample was collected in sterilized glass bottle and immediately brought to the laboratory of College of Veterinary and Animal Sciences, Parbhani by maintaining cold chain (4°C) using icebox and then they were analyzed for microbial assessment.

Determination of Total Viable Count (TVC)

As per the method described by Association of Official Analytical Chemists (AOAC, 1997) total viable count of milk samples was calculated.  Pour plate method was used for determination of TVC in which 0.1 ml of 10^-5 dilutions was added.  Molten plate count agar was added to the petri plates and the plates were incubated for 24 hr at 37°C. Incubation was done at 37°C. After 24 hrs, the colonies were counted using digital colony counter. A separate plate was used for each dilution of milk samples. The TVC of each milk sample was calculated by using following formula.

Where,

Σ C = Total number of colonies counted from all plates

n₁       = No. of plates of lower dilution

n₂       = No. of plates of higher dilution

d      = Dilution factor

Determination of Differential Count

Collected milk samples were processed for determination and isolation of different food borne pathogens. Selective media from Hi-media were used for isolation of differential organisms viz. E. coli, Streptococcus spp, Staphylococcus spp, Enterobacter spp, Clostridium spp and Salmonella. E. coli, Streptococci, Staphylococci, Enterobacter spp and isolation was carried out as per the method of American Public Health Association (APHA, 1984).

Preparation of Dilutions

Representative milk sample of 10 ml was pipette out using a sterile pipette from each collected milk sample and homogenized in sterile bag containing 90ml normal saline solution. These diluents were used for the preparation of subsequent decimal dilutions.

Isolation of E. coli

Organism E. coli was isolated by using 0.1 ml inoculum of 10^-4 and 10^-5 dilutions of milk samples on eosine methylene blue (EMB) agar plates. The inoculum was spread by means of L-shape spreader and plates were kept overnight at 37°C for incubation. Bluish purple coloured colonies with greenish metallic sheen were considered indicative of E. coli.

Isolation of Streptococci

A quantity of 0.1 ml inoculum of 10^-4 and 10^-5 dilutions was inoculated on Slantez and Bartley Medium (SBM) plates. The inoculum was spread by means of L-shape spreader. The plates were kept at 37°C for 24 hrs for incubation. Maroon red colored colonies were considered as Streptococci colonies.

Isolation of Clostridium spp

Sodium polymyxin sulpha-diazine (SPS) agar was used as selective medium for the Clostridia spp and was isolated as per the method described by Angelotti et al. (1962). The organisms Clostridia were grown anaerobically in the tubes of SPS agar. A quantity of 0.1 ml inoculum 10^-4 and 10^-5 dilutions was allowed to trap in between the two layers of SPS agar to maintain the anaerobic conditions in the test tube. The tubes were sealed by paraffin films and incubated in the incubator for 24 hrs at 37°C. Black cottony wool growth in between two layers indicates presence of Clostridia in the samples.

Isolation of Staphylococcus spp

A quantity of 0.1 ml inoculum of 10^-4 and 10^-5 dilutions out of six serial dilutions was inoculated on Baired Parker Agar (BPA) plates. The inoculum was spread by means of L-shape spreader. The plates were kept at 37°C for 24 hrs for incubation. After incubation grey black colonies on the plate were considered as positive.

Isolation of Enterobacter spp

A quantity of 0.1 ml inoculum of 10^-4 and 10^-5 dilutions was inoculated on Eosin & Methylene Blue (EMB) plates. The inoculum was spread by means of L-shape spreader. The plates were kept at 37°C for 24 hrs for incubation. Red purple colour colonies without greenish metallic shine were considered as colonies of Enterobacter spp.

Isolation of Salmonella spp

Isolation of Salmonella spp was done by using the method of Andrews (1992). The procedure was followed as pre-enrichment, enrichment and incubation period. Pre enrichment was done by using 1 percent Buffered peptone water (BPW) at 37⁰C for 24 hours. Pre-enriched sample was inoculated in Tetrathionate Broth (TTB) for enrichment and incubation was done for 24 hours at 42⁰C. Streaking of TTB grown bacteria was done on Xylose Lysine Deoxycholate (XLD) agar and incubated for 24 hour at 37⁰C. Pink colonies with or without black centers were considered as positive for Salmonella.

Result and Discussion

The finding with respect to the microbiological quality was found to be poor. An observation with respect to the mean value of Total viable count of market milk is presented in Table 1 and prevalence rate of food borne pathogens were presented in Table 2.

Table 1:  Mean value of total viable count of raw milk

Table 2:  Prevalence rate of differential microorganisms

The microbial content of milk indicates the hygienic level during milking and wholesomeness of udder of individual animal (Speer, 1998). Tested milk samples showed 6.61 ± 0.13×10⁵cfu/ml, mean ± standard deviation of total plate count with minimum and maximum count 2.73 ±0.15 x10⁵cfu/ml and 8.83 ±0.08 x10⁵cfu/ml respectively. The details of TVC counts was shown in Fig. 1.

Fig 1: Plate showing total viable counts (TVC)

The TVC of tested market milk was higher than the permissible limit in the raw milk i.e. 2×10⁵ cfu/ml, since samples were put in the very poor quality and marked as grade-D. The animal may be the main source of contamination that probable result in higher number of microbial flora in raw milk. Bacteria can also easily enter through the dairy utensils due to the contact of surfaces with water, soil, manure etc. and further transferred to milk. Therefore, possible reason for the higher counts could be infected udder of milking animals, unhygienic milking procedure and/ or equipment and unclean water having high microbial count used for cleaning of utensils & animal.  Inappropriate storage of milk can also be a cause for increment in microbial load (Sahu et al., 2016). Similarly, Lingathurai et al. (2009) reported 5.84 log cfu/ml total counts in cow milk of Madurai, Tamil Nadu. Monika and Poonam (2013) noticed lower total viable count in milk sample during the preservation period i.e. 4.19+0.69 to 6.35+0.11 log cfu/ml.  Higher counts were observed by Manzoor et al. (2012) in milk samples collected from market milk sample collected during transportation to consumers from three different areas of Abbottabad city.

The findings obtained with respect to the prevalence study after isolation and identification of differential organisms revealed the presence of organisms E. coli, Staphylococcus spp, Streptococcus spp and Enterobacter spp respectively in 38, 4, 58, 66 percentages of milk samples. Kumar and Prasad (2010) reported presence of E. coli and Staphylococcus spp in 26 percent milk and milk products samples collected from Pantnagar, these findings are almost analogous with the present study for E. coli organism, but lower for Staphylococcus spp. Contrarily, Lingathuria et al. (2009) found E. coli and Stahyloccous spp in 80 percent cow milk sample collected from different locations of Madurai city, Tamil Nadu, which reflect higher percentage of positive samples than the present findings. Kivaria et al. (2006) observed lower results during quality evaluation of raw milk marketed by milk selling points (MSPs) in Dar es Salaam region and reported 8.2 ± 1.9 x 10⁶ cfu/ml mean value for TBC and major bacterial isolates i.e. Escherichia coli, Bacillus cereus, Staphylococcus aureus, Streptococcus agalactiae in 6.3 percent, whereas Enterobacter aerogenes and Enterococcus faecalis were 5.6 percent and 4.7 percent in milk samples respectively. Pathak et al. (2012) were found significantly lower values for presence of pathogenic organisms such as Enterobacter spp in 6.92, 15.3, 5.88 and 8.06 percent, Escherichia coli in 18.46, 18.37, 17.65, 20.97 percent and Staphylococcus spp in 3.08, 6.12, 7.84, 8.06 percent in boiled bovine milk collected from urban consumers and open market areas of Jabalpur during summer, monsoon, post monsoon and winter season respectively than the present findings.  Presence of these organisms is a horizontal source of human health deterioration due to food poisoning. Source of these pathogenic organisms may be due to the fecal contamination, unhygienic conditions during milk production, poor sanitation and cleanliness of environment as well as utensils.

None of the sample from local market of Parbhani city was found positive for Salmonella spp and clostridium spp. which was in agreement with the Chandrashekhar et al. (2010) for Salmonella spp  in milk sample of Kumargani, Faizabad, whereas Lingarthurai and Vellathurai (2010) for clostridium spp in milk samples from Madhurai.

Conclusion

The milk is considered to be as the richest medium for the growth of microorganisms. Improper rearing condition, processing practices and slackness in storage and distribution leads to rise in the growth of pathogenic microorganism. Collected samples were found positive towards presence of E. coli, Streptococcus, Staphylococcus and Enterobacter. Effective measures such as good hygienic practices at production point, maintenance of cold chain during storage, transportation and proper pasteurization of milk before consumption should be done to avoid spoilage of milk and to prevent the consumers from risk.

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