PREVALENCE OF PSYCHROTROPHIC BACTERIA IN KULFI SOLD IN CHENNAI MARKET

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1 J. Dairying, Foods & H.S., 29 (2) : , 2010 AGRICULTURAL RESEARCH COMMUNICATION CENTRE / indianjournals.com PREVALENCE OF PSYCHROTROPHIC BACTERIA IN KULFI SOLD IN CHENNAI MARKET A. Elango, V. Jayalalitha, T.R. Pugazhenthi and B. Dhanalakshmi Department of Dairy Science Madras Veterinary College, Chennai , India ABSTRACT Twenty four samples of kulfi, eight from each of the following sources viz. road side, small shops and organised sectors, were collected aseptically and subjected to estimation of total count, coliform count and psychrotrophic count to determine the microbiological quality of kulfi sold in Chennai. The level of psychrotrophic count was found to be more in road side local vendor samples (1.8 x 10 3 cfu/g) followed by that of samples of small scale producers (1.01 x 10 3 cfu / g) and branded samples from organised sector (3.5 x 10 2 cfu / g). Road side local vendor samples contributed % of organisms to the psychrotrophic count, followed by small scale producers (32.03 %), while organised sector contributed the least (11.07 %). Pseudomonas fluorescens was found to have greater proteolytic and lipolytic effect when compared with the activity of Pseudomonas aeruginosa and Pseudomonas putrifaciens. The mean total count and coliform count were found to be maximum in road side local vendor samples followed by samples of small scale producers The microbial quality of most of the kulfi samples needs to be improved in order to avoid any food poisoning and to safeguard the health of the consumers. Key words : Kulfi, Psychrotrophs, Prevalence, Proteolysis INTRODUCTION Psychrotrophs are the microorganisms which will grow at 7 C regardless of their optimum growth temperature. These bacteria do not constitute a taxonomic group but are found scattered in several unrelated genera such as Pseudomonas, Achromobacter, Flavobacterium, Micrococcus, Serratia, Aeromonas, Acinetobacter, Enterobacter, Escherichia, Lactobacillus, Bacillus, Streptococcus, Alcaligenes, Proteus Staphylococcus, Corynebaterium, Arthrobacter, Clostridium and yeast and moulds (Buchanan and Gibbons, 1974). Kulfi is a popular Indian ice cream made with boiled milk typically from water buffalo. It comes in many flavours, including pistachio. malai, mango, cardomom(elaichi)apple, orange, pea nut, avocado,anchovie and saffron. Kulfi differs from western ice cream in that it is richer in taste and creamier in texture as well,where as westerrn ice creams are whipped with air or over run, kulfi contains no air and it is a solid dense milk. It is one of the widely consumed milk products in India. Since this milk based food forms an excellent culture media the growth and reproduction of saprophytic and pathogenic micro-organisms are possible. The most contaminants of kulfi are coliforms and psychrotrophic group of organisms (Yadav et al.,1993) The presence of these organisms in milk and milk products indicate not only unsanitary conditions but also the keeping quality of the products. It is well known that the condition, under which kulfi is produced, stored, transported and distributed are far from satisfactory in India. Such a situation warrants a strict vigilance and exposes the need for suitable sanitary standard for the manufacture and sale of kulfi. The present study is contemplated with the objectives of finding out the incidence of psychrotrophic bacteria in kulfi and studying the proteolytic and lipolytic effect of isolates of Pseudomonas spp. in spiked samples of milk owing to their involvement in spoilage of dairy products.

2 98 J. DAIRYING, FOODS & H.S. MATERIAL AND METHODS Collection of samples Twenty four samples of kulfi, eight from each of the sources viz. road side, local vendors, small scale producers and branded kulfi from organised sectors were collected aseptically. Estimation of plates counts The procedure given in the standard methods for examination of dairy products, APHA (1992) using Standard plate count agar was used to estimate the total count, coliform and psychrotrophic count. lsolation and identification of psychrotrophic bacteria Representative colonies that had developed on the psychrotrophic plates after incubation were isolated and inoculated into the nutrient broth (Cowan, 1974). The purification of culture was done by plating and sub culturing in nutrient broth. The cultures were examined under the microscope for purity and confirmed by Grams staining techniques (Cruickshank et al., 1975). The isolates were identified by morphological, cultural and biochemical tests following the methods described by Buchanan and Gibbons (1974) and Macfaddin (1980). Estimation of proteolysis and lipolysis. Sterilised whole milk samples (Fat 4% ; SNF 8.5%) were inoculated with P.aerugionsa, P.putrifasciens and P.flourescens at 1% level and held at 7 C. At intervals of 0,5,6, and 7 days, samples were drawn and tested for proteolytic and lipolytic activity according to the protocols of Lowry et al. (1951) and Deeth et al. (1975) respectively. RESULTS AND DISCUSSIONS Total bacterial count : The level of total count means found to be maximum in road side local vendor samples (1.55 x 10 7 cfu / g) followed by that of small scale producer (1.62 x 10 6 cfu / g) and organised sector (1.2 x 10 5 cfu / g) and these counts were much higher than the standards set by BIS (1953) (Table 1) Coliform count : The level of coliform count means found to be highest in local vendor samples (1.7 x 10 3 cfu / g) followed by that of small scale producer (7.7 x 10 1 cfu / g) and organised sector (2.4 x 10 1 cfu / g). Local vendor samples contributed per cent of organisms to the coliform count, followed by small scale producer (4.28 per cent) while organised sector contributed the least (1.33 per cent). The level of coliform encountered very very high in road side local vendor samples and very high in samples of small scale producer and high in brands of organized sector when compared to the standard of less than 100/g of Bureau of Indian Standards. The presence of coliform bacteria indicated the presence of fecal contamination and other intestinal pathogens in kulfi. Singh(1970) reported that 80% of kulfi samples contained E.coli with a count ranged from 18.8 x x 10 5 (ful/g). Doraisamy and Elango (2009) found that kulfi samples had a mean of total bacterial count and coliform count of 218 x 10 3 ±28.02 cfu / g and 0.7 x 10 2 ±0.28 cfu / g respectively. Psychrotrophic count : The level of psychrotrophic count was found to be more in local vendor samples (1.8 x 10 3 cfu/g) followed by that of small scale producer (1.01 x 10 3 cfu / g) and organised sector (3.5 x 10 2 cfu / g). Local vendor samples contributed per Table 1 : Average of total count, coliform count and psychrotrophic count of kulfi from different sources Sl.No. Sources Mean counts Total Count* Coliform Count** Psychrotrophic Count** 1 Road side local vendors 1.55 x 10 7b ± x 10 3b ± x 10 3a ± Small scale producers 1.62 x 10 6ab ± x 10 1a ± x 10 3ab ± Organised sectors 1.2 x 10 5a ± x 10 1a ± x 10 2b ± 0.45 Means ± S.E bearing at least one common superscript do not differ significantly. *P < 0.05, ** P < 0.01.

3 Vol. 29, No. 2, TABLE 2 : Morphological and Biochemical Chracterstics and Identity of Psychrotrophic Bacteria Isolated from Kulfi Samples No of Isolates Size of Cells Shape Gram reaction Matility Arakinose Maltose Glucose Marital Xylose Raffimere Salin Frutase Starch hydrolysis Citrate Utilisation Nitrate Reduction Voges - proskavertest Indole test Gelatin Liquefaction Catalase test Arginine Metabolisis Hydrogen sulfide test Oxidase test Metthyl Red Test Name of the Bacterial μm Rods Pseudomans fluorescens x μm Rods Pseudomonas aeruginosa 1 1-4μ long Rods Pseudomans putrifaciens x μm μm in diameter Rods Flavobacterium aquatile Sphe res Staphylococcus aureus μm Rods Aeromonas hydrophila x μm Rods x μm Bacillus cereus Rods Escherichia coli

4 100 J. DAIRYING, FOODS & H.S. cent of organisms to the psychrotrophic count, followed by small scale producers (32.03 per cent) while organised sector contributed the least (11.07 percent). From the 24 samples of kulfi, 42 isolates of psychroprophic bacteria were identified by defined morphological and biochemical characteristics (Table 3). Sixteen isolates were identified as Pseudomonas flourescens, 4 as Pseudomonas aeuruginosa, one as Pseudomonas putrifaciens, 3 as Flavobacterium aquatile, 6 as Staphylcoccus aureus, 3 as Aeromonas hydrophila, 3 as Bacillus cereus, and 6 as Escherichia coli. Among the psychrotrophic organisms isolated from kulfi, P. flourescence was predominantly present (38%) followed by S.aureus and E.coli (each 14%) and P. aeruginosa (9.5%), F.aquatile,A.hydrophila and B.cereus(each 7%) and the least is P. putrifaciens with a prevalence of 2.3%.The distribution of psychrotrophs in kulfi from different sources is shown in Table 3. It is interesting to observe that the highest number of Pseudomonas isolates were found in samples from organized sector followed by the others. However gram positive isolates and E.coli were totally absent in branded samples. Kahlos and Grover(1984) analysed 31 samples of kulfi and reported that total count and staphylococcal count of kulfi were 70 x 10 5 cfu/ml and 14 x 10 3 (cfu/g) respectively. Batish et al., (1984) studied the incidence of enterococci in milk and milk products and reported that out of 728 isolates, 63.4% of enterococci was contributed by kulfi samples only. Sharma and Joshi (1992) observed that out of 38 samples of milk products analysed the number of isolates of Pseudomonas sp. in burfi, ice cream and kulfi were 8, 5 and 2 respectively. The results of the present study suggest poor sanitation during the preparation/sale of kulfi was responsible for high count. These included the observed dirty premises, used utensils, quality of water and the use of bare hands in preparing the products. This can be substantiated by the findings of Grover and Srinivasan (1988) who opined that water contamination and uncleaned equipments were the major sources of psychrotrophic bacteria in raw milk, cream and paneer. Sharma and Joshi (1992) also reported that the chief sources of psychrotrophs in milk and milk products were contamination of milk from contaminated containers, water or due to unhygienic handling. Table 3 : Distribution of psychrotrophs in kulfi from different sources Psychrotrophs Total No. Percent Sources isolated of isolates Road side small scale Branded local vendors producers Kulfi P. fluorescens P. aeruginosa P.putrifaciens F. aquatile Ahydrophila S. aureus B. cereus E. coli Total Per cent Table 4 : Tyrosine values and free fatty acid content of P.aerugionsa, P.putrifasciens and P.flourescens inoculated milk Days Tyrosine (g/ml) Free fatty acid(μ Equiv / ml) Control P.aeruginosa P.putrifaciens P.fluorescens Control P.aeruginosa P.putrifaciens P.fluorescens

5 Tyrosine and free fatty acid Tyrosine value of milk samples inoculated with P. aeruginosa, P. putrifaciens and P. fluorescens increased from 0.002( 0 day) to 0.041,0.05 and mg/ml respectively. P. fluorescens was found to have the highest proteolytic effect when compared with the activity of P.aeruginosa and P. putrifaciens. O Connor et al.,(1986) reported a predominance of Pseudomonas spp. among the isolates of proteolytic psychrotrophs. Fatty acid content also increased from 0.06 (0 day) to 0.3, 0.45 and 0.88 (7 th day )μ Equiv / ml respectively (Table 4) P. fluorescens was found to have the highest lipolytic activity when compared with the activity of P.aeruginosa and P.putrifaciens. Vassiliadu(1984) isolated the Pseudomonas sp. Vol. 29, No. 2, and found it had higher lipase activity than other psyhrotrophic bacteria. CONCLUSION The results suggest that the heavy bacterial contamination of psychrotrophs and total bacterial count were found in all samples of kulfi. The presence of coliform bacteria indicates the presence of fecal contamination of food. This suggests possibility that other intestinal pathogens may also present in food. The high level of bacterial contamination can be attributed to poor hygienic conditions under which kulfi is produced, stored, transported and distributed, as it is largely in the hands of the road side/ small local vendors, which warrants a stringent control and exposes the need for suitable sanitary standard for the manufacture and sale of kulfi. REFERENCES APHA(1992) American Public Health Association: Standard Methods for Examination of Dairy Products, ed..marshal, R.T., Washington, D.C. Batish,V.K., et al., (1984) Incidence of enterococci thermonuclease in milk and milk products J. Food. Sci. 49 (6) BIS(1983) Bureau of Indian Standards- kulfi IS 10501:1983 Buchanan, R.E and Gibbons,N.E (1974) Bergy s Manual of Determinative Bacteriology, 5 th edition. The Williams and Wilkins Company, Baltimore, USA Cowan, S.T(1974) Manual for Identification of Medical Bacteria, 2nd Edn. Cambridge University Press, Cambridge, London. Cruickshank, R., et al., (1975) Medical Microbiology, Vol. I, XII Edn., Churchill Livingstone, Edinburgh, London and New York. Deeth, H.C, et al.,(1975) Aus. J. Dairy Technol. 9: Doraisamy, K.A and.elango,a (2009) Egyptian J.Dairy Sci., 97: Grover,S and Srinivasan, R.A (1988).Indian J. Dairy Sci., 41 : Kahlon, S.S.and Grover, N.K.(1984) Indian J. Dairy Sci.,.37: 381. Sharma, D.K and.joshi, D.V (1992) J. Food. Sci., Technol., 29 : Singh, R.S., (1970), Ph.D. Thesis. NDRI, Punjab University, Chandigarh, India. Lowry, O.H., et al.,.(1951) J. Bio. Chem., 193 : Macfaddin, J.F(1980) Biochemical Tests for Identification of Medical Bacteria. 2nd Edn. Waverly Press, Baltimore, U.S.A. O Connor, R.E. et al.,(1986) J.Appl.Bacteriol., 61: Vassiliadou, O.K (1984).Milchwissnschaft., 39: 601. Yadav, J.S., et al. (1993). A Comprehensive Dairy Microbiology, Metropolitan press New Delhi.

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