PKISSN 0006 – 3096 (Print) ISSN 2313 – 206X (On-line) BIOLOGIA (PAKISTAN) December, 2015, 61 (2), 271-277 Morphological and Biochemical Characterization of Bacteria isolated from Milk Products * AMNA ALI, NAUREEN AKHTAR, UZMA BASHIR, RAHILA HAFEEZ, & MUHAMMAD SALEEM HAIDER Institute of Agricultural Sciences, University of the Punjab, Quaid-e-Azam Campus, Lahore, Pakistan ABSTRACT Microorganisms have been handed down from one generation to the next for use in fermented dairy products. In addition some harmful bacteria are also present in dairy products which have detrimental effect on human nature. The diversity of bacterial communities in dairy milk, dry milk, butter and yogurt samples collected from different markets of Lahore Pakistan was evaluated in present study. Lactobacillus sp. was isolated from dairy milk and yogurt while other isolates were identified as Citrobacter freundii, Ralstonia pickettii, Salmonella typhii, Klebsiella pneumoniae, Salmonella gallinarum, Pasteurella sp. (hemolytical type T and A), Shewanella putrefaciens, Acinetobacter jansani and Pantoea sp. and these are possible pathogens of humans that cause infections. All strains were identified on the basis of morphological features and metabolic processes. Maximum numbers of bacterial species were isolated from dry milk (brand A). The results demonstrate that traditional fermented dairy products have diverse range of bacterial species which are harmful for humans. Key words: Identification, dairy products, gram negative, rods INTRODUCTION Milk is the basic dairy product and people use it as basic supplement in food stuff. Liquid fresh milk can be utilized in drinking purpose while after processing cheese, cream, butter, yogurt, buttermilk and other products are also used. It is truly an amazing food when it is non-contaminated with any other microorganisms. Conversely, it is a main source for dispersal of food-borne pathogens especially gram-negative bacteria in human life and environment. Moreover, most of the dairy products are unhygienic due to improper processing conditions. Bacteria are ubiquitous in nature and have the ability to colonize a wide variety of substrates (Cleenwerck & Paul, 2008). Identification of bacterial strains is not only valuable for microbiologists but also for people associated with medicines, as control and treatment of the pathogenic bacteria is possible for properly identified strains (Hasibe & Dilek, 2011). In the absence of molecular data, morphological features combined with certain biochemical studies provide sufficient data required to identify the bacterial unknowns (Ali & Naseem, 2012). The goal of present work is to isolate and identify bacteria from different products of dairy sold in Lahore markets and to show the level of prevalence of pathogenic bacteria in dairy products. MATERIALS AND METHODS Samples Collection A range of dairy products: dairy milk *Corresponding author: [email protected] (pasteurized specifically in tetra packs or a milk processing by plant), liquid milk (non- pasteurized obtained from milk men), dry milk - brand A (manufactured by local company), dry milk - brand B (product of international company), loose packed butter and yogurt were purchased randomly from selected markets of Lahore. Properly labeled samples, with sample type, collection site, date of collection etc., were transported to the laboratory. Isolation of bacterial species Isolations were carried out using the freshly collected samples by pour-plate technique on Luria Bertani Agar (LBA) and Nutrient Agar (NA) media (Ali & Naseem, 2011). The dry samples (butter, yogurt, dry milk) 0.5 gm and liquid sample (liquid milk) 0.5 ml were inoculated on prepared media plates and incubated at 37°C for 3 days under aerobic conditions. Purification was done by streak plate technique, transferred the emerging colonies on fresh media Petriplates under aseptic conditions (Beishir, 1991). Pure bacterial cultures were stored in 20% sterile glycerol at -20°C until further analysis. Identification of bacterial species First morphological and cultural features of the bacterial colony/cells were recorded then certain biochemical tests were performed as the routine steps of bacterial identification. Morphological Features: Morphological parameters recorded for identification were cell shape, Gram type, capsule stain, motility and pigmentation. Growth on osmotic medium i.e., containing 2% NaCl 272 A. ALI ET AL was also observed. Finally the ability of bacteria to o o grow at 25 C and 40 C was also studied (Konem et al., 1997). Biochemical Analysis Using the commercially available bacteria identification kit, Microgen-TM GnA+B-ID Identification System (Microgen Bioproducts Ltd, Surrey, UK), pure colonies was differentiated by biochemical test. Initially preference of carbon source of isolated bacteria was analyzed by providing a wide range of carbohydrate sources that include glucose, lactose, sucrose, inositol, sorbitol, mannitol and xylose while sterile water was used as control. Other biochemical analysis included study of enzymatically catalyzed metabolic reactions such as citrate, Indole, Methyl red, nitrate reductase, oxidase, catalase, urease, malonate and gelatinase, hydrogen sulphide, arginine and lysine (Holt et al., 2000; Benson, 1996). Bacteria were identified by providing the results of all above mentioned biochemical tests to Microgen Identification System software. RESULTS AND DISCUSSION In nutrition balancing science, dairy products can play a definite and important role. Therefore dairy products should be raw and preferably certified. This cross-sectional study of Gram-negative and Gram-positive staining bacterial contamination of milk meant for human consumption was carried out in some areas of Lahore, Pakistan. Milk sampling points included smallholder’s milk producers, dairy co-operatives, a milk processing plant, and supermarkets. The hygienic procedures applied during milking, milk collection, transportation, pasteurization, and postpasteurization storage conditions in these specified dairy products were evaluated. Standard bacteriological cultivation and biochemical assays were used to isolate and identify bacterial pathogens in the milk samples. During present study, a total of thirteen different bacterial species were isolated from various sources of dairy products (Table I). Percentage occurrence of each species was calculated. However maximum percentage of bacterial species was obtained, six from dry milk (brand A), two species from dry milk (brand B) and butter whereas one species was isolated from dairy milk, liquid milk and yogurt each (Figure 1). Thirteen bacterial were isolated; 2 Gram-positive bacteria BIOLOGIA (PAKISTAN) and 11 Gram negative. Each isolate was given a reference number (DP1 - DP13) that was used throughout this study to represent the results of that particular bacterium. Table I: Detail of the substrates used to isolate the bacterial strains. Sample No. Substrate S1 Dairy milk S2 Dry milk (Brand A) S3 Dry milk (Brand B) S4 Liquid milk S5 Butter S6 Yogurt All the strains studied for this present work were rod-shaped except DP1 and DP13 Gram negative bacteria, likewise all strains were unable to produce pigment except DP12. Furthermore only DP12 managed to grow at 25°C although all strains showed similar pattern of growth at 37°C. Only DP6 and DP7 were capsulated. Data recorded while studying the morphology of bacteria is presented in Table II. Naturally different bacterial strains have different metabolic path ways (Sathishkumar et al., 2008). Result demonstrated that all isolates gave positive results with the carbohydrates (glucose) except DP3 and DP7. In addition, DP1, DP2 and DP13 had the ability to ferment lactose also while others did not. Isolate DP2 exhibited positive result with inositol among all species. Bacteria are referred to as individuals or groups based on their patterns of growth under various chemical (nutritional) or physical conditions. Like all other living organisms, different groups of bacteria utilize different sources of energy to generate ATP, required for their maintenance and reproduction. Most of the bacteria use monosaccharides, for example glucose, as energy source while few prefer disaccharides or polysaccharides (Richard et al., 2011). Furthermore, Fish (2002) reported that bacteria frequently secrete chemicals into their environment in order to modify it favorably and these secretions are often proteins and may act as enzymes that digest some form of food in the environment. Therefore, each species of pathogen has a characteristic spectrum of interactions with its human hosts. Capacity of different bacterial strains to use various carbon compounds as energy source is summarized in Table III. VOL. 61 (2) BACTERIA FROM MILK PRODUCTS 273 Table II: Morphological and cultural features of bacteria studied. Strain No. DP1 DP2 DP3 DP4 DP5 DP6 DP7 DP8 DP9 DP10 DP11 DP12 DP13 Gram type + + Capsule stain + + - Motility Pigment + + + + + + + + - Growth on 2% NaCl + + + + - Growth o at 25 C + - Growth o at 40 C + + + + + + + + + + + Table III: Carbohydrate source preference analysis of bacterial isolates. Strain No. DP1 DP2 DP3 DP4 DP5 DP6 DP7 DP8 DP9 DP10 DP11 DP12 DP13 Glucose + + + + + + + + + + + Lactose + + + Sucrose Inositol + + + + + + + - Biochemical tests actually exhibit the ability of an enzyme to utilize different substrates. Such ability can be assessed by the presence of products in a biochemical reaction. Results of these tests help in Sorbitol + + + - Mannitol + + + + - Xylose + + + + + + + identification of bacteria (Harley, 2008). Enzymatic activities of bacterial isolates are tabulated in Table IV. Table IV: Enzymatic activities of bacterial isolates. Biochemical test Citrate Indole Methyl red Nitrate reductase Oxidase Catalase Urease Malonate Gelatinase H2S Lysine Arginine DP1 + - DP2 + + DP3 - DP4 + DP5 + + + - + - + - + + - + + + - Reference Strain No. DP6 DP7 DP8 DP9 + + + + + + + + - + + - + + + + - + + - DP10 + DP11 + DP12 + - DP13 + - + + - + - + + - - 274 A. ALI ET AL BIOLOGIA (PAKISTAN) % of Bacterial species 50 40 30 20 10 0 Dairy milk Dry milk (Brand A) Dry milk (Brand B), Butter Yogurt Liquid milk Dairy Products Fig., 1: Percentage of Bacterial Strains Isolated from each Sample of Dairy products The identification of bacteria is essential in microbiology by various aspects (Nitesh et al., 2011). Identification was made by using the Microgen Identification System software. Cultural and biochemical data recorded for the isolates (Table I-IV) was entered in the software to key out the unknown bacteria. Lactobacillus sp. (n=2), Citrobacter freundii (n=1), Ralstonia pickettii (n=1), Salmonella typhii (n=1), Klebsiella pneumonia (n=1), Salmonella gallinarum (n=1), Pasteurella sp. (hemolytical type T) (n=2), Pasteurella sp. (hemolytical type A ) (n=1), Shewanella putrefaciens (n=1), Acinetobacter jansani (n=1) and Pantoea sp. (n=1) were the identified species. Identified bacterial species were deposited in First Fungal Culture Bank of Pakistan (FCBP). All species versus their reference no as well as their FCBP accession numbers are given in Table V. Occurrence of each bacterial strain in each sample is also shown in Figure 2. Table V: Bacterial species identified from dairy products Sr. No. S1 S2 Substrate Dairy milk Dry milk (Brand A) Ref. No. of Strain DP1 DP2 C. freundii DP3 DP5 DP6 DP8 R. pickettii S. typhii K. pneumoniae S. gallinarum Pasteurella sp. (hemolytical type T) Pasteurella sp. (hemolytical type T) Pasteurella sp. (hemolytical type A) S. putrefaciens A. jansani Pantoea sp. Lactobacillus sp. DP9 S3 Dry milk (Brand B) DP4 DP10 S4 S5 Liquid milk Butter S6 Yogurt Species Identified Lactobacillus sp. DP7 DP11 DP12 DP13 FCBP accession No. FCBP004 FCBP063 FCBP065 FCBP067 FCBP068 FCBP073 FCBP078 FCBP066 FCBP079 FCBP071 FCBP090 FCBP100 FCBP139 VOL. 61 (2) BACTERIA FROM MILK PRODUCTS Pantoea sp. 275 Lactobacillus sp. A. jansani Pasteurella sp. (type A) C. freundii S.gallinarum R. pickettii S. putrefaciens Pasteurella sp. (type T) K. pneumoniae S. typhii Fig., 2: Occurrence of Bacterial Strains in various Sample of Dairy products Ranadheera et al. (2012) revealed that some beneficial bacteria were used in multiple sectors i.e., industry (Lactobacillus sp. in food, plant and dairy fermentation) and agriculture (Lactobacillus spp. as probiotic microorganism, Azotobacter sp. as nitrifying agent, Pseudomonas sp. as phosphate solubilizing agent). Results of Wang et al. (2008) and Duskoval et al. (2012) studies were similar to recent findings of bacterial isolation from dairy product. Furthermore, it shows that it is possible for dairy products to be contaminated with pathogenic bacteria. This implies that attention should be given to sanitary behavior of food handlers. These pathogenic organisms release toxins, which are the agents responsible for illnesses such as diarrhea, dysentery, nausea and vomiting, caused by these organisms upon consumption of the contaminated foods (Okolie et al., 2012). Unfortunately, even quite small numbers of microbes can grow quickly into dangerous hordes when products are not properly stored. The findings of this study have confirmed that pathogenic bacteria can exist in dairy products even though they may physically appear to be quite wholesome; thus, proper steps should be taken to ensure that the occurrence of such organisms in products is kept within limits (Okolie et al., 2012). In addition present results also highlighted that most of the sampled dairy milk was collected under unclean environmental conditions and poor preparation. Therefore, early study of Connor & Charles (Connor & Charles, 1995) was also focused on contamination in raw milk from soil, manure and soiled bedding as well as direct contact with fecal material during milking. Milk contains important nutritional components for bacterial growth, and, therefore, it is also an ideal medium for the growth of many different bacteria. Temperature plays an important role in bacterial growth. Many bacteria prefer to grow at high temperature. As we know dry milk is usually stored at room temperature while liquid milk at cold temperature that’s why highest percentage of bacteria was isolated from dry milk (brand A) as compared to liquid milk. Further findings of Champagne et al. (1994) and Jayaroo & Wang (1999) about untreated water used in equipment cleaning process, hands of milk producer or improper udder preparation before milking were main source of bacterial contamination in milk and that might hold true in this study. Moreover, it is necessary that the equipment be washed using detergents then with cold water to remove as much previous milk and dirt as possible followed by washing with warm water to remove fatty deposits. Afterwards, the equipment has to be washed again with warm water and stored in a clean, dry and dust free area (Robinson, 2002). Different bacterial species isolated from the Dry milk (brand A) were coliform bacteria including, K. pneumoniae, S. 276 A. ALI ET AL gallinarum and S. typhii with frequency of 7.69%. It has previously reported that dominant Gramnegative staining bacteria isolated from raw milk of bulk tank milk were Escherichia coli and Pseudomonas aeruginosa species (Khan et al., 2008). Therefore these findings are similar to our results. Additionally frequencies of bacterial isolation observed in present investigation are also in agreement with the studies performed to assess bacteriological quality of raw milk in Ethiopia (Tassew & Seifu, 2011). On the contrary, the additional bacterial species isolated as Acinetobacter sp., Pantoea sp. and Citrobacter sp. which were not isolated previously might be attributed to higher environmental contamination during transportation and/or contamination during waiting along the roadside. Such kind of results indicates that there are weaknesses in milk transportation and processing. Ashenafi & Beyene (1994) indicated that all microorganisms are not killed by pasteurization especially when their number is very high in raw milk. Therefore, it is suggested that pasteurized milk should not be kept at room temperature for days. Recommendations It is hereby recommended that routine microbial analysis of dairy products sold at public places be carried out to prevent outbreak of human diseases. Also appropriate handling and hygienic practices should be ensured by product vendors in markets. Furthermore, product handlers should be educated on appropriate storage temperature for processed products. 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