Isolation and Identification of Lactic Acid Bacteria and Their Exploration in Non-Dairy Probiotic Drink
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Int.J.Curr.Microbiol.App.Sci (2017) 6(4): 1023-1030 International Journal of Current Microbiology and Applied Sciences ISSN: 2319-7706 Volume 6 Number 4 (2017) pp. 1023-1030 Journal homepage: http://www.ijcmas.com Original Research Article https://doi.org/10.20546/ijcmas.2017.604.127 Isolation and Identification of Lactic Acid Bacteria and their Exploration in Non-Dairy Probiotic Drink Mamta Thakur1*, H.W. Deshpande2 and M.A. Bhate3 1Department of Food Trade and Business Management, College of Food Technology, Vasantrao Naik Marathwada Krishi Vidyapeeth, Parbhani - 431 402 (Maharashtra) India 2Department of Food and Industrial Microbiology, College of Food Technology, Vasantrao Naik Marathwada Krishi Vidyapeeth, Parbhani - 431 402 (Maharashtra) India 3Department of Microbiology, Shivaji College, Parbhani- 431 402 (Maharashtra) India *Corresponding author ABSTRACT Lactic acid bacteria were isolated from the curd and pickle samples. The K e yw or ds morphological and biochemical characteristics and sugar fermentation pattern were employed to identify lactic acid bacteria. The isolates from both curd and Biochemical tests, pickle were identified as rods/bacilli. Both the isolates were Gram positive, non- Carbohydrate fermentation, motile, catalase negative and exhibited negative pattern of citrate utilization, H2S formation, indole production, oxidase test, urease activity, NH production from Identification, Lactic 3 acid bacteria, Probiotic arginine and VP reaction. Phenotypic methods, especially determination of sugar pomegranate drink. fermentation patterns, were found to be more effective than biochemical tests for the differentiation of bacilli isolates at the strain level using Bergey's Manual of Article Info Determinative Bacteriology. Fermentation profile of nine different sugars was Accepted: taken as base for identifying isolates. The isolate from curd was identified to be L. 12 March 2017 delbrueckii ssp. bulgaricus while other was Lactobacillus plantarum. The Available Online: genotypic identification also supported the results of phenotypic methods. Thus 10 April 2017 identified cultures were then incorporated in pasteurized pomegranate juice and allowed to ferment for preparing the drink with optimum level of probiotic culture. Introduction Industrialization of biotechnological distributed in nature and able to promote transformation of the foodstuffs has increased fermentation by utilization of food nutrients the economic importance of lactic acid and produce a variety of substances like bacteria (LAB) because they play a crucial organic acids, aromatic compounds, health role in the sensorial and safety aspects of benefit-substances, etc (O'Shea et al., 2011). fermented foods. Further, interest in the use of LAB is characterized as gram-positive, non- LAB has significantly increased globally due motile, non sporulating bacteria that produce to their nutraceutical role (Leisner et al., lactic acid as a major product of fermentative 1999). Lactic acid bacteria are widely metabolism (Pelinescu et al., 2009). They are 1023 Int.J.Curr.Microbiol.App.Sci (2017) 6(4): 1023-1030 Generally Recognized as Safe and can be Isolation of lactic acid bacteria used well for medical and veterinary applications (Hoque et al., 2010). Some After, weighing and homogenization of each species of the genus Lactobacillus, sample, 1:10 dilution was subsequently made Lactococcus, Pediococcus and Leuconostoc using sterile normal saline (0.85%) followed are included in this group. by making a 10 fold serial dilution. The 0.1 mL from each dilution was then sub-cultured To comprehend the emerging requirement of aseptically into MRS (deMan Rogosa and probiotics, many studies focused on isolating Sharpe) agar (Guessas and Kihal, 2004) using the probiotic potential LAB from different pour plate technique, all plates were then resources of fermented foods (Kalui et al., incubated at 37˚C for 24-48 hours in 2009). anaerobic condition to provide an optimal environmental for growing Lactobacilli. Dairy is a very nutritious substrate for LAB but expanding trend of vegan lifestyles; the Only the Gram positive and rod shape isolates issues of lactose intolerance and demand for were then purified by streak plating using the low-fat and low-cholesterol foods have MRS medium. After several subcultures, created a need for non-dairy probiotic finally the single colony of LAB was isolated products (Ranadheera et al., 2010). by observing their colony morphology and Gram staining, catalase and motility test. The Fresh fruits and vegetables are considered culture was kept in MRS agar slant and stored good matrices and can provide ideal at 4 °C for long term storage (Hawaz, 2014). substrates for probiotics, since they contain minerals, vitamins, antioxidants and fibers Identification of lactic acid bacteria and do not contain any dairy allergens (Luckow and Delahunty, 2004). Pomegranate The isolated colony was identified using (Punica granatum L.), known to be a Gram staining, biochemical tests and superfruit, has health-promoting properties carbohydrate fermentation profile (Chris et with antimicrobial, anticancer, antioxidant al., 2006). and antimutagenic effects (Negi et al., 2003). Biochemical identification In the present investigation, lactic acid bacteria were the isolated and identified from Strains with Gram positive, catalase negative curd and pickle in order to use them as starter and no motility were further used for for developing probiotic drink based on identification (Sharpe, 1979). Utilization of pomegranate juice. citrate, hydrolysis of arginine, production of hydrogen sulfide and indole were determined Materials and Methods (Samelis et al., 1994). Further, methyl reduction test, oxidase test, urease test and VP The curd, mango pickle and freshly harvested reaction were also performed. pomegranate fruits (var. Bhagwa) were procured from local market of Parbhani Carbohydrate fermentation profile (Maharashtra) India. Pomegranate juice was prepared by blending the juicy arils in the All the strains were also tested for domestic mixer. Its total soluble solids was fermentation of sugars viz. glucose, fructose, maintained to 13oBx and stored at 4oC before lactose, sucrose, maltose, mannitol, salicin, use. 1024 Int.J.Curr.Microbiol.App.Sci (2017) 6(4): 1023-1030 agarose and sorbitol by adding the specific was allowed to ferment in incubator at 37oC sugar to the basal carbohydrate media. Acid for 7 h. After incubation, the drink was kept at production was indicated by a change in color refrigeration temperature for future use. and gas production was detected by observation of gas collection in the inverted Microbial analysis of probiotic Durham tubes (Tserovska et al., 2002). pomegranate drink Molecular identification of lactic acid The viable count of mixed culture was bacteria determined by the standard plate count method using Man-Rogosa-Sharpe agar The isolated bacteria were sent to Agharkar (MRS agar) and the results were expressed as Research Institute, Pune where strains of CFU/ml juice. The yeast and mold count of promising probiotic LAB were identified by drink was determined using potato dextrose 16S rRNA multiplex PCR analysis agar medium. The coli-form and basically E. (Markiewicz and Biedrzycka, 2005). This was coli are the indicator microbes of water in confirmation to the morphological, contamination by feces. The coli-form gives biochemical and carbohydrate fermentation red pink color colonies on the MacConkey tests performed in Department of Food and agar (Chris et al., 2006). Industrial Microbiology, College of Food Technology, V.N.M.K.V., Parbhani Results and Discussion (Maharashtra) India. Isolation of lactic acid bacteria Preparation of starter culture Bacteria were isolated with MRS agar media L. plantarum and L. bulgaricus was cultivated at 37°C under anaerobic conditions for 48 separately in the MRS broth for 24-h at 37oC. hours. The isolated colonies from curd were To obtain the biomass, 10 mL of the named as Isolate A and from pickle were as separately cultivated MRS broths were mixed Isolate B (Fig. 1). The isolated colonies were in equal proportion (1:1) and centrifuged at then screened for further identification. 4000 rpm for 10 min. The obtained biomass was washed with sterile saline solution twice Identification of lactic acid bacteria species to remove the residual MRS media. Thus, inoculum was prepared. The complete findings of morphological and biochemical characterization of the isolates It was then introduced into pasteurized are presented in table 1. pomegranate juice (100 mL) for making it 10% concentration of probiotics. The Both the isolates gave purple/violet color with inoculated juice was then incubated at 37oC staining showing Gram positive nature (Fig. for 24 h and was treated as starter culture for 2). Because Gram positive cell wall contain preparation of final drink. thick layer of peptidoglycan with numerous teichoic acid cross linking which resists the Preparation of probiotic pomegranate decolorization, thus remaining purple in color. drink The isolates coming from MRS plates were all rods with long and rounded ends. They Above prepared starter culture (10mL) was appeared mostly as a chain of 3-4 cells or then added to the pasteurized pomegranate single or in pairs. juice (100 mL) to obtain 10% inoculation. It 1025 Int.J.Curr.Microbiol.App.Sci (2017) 6(4): 1023-1030 The hanging drop method showed that the were found by Forouhandeh (2010) in the bacteria were non-motile which one of the isolation