722 BACTERIA ASSOCIATED WITH NON-ALCOHOLIC FERMENTED BAMBOO SHOOT FOOD PRODUCT Asifa Qureshi* 1 , Yogeshwari B. Itankar 1 , D.Saikia 2 , M.Mandal 2 , Hemant. J. Purohit 1 Address(es): Dr Asifa Qureshi, 1.Environmental Genomics Division, CSIR-NEERI, Nehru Marg, Nagpur 20, India. 2. Tezpur University, Assam, India. *Corresponding author: a_qureshi@neeri.res.in ABSTRACT Keywords: Non-alcoholic fermentation, bamboo shoots, bacteria INTRODUCTION Bamboo shoots as food products are rich source of various macro and micro nutrients but needs processing before consumption. Fermentation is one of the processing method which increases the nutritional quality of the bamboo food product in terms of its amino acids, sugars, esters and organic acid contents (Jeyaram et al., 2008). Although fermentation is one of the oldest and most economic methods of food preservation and ethnic people of Sub-Himalayan region of North East India, especially Assam and Andhra Pradesh prepare and consume a variety of domesticated and wild bamboo tender shoots and their fermented products, there is need to increase the probiotic attributes to the food for betterment of health (Jeyaram et al., 2008, Qureshi et al., 2014). Some popular fermented bamboo tender shoots of North East India are Mesu of Sikkim, Arunachal Pradesh, Henoop, Khorisa of Assam, Hikung ,Mesu, Bastenga, Hiring of Arunachal Pradesh, Lung-siej of Meghalaya, Soibum, Soidon, Soijm of Manipur (Tamang et al., 2008). Henoop, Khorisa, Mesu, Hikung, Bastenga are traditional non-alcoholic fermented food products of Assam and Arunachal Pradesh consumed by Kharbi, Sonowal, Assamese community of Assam and Monpa, Nepali community of Arunachal Pradesh of North-East India (Das et al., 2012; Tamang et al., 2012). Fermented food products are not only rich in nutrients such as proteins, vitamins, essential amino acids, sugars, fatty acids but are also good for digestion (Jeyaram et al., 2008, Das et al., 2012). Most of the bacterial species associated with the fermented food do not possess health risk, they are designated as GRAS (generally recognized as safe) organism (Hansen, 2002, Das et al., 2012). The objective of our study was to demonstrate culturable microbial diversity of traditionally processed fermented bamboo shoot products of Assam and Arunachal Pradesh, India using molecular approach and designate probiotic attributes of each isolate as safe and healthy food for consumption. MATERIAL AND METHODS Total six food samples of fermented bamboo shoot products were collected from different parts of Assam and Arunachal Pradesh in India. Out of six, three samples were from Erdangte, Khowang, North-Lakimpur, Dibrugarh locations of Assam and three samples were from Ziro, Bomdila, Bhalukpung of Arunachal Pradesh. All the samples were collected aseptically into sterile containers and transferred to the laboratory for analysis. . Fermented bamboo shoot products were prepared by defoliating and finely chopping the young edible shoots of the bamboo plant. The small pieces of bamboo shoots were placed tightly into green hollow bamboo stem and were covered with leaves of bamboo. This was then allowed to ferment at room temperature for 7-14 days. These fermented bamboo shoots were extensively used as pickles, curries and as additives in various recipes Characterisation of fermented bamboo shoot products Microbiological analysis Each food product (5gms) was homogenized with 45ml of 1X PBS. It was diluted serially in the same diluents. Appropriate decimal dilution (100µl) of the homogenate was spread over different media plates such as Man, Rogosa and Sharpes (MRS) agar plate (Himedia), nutrient agar, 30 different media plates having different media components and incubated at 30 o C for 24-48 hrs. Morphologically different colonies were selected and pure cultured by streaking repeatedly on respective agar media plates. The isolates were grown in respective broth and pure cultures were preserved in 30% glycerol at -80⁰C (Sanyo, Ultralow deep freezer). The average number of microbes present per gram of different types of samples were also calculated, which was expressed in terms of CFU (colony forming units) per gram of sample (Table 1). GC-MS based metabolite profiling of fermented bamboo shoot products Sample preparation was carried out by modifying the method described earlier by various researchers (Ojinnaka et al., 2013, Lee et al., 2007). Fermented bamboo shoot products were finely ground in mortar and 500mg of the grinded samples were extracted with 5ml of methanol at 60 º C in a heated water bath for 30 min. Samples were cooled to ambient temperature for 30 min and then vortexed for 30-60seconds prior to centrifugation at 3000rpm, at 4ºC for 10 min. Supernatant obtained was passed through the oven-dried anhydrous sodium sulfate. The filtrate obtained were used for GC-MS analysis. GC-MS analysis (GC Clarus 500 Perkin Elmer system) of a food sample each from Assam and Arunachal-Pradesh was performed employing the following conditions: column Elite-1 fused silica capillary column (30 x 0.25 mm ID composed of 100% Dimethylpolysiloxane), operating in electron impact mode at 70 eV; helium (99.999%) was used as carrier gas at a constant flow of 1 ml /min and an injection volume of 0.5 μl was employed (split ratio of 10:1) injector temperature 250ºC; ion-source temperature 280ºC. The oven temperature was programmed from 150ºC (isothermal for 2 min), with an increase of 6ºC/min, to 200ºC, then 5ºC/min to 300ºC, ending with a 10 min (isothermal at 300ºC). Mass Pure bacterial cultures were isolated from fermented products collected from two locations of North East India (Assam and Arunachal Pradesh). Forty four variants were identified by 16S rRNA gene sequencing. The dominant microbial genera found were Bacillus, Paenibacillus followed by Oceanobacillus and Lactobacillus in Assam and Bacillus, Enterococcus, Lactobacillus in Arunachal-Pradesh food products. Bacillus isolates showed extracellular enzyme production (amylases, proteases and lipases) as analyzed through plate assay. GC-MS analysis showed beneficial chemical components like organic acids, esters, aminoacids, vitamins in fermented bamboo shoot products. Probiotic attributes of culturable bacterial isolates from bamboo fermentations gives promiscuity for human consumption. Beneficial microorganisms from bamboo shoot fermented food products of North-Eastern region of India were explored and compared using bioinformatics tools. ARTICLE INFO Received 18. 9. 2014 Revised 5. 11. 2015 Accepted 19. 4. 2016 Published 1. 8. 2016 Regular article doi: 10.15414/jmbfs.2016.6.1.722-729 brought to you by CORE View metadata, citation and similar papers at core.ac.uk provided by IR@NEERI (CSIR)