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Native Microorganisms in the Fermentation of Kinema

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Native Microorganisms in the Fermentation of Kinema IndiJIn Journal ofMicrobiology Vol43, No. 2, June 2003, pp 127-130 Native microorganisms in the fermentation of kinema Jyoti Prakash Tamang Food Microbiology Laboratory, Department of Botany, Sikkim Government College, Gangtok, Sikkim 737102, India Kinema is a naturally fermented indigenous soybean food, common to the eastern himalayan regions of India, Nepal and Bhutan. Microbial diversity in kinema was assessed to establish the source of inocula during spontaneous fermentation. Microorganisms associated with kinema were present in or on the ingredient, equipment, ash and wrapping materials, which also contributed significant genetic resources. Species of Bacillus, Enterococcus, Geotrichum, Candida were recov­ ered. Key words: Kinema, microbial diversity, spontaneous fennentation, Bacillus, Enterococcus Kinema is a popular fermented soy bean-based, gray tan purchased from Gangtok market, aseptically kept in ice-box coloured, slightly alkaline, sticky food having ammonical and transported to laboratory for analyses. Fresh leaves of odour, which serves as cheap source of high protein food in fern [locally called pireunion, (Glaphylopteriopsis erubescens a local diet. Preparation and consumption of kinema reflect (Wellex. Hook.) Ching}] and fig plant (locally called neuara, deep rooted food culture of ethnic people of the eastern Ficus hookeriana Corner.) and wood-ash were collected from himalayan regions of Darjeeling hills and Sikkim in India, villages in Sikkim, where kinema is made. Nepal and Bhutan. The preparation of kinema is exclusively Microbial analysis: Ten g of well-mixed sample was blended practiced by mountain women using their indigenous in 90mLsterile physiologicalsaline in a stomacher lab-blender knowledge. Overnight-soaked soybeans are cooked until 400 (Seward, UK)for 10 min. For aerobic endospore-counts, they can be pressed easily, then cracked lightly in wooden 1 mL dilution was mixed with 9 mL sterile physiological mortar (locally called okh1r) and pestle (locally called muslo). saline, and heated for 2 min in continuously boiling water Grits are placed in a bamboo basket, lined with locally grown (4). Decimal dilution series was prepared in sterile diluent fresh fern (or sometime, fig) leaves, about 1% of fire-wood and 1 mL of appropriate diluted suspension was mixed with ash is dusted, covered with a jute-bag and left to fennent molten media and poured into plates. naturally at ambient temperatures (25 - 400 q for 2 - 3 d above earthen-oven in kitchen (1). Kinema is similar to natto Wooden mortar and pestle used during kinema of Japan, chungkok-jang of Korea, ihua-nao of Thailand, dou­ preparation were rinsed with sterile distilled water, which chiofChina, pe-poke of Myanmar;hawaijar of Manipur, tUl1choni was collected in sterile bottles, and transported immediately of Nagaland, turangbai of Meghalaya and bekanihu of to laboratory for microbial analysis using dilution plate Mizoram in northeast India (2,3). Microbiota associated with method as described above. natural fennentation of has not been assessed. We kinema Circular discs (0.5 cm diameter) of fresh leaves of fern examined the materials and equipment involved in kinema and figwere cut with flamed sterilised cork borer and washed preparation, such as soybean, wooden mortar and pestle, in sterile physiological saline for isolation of phyUoplane fire-wood ash, fresh leaves usedas wrapping materials so as microflora (5). One mL of leaf-wash was inoculated into ~urce to establish the of microorganisms associated during plate containing nutrient agar (HiMedia M001) for natural fennentation of kinema. enumeration of endospore-fonning bacteria and incubated Materials and Methods at 370 C for 24 h. Lactic acid bacteria were enumerated on de Man, Rogosa and Sharpe (MRS) agar (HiMedia Sample collection: Local variety of soybean having Yellow­ M641) supplemented with 1% calcium car~nate and seed [Glycine max(L.)Merrill] as well as kinema sampleswere incubated anaerobically in an Anaerobic Gas-Pak system *Corresponding author; E-mail: [email protected] (HiMedia LEOO1) at 300C for 72 h. Total viable counts were Tel: 03592-231053 determined using plate count agar (HiMedia M091A) after 128 Indian! Microbiol, [une2003 incubating at 30° C for 48 h. Presence of the members of during soaking of soybeans, indicating their entry through Enterobacteriaceae was tested by using selective violet red water source. E.[aecium appears as non-faecal contaminant bile glucose agar (HiMedia M581) and incubating at 30° C (17). Mulyowidarso et al (18) reported the presence of E. for 48 h. Samples were examined for the presence of yeasts faecium during soaking of soybeans in iempeh fermentation. and moulds, using malt extract ag~r (HiMedia M924) Level of Enterobacteriaceae was <1Q4 cfu g.l in ovemight­ supplemented with 100 mgl.:' chloramphenicol and soaked soybeans. Mortar and pestle also supplemented incubating aerobically at 28° C for 72 h. Typical colonies population of Enterobacteriaceae, which was detected at a were isolated from different samples and subjected to levelof 1()2cfu g". Cells of microorganisms were killed during taxonomical identification. cooking of soaked soy beans except heat-resistant spore­ formers. B. subtilis was found in all sources indicating its Phenotypic characterization and identification: Initial main role in fermentation. Source of E.faecium, C.parapsilosis characterizationof bacterial isolates included colony and cell and G.candidumwas mainly from wooden mortarand pestle morphology, Gram staining and catalase reactions. Motility commonly used to crack cooked soybeans before wrapping test was observed in a phase contrast microscope (Olympus during kinema fermentation. Usually wooden mortar and CH3-BH-PC, Japan) following the method of Harrigan (6). pestle are not washed properly by the rural kinema-makers All other phenotypic characterizations of bacterial isolates after cracking soybeans. This equipment is the main source were carried as per standard procedures (7,8). Ability of the of inocula for spontaneous fermentation of kinema. bacterial isolates to ferment carbohydrates was studied using Phylloplane studies of fresh fern fronds and fig leaves used API 50 CHB and API 20Strep (biolvlerieux, France) system. as wrapping materials also supplement B. subiilis and E. Endospore-forming bacteria were identified according to faecium. Yeasts and moulds were not recovered from these the keys based on Claus and Berkeley (9),and Slepecky and leaves. Singh and Umabati Devi (19) reported the presence Hemphill (10).Lactic acid bacteria were identified following of Bacillus and Xanthomonas spp. in fig leaves, used as the taxonomic keys laid down in Bergey's Manual of wrapping material during hauraijar production. The Systematic Bacteriology, (11) and by Wood and Holzapfel populationof yeasts as well of lactic acid bacteria were found (12). Yeast-isolates were identified by the standard to increase remarkably in kinema, stored at room temperature morphological and biochemical tests (13). after desirable fermentation (Fig 1). Enzymaticprofiles: The enzymatic profile of bacterial isolates was assayed following the standard method (14) using API .8 • Spcwe-bmer aLAS • v.-t D Tot.. c:an zym (biolvlerieux, France) galleries based on the manufacturer's colour chart. 8 Results and Discussion Microbial load in different sources during spontaneous fermentation of kinemais shown in Fig 1. Fifty six strains of rod-shaped, Gram-positive, endospore-forming bacteria were identified as Bacillus subiilis (Ehrenberg) Cohn according to the criteria laid down by Claus and Berkeley (9).Following the taxonomical keys (12,15), 30 strains of cocci-shaped, Gram-positive, catalase-negative, non-spore-forming lactic ~ ~ acid bacteria were identified as Enterococcus fuecium (Orla­ .1 .. ~ Jensen) Schleifer and Klipper-Balz. Two types of yeasts were recovered and were identified as Candida parapsilosis (Ashford) Langeron and Taliceand Geotrichum candidum Link Source of inocula following the taxonomical keys (13). Fig 1. Load of microorganisms associated during spontaneous Microbial analysis of raw soybeans showed the presence fermentation of kinema. Data represent the means of three replications. of Bacillus subtilis spores. Hesseltine (16) also reported p:esence of B. subtilis on raw soybeans. Besides B. subtilis, Bacillus-strains produced a wide spectrum of population of E.faecium and yeasts predominantly occurred enzymes, whereas strains of Enterococcus exhibited Fermentation of kinema 129 Table 1. Enzymatic profiles of bacterial strains isolated from different sources during spontaneous fermentation of kinema Enzyme Substrate Activity (nanomoles) Bacillus Enterococcus subtilis faecium (n = 10) (n = 6) Phosphatase alkaline 2-Napthyl phosphate ~ 40 o Esterase (C4) 2-Napthyl butyrate 5 5 Esterase lipase (CB) 2-Napthyl caprylate 5 5 Lipase (C14) 2-Napthyl myristate o o Leucine arylamidase L-Leucyl-2-napthylamide ?-'.40 10 Valine arylamidase L-Valyl-2-napthylamide o o Cystine arylamidase L-Cystyl-2-napthylamide o o Trypsin N-Benzoyl-DL-arginine-2-napthylamide o o Chymotrypsin N-Glutaryl-phenylalanine-2-napthylamide o o Phosphatase acid 2-Napthyl phosphate > 40 20 Naphthol-A5-BI-phosphohydrolase Napthol-AS-BI-phosphate ~ 40 5 a-galactosidase 6-Br-2-napthyl-aD-galactopyranoside o o ~-galactosidase 2-Napthyl-pD-galactopyranoside o o ~-glucuronidase Napthol-AS-BI-PD-glucuronide o o a-glucosidase 2-Napthyl-aD-glucopyranoside 5 o ~-glucosidase 6-Br-2-napthyl-PD- glucopyranoside o
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