Indian Journal of Traditional Knowledge Vol. 11(1), January 2012, pp 139-142 Microbial and chemical changes during preparation in the traditionally fermented soybean product Tungrymbai of ethnic tribes of Meghalaya Sharmila Thokchom & S R Joshi* Microbiology Laboratory, Department of Biotechnology & Bioinformatics North-Eastern Hill University, Shillong-793022, India E-mail: [email protected] Received 20.12.10; revised 15.08.11 In the present investigation, Tungrymbai, an ethnic fermented soybean food of the ethnic tribes of Meghalaya, India was analyzed for the proximate microbial and chemical changes occurring in the fermented product due to preparation method. Among the aerobic mesophilic forms, lactic acid bacteria (LAB), Enterobacteriaceae, spore formers, yeast and fungal counts; the microbial loads of spore forming bacteria count were not affected in the post-cooked Tungrymbai as compared to other counts. The pH and total titratable acidity were higher in the pre-cooked Tungrymbai while moisture content was higher in the post-cooked sample. The microbes that were prevalent in both the pre-cooked and post-cooked fermented samples were Bacillus subtilis, Enterococcus durans, Vagococcus lutrae, Staphylococcus equorum and Saccharomyces sp. However, probiotic bacteria like Lactobacillus were not detected in post-cooked samples indicating that the preparation method significantly altered the composition of lactic acid bacteria. Keywords: Traditionally fermented soybean, Tungrymbai, Pre-cooked, Post-cooked, Microbial, Chemical, LAB IPC Int. Cl8: A61K36/00, A01G1/00, A01G17/00, A47G19/00, A23L1/00, A23L1/06 Food fermentation is practiced by human cultures all or bacteria which are either sourced from the over the world and it serves as a major component of environment, or carefully kept in cultures maintained human survival in places where preserved food is a by humans. Tungrymbai is an ethnic fermented necessity. The preparation of many indigenous or soybean food prepared by the indigenous Khasi tribes traditional fermented foods and beverages remain of Meghalaya in India. It forms an intricate part of the today as a household art. It is the diversity of raw diet and serves as a cheap source of high protein food materials used as substrates, methods of preparation in the local diet. It is a sticky food that exhibits unique and sensory qualities of finished products that are so flavour and texture that may not be palatable to astounding as one begins to learn more about the everyone. Preparation and consumption of this food eating habits of various cultures. Production of reflect deep-rooted food culture of the ethnic fermented food does not require knowledge of the communities. It is exclusively prepared by the local biologically mediated nature of fermentation as people using indigenous technology. Dried soybean the biota that carryout fermentation are present seeds are cooked until soften, excess water is drained worldwide1. The main components of the off, are placed on fresh leaves of Pyrnium pubinerve fermentation ecosystem include: microbes (yeast and Bl. (Marantaceae) locally called ‘slamet’ and are left to ferment naturally in ambient temperature (25-30°C) bacteria), organic material to be fermented, a solution 2 in which the fermentation takes place, a vessel with preferably near/over the fire place for 3-4 days which a controlled gate, and various tools. This is an is ready for further preparation and at this stage it is ecosystem with a complex of living and non-living indicated as pre-cooked Tungrymbai. This fermented components that are viewed in terms of their food is then cooked by supplementing various interactions in a specific place1. ingredients like mustard oil, ginger, garlic, black beefsteak seeds until completely fried which is made Fermented foods are generally produced using into ready to eat (post-cooked) Tungrymbai and is plant or animal ingredients in combination with fungi consumed along with routine meals. ——————— In this investigation, the pre- and post-cooked *Corresponding author Tungrymbai were analyzed for their microbial 140 INDIAN J TRADITIONAL KNOWLEDGE, VOL 11, NO 1, JANUARY 2012 composition and chemical properties for a as ready-to-eat product. Firstly, mustard oil is heated at comparative assessment to find out the changes 100° C, then paste of garlic ginger and beefsteak are brought about by cooking practices of the fermented added and fried until brown. Finally fermented food and to analyse the changes in the microbial load Tungrymbai is added and fried until cooked (Fig. 2). specially of LAB in fermented soybean present at This serves as ready-to-eat cooked Tungrymbai. two different stages of preparation. Mustard oil is heated at around 100° C in a pan Methodology Paste of spices such as ginger, garlic, black beefsteak Preparation of Tungrymbai seeds (Perilla ocymoides) are added and fried until brown Pre-cooked Tungrymbai (Fermentation of soybean) The local variety of soybean seeds [Glycine max (L.) Merrill] are washed in cold water and boiled until Fermented soybeans are then added and fried soften for about 45 min – 2 hrs at 100°C. The excess until cooked water is drained off and left open for the temperature to drop down to 25°-30° C. The boiled soybean seeds Ready-to-eat product and consumed with regular are transferred to bamboo basket aligned with fresh meals (Post-cooked Tungrymbai) leaves of Pyrnium pubinerve Bl. (Marantaceae) Fig.2—Preparation of post-cooked Tungrymbai locally called ‘slamet’ in which 2-3 hot charcoal are Pre- and post-cooked Tungrymbai samples were placed and are fully wrapped with the leaves. The collected aseptically in sterile containers from the local basket is placed inside a jute bag and kept for markets of Shillong and were processed within 24 hrs. fermentation at 25°-30° C for 3-4 days preferably The samples were analyzed for their pH, moisture near a fire place. The fermented soybeans, i.e. content, dry weight, total protein and total titratable Tungrymbai, are crushed lightly in wooden mortar acidity according to AOAC methods3. The titratable and pestle (Fig.1). This serves as pre-cooked acidity was calculated as per cent lactic acid. fermented Tungrymbai. Ten gram of the samples were blended in 90 ml of Dried soybean seeds washed in cool water physiological saline for 10 min. They were serially diluted in the same diluent and plated on plate count Boiled at 100° C for about 45 min to 2 hrs until agar (PCA), de Mann, Rogosa and Sharpe (MRS) softened agar, potato dextrose agar (PDA) and violet red bile glucose agar (VRBG) for total aerobic mesophilic Excess water drained off and left opened for count, lactic acid bacteria count, yeast and mould temperature to drop down to 25°-30° C count and Enterobacteriaceae count respectively. For the spore forming bacteria count, 1 ml of blended Transferred to bamboo basket aligned with leaves samples were heated for 2 min in boiling water, (Pyrnium pubinerve Bl.) in which 2-3 hot charcoals serially diluted and plated on tryptone soya agar are placed. The seeds are then fully wrapped with (TSA). The PCA, VRBG, TSA plates were incubated the leaves at 37° C for 24 hrs whereas MRS and PDA were incubated at 25° C for 3 days. The basket was put into a jute bag and kept for fermentation at 25°-30° C for 3-4 days Characterization and identification Gram staining, spore staining, catalase, oxidase Fermented soybeans are crushed lightly in wooden and biochemical tests such as sugar fermentation, mortar and pestle ammonia production from arginine were done for the initial characterization of the bacteria isolated from Fermented Tungrymbai (Pre-cooked) both the samples. Molecular analyses were performed for the Fig.1—Preparation of Tungrymbai confirmation of the isolates. PCR of the 16S rRNA Post-cooked Tungrymbai gene was performed using universal primer 27F For consumption purposes, a mixture of ingredients (5’-AGAGTTTGATCCTGGCTCAG-3’) and 1492R are supplemented to the fermented sample to prepare it (5’-TACGGYTACCTTGTTACGACTT-3’) (Thomas THOKCHOM & JOSHI: MICROBIAL AND CHEMICAL CHANGES IN TRADITIONALLY FERMENTED 141 SOYBEAN PRODUCT TUNGRYMAI 2004). The final reaction mixture (50µl) contained Table 1—Chemical analysis of pre- and post-cooked 10 mM Tris-HCl; 50 mM KCl; 1.5 mM MgCl2; 0.2 mM Tungrymbai samples Total (each) dATP, dCTP, dGTP, and dTTP; 0.2 µM Moisture Dry Total primers; 1.25 IU of Taq DNA polymerase and 6 µl of Titratable Sample pH content Weight Protein Acidity template DNA. The PCR was set as 94° C for 5 min; (%) (%) (g/100gm) (Molarity) then 30 cycles at 94° C for 1 min, 55° C for 1 min, Pre-cooked 6.95-7.05 88.31 11.69 46.50 0.0092 and 72° C for 2 min, followed by 72° C for 5 min. Tungrymbai The amplified products were purified and sequenced. Post-cooked 5.86-6.00 93.42 6.58 29.30 0.0064 Phylogenetic analysis was done according to Tungrymbai 4 prescribed method . Table 2—Microbial population counts of pre-and post-cooked Tungrymbai Results Microbial Enumeration Average microbial count per gm The chemical analyses showed changes in dry weight properties of pre- and post-cooked Tungrymbai Pre-cooked Post-cooked (Table 1). The moisture content was calculated as loss Tungrymbai Tungrymbai Aerobic Mesophilic on drying (LOD): 56.19 x 106 19.6 x 105 Count 100× wt. loss on drying, gm Enterobacteriaceae count 36.09 x 106 8.63 x 104 % (w / w) LOD = % (w / w) moisture = Spore Forming Bacteria wt. test portion, gm 46.3 x 106 41.4 x 106 Count Lactic Acid Bacteria % Dry matter = 100 - % LOD 31.9 x 106 31.7 x 104 Count The average microbial population count showed Yeast and Fungal count 7.9 x 106 4.7 x 104 significant difference between the pre- and post-cooked Tungrymbai samples (Table 2).