The study was carried out in February to July 2017 and the samples of starter culture, plant species and other ingredients were collected and identified (Figure 1) and the detailed indigenous method of making beer was determined during the field visit. The location of villages was chosen according to the presence of the particular ethnic tribal community. The survey were conducted in the village

Tuisindrai (Latitude-23˚82'42.99"N and Longitude-91˚61'97.73"E). under Tusindrai gram panchayat, Teliamura sub-division, Khowai district, State-Tripura, India (Figure 2).

The samples of starter culture, plant species and other ingredients were collected and the detailed indigenous method of making beer was documented during the field visit (Figure 1). The process of rice beer making involves whole rice grains which is cleaned and soaked in water for a few hours followed by cooking and is spread over banana leaf or a mat for 1 - 2 hours for cooling. The starter culture cake “Chuwanchwla” is used as inoculum for this preparation and

kept for natural fermentation. After cooling an appropriate amount of starter culture is mixed with cold cooked rice (approx50 gm in 1 kg). Then the mixture is placed in a container (batikasla) and covered with banana leaf so that minimum vapor can escape out of the container (Figure 1(J)). After that the container is covered with clean cloth and kept for 3 - 4 days for primary fermentation. After 2 - 3 days some water is added and kept again for 3 - 4 days for secondary fermentation. After secondary fermentation, the turbid milky watery extract is filtered and consumed as un-distilled rice beer (Gora). For preparing distilled rice beer, the mixture is heated and the vapor is collected in another container (batibakhrak) placed on the top of the container (Figure 1(L-ii)) and the vapour from batibakhrak is allowed to pass through a bamboo (batisabchaw) in another container containing water (patini) to collect the distilled one which is called “Chuwak” and this distilled beer can be kept for a week for later consumption (Figure 1). However in the laboratory, the same procedure was followed to make Gora and Chuwak under aseptic conditions followed by primary and secondary fermentation. During the fermentation samples were been drawn in regular intervals for analysis.

The industrial brew yeast S. Cerevisiae (MTCC 249) was collected from Kalyani University, Kalyani, West Bengal, India. The rice beer was made in the laboratory in the same manner using the industrial yeast instead of the traditional rice beer starter cake used by the Koloi tribe. During the fermentation, samples were also analysed in regular intervals side by side.

Total genomic DNA was extracted from the yeast cells according to Cenis (1992) with minor modification [15] . The internal transcribed spacer (ITS) region was amplified by polymerase chain reaction (PCR) using universal primers ITS1 (5’-TCCGTAGGTGAACCTGCGG-3’) and ITS4 (5’-TCCTCCGCTTATTGATATGC-3’) [16] . The amplification mix contained 40 mM dNTPs (NEB, MA, USA); 5X Phusion HF reaction buffers (NEB, MA, USA); 2 U/µl F-540 Special Phusion HS DNA Polymerase (NEB, MA, USA); and 5 ng DNA and Milli-Q water to make up 30 µl total volume. The PCR running conditions consisted of an initial 98˚C for 30 s followed by 30 cycles of 98˚C for 10 s, 72˚C for 30 s, and a final extension at 72˚C for 5 s. Successful amplification was confirmed by agarose gel electrophoresis and sequenced with Big Dye (3.1) terminator protocol using ABI 3500 Genetic Analyzer (Applied Biosystem, USA) in the Department of Biotechnology, Mizoram University.

The ITS gene sequences of the isolates were searched for their homologue sequences in public domain databases. Identification to the species level was determined with a sequence similarity of ≥97% with that of a prototype strain sequence in Gen Bank using Basic Local Alignment Search Tool (BLAST) [17] . Phylogenetic trees were constructed with MEGA version 6.0 using a neighbor-joining algorithm bootstrap analyses for 1000 replicates where the evolutionary distances were computed using the Tamura 3-parameter method and are in the units of the number of base substitutions per site [18] [19] [20] [21] .

The sensory evolution was carried out by a food technology scholar panel of 10 members. The final product was evaluated for the following attributes: aroma, appearance, flavor, purity of taste, mouth-feel, gustatory sensations (tingling, bitterness etc.) according to the Standard ISO 8586:2012 with slight modification [27] . Both the beer samples were kept at 4˚C - 8˚C for analysis. The rice beer samples in glass cups were given to the panelist in a single cabin with partition for evolution for different sensory characteristics. The samples were evaluated in random order and every time after testing panelist rinsed their mouth thoroughly between samples with 10 min of intervals. Both the samples were assessed three times. The grading system of the beer samples were on a 10 cm unstructured line scale from 0 to 10 for the testers [28] .

Interestingly after several sequential cultivations of samples we obtained only a single type of yeast strain from the sample of “Gora”. Morphological examination for cultural characteristics revealed a butyrous, white and light cream colour, round to elongate shaped with small lobes, smooth texture, flat elevation, entire marginal and transparent opacity in nature. In YPD broth culture pellicles was formed. Ascospore, pseudohyphae and budding stage was observed under the microscope and the results are illustrated in (Table 2).

The isolated yeast strain holds a high assimilating activity against glucose, glycerol and methanol but weak in maltose and citrate metabolism. Within the nitrogenous compound only L-lysine was found to be assimilated. In case of sugar fermentation the isolate was able to ferment glucose, fructose and mallic acid only. The capability to grow in different temperature, pH was determined comparing the measurement of growth in the media at OD₆₀₀. The other physiological characteristics—growth in different temperature, pH, Urea hydrolysis and starch hydrolysis are given (Table 2). All these preliminary morphological and physiochemical analysis showed similar characteristics with Pichia kudriavzevii [14] . The blast search of the ITS region of the isolated strain showed 99% - 100% similarity with the yeast strain Pichia kudriavzevii isolated from different parts of the worlds. All the conventional, molecular and phylogenetic analysis reviled that the isolated strain was Pichia kudriavzevii (Figure 4) [34] [35] .

The preliminary qualitative physical properties like taste, state, opacity of both the rice beer are mentioned in (Table 3). A trend of gradual decrease in the pH from initial range 5.5 ± 0.004 to 3.55 ± 0.002 and 5.5 ± 0.004 to 3.53 ± 0.003 during 6 - 7 days of fermentation has been observed using the indigenous and commercial yeast strain respectively. The degree of acidity (pH) of the rice beer plays a significant role in its stability during ageing as well as maintains its freshness, taste and flavor. The low pH or high acidity comes from the different degree of utilization of the carbon substrates (rice and plant varieties) used during fermentation by the different tribes. During the growth the yeast cell consume nitrogen source from the substrate and release hydrogen ions continuously leading to decrease in the pH of the solution [36] . This low pH ultimately helps in eliminating enteric pathogens, coliforms and spoilage organisms and enhances the growth of yeast cells in the process. The total acidity of the final product measured 2.20 ± 0.035 and 2.53 ± 0.059 for both the beers respectively. The volatile acidity of Gora also followed the same trend and found 1.34 ± 0.083 and 1.73 ± 0.017 respectively as fermentation progressed. As reported some yeast

also produce acid in alcohol fermentation and this acidity in beer plays a significant role in ageing of rice beer enhancing the flavor of the product [37] . The moisture content was measured after fermentation for the both rice beer was found to be 80.6 ± 0.001 and 78.5 ± 0.008 respectively, moisture content directly affects the food quality, microbial stability, and chemical aspects of the beer. It is also a pre requisite in food processing industries for operating with legal bindings and proper labeling of the product for marketing [38] . The total carbohydrates were analyzed during fermentation at an interval of 3 days from their initial stage shows a rapid decrease of carbohydrate (mg/ml) content from 5.2 ± 0.004 to 0.573 ± 0.053 and 0.56 ± 0.101 to 5.4 ± 0.013 for rice beer produced by the local and the industrial strain respectively. The rice varieties, different plant species and the microbial load of starter culture may be associated with the degree of carbohydrate decomposition in rice beer. The microbes present in the starter culture utilize the carbohydrate as substrates and plays an important role in their metabolism by partial decomposition of carbohydrate and this phenomenon can be beneficial for fermentation yields [39] . After a due course of time of fermentation the sugars starts rapidly metabolize in to acids, ethanol, carbon dioxide, biomass etc. required for the growth of microorganisms [40] . The reducing sugar was found to 1.19 ± 0.999 mg/ml and 1.04 ± 0.999 mg/ml whereas the non-reducing sugar was 0.77 ± 0.014 mg/ml and 1.04 ± 0.010 mg/ml respectively of the final product after 7 days in both the cases. The increase of reducing sugar may be due to hydrolysis of starch and oligosaccharides and the activity of amylases present in the cereal grains and the amylase is responsible for initial phases of fermentation by the microorganism [41] . In fermentation sugar catabolism is an oxidative process reduces the production of reduced pyridine nucleotides after that re-oxidized for further biological process to continue under anaerobic condition. In yeast, the NADH is regenerated by the reduction of pyruvic acid to ethanol [42] . Initially the reducing sugar level increases but ultimately it decreases with the time of ageing. This might be due to the microbes present in rice beer which utilize the reducing sugar for the production of alcohol and energy [22] . The protein content of the rice beer was found increased from 5.5 ± 0.041 to 11.96 ± 0.001 and 5.6 ± 0.034 to 11.63 ± 0.002 respectively in both the cases as shown in the Table 4. The protein level is directly proportional

to the microbial biomass and if the microbial biomass is high, fermentation will be more and the alcohol content will be more [22] . Higher the protein content, beneficial will be the product, resulting adequate protein intake by the consumers. Total alcohol content was measured for the final product and was found 6.40 ± 0.008 v/v for Gora and 6.30 ± 0.007 v/v for the beer prepared using the industrial strain. The distilled alcohol of “Chuwak” was found to 31.46 ± 0.014 v/v and 33.20 ± 0.028 v/v for beer prepared using both the strain respectively. The studied samples showed significant (p < 0.05) results for different parameters (Table 4). These findings show and provide sufficient hope and scope for the fermented rice beer to enhancement its quality, nutrient enrichment, increasing self-life and product stability through ageing. Further careful exploration and study is required and will be promising for its large scale production and commercialization.

The sensory analysis is to evaluate food products which includes evoking, measuring, analyzing and interpreting by senses of sight, smell, taste, touch and hearing by the individuals. In this study the ten scale grading system (between 0 - 10) was used to evaluate the beer based on their individual sensory feelings of the candidates participated. Results of five tested parameters (aroma, appearance, flavor, mouth-feel and overall impression) were shown in (Table 5). The fragrance or aroma of the beer depends on the yeast strain, substrate, hops or other plant parts and ingredients used during fermentation as well as its by-products [43] . Present study showed that the scores of aroma in both the beer samples were nearly equal (Figure 5). Appearance of both the beer ware also results in similar score (Table 5). Gora possess more foam stability then the industrial

strain which might be due to higher protein content. The color of Gora was slight misty then the industrial strain beer may be due to due to the presence of more phenolic compound in the beer [44] . Gora had slightly scored better than the industrial strain beer in respect of mouth-feel. The overall impression was the final opinion from the assessors, and the Gora was found as drinkable and they may prefer to drink more than one glass. Similar finding were also reported by the previous studies on rice beer sensory analysis [45] [46] .