Date Published: July 15, 2016
Publisher: Springer Berlin Heidelberg
Author(s): Sudipta Sankar Bora, Jyotshna Keot, Saurav Das, Kishore Sarma, Madhumita Barooah.
This is the first report on the microbial diversity of xaj-pitha, a rice wine fermentation starter culture through a metagenomics approach involving Illumine-based whole genome shotgun (WGS) sequencing method. Metagenomic DNA was extracted from rice wine starter culture concocted by Ahom community of Assam and analyzed using a MiSeq® System. A total of 2,78,231 contigs, with an average read length of 640.13 bp, were obtained. Data obtained from the use of several taxonomic profiling tools were compared with previously reported microbial diversity studies through the culture-dependent and culture-independent method. The microbial community revealed the existence of amylase producers, such as Rhizopus delemar, Mucor circinelloides, and Aspergillus sp. Ethanol producers viz., Meyerozyma guilliermondii, Wickerhamomyces ciferrii, Saccharomyces cerevisiae, Candida glabrata, Debaryomyces hansenii, Ogataea parapolymorpha, and Dekkera bruxellensis, were found associated with the starter culture along with a diverse range of opportunistic contaminants. The bacterial microflora was dominated by lactic acid bacteria (LAB). The most frequent occurring LAB was Lactobacillus plantarum, Lactobacillus brevis, Leuconostoc lactis, Weissella cibaria, Lactococcus lactis, Weissella para mesenteroides, Leuconostoc pseudomesenteroides, etc. Our study provided a comprehensive picture of microbial diversity associated with rice wine fermentation starter and indicated the superiority of metagenomic sequencing over previously used techniques.
The methodology of rice wine preparation is more or less similar among most of the ethnic communities of Assam, a north-eastern state of India; however, fermentation starters (Assamese name xaj-pitha) and the substrates differ resulting in variance in the quality of the final product (Tanti et al. 2010; Das et al. 2012). The use of fermentation starters is believed to have originated in China from where it spread to the other neighboring countries in Asia (Hanai 1992). Various local names for rice wine starters are used in Asian countries, such as banh men in Vietnam, chu in China, koji in Japan, nuruk in Korea, murcha in India, ragi in Indonesia, ragi tapai in Malaysia, and bubod in Philippines (Limtong et al. 2005). In Assam, different ethnic communities also have their own terms to refer to such fermentation starter cultures. The starters are mixed cultures of molds, yeasts, and bacteria that are maintained on substrate, such as rice powder, supplemented with various herbs.
The starter is prepared by concoction of rice flour and several herbs. These herbs are believed to impart intoxicating property to the liquor (Sarma 2002). Apart from contributing various organoleptic properties to the wine, these various plants are also said to have many other medicinal properties (Das et al. 2012). Some of the plant extracts may also provide certain nutrients for the survival and growth of the microflora present in the starter cakes (Thanh et al. 2008).
Rice wine is popular among most of the ethnic communities of Assam. They are prepared and consumed during various religious and harvest festivals. The preparation of rice wine involves the conversion of cooked rice by physical, microbiological, and biochemical operations, including steaming, inoculation with starter, and fermentation. A wide range of microorganisms are involved during fermentation processes, but only a few determines the quality of the endproduct. Three major microbial groups, namely molds, yeasts, and lactic acid bacteria, are reported to be involved in the traditional rice wine starters (Hesseltine et al. 1988; Steinkraus 1989; Lim 1991). Rice wine fermentation basically involves two major steps, viz., liquefaction and saccharification and alcoholic fermentation. In the first step, i.e., liquefaction and saccharification, fermentable sugars are produced from starch by the action of a-amylase and amyloglucosidase from molds which occurs as aerobic solid-state fermentation proceeds. Some yeast can also degrade starch, but this trait is not widespread (Laluce and Mattoon 1984; De Mot et al. 1985). The endproducts in this step are mainly glucose, and also to some extent dextrins and maltose (Crabb 1999). The second step of rice wine fermentation, i.e., alcoholic fermentation, involves conversion of fermentable sugars into alcohol by various yeasts. Some molds are also efficient in alcoholic fermentation. Different bacteria, mainly lactic acid bacteria (LAB), occur as opportunistic contaminants (Gandjar 1999; Thanh et al. 2008).
The MG-RAST analysis showed that the analyzed starter sample had an alpha-diversity of 68.84 species. Taxonomic hit distribution of the sample at domain level showed that 39.2 % sequences belonged to Bacteria and 60.4 % sequences belonged to Eukaryota. Taxonomic hits of distribution at phylum level showed that 38.7 % sequences belonged to Streptophyta, 23 % sequences belonged to Proteobacteria, and 15.8 % belonged to Ascomycota. According to the MEGAN analysis, the total 2,78,231 sequences were found to have maximum abundance of Rhizopus delemar followed by Mucor circinelloides, Lactobacillus plantarum, Meyerozyma gulliermondii, and so on. Along with some efficient molds and yeasts, a wide range of environmental (opportunistic) contaminants were also found, which may pose serious health hazards. At this point, we assume that the main difference between traditional and industrial starters is that traditional starters have a higher resilience over an industrial one. Therefore, it may be suggested that a study on changes of microbiota during spontaneous fermentation at different time points would throw more light into the role of the various microorganisms.