Diversity of Yeasts and Molds by Culture-Dependent and Culture-Independent Methods for Mycobiome Surveillance of Traditionally Prepared Dried Starters for the Production of Indian Alcoholic Beverages

Abstract

Marcha, thiat, dawdim, hamei, humao, khekhrii, chowan, and phut are traditionally prepared dried starters used for production of various ethnic alcoholic beverages in North East states of India. The surveillance of mycobiome associated with these starters have been revealed by culture-dependent methods using phenotypic and molecular tools. We identified Wickerhamomyces anomalus, Pichia anomala, Saccharomycopsis fibuligera, Pichia terricola, Pichia kudriavzevii, and Candida glabrata by ITS-PCR. The diversity of yeasts and molds in all 40 samples was also investigated by culture-independent method using PCR-DGGE analysis. The average distributions of yeasts showed Saccharomyces cerevisiae (16.5%), Saccharomycopsis fibuligera (15.3%), Wickerhamomyces anomalus (11.3%), S. malanga (11.7%), Kluyveromyces marxianus (5.3%), Meyerozyma sp. (2.7%), Candida glabrata (2.7%), and many strains below 2%. About 12 strains of molds were also identified based on PCR-DGGE analysis which included Aspergillus penicillioides (5.0%), Rhizopus oryzae (3.3%), and sub-phylum: Mucoromycotina (2.1%). Different techniques used in this paper revealed the diversity and differences of mycobiome species in starter cultures of India which may be referred as baseline data for further research.

Diversity of Filamentous Fungi Isolated From Some Amylase and Alcohol-Producing Starters of India

“…Drinking of cereal-based mild to strong alcoholic beverages produced by traditionally prepared amylase and alcoholproducing starters has been a traditional food culture of the ethnic people from the North East states of India for centuries. Traditionally prepared dry starters have consortia of co-existed microbiota containing filamentous molds, yeasts, and bacteria and are crudely sub-cultured through a "backslopping" process by traditional starter-makers Tamang and Sarkar, 1995;Tamang et al, 2007;Sha et al, 2018Sha et al, , 2019, for alcohol production by the Indian people. The pH of traditionally prepared dry starters from India were slightly acidic in nature, perhaps due to accumulation of metabolic organic acids (Ma et al, 2019).…”

“…Some traditionally prepared starters from North East India have been microbiologically analyzed in earlier works and several species of yeasts (Tsuyoshi et al, 2005;Jeyaram et al, 2008Jeyaram et al, , 2011Sha et al, 2017Sha et al, , 2018Sha et al, , 2019 and bacteria (Tamang et al, 2007;Pradhan and Tamang, 2019) were reported. However, detailed taxonomical studies of filamentous molds isolated from traditionally prepared dry starters from North East India have not been reported yet, except for marcha (Tamang et al, 1988;Tamang and Sarkar, 1995;Sha et al, 2017Sha et al, , 2019, thiat (Sha et al, 2017(Sha et al, , 2019, amou, perok-kushi (Das et al, 2017).…”

“…A total of 40 samples of traditionally prepared dry starters viz marcha from Sikkim, thiat from Meghalaya, humao from Assam, hamei from Manipur, chowan from Tripura, phut from Arunachal Pradesh, dawdim from Mizoram, and khekhrii from Nagaland (Table 1) were collected directly from local markets and the homes of local producers in North East India (Figure 1) in pre-sterile containers. Dry starter samples were transported to the laboratory and stored in desiccators at room temperature as traditionally prepared dry starters have a shelf life of more than 1 year (Sha et al, 2018).…”

“…Taxonomical identification of filamentous molds isolated from traditionally prepared dry starters from India have not been reported yet except from marcha (Tamang et al, 1988;Sha et al, 2017Sha et al, , 2019, thiat (Sha et al, 2017(Sha et al, , 2019, amou, and perok-kushi (Das et al, 2017). Mucor circinelloides, Rhizopus chinensis, and Rhizopus stolonifer were reported earlier from marcha samples collected from Nepal, Darjeeling, and Sikkim (Tamang et al, 1988;Tamang and Sarkar, 1995;Thapa and Tamang, 2006;Sha et al, 2017Sha et al, , 2018, Amylomyces rouxii and Rhizopus oryzae from samples of amou, and perok-kushi, traditional starters of Assam (Das et al, 2017). Sha et al (2017) reported fungal Phylum Ascomycota (98.6%) followed by Mucoromycota (1.4%), while in marcha samples only Phylum Ascomycota by high-through sequencing was reported.…”

“…Several species of filamentous molds Yang et al, 2011;Lv et al, 2012a;Chen et al, 2014;Das et al, 2017); yeasts (Hesseltine and Kurtzman, 1990;Tamang and Sarkar, 1995;Tsuyoshi et al, 2005;Jeyaram et al, 2008Jeyaram et al, , 2011Thanh et al, 2008;Fronteras and Bullo, 2017;Sha et al, 2017Sha et al, , 2018Sha et al, , 2019, and bacteria (Hesseltine and Ray, 1988;Tamang et al, 2007;Sha et al, 2017;Roslan et al, 2018) are found to coexist in traditionally prepared dry starters as "micro-resources" which have been sub-cultured to preserve essential microbiota for alcohol production by Asian people for centuries (Tamang et al, 2020). Filamentous fungi present in traditional starters from Asia have several functionalities such as saccharification (Lee and Lee, 2002;Thapa and Tamang, 2004), liquefaction (Suesse et al, 2016), and ethanol production (Dung et al, 2007;Chen et al, 2014) to produce different types of low-alcoholic beverages and highalcoholic distilled liquor.…”