Identification of yeast strains isolated from marcha in Sikkim, a microbial starter for amylolytic fermentation

Abstract

Marcha or murcha is a traditional amylolytic starter used to produce sweet–sour alcoholic drinks, commonly called jaanr in the Himalayan regions of India, Nepal, Bhutan, and Tibet (China). The aim of this study was to examine the microflora of marcha collected from Sikkim in India, focusing on yeast flora and their roles. Twenty yeast strains were isolated from six samples of marcha and identified by genetic and phenotypic methods. They were first classified into four groups (Group I, II, III, and IV) based on physiological features using an API test. Phylogenetic, morphological, and physiological characterization identified the isolates as Saccharomyces bayanus (Group I); Candida glabrata (Group II); Pichia anomala (Group III); and Saccharomycopsis fibuligera, Saccharomycopsis capsularis, and Pichia burtonii (Group IV). Among them, the Group I, II, and III strains produced ethanol. The isolates of Group IV had high amylolytic activity. Because all marcha samples tested contained both starch degraders and ethanol producers, it was hypothesized that all four groups of yeast (Group I, II, III, and IV) contribute to starch-based alcohol fermentation.

Introduction

In Oriental countries, microbial inocula, in the form of dry powders or hard balls made from starchy cereals, are used to prepare food and alcohol fermentations. These starter preparations have a variety of names such as marcha or murcha in the Himalayan regions of India, Nepal and Bhutan, ragi in Indonesia, loogpang in Thailand, bubod in the Philippines, Chinese yeast in Taiwan (Hesseltine et al., 1988), and nuruk in Korea (Park et al., 1977). Marcha is a mixed dough inocula used as a starter culture for production of various indigenous alcoholic beverages such as jaanr in the Darjeeling hills and Sikkim in India, Nepal, and Bhutan (Tamang et al., 1996). It is a dry, round-to-flattened, creamy white to dusty white, solid ball-like starter ranging from 1.9 to 11.8 cm in diameter and from 2.3 to 21.2 g in weight.

Marcha or murcha is a Nepali word. Different ethnic communities of the region call it by their own dialect such as khesung by Limboo, bharama by Tamang, bopkha or khabed by Rai, phab by Bhutia and by Tibetans, and buth/thanbum by the Lepcha. During preparation, glutinous rice is soaked in water for 6–8 h and crushed in a foot-driven heavy wooden mortar and pestle. In 1 kg of grained rice, ingredients added include roots of Plumbago zeylanica L., 2.5 g; leaves of Buddleja asiatica Lour, 1.2 g; flowers of Vernonia cinerea (L.) Less, 1.2 g; ginger, 5.0 g; red dry chili, 1.2 g; and previously prepared marcha as a mother culture, 10.0 g. The mixture is kneaded into flat cakes of varying sizes and shapes and placed individually on a platform suspended below the ceiling that is made up of bamboo strips above the kitchen, bedded with fresh fronds of ferns [Glaphylopteriolopsis erubescens (Wall ex Hook.) Ching], and covered with dry ferns and jute bags. These are left to ferment for 1–3 d. Finally, cakes of marcha are sun-dried for 2–3 d and stored in a dry place for more than a year (Thapa, 2002). Marcha is produced at home exclusively by women. This art is practiced as a hereditary trade that passes from mother to daughters.

The microflora of marcha consists of filamentous molds such as Mucor circinelloides and Rhizopus chinensis, yeasts such as Saccharomycopsis fibuligera and Pichia anomala, and bacteria such as Pediococcus pentosaceus (Tamang and Sarkar, 1996). Among these microbes, saccharifying yeast species belonging to Saccharomycopsis predominate, suggesting that they are involved in the amylolytic fermentation (Hesseltine and Kurtzman, 1990, Tamang and Sarkar, 1996) although alcohol producing microbes have not yet been recovered. The present study investigated the yeast diversity in marcha of Sikkim, and their possible roles in jaanr production are discussed.