Identification of yeast strains isolated from marcha in Sikkim, a microbial starter for amylolytic 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.
Section snippets
Sample collection
Three samples of marcha were purchased from different markets at Gangtok in Sikkim. Two samples were obtained from the Sang and Aho villages in East Sikkim. One sample was obtained from Jhosing village in North Sikkim.
Isolation of yeasts
Samples were cut into 5-mm cubes and homogenized with 500 μl of sterile distilled water. The suspension was serially diluted with sterile distilled water. A 0.1-ml aliquot of the diluted suspension was pipetted and spread onto YM agar plates (Difco Laboratories, Detroit, MI, USA).
Results
Twenty-two yeast strains were isolated from six samples of marcha (A–F; Table 1). There were 3.5×106 to 3.2×109 yeast colonies that developed from 1 cm3 of sample. On the other hand, filamentous molds recovered at less than 104 cfu (mostly <103) were omitted from further investigation. Twenty-two yeast strains underwent preliminary phenotypic characterization using the API test and were divided into four groups (Group I, II, III, and IV).
Representative strains from each group were randomly
Discussion
In this study, phylogenetic and phenotypic studies of the yeasts isolated from marcha demonstrated that the amylolytic starters constituted more diverse microflora than previously recognized. The yeast species identified were S. bayanus, C. glabrata, P. anomala, S. fibuligera, S. capsularis, and P. burtonii. Among them, the strains of S. bayanus, C. glabrata, and P. burtonii had not previously been isolated from marcha (Batra and Millner, 1974, Hesseltine and Kurtzman, 1990, Tamang and Sarkar,
Acknowledgement
We thank Dr. Jiro Kataoka and Dr. Hiroaki Nagasaki for their helpful discussions.
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