Int.J.Curr.Microbiol.App.

Sci (2019) 8(5): 1519-1527

International Journal of Current Microbiology and Applied Sciences

ISSN: 2319-7706 Volume 8 Number 05 (2019)
Journal homepage: http://www.ijcmas.com

Original Research Article https://doi.org/10.20546/ijcmas.2019.805.175

Exploring the Yeast Diversity of Common Alcoholic Beverages of Assam

B. Saikia*, M.S. Ali, P.D. Nath and P.C. Dey

Department of Plant Pathology, Assam Agricultural University, Jorhat-785013, Assam, India

*Corresponding author

ABSTRACT

Rice is a major crop of Assam. Using rice as a substrate, different communities in the state
Keywords produces peculiar fermented and non-fermented products like rice beer, sweets and snacks
Starter culture,
etc. Production of rice beer involves two steps; starter culture preparation and brewing.
Saccharomyces The starter culture is made of rice and herbal combinations and believed to act as
cerevisiae, inoculants for brewing. These products often contain mixed microbial population due to
Saccharomycopsis allowance of natural fermentation. Some amylolytic fungi plays important role in
fibuligera, Candida saccharification, whereas yeast is the dominant fermenting agent of glucose to produce
sp ethanol. Besides, these products often contain wild yeast and other microbial population
due to allowance of natural fermentation which have a significant impact on food quality.
Article Info A study was undertaken to systematically study the fungi associated with rice beer starter
Accepted: culture. Thirty two yeasts were isolated from twelve rice beer starter cultures collected
15 April 2019 from six different districts viz., Jorhat, Sivasagar, Golaghat, Lakhimpur, Dhemaji and
Available Online: Majuli. Morphological identification was carried out with the help of standard literature.
10 May 2019 Macro- and micromorphological studies revealed the most frequently associated yeasts
species viz., Saccharomyces cerevisiae, Saccharomycopsis fibuligera and Candida spp.

Introduction in the socio-cultural life of the people of the

state. As a traditional practice, rice is
Assam is one of the states in North East India, processed into rice beer, which is of both
situated south of the Eastern Himalayas. The ethnic and possible commercial importance
climate is temperate to sub-tropical with (Ahmed et al., 2010; Dutta and Mahanta,
summer maximum temperature 35-38 °C and 2014).
winter minimum 6-8 °C, high relative
humidity and annual rainfall of average 2500 Assam is inhabited by many indigenous tribes
mm to 4500 mm per annum. Assam is and as part of their socio-cultural lives; most
traditionally a rice growing state. Out of total of the tribes prepare their own household
4 million hectare cultivated area 25 lakh ha is alcoholic beverages using rice as a substrate.
under rice cultivation with a production of The alcoholic beverage is used in various
2093 kg/ha where national average is 104.32 traditional ceremonies, religious rituals and
mt, area 43.87 mha and productivity of 2381 also considered to posses medicinal and
kg/ha (Anon, 2016). Rice plays a pivotal role therapeutic properties. These products are

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similar to shaosingiju of China; sake of Japan; the traditional rice beer is necessary to take
chongju and takju of Korea; brem bali, tape- decisions regarding the optimization of
ketan and tapuy of Indonesia, khaomak of processes to eliminate unwanted yeast
Thailand and tapai pulul of Malaysia (Xie et contaminants and thus prevent unnecessary
al., 2007). Moreover, different tribes of beer spoilage.
Assam like; Ahom, Deori, Mishing, etc. have
their own unique rice based alcoholic Chakrabarty (2017) examined the yeast flora
beverages known as haj, suzen, apong, present in nduiyi, a traditional amylolytic
respectively. These three major tribal starter used to produce alcoholic beverage
beverages were focused in this study. called nduijao from Dima Hasao district of
Assam. Based on cell morphology and
Basically, rice wine manufacturing consist of phenotypic characterization isolates were
saccharification of steamed rice starch by identified as Candida glabrata and
fungal enzymes under aerobic solid state Saccharomyces cerevisiae. A metagenomic
fermentation and the product is mixed with study revealed the microbial community
water and is allowed to undergo submerged associated with haj, a traditional alcoholic
alcoholic fermentation by yeasts using beverage of Ahom tribe of Assam. The
traditional starter cakes (Sujaya et al., 2004; existence of ethanol producers viz.,
Dung et al., 2006). Meyerozyma guilliermondii,
Wickerhamomyces ciferrii, Saccharomyces
Variety of microorganisms naturally ferment cerevisiae, Candida glabrata, Debaryomyces
majority of global fermented foods and hansenii, Ogataea parapolymorpha and
beverages. Bacteria, yeast and mycelial fungi Dekkera bruxellensis, were found associated
are mainly associated with fermentation. with the starter culture along with a diverse
Yeast can be present alone or in a stable range of opportunistic contaminants (Bora et
mixed population with mycelial fungi or al., 2016). Literature is meagre with respect to
bacteria and have a significant impact on food yeast taxonomical study in north east India
quality. Modern breweries employ closed with special reference to state of Assam.
brewing system that reduces the risk of
contamination by ‘wild’ yeast. However, This investigation was aimed at the
traditionally fermented products contain morphological identification of indigenous
mixed microbial populations because of lack yeasts isolates from rice beer starter culture
of sterility and the use of natural fermentation samples. The samples were collected from
or mix culture fermentation starters (Robert Ahom, Mishing and Deori community
and Kofi, 2015). Wild yeasts can therefore be residing in seven different districts of Assam,
non-Saccharomyces yeast, Saccharomyces India.
yeast species other than brewing production
yeast, or even production yeast strains other Materials and Methods
than those intended for a specific
fermentation. In other words, yeast not Collection of samples
deliberately used and under full control
(Gilliland, 1971). Beer spoilage due to the Indigenously prepared rice beer starter
presence of wild yeast contaminants includes cultures viz., xaj, apong, suzen were collected
production of off-flavours, competition with in sterilized plastic bags and brought to
brewing yeasts for nutrients. Therefore, laboratory for isolation of associated
understanding of yeast mycoflora present in mycoflora.

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Isolation of yeasts and fungi from starter 1971; Barnett, 1990; Barnett, 2000) and CBS
culture yeast database.

Rice starter cultures were ground to fine Results and Discussion

powder with the help of electric grinder
(Bhuyan and Baishya, 2013) and 10g of Twenty eight yeasts were identified from all
sample is suspended in 90 ml sterilized water the starter culture samples collected during
and mixed thoroughly. 1ml solution was the investigation.
serially diluted to 10-4 and 10-5. Each dilution
was spread onto potato dextrose agar (PDA) Morphological identification
supplemented with 200ppm streptomycin
sulphate and incubated at 25±1°c for 48 Saccharomyces cerevisiae isolates were found
hours. Individual yeasts colonies with distinct to be dominant in all the samples. It showed
colony and morphological characters were white to creamy colonies with smooth and
picked up and repeatedly streaked in yeast butyrous growth on yeast extract peptone
extract peptone dextrose (YEPD) agar media dextrose agar. After three days at 25 ± 1° C
(Jeyaram et al., 2008) and maintained. cells are spheroidal, sub- globose, ovoid and
occur singly, in pairs or sometimes in small
Cultural and morphological identification clusters. Cell size of different isolates ranging
of yeasts from 1.3-3.2 µm to 4.0-5.8 µm. Ascus with
two to four round ascospores were typical of
Each of the purified colonies of yeasts was S. cerevisiae (Fig. 1D). Table 1 shows macro
grown on yeast extract peptone dextrose and micomorphology of S. cerevisiae isolates.
(YEPD) agar for assessing their colony Pseudohyphae or true hyphae were not
characteristics mostly shape, colour, margin, observed in any of the isolates. S. cerevisiae
texture (Chavan et al., 2009; Goralska, 2011; is the major fermenting agent of all the
Spencer et al., 2011). To induce ascospore globally fermented products.
formation yeasts isolates were cultured on V8
juice agar (Yarrow, 1998; Barnett et al., Three isolates showed typical tough, raised
2000). and farinose, partly or entirely hairy colonies
on YEPD agar (Table 2.). Based on the
Microscopic characteristics of yeasts viz., cell colony characters (Fig. 2), it was identified as
shape, size, presence/absence of budding and Saccharomycopsis (Lindner) Klocker. The
pseudohyphae, sexual stage, shape and size of identification was further confirmed based on
ascus and ascospore if present was studied by the micromorphological observations. The
lacto phenol cotton blue and basic fuchsin yeast was characterized by the formation of
staining under 40 x &100x light microscope. septate hyphae and multipolar budding cells.
Whole preparation of ascus was done by Presence of spherical to oval asci, situated at
moderately heating and tapping the structures, the ends of the mycelia hyphae or alongside
released ascospores were examined and them; bearing two to four hat shaped
measured. Microphotographs were taken to ascospores confirms the yeast upto species
show the typical morphology of the fungi. level as S. fibuligera. Clear zone formation
was also observed when subjected to starch
Identification and characterization of yeasts hydrolysis test, indicating the production of
were carried out with the help of relevant amylase enzyme by the yeast (Wickerham et
keys, monograph, standard literature (Lodder, al., 1944). S. fibuligera was reported to be the

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principal amylolytic yeasts found in µm. Pseudomycelium is abundantly formed

Indonesian cassava-tape and ragi tape and consist of long-stretched, branched
(alcoholic beverages made from cassava and pseudohyphae bearing blastoconidia and
ragi); that produces large amount of amylases, verticils of blastospores in branched or simple
acid protease and β-glucosidase which have chains. True mycelium occurs. Based on these
highly potential applications in fermentation morphological characters and comparison
industry (Chi et al., 2009). It is also used to with standard literature the yeast was
produce ethanol from starch, especially identified as C. tropicalis (Fig. 3).
cassava starch by co-cultures of
Saccharomyces cerevisiae (Chi et al., 2009). The other three isolates have cells of ovoid to
The presence of S. fibuligera is thus cylindrical shape, pseudomycelium present,
beneficial for the beer production process. true mycelium not observed, reproduce by
budding. The cell sizes varied between the
Four Candida spp. were identified based on three isolates, ranging from 2.2-3.5 x 2.8- 4.5
presence of pseudomycellium and true µm, 2.5-3.6 x 2.5- 4.8 µm and 10.2 -11.4 x
mycelium and absence of sexual stage. All the 10.4-15.5 µm (Table 3).
four isolates produced white to creamy,
smooth and butyrous colonies on YEPD agar. The study shows that different kinds of yeasts
However, the cell size and shape varies were associated with rice beer starter culture
among all the isolates. The cells of one isolate of Assam (Fig. 1–3).
were short-ovoid to ovoid 4.2 -5.4 x 6.5-8.5

Table.1 Macromorphology and micromorphology of Saccharomyces cerevisiae

Location Macromorphology Micromorphology

Colour Appreance Texture Cell shape Cell size (µm) Budding Sexual stage
Jorhat White Smooth Butyrous Round, 2.4-3.8 x 2.6 - Present Ascus with 1-4
oval to 3.6 thin walled,
cylindrical smooth,
spherical to sub-
spherical
ascospore
White Smooth Butyrous do 1.5-2.9 x 2.0 -3.6 Do do
White to Creamy Smooth Butyrous do 3.2 x 4.5-4.9 do Do
White to Creamy Smooth Butyrous do 1.9-3.1 x 3.1-4.5 do Do
Golaghat White Smooth Butyrous do 3.0-4.2 x 4.1- 5.2 do Do
and raised
White to off white Smooth Butyrous do 2.3 x 2.5-2.6 do Do
Sivasagar White Smooth Butyrous do 3.2 x 4.5-4.9 do Do
White to Creamy Smooth Butyrous do 1.9-3.1 x 3.1-4.5 do Do
Lakhimpur White to Creamy Smooth Butyrous do 2.0-2.9 x 2.8 -4.5 do Do
White to Creamy Smooth Butyrous do 3.8-3.9 x 5.9-6.0 do Do
White Smooth Butyrous do 2.9-3.0 x 5.1-5.2 do Do
Dhemaji White to Creamy Smooth Butyrous do 3.2-3.6 x 5.0-5.2 do Do
Majuli White to Creamy Smooth Butyrous do 4.0-4.7 x 5.5-5.8 do Do
Karbi White to Creamy Smooth Butyrous do 1.9-3.1 x 3.1-4.5 do do
Anglong

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Table.2 Macromorphology and micromorphology of Saccharomycopsis fibuligera

Location Macromorphology Micromorphology

Colour Appreance Texture Cell Cell Sexual stage True hyphae
shape size(um)
Jorhat Creamy Farinose Membranous Oval to 3.0-3.5 x Spherical to Septate mycelium
cylindrical 3.5-4.5 ovoidal asci present,
formed on hyphal originating from
structure, 2-4 hat spore. Sexual and
shaped ascospore asexual stage both
present together
White Farinose Membranous Oval to 2.5 -3.4X do do
to cylindrical 4.0 -4.5
creamy
Golaghat White Farinose Membranous Oval to 2.4 -3.2X do do
to cylindrical 3.9 -4.6
creamy

Table.3 Macromorphology and micromorphology of Candida tropicalis and Candida spp.

Colour Appearance Texture Cell shape Cell Budding Sexual

size(µm) stage
a) Candida tropicalis
Sivasagar White to Smooth Butyrous Oval to 4.2 -5.4 x Present Absent Present
Creamy cylindrical 6.5-8.5
b) Candida spp.
Jorhat White Smooth Butyrous Ovoid 2.5-3.5 x Present Absent Present
forming 2.5- 4.7
short chains
by budding
Sivasagar White to Smooth Butyrous Oval to 10.2 -11.3 x Present Absent Present
Creamy cylindrical 10.3-15.5
Dhemaji White to Smooth Butyrous Oval to 2.1-3.2 x Present Absent Present
Creamy cylindrical 2.6- 4.2

A B C D
Fig.1 Macro and micromorphology of Saccharomyces cerevisiae (A-B) pure culture on YEPD agar (C)
S. Cerevisiae cells, budding (D) ascus with ascospore/ tetrads

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10µm 10µm

A B C

10µm
10µm
D E F

Fig.2 Macro and micro morphology of Saccharomycopsis fibuligera

(A) farinose colonies on yepd agar (B) pseudo and true hyphae (C) budding cells
(D) ascus with ascospores (E) hat shaped ascospore (F) clear zone formation on iodine test

10 µm

B
A
Fig.3 Macro and micromorphology of Candida tropicalis (A) pure culture on
YEPD agar (B) budding cells, pseudohyphae and true hyphae

Isolates derived from all the seven districts In the present study S. fibuligera was found to
revealed the presence of S. cerevisiae. Many be associated with starter cultures from
worker reported S. cerevisiae as the dominant Jorhat, Golaghat and Sivasagar districts. S.
alcohol producing yeast in fermented products fibuligera was the principal amylolytic yeasts
worldwide (Sujaya et al., 2004; Thapa and found in Indonesian cassava-tape and ragi
Tamang, 2004; Xie et al., 2007;do Amaral tape (Kuriyama et al., 1997). Many workers
Santos et al., 2012; Jimoh et al., 2012; Miguel reported S. fibuligera from various fermented
et al., 2013; Chakrabarty, 2017). drinks (Tamang and Sarkar, 1995; Tamang,
2003; Thapa and Tamang, 2004; Tsuyoshi et

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al., 2005; Soka and Irene, 2013; Thakur et al., of S. cerevisiae and S. fibuligera may be a
2015). S. fibuligera produces large amount of possible way of exploitation of yeasts species
amylases, acid protease and β-glucosidase of the region to produce local wine of interest
which have high potential of applications in avoiding unwanted contaminants of the
fermentation industry (Chi et al., 2009). It is fermented products.
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How to cite this article:

Saikia, B., M.S. Ali, P.D. Nath and Dey, P.C. 2019. Exploring the Yeast Diversity of Common
Alcoholic Beverages of Assam. Int.J.Curr.Microbiol.App.Sci. 8(05): 1519-1527.
doi: https://doi.org/10.20546/ijcmas.2019.805.175

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