Isolation and Characterization of the Mycofloral Diversity in Traditional Assamese Alcoholic Fermentation from India

Fermentation is a spontaneous method of producing alcoholic beverages around the world. The process is dominated by strains of Saccharomyces cerevisiae and related species as well as non-saccharomyces yeasts from the genera Candida, Pichia, Kluyveromyces and Torulaspora, among others (Qi et al., 2015). Initially, the non-Saccharomyces yeasts are the dominant partners kick-starting the fermentation process generating various flavor compounds like terpenoids, higher alcohols, esters, acetic acid, glycerol, acetaldehyde and succinic acid (Jolly et al., 2014). The liquification and saccharification of rice based fermentation results from moulds that produce a-amylase that converts starch into dextrin, maltose and mostly glucose (Pervez et al., 2014). The fermentation is carried out in a time dependent manner and can take upto 3-10 days with varing conditions of residual sugars and sweetness. The process can further be regulated by using standardized starter cultures and to achieve better utilization of the fermentable sugars. Recently, various studies have applied monocultures or co-cultures of S. cerevisiae and non-Saccharomyces yeast to produce wine (Padilla et al., 2016; Zhang et al., 2018), sugarcane and pineapple beverage (Ribeiro et al., 2015), mango wine (Sadineni et al., 2012), etc.
       
Almost the entire human world has followed traditional methods of fermentation and alcoholic beverage production. Few well known beverages are shaosingiju and laochao of China (Ghosh et al., 2016), tapuy, brem bali and tape-ketan of Indonesia (Tamang, 2016), khaomak of Thailand, tapai pulul of Malaysia, chongju and takju of Korea and sake of Japan (Kitamura et al., 2016). Such beverages form an integral part of the producing communities and are used in various traditional ceremonies, religious rituals and also considered to possess medicinal and therapeutic properties. The fermentation process, in almost all cases has the same protocol with variations in the starter culture composition and fermentation period. Previous studies point out to the fact that Saccharomyces and non- Saccharomyces cultures may act synergistically making it important that the starter inoculants are explored for their mycoflora. Selection of the appropriate strains is generally based on criteria such as fermentative power, tolerance to ethanol, ethanol yield, killer phenotype and low acetic acid production (Suarez-Lepe and Morata, 2012).
       
The aim of the present work was to isolate native strains of yeast from the alcoholic fermentation of rice leading to the production of rice beer. A comprehensive sampling survey was carried out in six different districts of Assam, India and a total of nine different starter inoculum samples were collected for the study. A detailed morphological and molecular characterization was done to unravel the yeast and other fungal communities present in the starter inoculums.  
 

Collection of samples
 
Indigenously prepared rice beer starter cultures (in the form of dry cake) viz., Xaaj pitha, Apong, Suzen (mod pitha) were collected during 2019-20 in sterilized high density polypropylene bags and brought to laboratory of Department of Plant Pathology, Assam Agricultural University, Jorhat for isolation of the associated mycoflora.
 
Isolation of yeasts and fungi from starter culture
 
Rice starter cultures were ground to powder in pestle and morter and 10 g of sample is suspended in 90 ml sterilized water. One ml solution was serially diluted to 10-4 and 10-5 dilutions. Each dilution was spread in different media namely yeast extract peptone dextrose (YEPD) agar, potato dextrose agar (PDA) and V8 juice agar supplemented with 200 ppm streptomycin sulphate to avoid bacterial growth and incubated at 25±1^oC for 48 hours (Jeyaram et al., 2008). Individual yeasts and fungal colonies with distinct colony and morphological characters were picked up, purified, sub-cultured and stored in mineral oil for future use.
 
Cultural and morphological identification
 
Identification and characterization of yeasts and filamentous fungi were carried out with the help of relevant keys, monograph, standard literature (Barnett, et al., 2000; Barnett et al., 1983; Lodder and Kreger-Van, 1952; Raper and Thom, 1949; Subramanian, 1971; Thom and Church, 1926) and CBS yeast database (http://www.cabri.org/CABRI/srs-doc/cbs_yeast.info.html).
 
Yeasts
 
Purified yeasts colonies of was grown on Yeast Extract Peptone Dextrose (YEPD) agar for assessing their colony characteristics mostly shape, colour and margin; texture (Spencer et al., 2011). To induce ascospore formation, yeasts isolates were cultured on V8 juice agar. Microscopic characteristics of yeasts viz., cell shape, size, presence/absence of budding cells and pseudo-hyphae, sexual stage, shape and size of ascus and ascospore if present were studied by lacto phenol cotton blue and basic fuchsin dye staining under 40x and 100x resolution of Magnus USB binocular microscope.
 
Filamentous fungi
 
Cultural characteristics of filamentous fungi were analyzed in PDA medium. Petri plates having 20 ml sterile media were inoculated aseptically with 5 mm mycelial disc of the fungus and incubated at 25±1^oC. Microscopic observations for head of Aspergillus, number of phialides, foot cells, spore characters like spore arrangement, size, colour of different isolates were studied from the pure culture of the isolates. Specimen were mounted with lacto phenol; lacto phenol cotton blue; basic fuchsin. Microphotographs were recorded to show the typical morphology of the fungi.
 
Starch hydrolysis test
 
Starch agar medium containing 0.2% w/v of starch, 0.5% peptic digest of animal tissue, 0.15% yeast extract, 0.15% beef extract, 0.5% sodium chloride, 1.5% agar was prepared and pH was adjusted to 7.5. Ten millimetre diameter discs of pure culture were inoculated to three corner of each plate and incubated at 30^oC for 48 hours. Visualization of starch degradation was done by flooding with a 0.25% Iodine solution. Clearing of the typically blue coloration of starch with iodine indicate starch degradation.
 
Phylogenetic analysis, molecular identification and sequencing
 
PCR amplification of the internally transcribed ribosomal spacer region and the 5.8S rDNA was done by colony PCR method as described by (Jeyaram et al., 2008). Universal primers (ITS1 5'-TCCGTAGGTGAACCTGCGG-3', ITS4 5'-TCCTCCGCTTAT TGATATGC-3') were used for amplification (Schoch et al., 2012). The products were visualized on 1.5% (w/v) agarose gel prepared in 1x TAE buffer using gel documentation system. PCR product of samples along with primer pairs were sent to Bioserve Biotechnologies India Pvt. Ltd. for sequencing. Codoncode aligner software was used to assemble the curated sequences into contigs. The sequences were submitted in NCBI and accession numbers were obtained.
       
Phylogenetic analyses of the sequences were done using MEGA 7 software by maximum likelihood method (Kumar et al., 2016) following Neighbour joining method. ITS sequences of S. cerevisiae, S. fibuligera, Candida sp.were obtained from National Centre for Biological Information to draw a comparative evolutionary profile of the sequenced strains.
 

Morphological identification of filamentous fungi
 
Two filamentous fungi were isolated and identified as Aspergillus oryzae and A. niger based on cultural and morphological characters Table 1. Colony growth of Aspergillus oryzae on Czapek Dox Agar is moderate attaining 4.5-5.5 cm in 10 days at 25±1^oC (Fig 1 C-D). On PDA mycelium white with green sporulation; reverse mustard yellow (154B, RHS), (149A, RHS) (Fig 1A-B). The fungus produced concentric rings on Malt Etract Agar (Fig 1 E-F). Conidial head radiate with globose to subglobose vesicles, uniseriate (Fig 1G). Conidia subglobose, rarely ellipsoidal or ovoid, 3.0-5.0 μm wide, with walls smooth to irregularly rough. Thom and Church (1926) described similar characteristics for Aspergillus oryzae, based on which the species was identified. However, the identification was confirmed by National Centre for Fungal Taxonomy (NCFT), New Delhi and given ID. No. 2001.17.




       
A. niger colonies on Czapek Dox agar, consist of a compact white or yellow (10C, RHS) basal felt covered by dense layer of black conidial heads (202 A, RHS) (Fig 2 A-B). On Oat Meal agar dark black sporulation occurred (Fig 2C). Conidiophores were smooth-walled, hyaline or turning dark towards the vesicle (Fig 2D). Conidial heads were large up to 250-300 µm in diameter, vesicle globose, dark brown in colour, becoming radiate and tending to split into several loose columns with age. Conidial heads were biseriate with the phialides borne on brown metulae. Conidia are globose to subglobose (3.5-5.0 µm in diameter), dark brown to black (Fig 2E). These characteristics were compared with standard description of Thom and Church (1926) and the species was confirmed as Aspergillus niger (Fig 2F).


 
Morphological identification of yeasts
 
Sixteen yeast strains were isolated from nine starter cultures and divided into three groups (Group I, II and III) based on cultural and morphological properties Table 2. Phylogenetic, morphological and physiological characterization identified the isolates as Saccharomyces cerevisiae (Group I); Saccharomycopsis fibuligera (Group II) and Candida sp. (Group III). Group I isolates belonging to Saccharomyces cerevisiae were found to be dominant in all the nine samples. Colonies white to cream colour, smooth and butyrous growth on YEPD agar (Fig 3 A); cells were spheroidal, sub-globose, ovoid and occurred singly, in pairs or sometimes in small clusters (Fig 3B). Cell size of different isolates ranging from 1.3-3.2 µm to 4.0-5.8 µm. Ascus with two to four round ascospores were typical of S. cerevisiae (Fig 3C). Pseudohyphae or true hyphae were not observed in any of the isolates.




       
Three isolates comprising group II showed typical tough, raised and farinose, partly or entirely hairy colonies on YEPD agar. Based on the colony characters it was suspected to be Saccharomycopsis fibuligera (Lindner) Klocker (Fig 4A). Further, the micro-morphological observations revealed the formation of septate hyphae (Fig 4B) and multi-polar budding cells, presence of spherical to oval asci, situated at the ends of the mycelia hyphae or alongside them; bearing two to four hat shaped ascospores confirming the yeast upto species level. Clear zone formation was also observed when subjected to starch hydrolysis test which indicated the production of amylase enzyme by the yeast (Wickerham et al., 1944).


       
Group III containing Candida sp. were identified based on presence of pseudo-mycelium and true mycelium and absence of sexual stage. The isolates produced white to creamy, smooth and butyrous colonies on YEPD agar (Fig 4C-D). The cells were short-ovoid to ovoid 4.2-5.4 × 6.5-8.5 µm; pseudo-mycelium is abundantly formed and consists of long-stretched, branched pseudo-hyphae bearing blastoconidia and verticils of blastospores in branched or simple chains, true mycelium occurs. Based on these morphological characters and comparison with standard literature the yeast was identified as C. tropicalis.

Phylogenetics and molecular characterization of yeasts
 
Representative strains from each group were randomly selected (strain Y-2 and Y-3 from group I; strain Y-6 from group II; strain Y-21 from group III) and their phylogenetic positions were examined based on evolutionary phylogenetic analysis. Fig 5 represents the phylogenetic position of the yeast strains based on the almost full-length 18S rDNA curated sequences. All the analyzed strains were in the class hemiascomycetes of division ascomycotina.


       
Molecular characterization of selected strains was carried out in support of the detailed morphological identification performed. Fig 6 depicts the gel electrophoresis results of the amplification of ITS in the isolated cultures. The sequenced strains viz., Y-32 (group I), Y-6 (group II) and Y-21 (group III) were highly homologous to the genera Saccharomyces cerevisiae, Saccharomycopsis fibuligera and Candida tropicalis, respectively. The sequences were submitted in NCBI under the accession numbers MK110643 (Saccharomyces cerevisiae), MK110642 (Saccharomycopsis fibuligera) and MK110644 (Candida tropicalis).


       
Morphological, phylogenetic and molecular analysis revealed the presence of diverse mycoflora associated with rice beer starter cultures (xaj pitha, suzen and apong). The filamentous fungi were identified as Aspergillus oryzae and A. niger and the dominant yeasts were Saccharomyces cerevisiae, Saccharomycopsis fibuligera and Candida tropicalis. S. cerevisiae as the dominant alcohol producing yeast in fermented products worldwide (Chakrabarty, 2017; Xie et al., 2007). However, the isolates of Saccharomycopsis fibuligera had not been identified from starter cultures of Assam. It is significant as there are a handful of reports which suggests an important role of S. fibuligera in the fermentation process. Earlier, Pirselova et al. (1993) suggested the co-culturing of S. fibuligera and S. cerevisiae for fermentation since  S. fibuligera  carries out the breakdown of starch into reducing sugars. These reduced sugar are then taken up by S. cerevisiae for ethanol production Pirselova et al. (1993). Lee et al. (2018) identified and quantified the bio-formation of various volatile and non-volatile metabolites by S. fibuligera KJJ81 strain Mi Lee et al. (2018). Pandian et al. (2016) carried out the co-cultivation of S. fibuligera NCIM 3161 and Zymomonas mobilis MTCC 92 and successfully achieved 93.75% of the theoretical yield of ethanol production using cassava peels (Pandian et al., 2016). 
       
It was generally assumed that three types of fungi were associated with xaj pitha, suzen and apong: Amylolytic fungi and yeast; alcohol producing yeasts and other fungi that acts as either flavour and odour enhancers or contaminants. Fermented rice batter itself contains 1.6 to 2.3 log10 cfu/g yeasts count (Angadi et al, 2021). Xie et al. (2007) identified species of A. oryzae and A. niger in wheat Qu, a Chinese alcoholic drink and recorded amylolytic activity of the former. Recently, a report from Korea indicated that the contents of all 18 amino acids detected were the highest in makgeolli fermented by S. fibuligera CN2601-09 and increased after combining with A. oryzae CN1102-08, unlike the contents of most fatty acids (Son et al., 2018). The studies clearly indicate that much work is to be done to understand the dynamics of filamentous fungi and metabolites produced by them. Also, the growth kinetics of these fungi have to be explored while in the fermentation systems (Abdul Manan and Webb, 2018). Yeast like S. fibuligera degrades starch and produce glucose upon which S. cerevisiae readily act to produce ethanol (Tamang and Sarkar, 1995; Tamang, 2016). Considering the presence of C. tropicalis, which is widely considered the second most virulen Candida species, preceded by C. albicans Zuza-Alves et al. (2017)  it can inferred that presence of this pathogenic fungus may lead to health hazard of the rural consumers.
       
The dominant yeast species associated with another Indian starter for rice wine from Manipur called Hamei were identified as S. cerevisiae, Pichia anomala, Trichosporon sp., Candida tropicalis, Pichia guilliermondi, Candida parapsilosis, Torulaspora delbrueckii, Pichia fabianii and Candida montana (Jeyaram et al., 2008) which is in agreement with Balinese rice wine starter ragi tape and Vietnamese rice wine starter mem (Dung et al., 2006). Interestingly the domination of S. cerevisiae, S. fibuligera and Candida spp. in rice beer starter culture of Assam is in agreement with Sikkimese rice wine starter Marcha (Tsuyoshi et al., 2005). Hence it is hypothesized that the yeast species associated with rice wine starter used in Himalaya regions particularly Assam and Sikkim (Himalaya biodiversity hotspot) are distinctly different from the starter used in Indo-Burma Biodiversity hotspot (includes Manipur, Vietnam and Indonesia of south eastern Asia).
 

The controlled fermentation processes is the main technology of fermentation industries. Selection of local effective yeast and filamentous fungal isolates and their maintenance for producing local alcoholic beverages can lead to the development of a small scale industry in the rural North-East states of India. Quality control along with this tradition specific fermentation has a potential to attract the consumers of international market. The current study revealed some good quality fermenting agents of the Assam state of India. Saccharomyces and Saccharomycopsis together in fermentation produce alcohol from sugars and break down complex sugars into simpler form, respectively. The native isolates along with the tribe specific fermentation recipes, their microbiota and herbs used should be explored extensively to develop marketable products.