Complete specification
1. Title of invention: Improved method of brewing "Xaj pani" -rice based alcoholic beverage
2. Field of invention: This invention relates to the development of a defined starter culture
consisting of fungi, yeast and plant parts for brewing of rice based alcoholic beverage "Xajpani".
3. Background of invention with regard to the drawback associated with known art:
Rice based alcoholic beverages are produced in solid state fermentation through starter cultures that act as saccharifying and fermenting agents. The general process of rice based alcoholic beverage brewing involves the breakdown of the starch present in the rice to sugar which are then converted to alcohol (Nout and Aidoo, 2002).This process involves the physical, biochemical and microbiological operations including steaming of rice followed by inoculation with a starter to allow the fermentation process (Dung et al., 2005; Hesseltine et al., 1988). The alcohol content and quality of alcoholic beverages vary depending upon the efficiency of the starter culture and the mode of preparation (Dung et al., 2005). Although the traditional brewers are aware of concocting good starter tablets for quality and yield of alcoholic beverage, they are unaware about the microbiological composition and their influence on the end product parameters (Luangkhlaypho et al, 2014).
Fermentation starters used for traditional rice based beverages are mostly manufactured at home-scale using starter cultures under non-sterile and marginally controlled conditions following village art techniques (Tamang et al., 2012). Fermentation starters are prepared using rice powder as substrate for the growth of microbes. Several cereal based alcoholic beverages are prepared by ethnic tribes and community viz. Chi (Lepcha), Haria (Adivasi), Jou (Dimasa and Mech), Haria (Adivasi community), Zu/Judima (Dimasa tribe), Arak/Hor-Alank (Karbi tribe), Jou Bishi, Jumai (Bodo tribe), Aapong (Mishing tribe), Photika (Kachari tribe), Laopani (Lalong tribe), Tsa-pe (Singpho community), Zu (Tiwa), Suze/Sujen (Deuri) , Choko or Jonga mod (Rabha) and Xaj/Xaj pani/Lao pani (Ahom community). These beverages play an important role in the spiritual and cultural lives of the indigenous communities ((Deka and Sarma, 2010; Das and Deka, 2012). All the ethnic community residing in this state use round to flattened solid ball-like mixed dough inocula or starter for preparation of their indigenous alcoholic beverages at home (Tamang et al, 2007; Jeyaram et al., 2008; Tanti et al., 2010; Srivastava, 2012 and Ghosh et al., 2015) which contain amylolytic fungi, alcohol producing yeasts and lactic acid bacteria (Dung et al., 2006). Microbial diversity in the rice-based alcoholic starters by both culture dependent and culture independent method have been reported (Akiyama, 1978, Dung et al., 2005; Tamang et al., 2007, Yang et al., 2011; Kitagaki and Kitamoto, 2013; Bora et al., 2016). Using culture-dependent method amylolytic fungal community and yeasts have been identified in rice wine starters from south East Asia. In case of Nuruk, a Korean cereal based beverage, six genera (Lichtheimia, Aspergillus, Rhizopus, Rhizomucor, Mucor and Syncephalastrum) and 17 fungal species were identified (Yang et al., 2011). Several bacteria viz.,Micrococcus, Escherichia, Pseudomonas, Enterobacter, Aerohacter and Achromobacter and non-Saccharomyces yeasts such as Pichia sp., Candida sp., Zygosaccharomyces sp. etc. have been isolated from Sake, the traditional rice-based alcoholic beverage of Japan (Kitagaki and Kitamoto, 2013; Akiyama, 1978; Ashizawa, 1976). Lactobacillales, Actinomycetales and Bacillales species were reported to be the dominant bacterial groups in starters of Fen Liquor of Chinese rice-based alcoholic beverage (Shi et al., 2009). The starter culture 'marcha' used in Sikkim contain the amylolytic microbes like Mucor

circinelloides, Rhizopus chinensis, and Penicillium burtonii; ethanol producing strains Saccharomyces bayanus, Candida glabrata and Pichia anomala (Tamang and Sarkar, 1995; Tsuyoshi et ai, 2005) and lactic acid bacteria Lactobacillus plantarum, Lactobacillus brevis and Pediococcus pentosaceus (Tamang et ai, 2007).
Culture-independent molecular approaches based on PCR-mediated Denaturing Gradient Gel Electrophoresis (PCR-DGGE) analysis of Ragi tape, fermentation starter of Indonesiahave shown the presence of Weissella sp., Pediococcus pentosaceus, Lactobacillus sp., Bacillus cereus, Clostridium perfringent, Eubacterium moniliforme, Clostridium sardiniensis, or Clostridium baratii and Enterococcus sp., were detected in Ragi tape (Balinese rice beer starter) samples (Sujaya et ai, 2010). The PCR-DGGE profile of 52 traditional Vietnamese alcohol fermentation starters (Bank men) detected 13 species of fungi including yeasts and 23 species of bacteria were identified (Thanh et ai, 2008). Pyrosequencing-based metagenomics study or Whole Genome Sequencing (WGS) study has been used to characterise and profile microbial populations associated with fermentation starters mostly of Asian types. Our metagenomics study of the starter culture Xaj pitha revealed the existence of amylase producers, such as Rhizopus delemar, Mucor circinelloides, and Aspergillus sp. Yeast species viz., Meyerozyma guilliermondii, Wickerhamomyces ciferrii, Saccharomyces cerevisiae, Candida glabrata, Debaryomyces hansenii, Ogataea parapolymorpha, and Dekkera bruxellensis were also present. Lactic acid bacteria (LAB) along with a diverse range of opportunistic contaminants such as Acinetobacter guillouiae, Microbacterium sp., Micrococcus sp., Staphylococcus sp., etc., were detected (Bora et ai, 2016). Acinetobacter guillouiae has received increasing attention in the context of serious underlying disease in immune-compromised patients from south-east Asia and tropical Australia (Dijkshoorn et al. 2007, Peleg et al. 2008). It is presumed that all contaminants in the starter were introduced along with the plant parts, or through unhygienic practices.
Fungi degrade starch into maltose and glucose by producing extracellular amylolytic enzymes (a-amylase and glucoamylase) and yeast convert these sugars into ethanol and carbon dioxide (Klein et ai, 1997; Ogue-Bon et ai, 2011). Fungi secrete amylases outside of the cells to carry out extracellular starch digestion. The natural isolates usually produce a low concentration of amylase. To maximize enzyme production and degradation of starch it is essential to have preliminary information on fungal growth characteristics for optimum enzyme production during fermentation, which helps in designing of proper fermentation conditions for economic production (Gupta et ai, 2003) Besides the extracellular enzyme production to degrade starch, many fungi produce toxic secondary metabolites. Fungal toxins or mycotoxins are potential problems for food producers as they adversely affect production. Fungi mainly Aspergillus flavus and A. parasiticus are responsible for aflatoxin production and aflatoxins are designated as AFB1, AFB2, AFG1 and AFG2. Since the discovery of the Aflatoxins (AFs) in the 1960s, International Agency for Research on Cancer (IARC) of the World Health Organization (WHO) accepted that aflatoxin should be classified as a Group 1 carcinogen in 1987, and then aflatoxin Bl (AFB1) is classified as Group 1 (carcinogenic to humans) by the WHO- IARC in 1993 (Li et ai, 2009; Williams et al, 2004 and Stefano et ai, 2014). Besides aflatoxin, ochratoxins (OTAs) is a potent group of mycotoxin produced by fungi Aspergillus and Penicillium species. The natural levels of OTA, OTB, AFB1, AFG1, AFB2 and AFG2 in 30 commercial sweet wines were investigated. Close monitoring, screening and elimination of mycotoxin producing fungi from food fermentation should, therefore, be done to prevent the risk of contamination (Stefano et/al., 2014).
Along with fungi yeasts play a pivotal role in ethanol fermentation in cereal-based alcoholic fermentation. Under anaerobic condition, yeasts carry out alcohol fermentation in

which they partially break down sugars and produce CO2 and ethyl alcohol (C2H5OH). Alcohol dehydrogenase (ADH), which catalyzes the inter conversion between acetaldehyde and ethanol, plays a central role in ethanol production and assimilation (Lin et al., 2010). Ethanol is an imperative metabolite of yeast. It serves as a source of calories and prevents the growth of disease or toxin producing microorganisms in food products. Yeasts contribute not only to ethanol and gas production, resulting in good texture, but also to sensory qualities like taste and aroma by producing a group of compounds such as glycosides, esters, fuel alcohols, acids and other compounds (Aidoo et al., 2006) as they produce many other enzymes such as esterases, decarboxylases, sulphite reductases, proteases and pectinases that, in various ways, could impact on wine flavour and other properties (Charoenchai et al., 1997; Fernandez et al., 2000; Strauss et al, 2001).
Criteria for the selection and development of yeasts for wine fermentation have evolved over many years and have been discussed by several workers (Degre, 1993; Rainieri and Pretorius 2000; Mannazzu et al, 2002; Preterms and Bauer, 2002; Bisson, 2004 and Schuller and Casal, 2005). Demand for selection of new wine yeasts enhanced for new technological properties (Berlese-Noble et al., 2014). Until about the 1980s, the contribution of yeasts to wine production was seen as a relatively simplistic concept and almost invariably, was dominated by strains of the yeast, Saccharomyces cerevisiae. Consequently, pure cultures of this yeast were isolated and developed as starter cultures for conducting wine fermentations (Benda, 1982; Lafon-Lafourcade, 1983; Reed and Nagodawithana, 1988). The use of mixed starters of selected non-Saccharomyces yeasts (such as Hanseniaspora, Candida, Pichia, Metschnikowia etc.) and Saccharomyces cerevisiae is of increasing interest for production of novel wines with more complex organoleptic characteristics and/or lower ethanol contents (Schlander et al., 2017). Growth characteristics, fermentative efficiency, agglutination capacity after fermentation, temperature, sugar and ethanol tolerance considered as physiological conditions for efficient fermentation (Fleet and Heard, 1993; Fleet, 2008; Zott et al., 2008 and Soare, 2012). Besides ethanol and aroma producing compounds yeasts are also reported to produce sulfites and biogenic amines in the fermentation medium which in high amount adversely affect the health of consumers (Lambrechts and Pretorius, 2000; Bisson, 2004; Swiegers et al., 2005).
Controlled and standard rice beverage brewing can be carried out using defined mixed starters containing well-characterized efficient fungal and yeast strains (Dung et al. 2005). Culture-dependent method is a useful method of isolation and characterization of cultivable microorganisms that can be isolated in pure form and can be used further.
The methodology of fermentation carried out by different communities is almost similar except for the different types and number of plant species used in starter culture preparation (Tanti et al., 2010). The communities in North-East India in different regions use different plant species based on their availability (Das 2012).Majority of the ethnic communities of North East India use several herbs and plants such as Albizia myriophylla, Amomum aromaticum, Plumbago zeylanica, Buddleja asiatica, Vernonia cinerea, Gingiber officinale, Glycyrrhiza glabra, Ananas comosus, Artocarpus heterophyllus, Calotropis gigantea, Capsicum frutescens, Leucas aspera, Piper nigrum, Scoparia dulchis etc. (Saikia et al., 2007; Das et al., 2012) for fermentation starter production. Bodo tribe of Assam use leaves of Xanthium strumarium, Scoparia dulcis and either roots or leaves of Clerodendrum viscosum. Fermentation starter culture perok kushi used by the Deori tribe for alcoholic beverage Sujen preparation is prepared by using the leaves of Jasminum sambac, Cinnamomum byolghata, Zanthoxylum hamillonianum, Lygodium flexuosum, Cyclosorus exlensa, Alstonia scholaris and roots of Alpinia malaccensis and the stem and rhizome of the plant Costus speciosus (Das et al., 2012).

Leaves from a number of plants such as Centella asiatica, Oldenlandia corymbosa, Clerodendrum viscosum, Saccharum officinarum, Cyclosorus externa, Scoparia dulcis, Drymeria cordata,Capsicum annuum, Ananas comosus, Lygodium flexuosum, Zanthoxylum hamiltonianum, Piper nigrum, Pteridium aquilinum, Phogocanthus thyrsiflorus and Piper longum are used in the preparation of starter culture "apop" for preparation of alcoholic beverage "Apong" of Missing tribe (Handique and Deka, 2016) Most of the plants used in fermentation starter preparation are also used in local medicaments and as such the rice based alcoholic beverages are believed to prevent headache, insomnia, body ache, urinary troubles and cholera (Saikia et al., 2007; Singh and Singh 2006; Deka and Sarma, 2010).
Several spice plants such as Capsicum sp., Alpinia sp., Piper sp., Allium sativum, Zingiber officinale, Illicium verum, Amomum krervanh, Cinnamomum sp. including plants that impart sweetness viz.,Albizia sp., Cinnamomum sp. and Saccharum officinarum are reported to be commonly used in preparation of starter culture (Das et al., 2012; Seesuriyachan, 2011). Some of the plant extracts also provide certain nutrients for the growth of the microflora present in the starter cakes (Phuc, 1998; Dung et al., 2005). Leaves, bark and fruits of Holarrhena pubescens, Wattakaka volubilis, Ichnocarpus frutescens and Clerodendrum viscosum are used in the preparation of Chullu starter culture of West Bengal and prepared Chullu is proved to have great ethnomedicinal value in the locality of Malda district (Sana et al., 2015).
Besides plant species, rice varieties used in fermentation starters vary from communities to communities. In Assam, 106 indigenous landraces of glutinous rice have been cultivated and classified into Bora and Chakuwa depending on the amylose content that varies between 15-20% (Rathi and Sarma, 2012). One of the reasons of variation of the quality and attributes of alcoholic beverages produced at different places of Assam may be due to the variations of variety rice used in alcoholic beverage preparation. This practice produces variable quality of beverage with alcohol content.
4. Object of invention
Traditional fermentation process of rice based alcoholic beverage brewing is a spontaneous fermentation process resulting in products that often lack uniform quality. There is no control in fermentation and stability of traditionally prepared alcoholic beverages is very low due to the presence of high microbial load as well as suspended hazes (Nout and Sarker, 1999). The plants used for organoleptic augmentation bear phytoplane microbial loads along with several plant pathogens (Schaad et al., 1975).These microbes are often opportunistic contaminants and deteriorate the product quality. Growth and contamination of undesirable yeasts, spoilage fungi and other microorganisms during fermentation, maturation and after packaging lead to off-flavours or foul smell and turbidity, leading to beer spoils (Gandjar, 1999; Haard et al., 1999). Diverse groups of microbiota from plant origin as endophytic organisms are the functional microbes for mixed culture and multistage fermentation of rice as selective species of plants are used in starter preparation (Sekar and Mariappan, 2007). The Colony Forming Unit (CFU) counts for different microbes associated with Bubod; a Philippians rice beer starter culture ranged from 2.1 x 103 to 2.3 x 107 fungi, 7.4 x 104 to 3.0 x 107 yeasts and 2.9 x 105to 4.7 x 107 LAB per gram of the sample (Hesseltine et al., 1988). In Ragi, an Indonesian starter culture, colony forming counts ranged from 3.2 x 104 to 4.0 x 104 fungi and 5.6 x 10 to 1.4 x 107 yeasts per gram of the sample (Hadisepoetro et al., 1979). Leuconostoc mesenteroides is reported to be present only in Tapuy; a Philippian rice beer (Steinkraus, 1989). Microbial diversity of the fermentation starter Xaj-pitha was enumerated through the culture-dependent approach in laboratory. The starters contained high counts of fungi, lactic acid bacteria (LAB) and aerobic

bacteria. The colony forming unit for yeast count ranged from 6.1x10 - 6.8 x 107, fungal counts ranged from 2.4 x 107 to 3.7 x 107 aerobic mesophiles counts ranged from 1.5 x 107- 2.7 x 107and LAB were present in the highest amount and ranged from 7.8 x 107 - 8.7 x 107. Enterobacteria count ranged from 1.1 x 107 to 1.5 x 107(Keot, 2017). Elucidation of the microbial population dynamics during the fermentation process is therefore crucial. The traditional way of producing starters has many drawbacks such as lack of quality control on types and numbers of each microorganism in the starter. The new starters are produced by mixing starch, herbs, water and bits of the old starter. Thus, it increases the risk of contaminating microorganisms or losing useful microorganisms in the starter every time a new one is made. Hence, development of a control formulated defined starter culture mixture is desired for standard quality and consistency in the production of traditional beverages (Holzapfel, 1997, 2002). A series of studies by Dung et al. (2005,2006 and 2007) addressed the variable quality of Vietnamese rice wine fermentation starter cultures and assessed the option of defined starter cultures with compatible fungal and yeast strains. Defined starter culture with Amylomes rouxii CBS111757 and Saccharomyces. Cerevisiae LU 1250 was produced, which was able to produce rice wine with superior flavor and overall acceptability. The feasibility of the defined starter in wine production using different starchy resources of rice , glutinous and purple glutinous rice were investigated and defined starter was found well performed(Dung and Phong, 2011)..Luangkhlaypho el al., (2014) identified the need for defined starter culture mixture with the composition of the microorganisms identified in the NPl starter for sato fermentation that lead to as an alternative starter for producing a constant high quality sato for industrial production . They used the microbial consortia consisting of fungi yeast and LAB. Rhizopus oligosporus, Mucor racemosus, S. cerevisiae, Saccharomycopsis flbuligera, Pichia anomala and LAB species Pediococcus pentosaceus isolate NP102. Sato brewed with defined starter culture mixture yielded a high quality sato of comparable quality with the original NPl starter with regard to all aspects of evaluation (i.e., colour, odour, flavour, and impression). The profiles of oenological parameters, such as the levels of the organic acids and the volatile compounds produced by NPl and defined starter culture were largely similar, except for the difference in the levels of a few volatile compounds, few organic acids and glycerol; although this did not appear to significantly affect the result of the sensory evaluation.
Keeping the above facts in view, the present patent relate to development of a defined starter culture for brewing of rice based alcoholic beverage of Assam that can serve as a suitable alternative starter for "Xaj " fermentation in place of the original starter "Xaj pitha " which will allow for the controlled fermentation resulting in product with uniform quality. 5. Statement of invention
As off date, no defined starter culture has been developed with known compatible microbial food culture for standard production of "Xaj panf, rice based alcoholic beverage of Assam. In this study we claim the development of a starter culture with compatible microbial food cultures consisting of fungal isolate Amylomes rouxii ABTSJ 82 (NCBI accession number KP790015) strain with yeast isolates Saccharomyces cerevisiae strain ADJ5 (NCBI accession number KX904349) and Wickerhamomyces anomalus strain ADJ2 (NCBI accession number KX904346) isolated from the traditional fermentation starter 'Xaj pitha' . Five representative plants viz., Leucas aspera (coded as PI), Lygodium jlexuosum (P2), Polygonum strigosum (P3), Centella asiatica (P4) and Alstonia scholaris (P5) were selected based on their common use during preparation of "Xaj pitha". Leucas aspera (Use value=0.73), Centella asiatica (Use value=0.45) and Alstonia scholaris (Use value=0.43) were frequently reported to be involved in starter nourishment (promotion of microbial growth), while Lygodium jlexuosum (Use value=0.18) and Polygonum strigosum (Use value=0.29) were reported to be associated with

organoleptic contribution and starter protection respectively. These plants also could have nourishing properties that encourage microbial growth. Antioxidant activity (2.2-diphenyl-l-picrylhydrazyl, DPPH activity, Ghatak et al. 2015) of these plant species were evaluated and found that methanolic extracts of Polygonum strigosum showed the highest DPPH inhibition of 84.90% followed by Leucas aspera (83.58%). All the plant extracts together showed the 83.27% DPPH inhibition. Due to the high free radical scavenging activity of these plants, these were selected for used in starter protection and stability of Xaj.
The defined starter culture is able to produce standard quality of rice based alcoholic beverage of Assam with 10 % alcohol. Thus, this is the first report of defined starter culture production for standard production of rice based alcoholic beverage. 6. Summary of invention
To the best of our knowledge, no defined starter culture has been developed and validated for production of rice based alcoholic beverage Xaj/ Xaj pani/ Lao pani of Assam. We developed a defined starter culture with compatible fungus Amylomes rouxii ABTSJ 82 and yeasts Saccharomyces cerevisiae ADJ5 and Wickerhamomyces anomalus ADJ2 along with selected plant parts viz., Leucas aspera , Lygodium flexuosum , Polygonum strigosum , Centella asiatica and Alstonia scholarisfor production of rice based alcoholic beverage of Assam. Compatible mix culture was able to produce higher amount of ethanol and liquid (ethanol: 10.423 % v/v, liquid: 51.0% w/v respectively).
Brief description of drawings
Fig.l- Schematic diagram showing the development of the assay
Fig.2- Amylase activity of fungal isolates
Fig.3- Glucoamylase activity of fungal isolates
Fig.4- Alcohol production and tolerance of yeast isolates
Fig.5- Osmotolerance of yeast isolates
Fig.6- Compatible growth of A.rouxii and W. anomalus on plate medium
Fig.7- Effects of fungal and yeast cultures on saccharification of gelatinization of rice starch
Fig.8- Method of plant extracts preparation
Fig.9- Colony Forming Unit of yeast isolates on different herbal media
Fig. 10- Colony diameter of fungal isolates on different herbal media
Fig. 11 - Production of alcoholic beverage using defined mix culture starter
Fig. 12- Turbidity removal of alcoholic beverage
Fig. 13- Laboratory prepared alcoholic beverage for sensory evaluation
Fig. 14- Bottling of laboratory prepared alcoholic beverage

Table 8. Stability of Xajpani during storage period

Note: LSD test: Means with the same letters within the days in each column are not significantly
different at p value (<0.05)level
7. Detailed description of the invention
Present patent claims the development of a defined starter culture consisting of compatible fungal and yeast culture along with five representative plants (parts) for production of rice based alcoholic beverage "Xaj pani" of Assam. Development of a defined starter culture for rice based alcoholic beverage production requires efficient starch degrading fungi and alcohol producing yeast cultures along with representative herbs. Therefore, isolation and functionality screening of these two types of candidate microbes were very important. Besides this effect of plant extracts on the growth of microorganisms were studied. The detailed methodology is described below:
7.1. Selection of candidate fungi for defined mixed starter preparation
Twenty morphologically different fungal strains were isolated from the collected ''Xaj pitha" (starter cultures). Fungi were enumerated on Rose Bengal Agar. All plates were incubated at 30° C for 2-4 days. The colonies that appeared after incubation were counted and calculated as Colony Forming Units (CFU) per gram of dry weight samples. Resulting colonies were purified from the primary plates by taking the representatives of each different colony and sub cultured on to sterile PDA plates. The starch hydrolyser were screened for extracellular amylase production by the starch agar plates (Williams et ai, 1970).The plate medium contained 20.0 g soluble starch as sole carbon source, 1.0 g NaNO3, 1.0 g K2HPO4, 0.5 g MgSO4.7H2O, 0.01 g FeSO4, and 20 g agar per liter. The initial pH of the medium was adjusted to 6.5. Pure fungal agar blocks from 7 days old culture plates were incubated on Starch Agar plates for 4-5 days at 30°C. Starch degrading activities were detected as clear zones after exposure to iodine solution (2% [w/v] KI, 1% [w/v] I2 and distilled water) and the size of their clear zone was measured. Four fungal isolates viz., ABT SJ23 (1.2 cm), ABT SJ63 (0.4 cm), ABT SJ72 (0.7 cm) and ABT SJ82 (0.5 cm) produced greatest clear zone on the starch agar plates and were thus selected for further analysis. Based on molecular ITS sequencing and morphological observation, the four

selected fungi were identified as Penicillium sp, Rhizopus oryzae, Mucor guilliermondii and Amylomyces rouxii (NCBI Accession no. KP790012- KP790017/ Under solid state fermentation (SSF) conditions actual amylase activity was determined for the amylase positive fungal strains using substrate glutinous rice grain (Gessesse and Mamo, 1999; Singh et al., 2009). Glutinous rice (5 gms) was taken into Erlenmeyer flasks and 15 mL of nutrient salt solution (NSS) (5g/L KH2PO4, 5g/L NH4.NO3, lg/L NaCl and 0.5 g/L MgSO4) was added to it. These flasks sterilized and inoculated with two discs of fungal isolates (each of a diameter of 5 mm), and subsequently incubated at 25 °C. After 3 days of incubation, 15 mL of distilled water was added. The contents were then crushed and agitated for 10 minutes at 55 °C on a rotary shaker at 200 rpm. The slurry obtained was squeezed through four layers of cheese cloth. The extract was filtered with a Whatman filter paper no.l (Whatman, Maidstone, England) and then centrifuged at 5000 rpm for 10 minutes (Singh et al, 2009). The filtrate obtained was treated as crude enzyme and the amylase activity was determined by DNS method at 540 nm and reported as U/mL using glucose as a standard. One unit of amylase is defined as the amount of enzyme which releases 1 μmole of reducing sugar per minute (U/mL), with glucose as standard (Miller, 1959). Glucoamylase activity was determined by incubating a mixture of 0.5 ml of the enzyme aliquots with soluble starch (1% dissolved in 0.1 M sodium acetate buffer, pH 5) at 55°C for 15 min (Bemfield, 1955). One unit (U) of glucoamylase activity is defined as the amount of enzyme that releases 1 μmole of reducing sugar as glucose, per minute, under assay conditions and expressed as U/g of dry substrate (gds) (Miller, 1959). Activity of amylase during solid state fermentation (SSF) under different pH (4-7.5), incubation temperature (20°C to 55°C) and time (24 hrs to 120 hrs) were determined (Fig.2 and Fig.3). Out of the four efficient fungal strains viz. Penicillium sp. ABTSJ23, Rhizopus oryzae ABTSJ63, Mucor guilliermondii ABTSJ72 and Amylomyces rouxii ABTSJ82, Amylomyces rouxii ABTSJ82 was selected as the candidate fungal strain for defined mixed starter preparation. Amylomyces rouxii ABTSJ82 was found to have alpha-amylase (5.92 U/ml) and glucoamylase activity (7.50 U/ml) useful in saccharification of rice starch. It has also been reported to be involved in most the amylolytic fermentation starters of Asian countries (Hesseltine, 1983; Hesseltine et al., 1988, Saono et al., 1996; Dung et al., 2005 ).Aflatoxins production by fungi were detected through a commercially available immunoassay kit "Veratox" (Neogen Food Safety, USA). The assay kit is based on Competitive Direct Enzyme Linked Immunosorbent Assay (CD-ELISA) (Stoloff et al, 1991).Portions of the filtrate and the residue (must) from each fermentation flasks of the quantitative amylase test were kept aside at 4°C for total aflatoxin determination. Samples (10 g/lo ml) were taken in 50 mL of 70% methanol for aflatoxins analysis through Enzyme Linked Immounosorbent Assay technique (ELISA). The samples were blended individually with high speed blender for three minutes. The blended material was filtered through Whatman filter paper number 1 and the filtrate was further analysed. Hydrogen peroxide was used as a substrate for color development. The reaction was terminated by addition of the terminating solution. The color intensity was inversely proportional to the mycotoxin concentration and measured with the ELISA reader. None of the isolates viz., ABTSJ 23, ABTSJ 63, ABTSJ 72 and ABTSJ 82 produced Aflotoxins (Bl, B2, Gl, and G2) when tested. However, several strains of Penicillium sp. have been reported to secrete mycotoxins such as extrolite, citrin and citreoviridin (Adeyeye, 2016). Mucor circinelloides can potentially cause primary invasive cutaneous and maxillofacial zygomycosis (Iwen et al., 2007). Rhizopus oryzae occasionally causes the human disease mucormycosis (Ribes et al., 2000).Therefore, presence of Mucor, Rhizopus and certain strains of Penicillium sp. in ethnic starters could also be a threat to human health. Saito et al. (2004) compared Amylomyces rouxii and R. oryzae in lactic acid fermentation of potato pulp and other agricultural by products into food materials and preferred A. rouxii to R. oryzae. Considering the facts, Amylomyces rouxii

was selected as liquefying and saccharifying agent while developing the defined mixed starter culture for controlled rice beer production.
7.2 Selection of yeast for defined mixed starter preparation
For isolation of yeasts Yeast Extract Agar (YEA) was used. Serially diluted starter samples were poured on to YEA (Yeast extract 5gm/L, glucose 20 gm/L and gar 12gm/L) medium (Himedia, India). Morphologically different colonies were picked up and were repeatedly sub-cultured to obtain the pure culture. The cultures were identified based on colony characters, microscopic examination. Saccharomyces cerevisiae ATCC 9763 strain (procured from American Type Culture Collection & Gene Bank, LGC Prochem, Banglore, India) was used as a reference strain.
Based on the data obtained from characterisation studies on ability of the yeast isolates to tolerate high alcohol concentration (Ali et al, 2014)(Fig. 4), production of high ethanol (Thammasittirong et al., 2013)(Fig. 4), high osmotolerance (Ali et al, 2014)(Fig. 5) and safety related issues associated with the undesirable production of biogenic amines (Bover- Cid and Holzapfel, 1999)(Table. 2) and sulphur dioxide (AOAC, 1970), the yeast strain Saccharomyces cerevisiae ADJ5 and Wickerhamomyces anomalus strain ADJ2 were selected for developing a defined starter culture production. S. cerevisiae is wine yeast known for alcoholic fermentation. W. anomalus strain ADJ2 showed high osmo (20%) and thermo (upto 40°C) tolerance. The yeast isolates did not produce sulphur dioxide and any one of the biogenic amine (viz. Tryptamine, Tyramine, Cadaverine, Spermine and Spermidine/ Sigma- Aldrich, Germany)s tested. Association of W. anomalus has been reported in Asian rice beer fermentation (Jeyarama et al., 2008, Lee & Fujio, 1999; Xie et al., 2007). W. anomalus strain AS1 was able to improve wine aroma (Sabel et al, 2014). It reported that fermentation with specialized starter culture with co cultured W. anomalus and S. cerevisiae can improve aroma of alcoholic beverage (Maturano et al, 2012 and Sadineni et al, 2012). Thus the isolated indigenous isolates viz., W. anomalus strain ADJ2 and S. cerevisiae ADJ5 were selected as yeast candidate for defined starter preparation.
7.3 Compatibility of fungus with yeasts
Defined starter cultures were prepared with the compatible fungal culture Amylomyces rouxii (NCBI accession number KP790015) yeasts, Saccharomyces cerevisiae and Wickerhomomyces anomalus (NCBI accession number KX904349 and KX904346 respectively) isolated from Xaj pitha. Yeast and fungus were grown together on the same plate to check their compatibility. The strains did not display any inhibition as visible from the plate (Fig. 6). Free glucose, ethanol contents, and volumes of liquid produced with the combination of fungus (Amylomyces rouxii) with yeasts (Saccharomyces cerevisiae and Wickerhomomyces anomalus) during solid-state fermentation of rice starch at 30°C for 5 days are presented in Fig. 7. Individual culture of S. cerevisiae and W. anomalus produced ethanol (1.3% w/v and 2.1); glucose (0.8% w/v and 1.3 %) and liquid 3.0% and 6.1 % respectively. On the other hand in mixed cultures of MY1 (A. rouxii + S. cerevisiae) and MY2 (A. rouxii + W. anomalus) glucose, alcohol and liquid productions were significantly enhanced (glucose: 11.24% w/v and 10.31% w/v; ethanol: 5.3 % w/v and 6.9 % w/v; liquid: 42.0% w/v and 48% w/v respectively.) than individual cultures. Compatible mix cultures of MY1Y2 (A. rouxii + S. cerevisiae + W. anomalus) was able to produce higher amount of glucose, ethanol and liquid (glucose: 10.91% w/v; ethanol: 7.5% w/v, liquid: 51.0% w/v respectively). In mixed cultures containing both fungus and yeast, glucose produced by fungus was consumed and fermented into ethanol by yeast. Dung (2006) checked the compatibility of yeast S. cerevisiae on fungi Amylomyces rouxii

in mix culture fermentation of purple glutinous rice. The compatible culture produced 8.6% (w/v) ethanol, 19.2% (w/v) glucose and 48% (w/v) liquid after 3 days of fermentation at 30°C.
7.4 Selection of plants for defined starter preparation
Based on the field survey, it was observed that 21 different plants are regularly used for starter preparation by the Ahom community. These plants are used with relevance to organoleptic properties, starter nourishment and protection. Five representative plants viz., Leucas aspera (coded as PI), Lygodium flexuosum (P2), Polygonum strigosum (P3), Centella asiatica (P4) and Alstonia scholaris (P5) were selected based on their common use value during preparation of Xaj pitha. Leucas aspera (Use value=0.73), Centella asiatica (Use value=0.45) and Alstonia scholaris (Use value=0.43) were frequently reported to be used in starter nourishment (promotion of microbial growth), while Lygodium flexuosum (Use value=0.18) and Polygonum strigosum (Use value=0.29) were reported to be associated with organoleptic contribution and starter protection respectively. These plants also have nourishing properties that encourage microbial growth. Sterile herb extract of each tested herb was prepared by dissolving 5 gm in 100ml distilled water and extraction was done in a water bath at 50° C for 1 hour. The extract solution was filtered over a filter paper and finally filtered over a 0.2 u sterile membrane filter. Mixtures of extracts were made by pooling equal aliquots of each herb extract (Dung, 2006) (Fig. 8).
Antioxidant activity of these plant species were evaluated which revealed methanolic extracts of Polygonum strigosum with the highest DPPH inhibition (84.90%) followed by Leucas aspera (83.58%). All the plant extracts together showed the 83.27% DPPH inhibition (Table 3). Due to the high free radical scavenging activity of these plants, these could be used in starter protection and stability of Xaj These plants were selected for used in starter culture preparation. DPPH free radical scavenging activity of 60 ul Centella asiatica L. methanolic extract was 85.98%; whereas 80 ul samples showed increased 95.78% activity (Pittella et ai, 2009). In our study 100 ul methanolic extract was taken for each sample. However Centella asiatica L. showed DPPH radical scavenging activity of 77.70%.
All the plants selected for this study showed ability to promote the growth of yeast (Fig. 9). Polygonum strigosum L. (P3) showed highest growth stimulation of yeasts S. cerevisiae ATCC culture and W. anomalus ADJ2, whereas Leucas aspera (PI) showed the strongest stimulatory effect on the yeast species S. cerevisiae ABT Y1J. The chemical compound(s) in these aqueous extracts that caused growth stimulation may be the mineral contents. Because of their beneficial properties, these three herbs were included in preparation of defined starter cultures. The plant extracts of all the plants together inhibited the colony growth of the fungi,however, the extracts of Lygodium flexuosum (P2) showed a stimulatory effect on the fungal colony growth and it was very effective in stimulating the growth of desired fungal isolate Amylomyces rouxii (Fig. 10). The effects of the herbs used in traditional starter preparations on the starter microflora have been studied (Phuc, 1998; Dung et ai, 2005).The herbs "Tieu Hof (Fennel: Foeniculum vulgare Miller) and "Dinh Huong" (Clove: Syzygium aromaticum L.) used in Vietnamese starter culture also have been proven to be stimulatory in biomass production of fungi and yeasts (Dung et ai, 2005). Certain plants have been known for their medicinal andantimicrobial properties since ancient times. Biologically active compounds that effectively control various pests and pathogens are known from approximately 2400 plant species (Saleem, 1988).

7.5 Laboratory preparation of rice based alcoholic beverage using defined starter
culture
Alcoholic beverages were produced by using defined starter culture and compared to that of the traditionally prepared starter culture beverage. Biochemical evaluation of the standard "Xaj pani" with defined mixed-culture starter and the traditional starter prepared samples were carried out. These were stored upto six months and biochemical analysis performed. The samples were analysed for their pH, acidity, TSS, sugars, alcohol volume etc. Alcoholic beverages using defined starter culture were prepared using substrates waxy Bora rice and mixture of equal amount of Bora and Ranjit rice. The physiochemical analyses of beverages after 7 days of fermentation are listed in the Table 4.
The pH and TSS decreased during the storage period for all the "Xaj pani" samples prepared with different starters. The pH of rice beer prepared by the defined starter culture with glutinous rice decreased from initial 3.84 to 3.14 after 90 days. Whereas in case of traditional rice based alcoholic beverage pH decreased from initial 3.33 to 2.65 after 90 days. Acidity was highest (0.78 %) for the traditional rice based alcoholic beverage that defined starter produced Xaj pani (0.43 %). TSS of the rice based alcoholic beverage prepared with defined starter culture decreased from initial 17 at 15 days to 6.6 90 days. Alcohol content 10.42 % (v/v) produced by defined starter culture after 15 days of fermentation was higher than that of traditional alcohol content of 9.6 % (v/v). In case of defined starter prepared rice based alcoholic beverage protein content 2.3 mg/mL was higher than that of traditional rice based alcoholic beverage 2.20 mg/mL. Antioxidant activity was highest (61.12 %) in the alcoholic beverage sample produced by using defined starter culture and glutinous rice as substrate (Table 4).
7.6 Removal of turbidity from alcoholic beverage
Turbidity is due to the suspended 'haze'; insoluble or semi soluble particulate matter which is small enough to form a colloidal suspension in alcoholic beverages. Clarity or haze is a very important analytical parameter as well as for alcoholic beverage quality attribute (Mundy & Boley 1999; Smythe etal. 2002).
Enzyme preparation (1-3 gm/liter Xaj-pani) was used on post fermentation and storage stage to improve wine clarity, alcohol yields and to assess sensory properties. Use of extracellular enzyme help in clarification and stabilization of beer, breakdown the cellular structures of starch and release colour, flavour and aroma precursor compounds. The main advantage of using commercial enzymes is to speed up the winemaking process (Byarugaba-Bazirake et al, 2013). A-amylase (Himedia, Germany, Malt based) used in Xajpani brewing process is authorized for wine and juice treatment by FDA (GRAS, advisory dated 8/18/83).
Fresh alcoholic beverages was filtered after one week using a clean muslin cloth, mixed with enzyme powder thoroughly and allowed to sediment gravitationally. After one week, filtrate was transferred to a new bottle to reduce the amount of suspended particles and yeasts. It was found that intensity of colour in EBC turbidity unit increased from initial 3.79 to 4.8 after 6 months (Table 5).
7.7 Sensory evaluation of laboratory prepared rice based alcoholic beverage
Sensory evaluation of traditional Xaj and filtered laboratory rice beer produced by defined starters was carried. Both fresh and 1 month old Xaj and standard rice based alcoholic beverage were taken for evaluation of the quality attributes viz., taste, colour, flavour, acidity, mouthfulness, strength and overall acceptability. Nine point hedonic scale from 1-9 with scale 1-dislike extremely, 2- dislike very much, 3- dislike moderately, 4- dislike slightly, 5- neither like

or dislike, 6- like slightly, 7- like moderately, 8- like very much and 9- like extremely was used for sensory evaluation. For each test, each panelist was served with 10 ml of rice beer in wine glass. Coded rice based alcoholic beverage samples that had been cooled at 4°C for 24 hrs and after that kept for 4 hours at ambient condition were served (Fig. 13). The panelists were instructed to rinse their mouths with water after tasting each of the wine samples provided (Table 6).
Traditional rice based alcoholic beverage and standard mash filtered alcoholic beverage scored lowest for colour parameter. Both the beverages scored non-significant 5.73 and 5.37 respectively after 7 days of fermentation. Strength of traditional fresh, 1 month old and 1 month old mash filtered standard beverage did not differ significantly. Mouthfulness of fresh, 1 month old traditional beverage and filtered standard rice based beverages were almost same which did not differ significantly (p<0.01). Acidity score for 1 month old traditional rice based beverage was lowest; i.e., 5.1.
The flavor score was lowest (6.82, p<0.05) in fresh laboratory made standard rice based alcoholic beverage. This reason for the flavour scores being higher in traditional product may be because of the starter cultures. In the traditionally prepared starter cultures several herbs are used that may aid in enhancing the flavor (Dung et al., 2005).
7.8 Stability of alcoholic beverage during storage
The stability of cereal based alcoholic beverages up to several months can be achieved if the spoilage organisms are destroyed. Sterile filtration was used as an alternative to pasteurization for many years. It has the advantage over pasteurization in that the risk of flavor damage by heat is eliminated. However, many brewers report that there is little difference in flavor stability between sterile-filtered and properly pasteurized beer. Moreover, sterile filtration require expensive replacement (single source); expensive formulated cleaning agents and have relatively short service life (Leeder et al., 2011). Xaj filtration with sterile syringe driven 0.22μm filters was not possible as it is expensive, time consuming, for single use only and little volume recovery.
A common practice is the addition of sulfur dioxide (SO2) to wine and beer which is added to reduce the microbial population and to preserve the final product for long period of time (Gayon et al., 2006). Sulfur dioxide acts as both an antimicrobial agent and an antioxidant in wine (Amerine et al, 1967; Romano & Suzzi, 1993). Sulfites occur naturally in a number of food and beverages as a result of fermentation. Sulfites have been used as a food additive since 1664 and have been approved for use in the United States since the 1800s (Lester, 1995). With such a history of widespread application, sulfites have been generally regarded as safe (GRAS) by the FD; however, it is suspected that a low percentage of the population is sensitive to sulfites. The European Commission's Scientific Committee on Food (SCF) put forward in 1994 an acceptable daily intake (ADI) of 0.7 mg/ kg body weight/day for sulfur dioxide and sulfur dioxide equivalents (SCF, 1994). The ADI is the amount of food additive, expressed on a body weight basis, which can be ingested daily over a lifetime without appreciable health risk (Guido L.2016). The inhibitory effect of sulfur dioxide on malolactic fermentation or secondary fermentation by the contaminated bacterial species is much greater than it is for the alcoholic fermentation that is performed by Saccharomyces yeast. The addition of sulfites helps in protection of the wine against oxidation and re-fermentation. The sulfites act as a stabilizer and inhibit yeast growth which ensured that refermentation does not occur during sweetening. This process does not kill off the yeast or bacteria but prevents further growth which stops the fermentation in the bottle.

Re-fermentation and malolactic fermentation was halted by the addition of potassium meta- bisulphite after 1 month of fermentation After filtration and removal of residues from the bottom of the fermenter, it was applied to the beverage to stop the fermentation by the bottom fermented yeasts. Sulphur dioxide solution was added in accordance with the pH and TSS (Blvd and Rafael, 2014) values of the beverage samples (Table 7).
After addition of the sulphur solution, rice based alcoholic beverage samples were stored at ambient condition. During stabilisation stage chemical parameters of rice based alcoholic beverage was analysed and it was found that up to 3 months all the parameters differed non- significantly (Table 8). Further, it. was observed that sulphur dioxide also restricted the growth and cultivability of yeasts and other contaminated microorganisms. No growth on YPD agar, NA and PDA plates were seen.
7.9 Proper bottling and storage
Filtered, turbid free "Xaj pani'' samples were allowed for maturation at room temperature for one month before bottling. Beverages were packaged in glass bottled (Fig. 14) . Glass is the trusted and proven packaging for health, taste and the environment. It is also the only widely-used food packaging granted the FDA status of "GRAS" or generally recognized as safe (Girling, 2003). Empty glass bottles bought from local market were washed thoroughly with detergent powder and sterilized by rinsing with diluted 10% potassium metabisulphite solution. By siphon filling method, bottles were filled and capped using bottle capper and stored at ambient temperature at laboratory condition and one set was stored at refrigerator
8. Conclusions
We isolated and screened functional fungi Amylomyces rouxii (NCBI accession number KP790015) Wickerhomomyces anomalus (NCBI accession number KX904346) and yeasts Saccharomyces cerevisiae (NCBI accession number KX904349) from traditional amylolytic starter culture "Xaj pitha" associated with rice based alcoholic beverage "Xaj panf preparation. Selected plant extracts of' Leucas aspera Spreng. Lygodium jlexosum Vent. Polygonum strigosum L., Centella asiatica Urban and Alstonia scholaris (L.) R. Br. were added as representative herb in defined starter culture preparation based on their frequency of use. The rice based alcoholic beverage brewed using defined starter culture contained 10.42% (v/v) alcohol with 23.1 (mg/l00ml) protein, 61.12 % antioxidant activity, 0.43 % acidity and a pH value of 3.84 .Fermentation was stabilised in the laboratory prepared rice based alcoholic beverage through the addition of sulphur dioxide (20ppm/L) and turbidity of standard alcoholic beverage was removed by enzyme treatment that could be stored for 3 months without any major changes in the physical and chemical properties or taste. It also scored higher in Hedonic sensory attributes such as acidity, colour and overall acceptability. This patent claims for a defined starter culture for "Xaj pani" production in Assam. This defined starter produces cereal based alcoholic beverage with standard quality alcohol 10%.
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We claim;
1. Development of a defined starter culture for production of rice based alcoholic beverage, clarification and stabilisation of alcoholic beverage Xaj pani
2. The defined starter culture of claim 1, wherein said defined starter was produced consisting of amylolytic fungi Amylomyces rouxii (NCBI accession number KP790015) with alcohol producing yeasts Saccharomyces cerevisiae NCBI accession number KX904349) and Wickerhomyces anomalus (NCBI accession number KX904346) isolated from traditional starter tablet "Xaj pitha".
3. The method of claim 1, wherein said could produce Xaj pani with 9.45-10.33(v/v, %) alcohol, 16.67-18°Brix, pH 3.68- 3.79, 22. 89- 23.99% protein, 60.78-61.79% antioxidant activity and 0.39- 0.49 % acidity.The method of claim 1, wherein said could be used for cereal based alcoholic beverage production.

4. The method of claim 1, wherein said enzymatic treatments of A- amylase (1-3gm/liter beverage) could produce clear, haze free "Xajpani" without the changes in the sensory properties of the beverage.
5. The method of claim 1, wherein said "Xaj pani" could be stabilized with addition of sulphur solution(20ppm) and stored 3 months without significant changes in biochemical parameters .