Title of Invention

A PROCESS FOR PRODUCING KINEMA USING A PURE STARTER CULTURE

Abstract

Kinema is a traditional fermented soybean food of the Eastern Himalayan regions. Pulverised starter using selected strain of Bacilus subtilis KK2:B10, previously isolated from traditionally prepared kinema, was developed for kinema production. Kinema prepared by Bacillus subtilis KK2:B10 strain was grown and harvested in soybean extract broth, was dried in an oven and grounded aseptically. The starter was added aseptically to steamed soybeans and fermented under 85% relative humidity to get kinema.

Full Text

FIELD OF INVENTION This invention relates to a process for producing kinema using a pure starter culture. EXISTING STATE-O-ART Kinema is a fermented soybean-based, gray tan coloured, slightly alkaline sticky product having ammoniacal odour, and it serves as cheap source of high protein-rich food in a local diet It is commonly consumed as a flavoured curry with boiled rice. During the known method of kinema preparation, soybeans are soaked in water overnight and cooked until they can be pressed easily, then cracked lightly by a wooded pestle (locally called muslo) in a wooden mortar (locally called okhli). Grits are placed in a bamboo basket lined with locally grown fresh fern Glaphylopteriopsis erubescens fronds, ~ 1% of firewood ash is dusted, covered with a jute-bag and left to ferment naturally at ambient temperatures (25-40°C) for 2-3 days above earthern-oven kitchen. Distinctive flavour, stickyness and taste of kinema are all attributing to fermentation of soybeans mainly by Bacillus subtilis, which produce high amount of proteolytic enzyme that hydrolyse soya-proteins to amino acids enhancing digestibility. Other microflora associated with kinema are Enterococcus faecium, Candida parapsilosis and Geotrichum candidum. Traditional method of kinema production by natural fermentation is not uniform, varying from place to place and in quality affecting consistency of the product due to microbial profile, fermentation time and temperature. OBJECTS OF THE INVENTION An object of this invention is to propose a process for producing kinema using a pulverised starter. Another object of this invention is to propose a process for producing kinema using a pulverised starter which is inexpensive. Still another object of this invention is to propose a process for producing kinema using a pulverized starter which is stable over a considerable period. Yet another object of this invention is to propose a process for producing kinema using a pulverized starter and wherein the processing period is considerably reduced in comparison to that of the prior art. DESCRIPTION OF THE INVENTION According to this invention, there is provided a process for producing kinema using a pure starter culture comprising in steps of: a. subjecting water soaked soybean to the step of autoclaving was carried out in the presence of steam for a period such as 30 minutes at 121°C; b. inoculating said pulverized starter into said autoclaved soybean; c. subjecting the mixture of step (b) to the step of incubation to produce kinema at 40°C for 20 hrs under 85% relative humidity. The pulverized kinema starter is prepared by subjecting water soaked , soybean to the step of autoclaving, inoculating the soaked soybeans with an a cell suspension of Bacillus subtilis KK-2:B 10 harvested in a nutrient broth, subjecting such a mixture to the step of incubation to produce kinema, drying the kinema and then pulverizing it to produce kinema starter. Reference to the numerical values made herein are only by way of example and without intending to imply any limitation on the scope of the invention. Materials and Methods Soybean used: Small (~ 6mm) with smooth yellow seed coat and dark brown helium 'local yellow' variety of soybean employed. Microorganism: Bacilus subtilis KK-2:B10 (MTCC-2756) was previously isolated from naturally fermented kinema samples, and selected as best starter cultures for improved kinema production. Sample collection: Kinema samples were collected in aseptically pre-sterile bags, which were kept in an icebox and transported immediately for analyses. Soybean extract broth: Overnight soaked soybeans (100g) were autoclaved in a beaker containing 200 ml tap water at 1210C for 30 mins.. Sedimentof soybean extract collected in beaker was filtered using Whatman filter paper No.l. The final pH of filtered soybean extract was adjusted to 7.0 with 1(N) NaOH using pH-meter (Systronics 335, India). Kinema preparation: soybeans were cleaned, washed and soaked in tap water overnight at room temperature. Soaked soybeans were, autoclaved at 121°C for 30 mins. and inoculated with cell suspension of Bacillus subtilis KK2:B10, harvested in nutrient broth (HiMedia M002), phytone-sucrose broth (phytone peptone 10.0g, sucrose 10.0g, sodium chloride 10.0g, agar 20. 0g, distilled water 1L, pH 7.0), soybean extract broth and soybean extract-sucrose broth (in soybean-extract, added.sucrose 1.0%, NaCl 0.5% pH 7.0) at 37°C for I8h, respectively, at 104 cfu/g of cooked soybeans while the temperatures of soybeans was above 80°C. Inoculated soybeans were put into pre-sterile petri-dish (outer lid is replaced by perforated polyethylene film), and incubated at 40°C for 20h under 85% relative humidity. Microbial analysis: Culture of Bacillus subtilis KK-2B10 was transferred onto the nutrient broth, phytone broth, soybean extract broth and soybean extract-sucrose broth, separately, and incubated at 37°C for 18h to employ as purified kinema starter. Decimal dilution series were prepared in sterile physiological saline (0.85% w/v sodium chloride in water) and 1ml of appropriate diluted suspension was mixed with molten Tryptone Soya agar (HiMedia M424) and incubated at 37°C for 24h. Colonies appeared were counted as colony forming unit per ml (cfu/ml). For viability test, 10g of pulverised starter was mixed wit' 90ml of sterile physiological saline for 10 mins. and decimal series were prepared as described above. The total viable count of Bacillus subtilis in pulverised starter was determined in every month till 6 months. Sensory evaluation: The sensory attributes of kinema fermented by Bacillus subtilis KK2.B10, harvested in nutrient broth, phytone-sucrose broth, soybean extract and soybean extract-sucrose broth, respectively, were evaluated for flavour, taste, stickiness, texture and colour after sampling. Chemical analysis: Total nitrogen and water-soluble nitrogen and formol nitrogen of samples were determined by micro-Kjeldahl method (AOAC, 1990). Statistical analysis: Data obtained were analysed by determining errors of the mean and analysis of variance using the least square design. Results The soybean extract was developed as an economical Soybean extract broth for production of B.subtilis spores. The number of cells of Bacillus subtilis KK2:B10 was significantly (P 108 cfu/ml) as compared to nutrient broth (0.4 x 108 cfu/ml) and phytone- sucrose broth (P compared to soybean extract-sucrose broth. Kinema prepared by starter culture harvested in soybean extract broth had significantly (P scores in ail sensory attributes than that of kinema starter harvested in other broth media (Table 1). Hence, soybean extract after adjusting pH to 7.0 was selected as an inexpensive broth medium for producing B.subtilis spores for pure culture fermentation of kinema at the laboratory-scale. Ready-to-use starter culture for kinema production was prepared following the flow sheet as shown in Fig.l of the accompanying drawings. Kinema prepared by using B.subtilis KK2.B10 strain which was harvested in soybean extract broth, was dried in an oven at 70°C for 10h and grounded aseptically to make pulverised starter. The 1% of pulverised starter (instead of B.subtilis, as in Fig.l) was added aseptically to autoclaved soybeans and fermented to get kinema (Fig.l, Stage A). The total viable count of B.subtilis in pulverised starter was found constantly maintained at the level of ~I09 cfu/g. till 6 months (Fig.3). This was due to survival of endospores of B.subtilis for longer period at room temperature. No other microorganisms were recovered from pulverised starter kept in pre-sterile polythene bag at room temperature. The consumers' preference trials showed that kinema prepared by using pulverised starter under optimized conditions was more acceptable than market kinema. Market kinema was liked extremely (score, 9) by 15%, very much (score, 8) by 30% and moderately (score,7) by 55%, while kinema prepared by using pulverised starter was liked extremely by 30%, very much by 40% and moderately by 30% of the consumers. Water-soluble nitrogen and formol nitrogen contents were higher in kinema prepared by using pulverised starter than market kinema (Table 2). Increased water-soluble nitrogen in kinema helps in digestibility and high amount of formol nitrogen which contains free amino acids supplements that impart better taste to kinema. Kinema prepared by using pulverised starter had more advantages over traditional method due to shorter fermentation time that eliminates the chance of growth of contaminants, hygienic conditions, maintaining consistency with better quality and flavour. Use of readymade pulverised starter appears more appropriate in kinema production at household level. We Claim: 1. A process for producing kinema using a pure starter culture comprising in steps of: a. subjecting water soaked soybean to the step of autoclaving was carried out in the presence of steam for a period such as 30 minutes at 121°C; b. inoculating said pulverized starter into said autoclaved soybean; c. subjecting the mixture of step (b) to the step of incubation to produce kinema at 40°C for 20 hrs under 85% relative humidity. 2. A process as claimed in claim 1 wherein 1% of pulverized starter was added to the water soaked soybean. 3. A process as claimed in claim 1 wherein the pulverized kinema starter is prepared by subjecting water soaked soybean to the step of autoclaving, inoculating the soaked soybeans with an a cell suspension of Bacillus subtilis KK-2:B10 harvested in a nutrient broth, subjecting such a mixture to the step of incubation to produce kinema, drying the kinema and then pulverizing it to produce kinema starter. 4. A process as claimed in claim 3, wherein the kinema is dried and then pulverized. 5. A process as claimed in claim 1, wherein the dried kinema is aseptically ground. 6. A process as claimed in claim 1 and 3, wherein the soybean is soaked in water and the excess water drained off prior to the step of autoclaving. 7. A process for producing kinema using a pure starter culture substantially as herein described. Kinema is a traditional fermented soybean food of the Eastern Himalayan regions. Pulverised starter using selected strain of Bacilus subtilis KK2:B10, previously isolated from traditionally prepared kinema, was developed for kinema production. Kinema prepared by Bacillus subtilis KK2:B10 strain was grown and harvested in soybean extract broth, was dried in an oven and grounded aseptically. The starter was added aseptically to steamed soybeans and fermented under 85% relative humidity to get kinema.

Documents:

100-kol-2003-abstract.pdf

100-kol-2003-claims.pdf

100-KOL-2003-CORRESPONDENCE 1.1.pdf

100-kol-2003-correspondence.pdf

100-kol-2003-description (complete).pdf

100-kol-2003-drawings.pdf

100-KOL-2003-EXAMINATION REPORT 1.1.pdf

100-kol-2003-examination report.pdf

100-kol-2003-form 1.pdf

100-KOL-2003-FORM 18 1.1.pdf

100-kol-2003-form 18.pdf

100-kol-2003-form 2.pdf

100-KOL-2003-FORM 26.pdf

100-KOL-2003-FORM 3 1.1.pdf

100-kol-2003-form 3.pdf

100-KOL-2003-GRANTED-ABSTRACT.pdf

100-KOL-2003-GRANTED-CLAIMS.pdf

100-KOL-2003-GRANTED-DESCRIPTION (COMPLETE).pdf

100-KOL-2003-GRANTED-DRAWINGS.pdf

100-KOL-2003-GRANTED-FORM 1.pdf

100-KOL-2003-GRANTED-FORM 2.pdf

100-KOL-2003-GRANTED-SPECIFICATION.pdf

100-KOL-2003-REPLY TO EXAMINATION REPORT.pdf

100-kol-2003-specification.pdf