Accepted Manuscript

Naturally Fermented Ethnic Soybean Foods of India

Jyoti Prakash Tamang

PII: S2352-6181(15)00004-9 DOI: 10.1016/j.jef.2015.02.003 Reference: JEF 11

To appear in: Journal of Ethnic Foods

Received Date: 15 January 2015 Revised Date: 22 January 2015 Accepted Date: 6 February 2015

Please cite this article as: Tamang JP, Naturally Fermented Ethnic Soybean Foods of India, Journal of Ethnic Foods (2015), doi: 10.1016/j.jef.2015.02.003.

This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. ACCEPTED MANUSCRIPT 1 Naturally Fermented Ethnic Soybean Foods of India

2

3 Jyoti Prakash Tamang

4

5 Department of Microbiology, School of Life Sciences, University, 6 th Mile,

6 Tadong 737102, Sikkim, India

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12 Corresponding author: Professor Dr. Jyoti Prakash Tamang*, Department of Microbiology,

13 Sikkim University, 6 th Mile, Tadong 737102, Sikkim, India (e-mail: 14 [email protected]; Mobile: +91-8016099902; MANUSCRIPT Tel: +91-3592-251188) 15

16 Running title: Indian fermented soybeans

17 Keywords : Ethnic foods, naturally fermented soybeans, kinema, Bacillus

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ACCEPTED

1 ACCEPTED MANUSCRIPT 19 Abstract

20 Kinema , hawaijar , tungrymbai , bekang, aakhone and peruyaan are naturally fermented ethnic

21 soybean foods of India popular among the Mongolian-origin races in the Eastern .

22 Bacillus subtilis is the dominant functional bacterium in all naturally fermented soybean foods

23 of these regions. Though there is a good demand of ethnic fermented soybeans foods among

24 the local consumers in North East India, the production is limited to household level. Ready-

25 to-use pulverised starter culture for kinema production can be introduced to kinema -makers or

26 similar sticky fermented soybean foods of North East India adapted to local conditions for

27 more income generation. Ethnic fermented soybeans are one of the major food resources in

28 the Eastern Himalayas to supplement inexpensive, high digested plant protein with low

29 fat/cholesterol content and high nutritive value with antioxidant and other health-promoting

30 properties in the local diet as functional food.

31 MANUSCRIPT

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2 ACCEPTED MANUSCRIPT 32 Introduction

33 In the Eastern Himalayas, soybean [Glycine max (L.) Merrill, family Leguminosae, sub-

34 family Papilionaceae] is grown under rain-fed conditions in upland terraces as a sole crop as

35 well as a mixed crop with rice and maize up to an elevation of 1500 m (Fig 1). Soybean,

36 locally known as bhatmas in , is traditionally used to prepare various

37 fermented and non-fermented recipes in the Eastern Himalayan regions of , India and

38 [1]. Soybean was probably introduced to India from China through the Himalayas

39 several centuries ago and some believe that soybeans were also brought via Myanmar by

40 traders from Indonesia [2]. Two indigenous varieties of soybeans ‘yellow cultivar’ and ‘dark

41 brown cultivar’ (Fig 1) are grown in between May and June and harvested in November.

42 Locally grown soybeans are harvested and dry seeds of soybeans are naturally fermented into

43 flavoursome and sticky product in eastern parts of Nepal, hills, Sikkim, North

44 Eastern regions of India, and southern parts of Bhutan by the Mongolian races. Some of the 45 common ethnic non-salted sticky fermented soybean MANUSCRIPT foods of Eastern Himalayas are kinema 46 (Nepal, Darjeeling hills, Sikkim and south Bhutan), hawaijar (Manipur), tungrymbai

47 (Meghalaya), bekang (Mizoram), aakhone (Nagaland), peruyaan (Arunachal Pradesh) (Table

48 1). This article will examine kinema’s characteristics, microbiology of kinema and health

49 benefits of kinema including hawaijar, tungrymbai, bekang, aakone, peruyaan.

50

51 Kinema

52 Kinema is an ethnic fermented soybean food of Nepali community in the Eastern Himalayas

53 which is sticky,ACCEPTED slightly alkaline product with a slight ammoniacal flavour produced by

54 natural fermentation. It is a whole-soybean fermented food with sticky texture, gray tan

55 coloured and flavoursome [3]. During traditional production of kinema , the small-sized (~ 6

56 mm) ‘yellow cultivar’ soybean dry seeds are selected, washed, soaked overnight (8-10 h) in

57 water. Soaked soybeans seeds are taken out and put into the container with fresh water, and

3 ACCEPTED MANUSCRIPT 58 boiled for 2-3 h until they are soft. Excess water is drained off and the cooked soybean seeds

59 are filled into the wooden mortar locally called ‘okhli, and are cracked lightly by a wooden

60 pestle locally called ‘muslo’ to split the cotyledons. This practice of cracking cooked seeds of

61 soybeans is observed only during kinema production unlike other similar fermented soybean

62 foods of Asia and North East India probably to increase the surface areas for speed

63 fermentation by aerobic spore-forming Bacillus spp. About 1 % of firewood ash is added

64 directly to the cooked soybeans and mixed thoroughly to maintain the alkaline condition of

65 the product. Soybean grits are placed in a bamboo basket lined with locally grown fresh fern

66 called (Glaphylopteriolopsis erubescens ). The basket is covered in a jute bag and left to

67 ferment naturally at ambient temperatures (25-40 o C) for 1-3 days above an earthen oven

68 kitchen (Fig 2). During summer, the fermentation time may require 1-2 days while in winter it

69 may require 2-3 days. In eastern Nepal, local consumers prepare dark brown local varieties of

70 soybean seeds rather than yellow-coloured seeds for making kinema [4]. Similarly, they 71 commonly use Ficus (fig plant) and banana leaves MANUSCRIPT as wrapping materials instead of fern 72 fronds. Other methods remain the same. Completion of fermentation is indicated by the

73 appearance of a white viscous mass on the soybean seeds and the typical kinema flavour with

74 a slight odour of ammonia.

75 Shelf-life of freshly prepared kinema (Fig 3) remains for 2-3 days in summer and

76 maximum a week in winter without refrigeration. It may be prolonged by drying in the sun for

77 2-3 days. Dried kinema is stored for several months at room temperature. Preparation of

78 kinema varies from place to place and is still restricted at the household level. It is interesting

79 to note the mountainACCEPTED women using their indigenous knowledge of food production prepare

80 kinema . This unique knowledge of kinema -making has been protected as a hereditary right

81 and passed from mother to daughter, mostly among the Limboo.

82 Kinema is eaten as curry with steamed rice. Delicacy of kinema can be perceived by its

83 appealing flavour and sticky texture. Fresh kinema is fried in vegetable oil, with chopped

4 ACCEPTED MANUSCRIPT 84 onions, tomatoes and turmeric powder. Salt and sliced green chillies are added and fried for 3-

85 5 min. A little water is added to make thick gravy, and cooked for 5-7 min then the kinema

86 curry (Fig 3) is ready for serving with steamed rice. Dried kinema is sometimes mixed with

87 leafy vegetables to make a mixed curry. Consumers like kinema mostly due to its typical

88 flavour and sticky texture, and some people dislike the product due to its strong umami -type

89 flavour [5] and mucilaginous texture.

90 Kinema production is a source of marginal income-generation for many families in the

91 Eastern Himalayas. Kinema is sold in all local periodical markets locally called ‘haats’ in

92 eastern Nepal, Darjeeling hills, Sikkim and southern parts of Bhutan by rural women.

93 Usually, it is sold by volume taking in a small silver mug containing 150-200 g of kinema ,

94 and pack in the leaves of fig plant ( Ficus hookeriana ) locally called ‘nevara’, and then tied

95 loosely by straw. Poly-bags are not used to pack kinema. One kg of kinema costs about Rs.

96 100. Average 5 kg of kinema is sold by each seller in a local market and about 60 % expenses 97 are incurred on purchase of raw soybeans, fuel MANUSCRIPT for cooking, transportation from village to 98 market, etc., and 40 % of profit is made [1]. This profit is spent on children’s education,

99 procuring essential commodities not locally available, and other domestic expenses. Though

100 there is good demand of kinema in the local markets, production of kinema is still confined to

101 home production, there is no organised processing unit or factory of kinema production.

102 Kinema making technology has not been recognised as small-scale industry for getting

103 financial support or loan by any public sector bank or financial institution, or, neither has it

104 been incorporated in the rural development programmes of the government in Nepal, India

105 and Bhutan. ACCEPTED

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107 Microbiology of kinem a

108 Several species of Bacillus have been isolated from kinema which include Bacillus subtilis, B.

109 licheniformis, B. cereus, B. circulans, B. thuringiensis and B. sphaericus at an average load of

5 ACCEPTED MANUSCRIPT 110 10 8 cfu/g [6,7,8]. However, B. subtilis is the dominant functional bacterium in kinema [9,10].

111 Besides Bacillu s, Enterococcus faecium (10 7 cfu/g) ; yeasts- Candida parapsilosis and

112 Geotrichum candidum (10 4 cfu/g) have also been reported from kinema [6,8].

113 It is observed that rich microbial diversity in various sources particularly soybean,

114 equipment and leaves as wrapping materials harness microbiota for natural fermentation of

115 kinema [8]. With the decline in protein nitrogen content, the non-protein and soluble nitrogen

116 contents increases during kinema fermentation [11] found a significant increase in relative

117 viscosity of kinema during maturation at 5 0 C and 10 0 C [12]. Keeping freshly prepared

118 kinema below 10 0 C for 1 day stabilizes the quality of the product by preventing the further

119 biological activity of microorganisms and shows better stickiness which is very important

120 sensory property of kinema [12]. Organoleptically, the monoculture fermentation of soybean

121 by B. subtilis MTCC 2756 produces the best kinema because of a pleasant nutty flavour and

122 highly sticky texture, and also minimizes the conventional fermentation time, maintains better 123 hygienic conditions, consistency, and increases lev MANUSCRIPTels of soluble protein [13]. 124 Inexpensive soybean extract broth after adjusting pH to 7 as medium is prepared for

125 enrichment of B. subtilis spores instead of discarding the soybean extract after autoclaving

126 soybeans [13]. Moreover, nutrient broth, conventionally used for enrichment of B. subtilis

127 spores, is composed of expensive beef extract, which is not acceptable to the majority of the

128 Hindu population in the Himalayas. Kinema prepared by using B. subtilis KK2:B10 strain

129 which is harvested in soybean extract broth is dried in an oven at 70 0 C for 10 h and ground

130 aseptically to make pulverized starter. The 1% of pulverized starter instead of B. subtilis is

131 added asepticallyACCEPTED to autoclaved soybeans and fermented to get kinema . The consumers’

132 preference trials show that kinema prepared by using pulverized starter under optimized

133 conditions is more acceptable than market kinema [13]. Water soluble nitrogen and formol

134 nitrogen contents are higher in kinema prepared by using pulverized starter than market

135 kinema [13]. Increased water soluble nitrogen in kinema helps in digestibility and high

6 ACCEPTED MANUSCRIPT 136 amount of formol nitrogen which contains free amino acids supplements that impart better

137 taste to kinema [14]. Application of ready-to-use pulverized starter may appear appropriate in

138 kinema production for marginal kinema producers in the Himalayas since it is cost-effective

139 and easy to handle [1]. In 2012, we obtained Indian Patent on “ A process for production of

140 kinema, fermented soybean food, using a pure starter culture ” (Patent No: 25346).

141 Although E. faecium does not add any sensory quality to the Bacillus fermentation of

142 soybeans, it is always encountered in naturally fermented kinema [1]. Presence and growth of

143 yeast during kinema preparation are associated with the development of rancidity in the

144 products. In fact, B. subtilis is the sole fermenting organism in kinema preparation.

145

146 Health Benefits of Kinema

147 Kinema has many health-promoting benefits including antioxidant, digested protein, essential

148 amino acids, vitamin B complex, low-cholesterol content, etc. [1] which can be considered as 149 a functional food. Kinema is the cheapest source MANUSCRIPT of plant protein as compared to milk and 150 animal products on the basis of protein cost per kg. It contains all essential amino acids [15],

151 and is rich in linoleic acid, an essential fatty acid [16]. Total amino acids, free amino acids

152 and mineral contents are increased during kinema fermentation [11,12,14]. Phytosterols

153 (cholesterol-lowering effect) is increased during kinema fermentation [16]. Riboflavin and

154 niacin increases in kinema during fermentation [17]. Kinema has antioxidant activities [18].

155 Due to large amount of Group B saponin contents, kinema claims to have health promoting

156 benefits [19].

157 ACCEPTED

158 Other Fermented Soybean Foods of North East India

159 Many kinema -like sticky naturally fermented soybeans are consumed by different ethnic

160 communities living in North Eastern part of India bordering with Bhutan, China and

7 ACCEPTED MANUSCRIPT 161 Myanmar (Fig 4) which include hawaijar in Manipur, bekang in Mizoram, peruyaan in

162 Arunachal Pradesh, aakhone in Nagaland and tungrymbai in Meghalaya (Fig 5).

163

164 Hawaijar

165 Hawaijar is a traditional fermented soybean alkaline food of Manipur. It is prepared from

166 local variety of small-seeded soybean grown in hilly terraces of Manipur [20]. It is similar to

167 kinema . Small sized soybean seeds are selected, washed and boiled in an open cooker for 2-3

168 h. Excess water is drained off, cooled to ~ 40 0 C and then packed the whole soybean seeds in

169 a small bamboo basket having lid. The basket is lined with fresh leaves of fig plant (Ficus

170 hispida ) locally called ‘assee heibong’ in Meitei language or banana leaves. After placing

171 cooled soybean seeds inside the basket, the lid is closed loosely and the basket is kept nearby

172 the kitchen or warm place for natural fermentation for 3-5 days (Fig 6). Emission of typical

173 ammonia odour and appearance of sticky texture on the cooked soybean seeds are determined 174 as good quality hawaijar by the Meitei. Shelf-life MANUSCRIPT of hawaijar is maximum 7 days without 175 refrigeration. Sometimes, it is sun dried for 2-3 days and stored for several weeks for future

176 consumption. Unlike kinema , the practice of cracking and addition of ash is not adopted by

177 the Meitei women in hawaijar production. Hawaijar is produced by the Meitei women, men

178 support in the process.

179 A special curry called ‘chagempomba’ is commonly prepared by the Meitei in

180 Manipur and is eaten with steamed rice. Hawaijar is eaten directly or used as a condiment or

181 mixed with vegetables to make curry in the Manipuri cuisine. Hawaijar is commonly sold in

182 local markets throughoutACCEPTED Manipur by the Meitei women. Despite of its popularity, there is no

183 organised food sector for mass scale production of hawaijar in Manipur. The product is still

184 prepared at home and many women are dependent upon the product for livelihood.

185

186

8 ACCEPTED MANUSCRIPT 187 Tungrymbai

188 Tungrymbai is an ethnic fermented soybean food of Khasi and Garo in Meghalaya. It is

189 similar to kinema . Soybean seeds are collected, cleaned, washed and soaked in water for about

190 4–6 h [22]. The seed coat of soybean is normally removed before cooking by rubbing the

191 soaked seeds gently. The soaked soybeans are cooked for about 1-2 h till all the water is

192 absorbed. Cooked beans are allowed to cool, and are packed with fresh leaves of Clinogyne

193 dichotoma locally called ‘lamet’ and are placed inside the bamboo basket, and covered by

194 thick cloth. The covered basket is kept over the fireplace and fermented naturally for 3–5 days

195 to get tungrymbai (Fig 7). Tungrymbai is mashed and put into a container with water and boil

196 till water evaporates, and stir continuously. It is mixed with fried onion, garlic, ginger, chilli,

197 grinded black sesame locally called ‘til’ and salt. A thick curry is made and is served as side

198 dish with steamed rice by Khasi in Meghalaya. Pickle is also made from tungrymbai . It is

199 commonly seen Khasi women selling tungrymbai packed in fresh leaves of ‘lamet’ or banana 200 at the vegetable markets of Shillong. MANUSCRIPT 201

202 Functionality: Bacillus subtilis TS1:B25 ( tungrymbai ) and B. subtilis BT:B9 ( bekang )

203 accounted for the highest production of PGA (2.8 mg/ml each) amongst the other strains

204 tested [24]. Though LAB showed antimicrobial activities, none of them produced bacteriocin

205 and biogenic amines under the applied condition. Enterococcus faecium TM2:L6

206 (tungrymbai ) and BAV:E2 ( bekang ) showed the highest degree of hydrophobicity of 72.7 %

207 and 71.6 %, respectively. LAB strains were able to degrade phytic acid and oligosaccharides,

208 showing their abilityACCEPTED to degrade anti-nutritive factors. Tungrymbai and bekang possess

209 antioxidant and free radical (DPPH and ABTS) scavenging activity [24].

210

211 Bekang

9 ACCEPTED MANUSCRIPT 212 Bekang is an ethnic fermented soybean food commonly consumed by Mizo in Mizoram. It is

213 also similar to kinema . During traditional method of preparation of bekang , small sized dry

214 seeds of soybean are collected, cleaned and soaked in water for 10-12 h. Excess water is

215 dewatered and beans are boiled for 2-3 h in an open cooker until the beans become soft.

216 Excess water is drained off and wrapped in fresh leaves of Calliparpa aroria ( Family:

217 Verbanaceae) locally called ‘nuhlhan’ in or leaves of Phrynium sp. (Family: Merantaceae),

218 locally known as ‘hnahthial’. The wrapped beans are kept inside the small bamboo basket.

219 The basket is then placed near the earthen oven or warm place and is allowed to ferment

220 naturally for 3-4 days. Sticky soybean with emission of ammonia odour is produced which is

221 liked by the local consumers. The product is called bekang in Mizoram (Fig 8). Bekang is

222 consumed as it is, or made into curry with addition of salt, green chillies and tomatoes. It is

223 consumed as side dish with steamed rice. Bekang is sold in the local markets by Mizo women,

224 who earn their livelihood [25]. 225 MANUSCRIPT 226 Aakhone

227 Aakhone or also called axone is an ethnic fermented sticky soybean food of Sema Naga in

228 Nagaland, similar to kinema . The preparation is same as other fermented soybean foods of

229 North East India. Soybean seeds are soaked, cooked and beans are wrapped in fresh leaves of

230 banana or Phrynium pubinerve Blume (Family: Marantaceae) or Macaranga indica Wight

231 (Family: Euphorbiaceae) and kept above the fireplace to ferment for 5-7 days [26]. The shelf-

232 life of freshly fermented aakhone is maximum a week. Fresh aakhone is moulded and made

233 into cakes and dryACCEPTED above the earthen oven. Sometimes, each fermented bean is separated by

234 hand, and dried in the sun for 2-3 days. Dried aakhone is stored in containers for future

235 consumption (Fig 9). Pickle is made from freshly fermented aakhone by mixing with green

236 chilli, tomato and salt. The dried aakhone cakes are cooked with pork and are eaten as side-

237 dish with steamed rice by Sema.

10 ACCEPTED MANUSCRIPT 238 Peruyyan

239 Peruyaan is an ethnic fermented soybean food of Apatani tribes in Arunachal Pradesh. The

240 word peruyaan has been derived from the Apatani dialect, perun means beans and yannii

241 means packing in leaves [25]. During the traditional preparation of peruyaan , soybean seeds

242 are collected, washed and cooked for 2-3 h till the beans become soft. The excess water is

243 drained off and is cooled for sometime. The cooked soybeans are kept in a bamboo basket

244 (vessel) lined with fresh ginger leaves locally called as ‘taki yannii’. The basket is loosely

245 covered with ginger leaves and is kept on the wooden rack above the fire place for

246 fermentation for 3-5 days. The stickiness of the product is checked, and if the product is

247 sticky enough then the product is ready for consumption (Fig 10). Peruyaan is consumed

248 mostly as a side dish with steamed rice by the Apatani tribes in Arunachal Pradesh. It is

249 mixed with hot water, chillies locally called as ‘tero’ and salt, and directly consumed without

250 frying or cooking unlike kinema curry preparation. 251 MANUSCRIPT 252 Similarity with other Asian non-salted Bacillus fermented soybean foods

253 Kinema is similar to other Asian Bacillus -fermented sticky soybean foods such as natto of

254 Japan [28], chungkukjang of Korea [29] and thua nao of northern Thailand [30] (Fig 11),

255 pepok of northern Myanmar [31] and sieng of Cambodia and Laos [32]. The preparation of

256 kinema is very similar to that of natto . In itohiki-natto whole soybeans are used for

257 fermentation, and in hikiwari-natto de-hulled soybeans cracked into 2 to 4 pieces are used

258 [33]. Some of the steps in kinema preparation do not resemble to those in natto and

259 chungkukjang, andACCEPTED thus make kinema a unique non-salted soybean fermented product. The

260 cooked beans are lightly crushed to de-hull most of the seeds. But, fermentation is carried out

261 with the kernels as well as the seed coats. Unlike natto and chungkukjang , kinema is always

262 fried in oil and made to curry. The practice of frying kinema may have developed to drive out

263 the unpleasant ammonia smell which masks the pleasant and persistent nutty flavour.

11 ACCEPTED MANUSCRIPT 264 Phylogenetic Similarity of Bacillus Strains from Asian Sticky Fermented Soybeans

265 The phylogenetic relationship among bacilli isolated from kinema (India), chungkokjang

266 (Korea) and natto (Japan), similar fermented sticky soybean foods of Asia on the basis of 16S

267 rDNA sequence has been studied [34]. Strains of Bacillus isolated from kinema and

268 chungkokjang show central to paracentral position of spores with few strains showing

269 negative nitrate reduction test, whereas Bacillus subtilis (natto ) isolated from natto show

270 central position of spores and all reduces nitrate [34]. However, all strains of Bacillus subtilis

271 isolated from kinema, chungkokjang and natto show stickiness on phytone agar and cooked

272 soybean, which are characteristics property of non-salty fermented soybean foods of Asia

273 [10]. However, type strain of B. subtilis JCM 1465 does not produce any stickiness [34].

274 In order to investigate the phylogenetic relationship of isolates to other bacteria, the

275 sequence of 16S rRNA gene PCR product is determined and found that strains B. subtilis

276 KD:B1 and KG:B1 isolated from kinema, B. subtilis CA:B1 and CK:B2 isolated from 277 chungkokjang , and B. subtilis JN-1 isolated from nattoMANUSCRIPT have identical sequences except that of 278 JA-1 ( natto ) which has one ambiguous nucleotide [34]. The evolutionary distance between

279 four strains CK:B1, KD:B1, JN-1 and JA-1, and Bacillus subtilis is 0.002 Knuc as calculated

280 by the ratio of nucleotide substitution per nucleotide site, indicating 99 % homology with

281 Bacillus subtilis type strain. However, the evolutionary distance between the strains CA:B1

282 and KG:B1, and Bacillus subtilis is 0.005 Knuc , showing approximately 99.5 % homology

283 with type strain [34]. The phylogenetic analyses reveal that all six strains belonged to B.

284 subtilis . This is the first report to describe the phylogeny of B. subtilis isolated from similar

285 non-salty fermentedACCEPTED sticky soybean foods of Asia [34]. The plasmid of B. subtilis (natto )

286 strain resembles that of B. subtilis strain in the partial nucleotide sequences [35,36,37].

287 Diversity of Bacillus subtilis -fermented soybean foods of Asia including that of the Eastern

288 Himalayas needs to be studied in details to find out the similarity. The probable source of

12 ACCEPTED MANUSCRIPT 289 common stock of similar sticky fermented soybean foods will help the food scientists to trace

290 the antiquity of fermented soybean foods in Asia.

291

292 KNT-Triangle Hypothesis

293 Non-salted fermented soybean foods are concentrated in a triangle with 3 vertices on Japan

294 (natto ), India and Nepal ( kinema ) and Indonesia ( tempe ). Nakao [38] named a ‘ natto triangle’

295 and included both bacilli and mould-fermented soybean products including tempe and

296 extended the triangle up to Indonesia. Tamang [1] renamed this hypothetical triangle ‘ kinema-

297 natto-thua nao triangle’ (KNT-triangle) and included only non-salty, Bacillus -fermented

298 soybeans foods with 3 vertices on India and Nepal (kinema and similar products), Thailand

299 (thua-nao ) and Japan ( natto ) (Fig 12). Within the proposed triangle-bound countries, many

300 fermented sticky non-salty soybean foods are consumed by the different ethnic groups of

301 people such as kinema (India, Nepal and Bhutan), natto (Japan), tungrymbai, bekang , 302 hawaijar , aakhone and peruyaan (India), thua nao MANUSCRIPT (Thailand), chungkokjang (Korea), pepok 303 (Myanmar) and sieng (Cambodia and Laos). Beyond this hypothetical ‘KNT-triangle’, there is

304 no report of kinema like products with sticky and ammonia flavoured fermented soybean

305 foods and the proposed ‘KNT triangle’ does not include salted, non-sticky and non-bacilli

306 fermented soybean products such as tempe, miso, sufu, soy-sauce, etc. [1]. Although the

307 method of production and culinary practices vary from product to product, all bacilli-

308 fermented Asian soybean foods have characteristic stickiness and typical flavor. Hara et al.

309 [35] reported that plasmid of B. subtilis (natto ) strain resembles that of B. subtilis isolated

310 from thua-nao ACCEPTED and kinema. The phylogenetic relationships among bacilli isolated from

311 kinema (India), chungkokjang (Korea) and natto (Japan), similar fermented sticky soybean

312 foods of Asia reveal that all bacilli strains belonged to B. subtilis [34]. This suggests that B.

313 subtilis strains responsible for fermentation of sticky soybean food in Asia might have

314 originated from the same stock. Another theory was proposed that non-salted fermented

13 ACCEPTED MANUSCRIPT 315 soybean foods were originated in Yunnan province of China, which was the center of the

316 hypothetical triangle [28].

317

318 Conclusion

319 It has been observed that flavoursome mucilaginous fermented soybean foods kinema ,

320 hawaijar , tungrymbai , bekang, aakhone and peruyaan are popular among the Mongolian-

321 origin races in the Eastern Himalayas. The Mongolian people prefer the umami flavoured

322 foods due to specific sensory development. Bacillus subtilis is the dominant functional

323 bacterium in all fermented soybean foods of these regions. Fermented soybean foods are

324 consumed only to the Eastern Himalayas, no such product is consumed in other parts of the

325 Himalayas. Though there is a good demand of ethnic fermented soybeans foods among the

326 local consumers in North East India, the production is limited to household level. The

327 scientific findings have correlated the indigenous knowledge of the ethnic people of the 328 Himalayas and acknowledged the innovative skills MANUSCRIPT of mountain women. which supplement 329 inexpensive, high digested plant protein and other health-promoting properties in the local

330 diet.

331 332 333 334 335 336 337 ACCEPTED 338 339 340 341 342 343

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382 contents of soybeans during kinema production. J Sci Food Agri 1998; 78: 498-502. 383 18. Moktan B, Saha J, Sarkar PK. Antioxidant activitiesMANUSCRIPT of soybean as affected by Bacillus - 384 fermentation to Kinema Food Res International 2008; 4(6): 586-593.

385 19. Omizu Y, Tsukamoto C, Chettri R, Tamang JP. Determination of saponin contents in raw

386 soybean and fermented soybean foods of India. J Sci Ind Res 2011; 70: 533-538.

387 20. Jeyaram J, Anand Singh Th, Romi W, Ranjita Devi A, Mohendro Singh W, Dayanidhi H,

388 Rajmuhon Singh N, Tamang JP. Traditional fermented foods of Manipur. Indian J

389 Traditional Knowl 2009; 8 (1): 115-121.

390 21. Jeyaram K, Mohendro Singh W, Premarani T, Ranjita Devi A, Selina Chanu K, Talukdar

391 NC, RohinikumarACCEPTED Singh M. Molecular Identification of dominant microflora associated

392 with ‘Hawaijar’ – a traditional fermented soybean (Glycine max L.) food of Manipur,

393 India. Int J Food Microbiol 2008; 122, 259–268.

394 22. Agrahar-Murungkar D, G Subbulakshmi. Preparation techniques and nutritive value of

395 fermented foods from the Khasi tribes of Meghalaya. Ecol Food Nutri 2006; 45: 27-38.

16 ACCEPTED MANUSCRIPT 396 23. Chettri R, Tamang JP. Bacillus species isolated from Tungrymbai and Bekang, naturally

397 fermented soybean foods of India. Int J Food Microbiol 2015; 197, 72-76.

398 24. Chettri R, Tamang JP. Functional properties of Tungrymbai and Bekang, naturally

399 fermented soybean foods of India. Int J Fermented Foods 2014; 3: 87-103.

400 25. Tamang JP, R Chettri, RM Sharma. Indigenous knowledge of Northeast women on

401 production of ethnic fermented soybean foods. Indian J Traditional Knowl 2009; 8 (1):

402 122-126.

403 26. Mao A A, Odyuo N. Traditional fermented foods of the Naga tribes of Northeastern,

404 India. Indian J Traditional Knowl 2007; 6(1): 37-41.

405 27. Singh TA, Devi KR, Ahmed, G, Jeyaram K. Microbial and endogenous origin of

406 fibrinolytic activity in traditional fermented foods of Northeast India. Food Res Int 2014;

407 55, 356–362.

408 28. Nagai T, JP Tamang. Fermented soybeans and non-soybeans legume foods. In Fermented 409 foods and beverages of the world , eds. JP Tamang, MANUSCRIPT K Kailasapathy, 191-224. New York: 410 CRC Press, Taylor & Francis Group, 2010.

411 29. Shin DH, Kwon DY, Kim YS, Jeong DY. Science and Technology of Korean Gochujang.

412 Public Health Edu , Seoul, 2012.

413 30. Inatsu Y, Nakamura N, Yuriko Y, Fushimi T, L Watanasiritum, S Kawamoto.

414 Characterization of Bacillus subtilis strains in Thua nao, a traditional fermented soybean

415 food in northern Thailand. Letters Appl Microbiol 2006; 43: 237.

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417 218-221. Tokyo:ACCEPTED Kenpakusha (in Japanese), 2008a.

418 32. Tanaka T. Sieng. In Advanced science on natto , ed. K. Kiuchi, T. Nagai and K. Kimura,

419 221-224, Tokyo: Kenpakusha (in Japanese), 2008b.

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421 D.K. Salunkhe, 85-95. Florida: CRC Press, 1986.

17 ACCEPTED MANUSCRIPT 422 34. Tamang JP, S Thapa, S Dewan, Y Jojima, R Fudou, S. Yamanaka. Phylogenetic analysis

423 of Bacillus strains isolated from fermented soybean foods of Asia: Kinema, chungkokjang

424 and natto. J Hill Res 2002; 15:56-62.

425 35. Hara T, Chetanachit C, Fujio Y, S. Ueda. Distribution of plasmids in polyglutamate-

426 producing Bacillus strains isolated from “natto”–like fermented soybeans, “thua nao,” in

427 Thailand. J Gen Appl Microbiol 1986; 32: 241–249.

428 36. Hara T, JR Zhang, S. Ueda. Identification of plasmids linked with polyglutamate

429 production in Bacillus subtilis (natto ). J Gen Appl Microbiol 1983; 29:345-354.

430 37. Hara T, S Hiroyuki I, Nobuhide, K. Shinji. Plasmid analysis in polyglutamate-producing

431 Bacillus strain isolated from non-salty fermented soybean food, “kinema”, in Nepal. J Gen

432 Appl Microbiol 1995; 41: 3-9.

433 38. Nakao S. Mame no ryori. In: Ryori no kigen. Japan: Japan Broadcast Publishing; 1972,

434 pp.115-126. (in Japanese). 435 MANUSCRIPT

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18 ACCEPTED MANUSCRIPT 436 Soybean 437 ↓ 438 Wash; boiled for 2-3 h 439 ↓ 440 Loosely pack in a bamboo basket lined with leaves 441 ↓ 442 Fermented (25-40 0 C, 2-3 d) 443 ↓ 444 HAWAIJAR 445 446 447 Fig 6. Traditional method of preparation of hawaijar in Manipur 448 Microorganism Bacillus subtilis (dominant functional bacterium) , B. licheniformis, B. cereus

449 and other non-bacilli bacteria- Staphylococcus aureus, S. sciuri, Alkaligenes spp . [21]

450 451 452 453 454 Soybean 455 ↓ 456 Soaked for 4-6 h 457 ↓ 458 Seed coat of soybean removed 459 ↓ 460 Cooked and dewateredMANUSCRIPT 461 ↓ 462 Cool down to room temperature 463 ↓ 464 Packed loosely in bamboo basket lined with leaves 465 ↓ 466 Basket is loosely covered with leaves and thick cloth 467 ↓ 468 Fermented (25-40 0 C, 3-5 d) 469 ↓ 470 TUNGRYMBAI 471 472 473 Fig 7. Traditional method of tungrymbai preparation in Meghalaya. 474 Microorganisms: On the basis of a combination of phenotypic and molecular 475 characterizationACCEPTED using ARDRA, ITS-PCR and RAPD-PCR techniques, species of Bacillus 476 isolated from tungrymbai were identified as Bacillus licheniformis (25.5%) , B. pumilus 477 (19.5%) and B. subtilis (55%) (dominant bacterium) [23]. 478 479 480

19 ACCEPTED MANUSCRIPT 481 Soybean 482 ↓ 483 Washed with water 484 ↓ 485 Boiled for 1-2 h 486 ↓ 487 Excess water drained off and cooled 488 ↓ 489 Cooked soybean placed in container lined with banana leaves and covered loosely 490 ↓ 491 Container kept near earthen-oven in kitchen 492 ↓ 493 Fermented (20-35 0 C, 6-8 d) 494 ↓ 495 AAKHONE 496 497 498 Fig 8. Traditional method of preparation of aakhone in Nagaland. 499 Microorganism : Bacillus subtilis, Proteus mirabil [27]. 500 501 502 503 Soybean 504 ↓ 505 Soaked overnight 506 ↓ 507 Boiled for MANUSCRIPT1-2 h 508 ↓ 509 Dewatered, cooled down 510 ↓ 511 Wrapped in leaves and placed inside basket 512 ↓ 513 Basket kept in warm place 514 ↓ 515 Fermented (20-35 0 C, 3-4 d) 516 ↓ 517 BEKANG 518 519 520 Fig 9. Traditional method of preparation of bekang in Mizoram. 521 Microorganism: On the basis of a combination of phenotypic and molecular characterization

522 using ARDRA,ACCEPTED ITS-PCR and RAPD-PCR techniques, species of Bacillus isolated from

523 bekang were identified as Bacillus brevis (2%) , B. circulans (7.5%) , B. coagulans (6.5%), B.

524 licheniformis (16.5%) , B. pumilus (9.1%) , B. sphaericus (4.6%) , B. subtilis (51.8%)

525 (dominant bacterium), and Lysinibacillus fusiformis (2%) [23].

526 527

20 ACCEPTED MANUSCRIPT 528 Soybean 529 ↓ 530 Cleaned and washed with water 531 ↓ 532 Cooked for 2-3 h 533 ↓ 534 Excess water is drained off and cooled 535 ↓ 536 Cooked soybeans packed in bamboo baskets lined with leaves of ginger 537 ↓ 538 Kept on the wooden rack above the fire place 539 ↓ 540 Fermented (20-35 0 C, 3-5 d) 541 ↓ 542 PERUYAAN 543 544 545 Fig 10. Traditional method of preparation of peruyaan in Arunachal Pradesh. 546 547 Microorganism : B. subtilis, B. amyloliquefaciens, Vagococcus lutrae, Ped. acidilactici, Ent.

548 Faecalis [27].

549 MANUSCRIPT

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21 ACCEPTED MANUSCRIPT 550

Table 1. Naturally fermented soybean foods of India Product Sensory features Microorganisms States in India References and nature Aakhone Alkaline, sticky, B. subtilis, Proteus Nagaland Singh et al. paste mirabil (2014)

Bekang Alkaline, sticky, B. subtilis, B. brevis, B. Mizoram Chettri and paste, curry circulans, B. coagulans , Tamang (2015) B. licheniformis, B. pumilus, B. sphaericus ,and Lysinibacillus fusiformis Hawaijar Alkaline, sticky B. subtilis, B. Manipur Jeyaram et al. licheniformis, B. (2008), Singh et amyloliquefaciens, B. al. (2014) cereus, Staph. aureus, Staph. sciuri, Alkaligenes sp. , Providencia rettgers, Proteus mirabilis Kinema Alkaline, sticky; B. subtilis, B. Sikkim, Sarkar et al. curry licheniformis, B. cereus, Darjeeling (1994), Tamang B. circulans, B. hills, Assam (2003) thuringiensis, B.MANUSCRIPT sp haericus, Ent. faecium, Cand. parapsilosis, Geotrichum candidum Peruyaan Alkaline, sticky, B. subtilis, B. Arunachal Singh et al. side dish amyloliquefaciens, Pradesh (2014) Vagococcus lutrae, Ped. acidilactici, Ent. faecalis Tungrymbai Alkaline, sticky, B. subtilis, B. Meghalaya Chettri and curry, soup licheniformis , B. Tamang (2015) pumilus 551 ACCEPTED

22 ACCEPTED MANUSCRIPT

MANUSCRIPT

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Fig 1. Soybean cultivation in Sikkim, India ACCEPTED MANUSCRIPT

Soybeans

Soaked

Cooked

Lightly cracked

MANUSCRIPTAsh added Wrapped

Fermented

ACCEPTEDKinema

Fig 2. Flow sheet of traditional method of kinema production in Sikkim village, India ACCEPTED MANUSCRIPT

MANUSCRIPT

ACCEPTED Kinema Kinema curry

Fig 3. Fresh kinema , sun-dried kinem a and kinem a curry ACCEPTED MANUSCRIPT

CHINA (TIBET) NEPAL Sikkim Kinema Arunachal Pradesh Darjeeling BHUTAN Peruyaan

N O R T H E A S T I N D I A Nagaland Assam MANUSCRIPT Aakhone Meghalaya Tungrymbai BAGLADESH Manipur Hawaijar MYANMAR Tripura Mizoram ACCEPTED Bekang

Fig 4. Diversity of naturally fermented ethnic soybeans foods in the Eastern Himalayas ACCEPTED MANUSCRIPT

Kinema of Sikkim and Hawaijar of Manipur Tungrymbai of Meghalaya Darjeeling MANUSCRIPT

ACCEPTED

Bekang of Mizoram Peruyaan of Arunachal Pradesh Aakhone of Nagaland

Fig 5. Naturally fermented ethnic soybeans foods of India ACCEPTED MANUSCRIPT

Natto of Japan Thua nao of Thailand MANUSCRIPT

ACCEPTED

Chungkok-jang of Korea

Fig 11 . Bacillus -fermented soybean foods of Asia ACCEPTED MANUSCRIPT

NATTO CHUNGKOK-JANG

DOUI-CHI

KINEMA SIENG

PE-POK THUA-NAO MANUSCRIPT Cambodia Vietnam

ACCEPTED

Fig 12. Kinema-Natto-Thua nao (KNT)-Triangle representing only Bacillus -fermented soybean foods of Asia (Tamang 2010)