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
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5 Department of Microbiology, School of Life Sciences, Sikkim 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 Himalayas.
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 Nepali language, is traditionally used to prepare various
37 fermented and non-fermented recipes in the Eastern Himalayan regions of Nepal, India and
38 Bhutan [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, Darjeeling 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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381 17. Sarkar PK, Morrison E, Tingii U, Somerset SM, GS Craven. B-group vitamin and mineral
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.
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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
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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
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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.
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404 India. Indian J Traditional Knowl 2007; 6(1): 37-41.
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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.
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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.
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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
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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
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Fig 12. Kinema-Natto-Thua nao (KNT)-Triangle representing only Bacillus -fermented soybean foods of Asia (Tamang 2010)