Phylogenetic Analysis of Bacillus Strains Isolated from Fermented Soybean Foods of Asia: Kinema, Chungkokjang and Natto

b Jyoti Prakash Tamang", Saroj Thapa', Sailendra Dewan', Yasuko Jojima , Ryosuke Fudou" and Shigeru Yamanaka'"

-Food Microbiology Laboratory, Department of Botany, Sikkim Govemment College, Gangtok, Sikkim 737 102, India

bCentral Research Laboratories, Ajinomoto Co Inc., 1-1, Suzuki-cho, Kawasaki-ku 210-8681, Japan

cFaculty of Textile Science and Technology, Shinshu University, Ueda 386-8567, Japan

(Received 19 August 2002; revised version received 27 November 2002; accepted 30 November 2002)

ABSTRACT

Kinema. chungkokjang and natto are sticky, non-salty.flavouresomefermented soybeanfoods ofAsia. A total 0/38 .strains ofdominant endospore-forming and rod-shaped hacteria were isolatedfrom thesefermented soybeanfoods andstudied phenotypically. On the basis ofselected criteria andAP/50CHB tests, all endospore-forming rods were identifiedas BaciIlus suhtilis. The identification was confirmed by phylogenetic analyses carried DuI on six randomly selected strains (?(Bacillus subtilis by sequencing ofthe J6S rRNA gene.

INTRODUCTION (Tamang, 2001). Chungkokjang is a fermented soybean paste, consumed as soup with boiled rice Preparation and consumption offermented soybean in Korea (Kim et al., 2000). Natto a highly sticky foods is a traditional art ofthe people ofSouth-East fermented soybean food is consumed in Japan Asia. Kinema is a whole-soybean non-salty (Nikkuni,1997). fermented food with sticky texture, gray tan in colour and a characteristic flavour. It is eaten as side-dish Conventional methods for identification art in the Eastern Himalayan regions of Nepal, the not sufficient to analyse the diversity among species Darjeeling hills and Sikkim in India and in Bhutan of bacteria. Hara et al. (1995) reported that the plasmid ofBacillus subtilis (nalto) strain resembles that ofBacillus subtilis strain, isolated from kinema * Corresponding Author in the partial nucleotide sequences. Sarkar et al. (2002) studied the genomic diversity of Bacillus Phenotypic characterization species particularly B. subtilis isolate, from kinema and soumbala, fermented locust bean of Africa by Gram staining was performed accordingly to RAPD-PCR technique. It was the purpose of the Bartholomew (1962). Cell morphology and motility present study identify predominant Bacillus spp. test were observed in a phase contrast microscope and to analyse the phylogenetic relationship on the '(Olympus CH3-BH-PC, Tokyo, Japan) following a basis ofsequencing ofthe 16S rDNA gene. Isolates standard method (Harrigan, 1998). Catalase activity was tested with 10 % (w/v) H~O~. Cultures were I from kinema, chungkokjang and natto were included. grown on sucrose glutamate agar plates containing (g/L): sucrose, 50; monosodium glutamate, 15; MATERIALS AND METHODS potassium dihydrogen phosphate, 2.7; disodium hydrogen phosphate, 4.2; sodium chloride, 0.5; Sample collection tnagnesium sulphate, 0.5; agar, 20 and 0.1 ug biotin! ml. Growth was observed in presence or absence of Six samples of kinema were purchased from biotin (Hara and Ueda, 1982). For observation of Darjeeling and Gangtok markets in India. Six natto stickiness, cultures grown on phytone agar (Nagai samples were purchased from supermarkets in et al., 1994) at 37° C for 24 h were pulledby touching Kawasaki in Japan. The samples were collected in with an inoculating needle. All other phenotypic sterile bags, transportedto the laboratory characteristics were carried out following the immediately and analysed. Four dried samples of methods described by Claus and Berkeley (1986) chungkokjang were collected from Kanghwa and and Schillinger and Liicke (1987). Ability of the Anshan regions of South Korea and were isolates to ferment carbohydrates was studied using refrigerated prior to analysis. API 50 CHB (Biolvlerieux, Lyon, Marcy-l' Etoile, France) system. Reference strain Bacterial strains and DiVA isolation Type strain Bacillus subtilis JCM 1465 was obtained from Japan Collection of Microorganisms (JCM, A total ofsix strains ofBacillus. KD:B I and KG:B I RIKEN, Wako-shi, Japan). isolated from kinema, CA:BI and CK:Bl from chungkokjang and IN-I and JA-l from natto, were selected randomly, and were grown aerobically at Microbial analysis 37° C in Luria-Bertani (LB) medium (1 % tryptone, Ten g ofwell-mixed sample were blended with 90 ml 05 % yeast extract, 1 % sodium chloride; w/v, pH sterile physiological saline (0.85 0/0) in Stomacher 7.0). Cells were harvested by centrifugation ofthe lab-blender400 (Seward Medical, London, UK). One cultures at the mid-exponential phase of growth, ml dilution was mixed with 9 ml sterile physiological washed with 0.9 % NaCI solution, and resuspended saline, and heated for 2 min in continuously boiling in pure water. The chromosomal DNA was prepared water for spore counts (Tamang and Nikkuni, 1996). by the method ofSaito and Miura (1963). Decimal dilutions were prepared in.sterile diluents and 1 ml ofappropriate dilutions were mixed with 16S rRNA gene sequencing and molten nutrient agar (MOO I, HiMedia Laboratories, Mumbai, India) and incubated at 37° C for 18 h for phylogenetic analysis enumeration of spores. After purification of The 16S rDNA fragments that corresponded to representative isolates they were kept on nutrient positions 8-1543 of Escherichia coli 16S rRNA agar in cryotubes at -20° C for further analyses. (Brosius et al., 1978) were amplified by the peR directly from cell lysates which were prepared by

57 treating cell suspensions with proteinase K (Hiraishi, been deposited in the DOBJ database with accession 1992; Hiraishi et al., 1994). The peRproducts were numbers AB072569, AB072570, AB072571, sequenced directly with a Sequi'Iherm Long-Read AB072572, AB072573 and AB072574, respectively. Cycle sequencing kit (Epicentre Technologies, Madison, WI, USA) and fluorescent primers and RESULTS AND DISCUSSION analyzed with a DNA sequencer (Pharmacia LKB Biotechnology, Uppsala, Sweden). The nucleotide sequences ofwere revealed using the Genetyx-Mac Morphology and cultural programme version 10 (Software Development Co. characteristics Ltd., Tokyo, Japan) and preliminary searches in the DDBJ (DNA Data Bank ofJapan, Mishima, Japan) Six samples of kinerna, four samples of database were performed with the programme chungkokjang and six samples of natto were BLAST (Altschul et al., 1997). Sequences of the collected from different places ofIndia, South Korea close relatives were retrieved from DDBJ. Multiple and Japan" respectively and analysed for microbial alignment ofsequence" calculation ofthe corrected load. Average load ofendospore-forming bacteria evolutionary distance based on nucleotide ofthe samples was 108 cfu/g (Table 1). A total of38 (K strains of aerobic, rod-shaped, Gram-positive substitution rates nuc values) (Kimura, 1980) and construction of a phylogenetic tree by the endospore-forming bacteria isolated from kinema, neighbour-joining method (Saitou and Nei, 1987) chungkokjang natto and were characterized (Table was performed using the Clustal W programme 2). All these endospore-formers were identified as .(Thompson et al., 1994). Branching patterns oftrees Bacillus subtilis (Ehrenberg) Cohn according to the were evaluated by bootstrapping w-ith 1000 criteria laid down by Claus and Berkeley (1986) as resamplings (Felsenstein, 1985). Alignment well as sugar-profile in API system. With respect to positions with gaps and unidentified bases were spore position and reduction of nitrate, Bacillus excluded for the calculations. The tree was subtilis strains isolated from kinema and illustrated by using the Treeview programme (Page, chungkokjang differed from Bacillussubtilistnatto) 1997). The 16S rDNA sequences ofsix strains CA: strains and type strain Bacillus subtilis JCM 1465. B I" CK: st. KD: B I" KG: B 1, IN-I and JA-I have All strains of Bacillus isolated from kinema and

Table 1. Microbial load ofspores ofBacillus isolated from kinema, chungkokjang and natto Sample with sample code Place Country Log cfu/g" Kinema(KD:B)J Darjeeling India 8.3 Kinema (k.G:BY Gangtok India 8.5 Chungkokjang (CA:Bt Anshan South Korea 7.6 Chungkokjang (CK:B)h Kanghwa South Korea 8.1 Natto (IN)a 'Naruse Co. Japan 8.8 Natto (JA)J dKawasaki Japan 8.5 -Data represent the means of 5 samples from each source. bDatarepresent the means of 3 samples, "Commercial natto starter "Asamshi Taro Amada Food Co. ~cfu" colony forming unit

58 Table 2. Differentiation characteristics of Bacillus strains isolated from fermented soybeans foods ofAsia Characteristic Kinema Chungkokjang Natto Bacillus subtilis KD:B KG:B CA:B CK:B IN JA JCM 1465

(n = 8)1 (n = 10) (n=4) (n=6) (n=4) (n=6) Cell width (urn) 0.7-1.0 0.5-1.0 0.5-0.8 0.5-0.8 0.8-0.9 0.8-0.9 0.8-0.9 Cell length (urn) 2.04.0 1.9-4.0 1.7-3.0 1.8-3.0 2.0-3.0 2.0-3.0 2.0-3.0

Spore position C/Cp2 c/cp c/cp c/cp c c c NO~ reduction -(6)3, - (7), - (2)~ - (2), + + + + (2)4 +(3) +(2) +(4) Biotin requirement + + + + + + Stickiness on phytone agar + + + + + +

In = number ofstrain le/pc, central/para-central "Number ofstrains showing negative result "Nurnberof strains showing positive result. All strains were rods, Grain-stained, catalase positive, aerobic, showed motility, produced acid from D-glucose, L arabinose and Dvmannitol but no gas from D-glucose; hydrolysed casein and starch; grew well at pH 5.7 and 6.8, at 0 7 % NaCI, no growth at 55-60 c.

chungkokjang showed central to paracentral of isolates to other bacteria, the sequence ofa 16S position of spores with few strains showing rRNA gene PCR product was determined and then negativenitrate reduction test, whereas natto strains analysed. A comparative analysis ofthe 16S rRNA showed central position of spores and all reduced gene sequences revealed that strains CA:Bl and nitrate (Table 2). However, all strains of Bacillus CK:B2 isolated from chungkokjang, K.D:B1 and subtilis isolated from kinema, chungkokjang and KG:B 1 isolated from kinema and IN-l isolated from natto showed stickiness on phytone agar and natto had identical sequences except that of JA-l cooked soybean, which are characteristics property (natto) which had one ambiguous nucleotide. The ofnon-sa": .fermented soybean foods ofAsia, and evolutionary distance between four strains CK:B I, their requirement ofbiotin from growth (Tamang and KD:B I, IN-I and JA-l, and Bacillus subtilis was

Nikkuni, 1996). These characters differed with type 0.002 Knuc as calculated by the ratio of nucleotide strain ofB. subtilis. substitution per nucleotide site, indicating 99 0/0 homology with Bacillus subtilis type strain. However, the evolutionary distance between the. Phylogenetic analysis strains CA:B 1 and KG:B l, and Bacillus subtilis was ' 0/0 In order to investigate the phylogenetic relationship 0.005 Knoc showing approximately 99.5 homology

59 KD:BI(AB072571) CK: BI(AB07257·0) IN-l(AB072573 ) JA-l(AB072574 ) . B.subtilis (X60646) CA:BI(AB072569) KG:BI(AB072572 ) B.amyloliquefaciens (X60605) B.arophoeus (X60607) B.popil/iae.(X6~33) .B. lentimorbie (X60622) .....- ...L_ B./awus (X60621) Group 1 B. lichenifa-mis(X68416) .....----- B.azacfo-mare (078309) B, cereus (016266) B.thuringiensis (016281 ) B. onthracis(X55059) B.medusa(X60628) B. mycoides(X55061) ---- B,cohnii(X76437) ----- Bfasudiosus (X 6061 5) B.benzoevorans (D78311) B. circuJans (D78312) B.lentus (D 16272) Bfusiformis (LI40 13) ] Group2 B.insolitus (X60642) B. marocconus (X60626) B.simplex (X60638) B.psychrosocchardyticus (X6063S) ~--- B.megaterium (DI6273) L.-.__ Bfirmus (016268) _----t~---- B.sp001 (X82436) Group 1 ------Amphlbocillus xylonus(08206S) 8.pantothenticu\· (DI627S) 8.51' (X60602) 8.badius (X777CX» -----..... B,"""lhii(Z26935) B. ('OUfU/PfU (016267) ~------'---- Sporolaa oaocillusinulinus(DI6283) B.stearothermophillls (XS7309) - Group 5 AIicyclobocillus acidocaldorius Anewin/bodltus areuriroiyticus (078455) (X60742) - Group 6 .....------Brevibocillus brevis(D78457) - Group 4 r-..__------_--- Paenibacillus poiymyxa (D 16276) - Group 3 .....----i------l.actdiocillus loctts (MS8823) ~------~Leoco~nocmese~eroides(X95~~

.....------SlrepIO('()('('US cecorum (X542'X» ~------Clostridiuminnocuum (M23732)

FIa. I. PbylOleDetle treeIbowml therelatlo.lblps of&cUi'" '''btills Itralnltootherstralnlof the leDUI &cUlus andrelated lenen buecl onpartialsequenee of 16SrRNAlene

60 with type strain. The phylogenetic analyses revealed Felsenstein, J. (1985) Confidence limits on phylog that all six strains belonged to Bacillus subtilis (Fig enies: an approach using the bootstrap. Evo I). This is the first report to describe the phylogeny lution 39: 783-791. ofBacillus subtilis isolated from similar non-salty Hara, T. and Ueda, S. (19R2) Regulation of fermented sticky soybean foods ofAsia. Diversity polyglutamate production in Bacillus ofBacillus subtilis-dominating fermented soybeans subtilis (natto): transformation ofhigh PGA foods ofAsia from the Eastern Himalayan regions productivity. Agricultural and Biological to far-east Asia needs to be studied in details to Chemistry 46: 2275-2281. trace the antiquity and similarity in the food culture ofAsia. Hara, T., Hiroyuki, S., Nobuhide.. I. and Shinji, K. (1995) Plasmid analysis in polyglutamate producing Bacillus strain isolated from non ACKNOWLEDGEMENT salty fermented soybean food, "kinerna", in The first author is grateful to Department of Nepal. Journal ofGeneral and AppliedMi Biotechnology, Government of India for research crobiology 41: 3-9. project. The authorswould liketo thankProf.Michio Hamgan,W.F.(1998-) Labora&ory MethGdI in Food Kozaki of Japan for helpful discussion and late Microbiology, 3rd Edition, 532 pp. Academic Jayant Prakash Tamang for sending the samplesof Press, London. chungkokjang from Korea. Hiraishi, A. (1992) Direct automated sequencing of 16SrDNA amplified by polymerase chain re REFERENCES action from bacterial cultures without DNA Altschul, S.F.,Madden, T.L.,Schaffer, A.A.,Zhang, purification. Letters in Applied Microbiol J., Zhang, Z., Miller, W. and Lipman, D. J. ogv 15: 210-213. (1997) Gapped BLAST and PSI-BLAST: a Hiraishi, A., Shin, Y. K., Ueda .. Y.and Sugiyama, J. new generation of protein database search (1994) Automated sequencing ofPCR-ampli programs. Nucleic AcidsResearch 25: 3389 tied 16S rDNA on 'Hydrolink' gels. Journal 3402. (~(Microbiological Methods 19: 145-154. Bartholomew, J. W. (1962) Variables influencing re Kim, J. I., Han. J. H., Kang, M. J. and Kwon, T. W. sults and the precise defmition of the steps (2000) The hypollycemie effect of in Gramstaining as a means ofstandardizing Chongkukjang in normal rats. la: the Pr0 the results obtained. Stain Technology 37: ceedings of The Third lntemationa! Soy 139-155. bean Processingtufd Utilization Conference Brosius, J., Palmer.J. L.,Kennedy,J.P.and NoUer, H. (Ed: 8aio, K.), pp. 171-172. The Japanese F. (1978) Complete nucleotide sequence ofa Society for Food Science and Technology 16S ribosomal RNA gene from Escherichia andISpuc-m, Tsukuba, October 15-20,2000, coli. The Proceedings ofNational Academy Japan. ofSciences USA 75: 4ROI-480.5. Kimura, M. (1980) A simple method for estimating Claus,D. and Berkeley, R. C. W.(1986)Genus Bacil evolutionary rates of base substitution lus Cohn 1872, 174. In: Bergev 's Manual 0.( through comparative studies of nucleotide Systematic Bacteriology (Eds: Sneath, P. H. sequences. Journal 0.( Molecular Biology A., Mair,N.S., Sharpe, M. E.and Holt,J. G.), 16: 111-120. Vol.2,pp. 1105-1139. WilliamsandWilkins, Nagai,T.,Nishimura, K.,Suzuki, H.Banba,Y.,Sasaki, Baltimore, MD. H. and Kiuchi, K. (1994) Isolation and char-

61 acterization of Bacillus subtilis strain pro... tional Journal ofFood Microbiology 77: ducing natto with strong Umami-taste and 175-186. high viscosity. Nippon Shokuhin Kogyo Schillinger, U.andLiicke, F. K.(1987)Identification Gakkaishi 41: 123-128. oflactobacilli from meat and meat products. Nikkuni, S. (1997) Natto, kinema and thua-nao: tra FoodMicrobiology 4: 199-208. ditional non-saltedfermented soybean foods Tamang, 1.P. (2001)Kinema.FoodCulture 3: 11-14. in Asia. FarmingJapan 31 (4): 27-36. Tamang, J. P. and Nikkuni, S. (1996) Selection of Page,R.D. M.(1997)TREEVIEWprogram version starterculture for production of'kinema, fer 1.5.1. University ofGlasgow, UK. mentedsoybeanfoodofthe Himalaya. World Saito, H. and M iura, K. (1963) Preparation oftrans Journal ofMicrobiology and Biotechnol forming deoxyribonucleic acid by phenol ogy 12 (6): 629-635. treatment. Biochemica et Biophysics Acta Thompson, J. D., Higgins, D. G. and Gibson, T. J. 72:619-629. (1994) CLUSTAL W: improving the sensitiv Saitou, N. and Nei, M. (1987) The neighbor-joining ity ofprogressive multiple sequence align method: a new method for reconstructing ment through sequence weighting, position phylogenetic tree. Molecular Biology and specific gap penalties and weight matrix Evolution 4: 406-425. choice. Nucleic Acids Research 22: 4673 4680. Sarkar,P. K.,Hasenack, B.and Nout, M. J.R (2002) Diversity and functionality ofBacillus and related genera isolated from spontaneously fermented soybeans (Indian Kinema) and locust beans (African Soumbala). Interna-