1. Department of Microbiology
Bacillus species isolated from Tungrymbai and Bekang, naturally fermented soybean foods of North East India
Rajen Chettri
Research Associate
Department of Microbiology
Sikkim University

2. Tungrymbai and Bekang are the indigenous fermented soybean product from Meghalaya and Mizoram states in North East India.
Tungrymbai (also pronounced as Turangbai) is prepared and consumed mostly by the ethnic tribes; Khasi, Jaintias and Garos of Meghalaya whereas Bekang is prepared and consumed by Mizos of Mizoram.
Bacilli are the predominant microorganism in both the products and was found at the level of 108 cfu/g.
Strains of bacilli isolated from turangbai were phenotypically identified as Bacillus subtilis, B. pumilus and B. licheniformis and bacillii isolated from bekang samples were identified as B. subtilis, B. pumilus, B. licheniformis, B. sphaericus, B. brevis, B. coagulans and B. Circulans.

3. Fig 1. Flowsheets of preparation of Tungrymbai and Bekang

4. Methods Applied For Molecular Characterization of bacilli
DNA Isolation:
Bacterial genomic DNA was isolated according to the slightly modified method of Zhang et al. (2002).
Polymerase Chain Reaction (PCR) :
Amplified 16S rDNA was obtained from each strain by PCR with the following universal primers; forward 5’–AGAGTTTGATCCTGGCTCAG-3’ and reverse 5’-AAGGAGGTGATCCAGCCGCA-3’ (Weisburg et al., 1991).
Amplified 16S ribosomal DNA restriction analysis (ARDRA):
The restriction digestion analysis of each PCR amplified product of 16S rDNA was separately carried out with HinfI (R6205, Promega), CfoI (R6241, Promega) and RsaI (Promega).
16S – 23S rDNA intergenic transcribed spacer (ITS) – PCR analysis:
The amplification of 16S–23S rDNA intergenic transcribed spacer (ITS) region was carried out.
RAPD – PCR Analysis
To differentiate at strain level, RAPD-PCR was carried out.
Gel Electrophoresis
The amplified DNA fragments was separated through gel electrophoresis.
16S rDNA sequence analysis
The sequencing reactions were performed using ABI 3100 DNA sequencer (Applied Biosystems) in both direction with universal primers used for amplification.

5. Amplification of 16S rDNA
Fig 2. Agarose gel of PCR products of various strains of Bacillus isolated from turangbai and bekang. Lane M: DNA size marker (Genei, RMBD 135, India); PC: positive control; NC: negative control
Fig 3 Agarose gel of PCR products of various strains of Bacillus isolated from turangbai and bekang. Lane M: DNA size marker (Genei,RMBD 135, India); PC: positive control; NC: negative control

6. Gel data of ARDRA analysis of 16S rRNA gene PCR amplimers
500
200 bp
1000
Fig 4a. ARDRA analysis of 16S rRNA gene PCR amplimers of various isolates of Bacillus from turangbai and bekang with restriction endonuclease Hinf I, Lane M: 100 bp DNA ladder (Genei, RMBD135).
Fig 4b. ARDRA analysis of 16S rRNA gene PCR amplimers of various isolates of Bacillus from turangbai and bekang with restriction endonuclease Hinf I. Lane M: 100 bp DNA ladder (Genei, RMBD135).
Fig 5a. ARDRA analysis of 16S rRNA gene PCR amplimers of various isolates of Bacillus from turangbai and bekang with restriction endonuclease Rsa I. M1: 100 bp DNA ladder (Genei,RMBD135), M2: 1 Kb DNA ladder (Promega, G5711).
M M2 bp
1000
500
200
M M2
Fig 5b. ARDRA analysis of 16S rRNA gene PCR amplimers of various isolates of Bacillus from turangbai and bekang with restriction endonuclease Rsa I. Lane M1: 100 bp DNA ladder (Genei,RMBD135), M2: 1 Kb DNA ladder (Promega, G5711).

7. Gel data of ARDRA analysis of 16S rRNA gene PCR amplimersbp
1000
500
200
Fig 6 (a). ARDRA analysis of 16S rRNA gene PCR amplimers of various isolates of Bacillus from turangbai and bekang with restriction endonuclease Cfo I. Lane M: 1 Kb DNA ladder (Promega, G5711).
Fig 6 (b). ARDRA analysis of 16S rRNA gene PCR amplimers of various isolates of Bacillus from turangbai and bekang with restriction endonuclease Cfo I. Lane M: 1 Kb DNA ladder (Promega, G5711).

8. Restriction digestion profile (bp)
Table 1. Grouping of Bacilli based on the band sizes of amplified ribosomal DNA restriction analysis (ARDRA)
ARDRA
GROUP
Restriction digestion profile (bp)
Strains
No. of strains
Hinf I
Rsa I
Cfo I
Group I
( II )
(II )
TM1:B1, TPI:B1, TB2:B8a, TB2:B8b, TB1:B10, BD1:B1, BT:B9, BK2:B6, BK1:B15, BT:B3, TM1:B5, TS2:B13, TP1:B4a, TP1:B4b, TB2:B13, TB1:B17, BT:B17, BME:B10, BAV:B1, TB1:B16, TB2:B11, TS1:B6,TM1:B12, BT:B20, BME:B23, BK1:B24, BME:B20, TSA:B15, BD1:B22, TP2:B12 30
600
370
340
200
500
480
420
150
890
430
230
Group II
(III)
TP1:B2, BT:B11, BK1:B13, TP2:B10, TP1:B5, TP1:B15 6
330
180
Group III
( I )
(I )
TSB:B17, BK2:B12, TS1:B25, TSA:B4, BK2:B8, BAV:B12, TM2:B6, TS2:B24, BME:B6 9
990
400
360
580
Group IV
BK1:B18 1
Group V
(III )
BAV2:B6, BAV:B3 2
Group VI
BT2:B18
Group VII
BAV:B15

9. ARDRA GROUP sub-group (bp) No. of bands Strains No. of strains
Table 2. Internal transcribed spacer (ITS) sub-grouping of ARDRA groups of bacilli isolated from turangbai and bekang
ARDRA GROUP
ITS
sub-group
ITS profile
(bp)
No. of bands
Strains
No. of strains
Group I
I A
650, 620, 500 3
TM1:B1,TB2:B8a, TB2:B8b, BD1:B1,
BT:B9, BK2:B6, BK1:B15, BT:B3, TM1:B5, TP1:B4a, TP1:B4b, BT:B17, BAV:B1, TS1:B6, TM1:B12 15
I B
650, 620, 580, 500 4
TPI:B1, TB1:B10, TS2:B13,TB2:B13, TB1:B17,BME:B10,TB1:B16, TB2:B11, BT:B20, BME:B23 10
I C
680, 580, 520
TSA:B15 1
I D
620, 500 2
BK1:B24, BME:B20
Group II
II A
700,670, 500
TP2:B10
II B
710, 680, 600, 550
BK1:B13, TP1:B2,
TP1:B15
II C
650, 580, 530
BT:B11
II D
600,500
TP1:B5
Group III
III A
710, 670, 550, 490
TSA:B4
III B
710, 670, 490
TSB:B17, TS1:B25, TM2:B6, TS2:B24, BME:B6 5
III C
490
BK2:B12, BAV:B12,
BK2:B8
Group IV
IV A
710, 670, 650, 490
BK1:B18
Group V
V A
700, 650, 500
BAV2:B6
V B
650
BAV:B3
Group VI VI
BT2:B18
Group VII
VII
BAV:B15

10. RAPD profiles bp bp bp bpM M bp
1000_
500
1000
500
Fig 7. RAPD profiles of major Bacillus subtilis group of isolates from turangbai and bekang using OPB-18 random primer. Lanes 1-26 belongs to Bacillus subtilis group of isolates. Lane M: 100 bp DNA ladder (Genei, RMBD135).
Fig 8. RAPD profiles of major Bacillus subtilis group of isolates from turangbai and bekang using OPN-13 random primer. Lanes 1-26 belongs to Bacillus subtilis group of isolates Lane M: 100 bp DNA ladder (Genei, RMBD135). M bp M bp
1000
500
1000
500
Fig 9. RAPD profiles of major Bacillus subtilis group of isolates from turangbai and bekang using E-11 random primer. Lanes 1-26 belongs to Bacillus subtilis group of isolates Lane M: 100 bp DNA ladder (Genei, RMBD135).
Fig 10. RAPD profiles of major Bacillus subtilis group of isolates from turangbai and bekang using OPD-5 random primer. Lanes 1-26 belongs to Bacillus subtilis group of isolates. Lane M: 100 bp DNA ladder (Genei, RMBD135).
M M bp
600
500
Fig 11. RAPD profiles of major Bacillus subtilis group of isolates from turangbai and bekang using M-13 random primer. Lanes 1-26 belongs to Bacillus subtilis group of isolates Lane M: 100 bp DNA ladder (Genei, RMBD135).

11. GROUP I
GROUP II
GROUP III
GROUP V
GROUP VI
GROUP VII
GROUP IV
Fig 12. Dendogram based on the UPGMA of Jaccard Similarity Coefficient (Si) of combined ARDRA and ITS profiles of various isolates of Bacillus from turangbai and bekang. Groups I and VII belongs to B. subtilis group, group II represents B. licheniformis group, groups III and IV belongs to B. cereus group, groups V and VI belongs to Lysinibacillus fusiformis group.

12. GROUP B
GROUP A
Fig 13. Dendogram based on the UPGMA of Jaccard Similarity Coefficient (Si) of combined RAPD and ITS profiles of various isolates of Bacillus subtilis (Group A and B) from turangbai and bekang.

13. Fig 14. Dendogram based on the UPGMA of Jaccard Similarity Coefficient (Si) of combined RAPD and ITS profiles of various isolates of Bacillus subtilis (Group A and B) from turangbai and bekang.
GROUP A
GROUP B

14. Bacillus subtilis sub sp. subtilis
Table S rDNA sequencing results of the representative bacilli of different ARDRA groups
Strain code
RDP database
NCBI database
Closest relative
S_ab score
% Similarity
TB2:B8
(I)
Bacillus subtilis sub sp. subtilis
0.921
98%
TB1:B10
0.953
99%
BK1:B13
(II)
Bacillus licheniformis
0.950
BK1:B18
(IV)
Bacillus cereus
0.859
97%
BAV2:B6
(V)
Lysinibacillus fusiformis
0.947
BAV:B3
0.968
BT2:B18
(VI)
0.926
BAV:B15
(VII)
0.972
Numerals in parenthesis denotes ARDRA groups

15. Conclusion
The Phenotypic identification of representative strains of Bacillus isolated from turangbai and bekang were reconfirmed by molecular identification tools based on ARDRA, ITS-PCR and RAPD-PCR techniques. Eight isolates (representing 7 ARDRA groups) were sequenced and their identity was confirmed as follows: Bacillus subtilis subsp. subtilis, B. licheniformis, B. cereus and Lysinibacillus fusiformis.
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