Molecular Diversity of Microbes Associated with Fermented

Bamboo Shoots

Authors:

Vijay Kumar*, Bindu Naik, Sachin Sharma, Akhilesh Kumar, Javed Masood
Khan and Mohammad Irfan

*Correspondence: vijaygkp@gmail.com

DOI: https://doi.org/10.21315/tlsr2022.33.3.9

Highlights

•• Fermented bamboo shoots are source of Bacillus safensis, B.

tequilensis, B. siamensis, B. nakamurai, B. subtilis and Enterobacter.

•• These isolates have not been reported previously from fermented

bamboo shoots except B. subtilis and Enterobacter.

•• Interestingly, no Lactobacillus species and molds were not detected

in any of the analysed samples.

•• Potent antibacterial activity was recorded against Klebsiella,

Staphylococcus aureus, Salmonella and B. cereus.

TLSR, 33(3), 2022

© Penerbit Universiti Sains Malaysia, 2022
Tropical Life Sciences Research, 33(3), 151–164, 2022

Molecular Diversity of Microbes Associated with Fermented Bamboo

Shoots
1
Vijay Kumar*, 2Bindu Naik, 3Sachin Sharma, 1Akhilesh Kumar, 4Javed Masood Khan and
5
Mohammad Irfan

1
Himalayan School of Biosciences, Swami Rama Himalayan University, Swami Rama
Nagar, Jolly Grant, Dehradun, Uttarakhand 248140 India
2
Department of Life Sciences, Food Technology, Graphic Era (Deemed to be University),
Bell Road, Clement Town, Dehradun, Uttarakhand 248002 India
3
Department of Food Technology, Doon (P.G.) College of Agriculture Science and
Technology, Selaqui, Dehradun, Uttarakhand 248011 India
4
Department of Food Science and Nutrition, Faculty of Food and Agricultural Sciences, King
Saud University, 2460, Riyadh 11451, Saudi Arabia
5
Plant Biology Section, School of Integrative Plant Science, Cornell University, Ithaca, New
York, USA

Publication date: 30 September 2022

To cite this article: Vijay Kumar, Bindu Naik, Sachin Sharma, Akhilesh Kumar, Javed
Masood Khan and Mohammad Irfan. (2022). Molecular diversity of microbes associated
with fermented bamboo shoots. Tropical Life Sciences Research 33(3): 151–164. https://
doi.org/10.21315/tlsr2022.33.3.9
To link to this article: https://doi.org/10.21315/tlsr2022.33.3.9

Abstract: Fermented bamboo shoots are rich in high protein, carbohydrates, fibre and
minerals while low in fat content. In the North-East region of India and other Asian countries,
they are mostly used in various food preparations. The present study was undertaken to
explore the diversity of bacteria associated with Bamboo shoots and to evaluate their
antibacterial profile. Based on the results the fermented bamboo shoots showed viable
counts ranging from 6.55 ± 0.91 log CFU/g to 7.86 ± 1.21 log CFU/g. The 16s rRNA
sequence analysis showed that these isolates belonged to the genus Bacillus (Bacillus
safensis, B. tequilensis, B. siamensis, B. nakamurai, B. subtilis) and Enterobacter. These
isolates have not been reported previously from fermented bamboo shoots except B.
subtilis. Surprisingly, no Lactobacillus species or molds were found in any of the samples
tested. Potent antibacterial activity was recorded against Klebsiella, Staphylococcus aureus,
Salmonella and B. cereus.

Keywords: Probiotic Bacteria, Fermented Bamboo Shoots, Bacillus Species, Antibacterial

Activity

*
Corresponding author: vijaygkp@gmail.com

© Penerbit Universiti Sains Malaysia, 2022. This work is licensed under the terms of the Creative
Commons Attribution (CC BY) (http://creativecommons.org/licenses/by/4.0/).
Vijay Kumar et al.

INTRODUCTION

Probiotics are microbial cell components or microbial cell preparations that

have a favourable influence on the host's health and well-being. They positively
influence gastrointestinal infections, hold antimicrobial activity, vitalise the immune
system, anti-mutagenic activity, anti-carcinogenic properties, anti-diarrhea activity,
etc. Probiotics are associated with the digestive tract of humans and animals.
Beneficial strains, which can be used as probiotic sources, are mostly found in the
genera Bifidobacterium and Lactobacillus, and some of these strains have anti-
inflammatory qualities (Isolauri et al. 2004; Behera & Balaji 2021). Fermentation
of foods is usually controlled action of micro-organisms to alter the texture
of food, upgrade their quality, preserve and produce characteristic flavour and
aromas and have additional advantages of giving bio-supplements and minerals.
Fermented food holds numerous microorganisms from the genera Lactobacillus,
Bifidobacterium, Saccharomyces, Enterococcus, Streptococcus, Pediococcus,
Leuconostoc and Bacillus and has been shown to have health advantages. The
human microbiome is gaining a lot of attention these days, and studies have already
shown that changing it can have far-reaching consequences. Despite preservation,
fermented foods can likewise have the additional advantages of achieving flavour,
expanded edibility, enhancing nutritious esteem and pharmacological qualities.
Each fermented food is connected to an exceptional consortium of micro-flora
which builds the levels of protein, vitamins and basic amino acids (Melini et al.
2019). Bamboos, sometimes known as the green gold, are a genus of giant woody
grasses that belong to the Poaceae family and subfamily Bambusoideae. They are
well-known for their environmental benefits (Goyal et al. 2010). Bamboo shoots, on
the other hand, have yet to be fully utilised as a food source. Fermented bamboo
shoots are a typical kind of food in north Indian cooking, particularly in Manipur,
Nagaland and Sikkim. It is also used as food in other parts of the world. They are
well-chosen healthiest foods because of their high protein, carbohydrate, vitamin,
fibre and mineral content and low-fat content. It promotes health benefits such
as antioxidant activity, cholesterol-lowering activity, anticancer activity, acts as an
immune booster, anti-aging, prevents cardiovascular disease, is helpful in weight
loss, decreases blood pressure, is rich in flavonoids, glycosides, and is rich in
probiotics (Thakur et al. 2016; Behera & Balaji 2021). Bamboo shoots, whether
fresh or fermented, are one of the most popular traditional foods among various
ethnic groups. Ushoi, soibum, rep, mesu, eup, ekhung, hirring and other bamboo-
based traditional meals are among the most popular (Choudhury et al. 2012). The
fermented bamboo shoots are the hub of various beneficial microorganisms which
may be of indigenous origin, starters, or may enter from utensils, containers, earthen
pots that biochemically and organoleptically modify substrate into edible products.
The type of microorganisms present during the fermentation is responsible for the
change in the chemical components of the raw materials, enhancing nutritional
value and providing health benefits (Tamang 2010; Tamang et al. 2016). In this
context, the present study was undertaken to explore the molecular diversity
of microbes associated with fermented bamboo shoots and evaluation of the
antimicrobial potential of these isolates against pathogenic microbes.
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MATERIALS AND METHODS

Sample Collection and Microbiological Analysis of Probiotic Bacteria from

Bamboo Shoots

In January and February 2018, 10 samples of fermented bamboo, as well as raw

bamboo samples weighing about 300 g each, were obtained from various locations
throughout Manipur. Total viable counts were determined after selective isolation
and microbiological examination. The samples were vortexed and serially diluted
in sterile normal saline (0.85% NaCl (w/v) before plating into triplicate plates on
MRS agar media (HiMedia, India) for the total viable count and selective isolation.
The colony-forming unit (CFU) was determined after 24 h at 37°C. Potato dextrose
agar media was used for the isolation of yeasts and molds.
Based on the colony colour, shape, size and appearance, and cell
morphology (cocci, bacilli, cell arrangement, etc.), the isolates were selected
(Tindall et al. 2010). Preliminary identification of isolates was done by colony
morphology and Gram staining. The purity of the isolates was checked, and
purified isolates were preserved in glycerol (20% w/v).

Proximate Analysis of Raw and Fermented Bamboo Shoot

The raw and fermented bamboo shoots were collected from the local people. The
procedure described by these people was used for fermentation. The bamboo
shoots were sliced and dried in the sun for 10 to 15 min before being placed
in an earthen pot to ferment for two months (25°C–27°C; room temperature)
with the addition of a tiny amount of water and salt. The shoots then are taken
out and dried in the sun until they are about 50% dry. The dried pieces are
then stored and combined in a cane/bamboo basket for additional drying. The
moisture content was determined by the hot air oven method (AOAC 1990, no.
947.05). The total phenolic in the sample was calculated using the Folin-Ciocalteu
reagent (Bray & Thorpe 1954). Ten grams of sample was homogenised in 90 mL
of sterile physiological saline (0.85%, w/v) and pH was determined using a pH
meter (Eutech-700, Thermo Scientific, India). A pH meter calibrated with standard
buffer solution was used to determine the pH. Ash content was determined by dry
ashing in a muffle furnace at 600°C until white grayish ash was obtained (AOAC
1990). Titratable acidity was estimated according to the standard method (AOAC
1990). The filtrate of homogenate was titrated with 0.1 N NaOH to an endpoint
of phenolphthalein (0.1% w/v in 95% ethanol). Reducing and total sugars were
determined by the method of Lane and Eynon (Gandhi et al. 2017).

Molecular Characterisation

Selected isolates (based on colony colour, size and shape on MRS agar
media and micromorphology such as cocci, bacilli, cell arrangement, etc.) were

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Vijay Kumar et al.

characterised using a molecular approach based on 16S rRNA gene analysis.

Genomic DNA was isolated as described previously (Kumar et al. 2010). The
primers used in the present study were F-5’-AGAGTTTGATCMTGGCTCA-3’ and
R- 5’- TACGGYTACCTTGTTACGACTT -3’. The PCR conditions were the same
as those described previously (Kumar et al. 2014). The BLAST (blastn) search
tool (http://www.ncbi.nlm.nih.gov) was used to analyse the sequences of selected
isolates, and the Ez-Taxon server (Chun et al. 2007) was used to calculate the
pairwise alignment score. The sequences were submitted to GenBank (NCBI).
The software contained in the MEGA version 7.0 package was used to conduct
phylogenetic and molecular evolutionary analyses (Kumar et al. 2016). The 16S
rRNA sequences were matched with corresponding nucleotide sequences of
bacteria retrieved from various nucleotide databases (GenBank, EMBL, DDBJ,
and RDP) using the CLUSTAL W program (Thompson et al. 1994). Sequence
analysis was performed according to the previous method (Kumar et al. 2014).

Antimicrobial Assay

For the determination of the antimicrobial potential of selected isolated, they were
grown in MRS broth (HiMedia, India) at 30°C for incubation for 24 h. The freshly
grown isolates were inoculated in the MRS broth and kept at 30°C. After 72 h,
these were centrifuged at 8000 rpm for 7–8 min. Sediments were removed after
filtration and a microbial filter (syringe filter, HiMedia-SF126, India) is used to filter
the fermented samples. Pathogenic bacteria were grown in nutrient agar media
(HiMedia, India). The bacterial inoculum of 0.5 McFarland (1.5 × 108 CFU/mL)
was prepared. The supernatant (50 µL/well) was checked for antibacterial activity
against S. aureus MTCC 96, Bacillus cereus MTCC 430, Escherichia coli MTCC
739, Salmonella enterica ser. Abony MTCC3858, Klebsiella pneumonie MTCC
4030 because these pathogens are mainly associated with food-borne illness as
well drug resistance among these pathogens are also a major concern. The wells
were made by using a cork borer (6 mm diameter). In secondary screening, the
fermentation conditions and extract preparation were performed according to the
method described previously (Gurban oglu Gulahmadov et al. 2006). The inhibitory
zone diameters were measured after 24-hour incubation period at 37°C.

RESULTS AND DISCUSSION

Microbiological Study

A good number of microbes was recorded in the fermented bamboo shoots.

Lactobacillus species and molds were not recovered in any sample analysed.
Most of the strains isolated from the fermented bamboo shoot belong to the genus
Bacillus and Enterobacter. In fermented bamboo shoots, the viable counts ranged
from 6.55 ± 0.91 log CFU/g to 7.86 ± 1.21 log CFU/g (Table 1). A total of 10 isolates

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 Molecular Diversity of Microbes

were selected based on morphology which is designated as DCAST 2, DCAST 3,

DCAST 5, DCAST 6, DCAST 8, DCAST 9, DCAST 11, DCAST 14, DCAST 16 and
DCAST 17, respectively. These samples were collected from the different districts
of Manipur. The highest number of the viable count was found in Churanchandpur
(7.86 ± 1.21 log CFU/g), Ukhrul (6.82 ± 1.121 log CFU/g), Noney (6.78 ± 0.23 log
CFU/g), etc. and the least amount of viable count was recorded in Senapati (6.55
± 0.91 log CFU/g) and Thoubal (6.65 ± 0.33 log CFU/g). Based on molecular study
all the samples were dominant Bacillus except sample 10 (Porompat) in which
Enterobacter was recovered. The findings of the present study are comparable
with those of Khunjan et al. (2017). They reported the viable count from 7.47–9.8
log CFU/g of the sample. Sharma and Barooah (2017) reported LAB bacteria in
the range of 4.66–7.87 log CFU/g from fermented bamboo shoots.

Table 1: Viable count of probiotic bacteria isolated from different fermented foods.

Sample Place Viable count No. of isolates selected Coding

no. Log CFU/g based on morphology
1 Bishnupur 6.65 ± 0.41 01 DCAST2
2 Thoubal 6.65 ± 0.33 01 DCAST3
3 Senapati 6.55 ± 0.91 01 DCAST5
4 Ukhrul 6.82 ± 1.121 01 DCAST6
5 Chandel 6.74 ± 0.52 01 DCAST8
6 Jiribam 6.68 ± 0.12 01 DCAST9
7 Churachandpur 7.86 ± 1.21 01 DCAST11
8 Kakching 6.76 ± 0.43 01 DCAST14
9 Noney 6.78 ± 0.23 01 DCAST16
10 Porompat 6.85 ± 0.84 01 DCAST17

Proximate Analysis

Bamboo shoots are rich in various nutrients such as proteins, carbohydrates,

vitamins, minerals fibers, phytosterols, phenol and fat (Nirmala et al. 2007). There
was no significant variability in data generated among all the raw and fermented
bamboo shoots in laboratory conditions. The combined proximate chemical
composition before and after fermentation including titratable acidity, pH, total
phenolic content, ash and reducing sugar is given in Table 2.

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Vijay Kumar et al.

Table 2: Fermented bamboo shoot sample: influence of fermentation (at 25°C–27°C for two
months) on the raw and fermented shoot.

Characteristics Raw bamboo shoots Fermented bamboo shoot

Titratable acidity 0.89 ± 0.01 4.09 ± 0.01
pH 6.74 ± 1.75 5.40 ± 0.40
Moisture content 68.42 ± 2.02 91.12 ± 3.68
Total phenolic content (mg/100 g) 97.50 ± 1.20 239.00 ± 1.00
Ash 0.86 ± 0.00 0.81 ± 0.01
Reducing sugar (g/100 g) 1.37 ± 0.01 0.26 ± 0.01
Note: Values are average of 10 samples ± standard deviation

Biochemical Changes by Isolates

After fermentation with isolated microorganisms, the acidity increases as

compared to raw bamboo shoots. The increase in fermentation duration is exactly
proportional to the drop in pH. The decrease in pH is mainly because of acid
production in bamboo shoots (Table 2). The increase in fermentation duration is
exactly proportional to the drop in pH. The fermentation of lactic acid in bamboo
shoots is mostly responsible for the decrease in pH. The above results are
comparable to several FBS products such as mesu, eup and hirring, which have
pH values in the range of 3.9 to 4.1 (Tamang et al. 2008; Tamang & Tamang
2009). During the production of Jiang-sun (a Taiwanese FBS product), there was
a decrease in pH from 4.2 (First day sample) to 3.5 (30-day sample)(Chen et al.
2010). According to previous reports, the pH of hardened tubers of yam (Dioscorea
dumetorum) fell from 5.5 to 4.8 during the first two days of natural fermentation,
and then to 3.9 after 14 days of fermentation (Medoua et al. 2008). Acidity was
calculated as a percentage of lactic acid. Lactic acid generation during bamboo
shoot fermentation could explain the relative rise in high acidity. Fermentation
duration had a significant effect on Titratable acidity. Dioscorea dumetorum tough
tubers showed a similar tendency (Medoua et al. 2008). TPC levels increased
from 97.5 to 239 mg/100 mL. The level of total phenol increased exponentially
as fermentation progressed. In contrast, the TPC of bamboo shoots gradually
decreased after storage at 10°C (Badwaik et al. 2014). On the other hand, the
TPC of control and treated bamboo shoots (salicylic acid) was found to be higher
in the treated bamboo shoots. Sugars such as glucose are the best substrate for
microbes in the fermentation process. Understanding the dynamics of lowering
sugar levels will also aid our understanding of the fermentation process. The
amount of reducing sugar is reduced after fermentation.

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Phylogenetic Analysis

The 16s rRNA was submitted to GenBank with accession number given in Table 3.
Among all isolates, Isolate number 2 is most closely related to Bacillus safensis
whose sequence identity diff/total nt is 98.57 (10/700) and the GenBank accession
number is MH532552. B. safensis is a non-dairy probiotic producing amino acids,
vitamins and cofactors. It can tolerate bile salt and exhibit aggregation and adhesion
(Saidumohamed & Bhat 2021). The probiotic properties of B. tequilensis have
been reported previously by Abid et al. (2019). Isolate number 3 is most closely
related to B. tequilensis whose sequence identity diff/total nt is 99.73 (2/753) and
the GenBank accession number is MH532553. Isolate number 5 is most closely
related to B. nakamurai whose sequence identity diff/total nt is 99.48 (4/763) and
the GenBank accession number is MH532554. Isolate number 6 is most closely
related to B. safensis whose sequence identity diff/total nt is 99.47 (4/750) and the
GenBank accession number is MH532555 and the isolate number 8 is most closely
related to B. siamensis whose sequence identity diff/total nt is 100.00 (0/781) and
the GenBank accession number is MH532556. Heo et al. (2021) reported that
B. siamensis is a safe strain with the potential to develop as a probiotic.
The isolate number 9 is most closely related to B. safensis whose
sequence identity diff/total nt is 98.43 (11/699) and the GenBank accession
number is MH532557 and the isolate number 11 is most closely related to
B. subtilis subsp. subtilis whose sequence identity diff/total nt is 99.87 (1/756) and
the GenBank accession number is MH532558. Isolate number 14 is most closely
related to Bacillus subtilis subsp. stercoris whose sequence identity diff/total nt
is 99.83 (1/594) and the GenBank accession number is MH532559, the isolate
number 16 is most closely related to B. safensis whose sequence identity diff/
total nt is 99.09 (7/771) and the GenBank accession number is MH5325510 and
the isolate number 17 is most closely related to Enterobacter bugandensis whose
sequence identity diff/total nt is 99.8 (1/770) and the GenBank accession number
is MH5325511. The phylogenetic analysis revealed that isolate 3 is clustered with
B. subtilis subsp. inaquosorum while isolates 11 and 14 form a new clad (Fig. 1).
Therefore, it may belong to the novel species of Bacillus. Similarly, isolates 8, 16
and 17 were clustered with B. amyloliquifaciencs, Enterobacter cancerogenus and
B. australimaris, respectively. Isolates 2, 6 and 9 form a new clad (Fig. 2) having
a boot level confidence of 67% showing that these isolates may belong to novel
species. However, to confirm it is novel further studies are required. These findings
suggest that fermented bamboo shoots of North East India harbour some novel
Bacillus species which were not previously reported to date. Jeyaram et al. (2010)
reported B. subtilis, B. cereus, L. plantarum and Carnobacterium sp. along with
L. brevis in soidon by using the molecular technique. In the metagenomic study
(fermented bamboo shoots) by Hu et al. (2021), the bacteria belonging to 8 phyla, 16
classes, 30 orders, 63 families, 92 genera and 156 species, with Lactiplantibacillus
accounting for up to 81% of the species, with 12 species, including L. plantarum,
were reported. This study suggests the huge diversity of bacteria associated with
fermented bamboo shoots.

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Vijay Kumar et al.

Table 3: GenBank accession number, sequence identity and most closet neighbours of
most promising isolates.

Sequence identity
Selected isolates GenBank accession no. Most closely related with
diff/total nt
Isolate 2 MH532552 98.57 (10/700) B. safensis
Isolate 3 MH532553 99.73 (2/753) B. tequilensis
Isolate 5 MH532554 99.48 (4/763) B. nakamurai
Isolate 6 MH532555 99.47 (4/750) B. safensis
Isolate 8 MH532556 100.00 (0/781) B. siamensis
Isolate 9 MH532557 98.43 (11/699) B. safensis
Isolate 11 MH532558 99.87 (1/756) B. subtilis subsp. subtilis
Isolate 14 MH532559 99.83 (1/594) B. subtilis subsp. stercoris
Isolate 16 MH5325510 99.09 (7/771) B. safensis
Isolate 17 MH5325511 99.8 (1/770) Enterobacter bugandensis

Detection of Antibacterial Activity

The emergence of drug resistance among S. aureus, B. cereus, E. coli, Salmonella

and Klebsiella species is a major concern in the recent era. To find new a chemical
entity to combat these pathogens will be an important step in the discovery of new
antimicrobials (Founou et al. 2016). In this context, the selected isolates were
screened for their antibacterial activity against S. aureus MTCC 96, B. cereus
MTCC 430, E. coli MTCC 739, Salmonella enterica ser. Abony MTCC 3858 and
Klebsiella pneumonie MTCC 4030. The antibacterial activity of promising isolates
is given in Fig. 3. The isolate 2 (B. safensis) demonstrated the most promising
antibacterial against all the tested bacterial pathogens (Fig. 3). Isolate number 3 (B.
tequilensis) showed activity against E. coli, Klebsiella and S. aureus. The isolate 5
(B. nakamurai) was effective against. E. coli, Bacillus cereus and S. aureus. The
isolates 11 (B. subtilis subsp. subtilis), 16 (B. safensis) and 17 (E. bugandensis)
demonstrated the most antibacterial intensity to Salmonella and Klebsiella. While
other isolates showed activity against one or more pathogens. As these isolates
have not been previously reported from bamboo shoots, hence no comparable
data is available.

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 Molecular Diversity of Microbes

Bacillus tequilensis KCTC 13622 AYTO01000043

Bacillus subtilis subsp. spizizenii NRRL B-23049 CP002905
Bacillus subtilis subsp. stercoris D7XPN1 JHCA01000027
Bacillus mojavensis RO-H-1 JH600280
45 54 Bacillus halotolerans ATCC 25096 LPVF01000003
Isolate3
Bacillus subtilis subsp. inaquosorum KCTC 13429 AMXN01000021

98 Isolate11
Isolate14
Bacillus nakamurai NRRL B-41091 LSAZ01000028
Bacillus velezensis CR-502 AY603658
Bacillus subtilis subsp. subtilis NCIB 3610 ABQL01000001
98 Bacillus siamensis KCTC 13613 AJVF01000043

46 Isolate 8
Bacillus amyloliquefaciens DSM 7 FN597644
Bacillus altitudinis 41KF2b ASJC01000029

70 Pseudescherichia vulneris NBRC 102420 BBMZ01000044

Yokenella regensburgei ATCC 49455 JMPS01000045
Isolate17
99 56 Enterobacter cancerogenus ATCC 33241 FYBA01000020
Enterobacter bugandensis EB-247 FYBI01000003
74 Klebsiella michiganensis W14 JQ070300

63 Klebsiella grimontii 06D021 FZTC01000044

Klebsiella oxytoca JCM 1665 AB004754
Bacillus zhangzhouensis DW5-4 JOTP01000061
Isolate16
Bacillus australimaris NH7I 1 JX680098
Bacillus safensis FO-36b ASJD01000027
Bacillus pumilus ATCC 7061 ABRX01000007

Figure 1: Molecular phylogenetic tree by maximum likelihood method.

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Vijay Kumar et al.

67 Isolate2
Isolate 6
Isolate9
Bacillussafensis FO36b ASJD01000027
76
Bacillus safensis FO-36b ASJD01000027
Bacillus australimaris NH7I 1 JX680098
100
53
Bacillus pumilus ATCC 7061 ABRX01000007
67 Bacillus zhangzhouensis DW5-4 JOTP01000061
Bacillusaltitudinis 41KF2b ASJC01000029
63

63
Bacillus xiamenensis HYC-10 AMSH01000114
Bacillus altitudinis 41KF2b ASJC01000029
Bacillus subtilis subsp. spizizenii NRRL B-23049 CP002905
Bacillus mojavensis RO-H-1 JH600280
Bacillus vallismortis DV1-F-3 JH600273
Bacillus tequilensis KCTC 13622 AYTO01000043
Bacillus subtilis subsp. subtilis NCIB 3610 ABQL01000001
Bacillus subtilis subsp. inaquosorum KCTC 13429 AMXN01000021
Bacillus amyloliquefaciens DSM 7 FN597644
Isolate5
Bacillus nakamurai NRRL B 41091 LSAZ01000028
Bacillus subtilis subsp. stercoris D7XPN1 JHCA01000027
Bacillus siamensis KCTC 13613 AJVF01000043
Bacillus halotolerans ATCC 25096 LPVF01000003
Bacillus velezensis CR-502 AY603658

Figure 2: Molecular phylogenetic tree by maximum likelihood method.

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 Molecular Diversity of Microbes

20

18 2
16
3
5
Inhibition Zone Diameter (mm)

14 6
12 8
9
10
11
8 14
6
16
17
4

-2
EC SAL BC KL SA
Bacterial pathogens

Figure 3: Antibacterial profile of isolates; bar indicates the mean of triplicates ± standard
deviation; EC = E.coli MTCC 739; SAL = Salmonella enterica ser. Abony MTCC 3858.; BC
= B. cereus MTCC 430; KL = Klebsiella pneumonie MTCC 4030 ; SA = S. aureus MTCC 96.

The production of natural metabolites by probiotics is responsible to

repress the growth of both Gram-positive and Gram-negative bacteria. In a study,
the bacterial strain B. tequilensis was isolated from various traditional Indian
fermented products such as fermented batter of Idli, Meduwada and Jalebi and
screened its activity against pathogenic Candida albicans and non-albicans
(Palande et al. 2015). Thai pickled vegetables (Phak dong) are used as a potential
feed supplement in aquaculture. They are mainly used for the growth and disease-
resistant alternative for hybrid catfish. B. siamensis was isolated from phak dong
and it was proved as a probiotic in catfish culture. This bacterium produced
bacteriocins-like substances and exhibited a broad-spectrum antibacterial activity
(Meidong et al. 2017). Among Indonesians, the traditional food ''tape" or fermented
Cassava is very popular. The quality of this fermented cassava is determined
based on the microorganism involved during its presence of fermentation. During
the research, 26 amylase-producing Bacillus spp. isolates were obtained; among
them B. subtilis subsp. subtilis was dominant. Thus, these isolates can improve the
taste and quality of fermented cassava (Barus et al. 2013). But none of the isolates

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of the present study has been previously reported from fermented bamboo shoots
except B. subtilis and Enterobacter sp. (Behera & Balaji 2021).

CONCLUSION

The present study revealed the presence of Bacillus safensis, B. tequilensis, B.

siamensis, B. nakamurai, B. subtilis and Enterobacter. These isolates have not
been reported previously from fermented bamboo shoots except B. subtilis and
Enterobacter. Interestingly, no Lactobacillus species and molds were not detected
in any of the analysed samples. Potent antibacterial activity was recorded against
Klebsiella, Staphylococcus aureus, Salmonella and Bacillus cereus. The results of
the present study are promising however, further studies are required to isolate,
purify and characterise the metabolites produced by these isolates.

ACKNOWLEDGEMENTS

The authors are grateful to the Researchers Supporting Project Number (RSP-
2021/360), King Saud University, Riyadh, Saudi Arabia. The authors are also
thankful to Doon P.G. College of Agriculture Science and Technology (DCAST)
management for providing the necessary facilities to carry out this work and to
Ms. Bindita for providing the samples of bamboo shoots from different locations in
Manipur, India.

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