Molecular Diversity of Microbes Associated with Fermented Bamboo Shoots

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.


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 antiinflammatory 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 bamboobased 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.

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 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 × 10 8 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.

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 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.

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.

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.

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.

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  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 diseaseresistant 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 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.