Tannin-Tolerant and Extracellular Tannase Producing Bacillus Isolated from Traditional Fermented Tea Leaves and Their Probiotic Functional Properties.

A total of 117 Bacillus strains were isolated from Miang, a culture relevant fermented tea of northern Thailand. These strains were collected from 16 sampling sites in north Thailand. In this collection 95 isolates were tannin-tolerant Bacillus capable of growth on nutrient agar supplemented with 0.5% (w/v) total tannins from tea leaves extract (TE). The strains were also positive for pectinase, xylanase and amylase activity, while 91 and 86 isolates were positive for cellulase and β-mannanase, respectively. Only 21 isolates producing extracellular tannase were selected for further characterization. Identification by 16S rRNA gene sequence analysis revealed that more than 50% (11 of 21 isolates) were Bacillus tequilensis, whereas the remaining were B. siamensis (3), B. megaterium (3), B. aryabhattai (3) and B. toyonensis (1). B. tequilensis K34.2 produced the highest extracellular tannase activity of 0.60 U/mL after cultivation at 37 °C for 48 h. In addition, all 21 isolates were resistant to 0.3% (w/v) bile salt, sensitive to gentamicin, erythromycin, vancomycin and kanamycin and also tolerant to acidic condition. Cell hydrophobicity varied from 9.4 to 80.4% and neutralized culture supernatants of some Bacillus isolates showed bacteriocin producing potentiality against Samonella enterica serovar Typhimurium TISTR 292. All tested probiotic properties indicated that B. tequilensis K19.3, B. tequilensis K34.2 and B. siamensis K19.1 had high probiotic potential. This is the first report describing tannin-tolerant Bacillus and their extracellular tannase producing capability in Miang, a traditional fermented tea of Thailand.


Introduction
Fermented tea leaves, called "Miang", is a traditional fermented food product made from Camellia sinensis var. assamica that has been important in the sociocultural lifestyle of northern Thailand for over several hundred years. A typical Miang production process mainly consists of fermentation of steamed tea leaves without adding other nutritional substances in a variety of containers such as bamboo basket or clay jar for several days or up to a year without the use of any preservatives. However, the steps of fermentation process of Miang are different depending on the ethnicity of the local producer [1]. Since tea leaves are rich in phenolic compounds, the microbial fermented tea develops unique functions and has multiple beneficial effects on human health. Previous investigation also confirmed that Miang contains similar bioactive compounds including polyphenols, flavonoids, catechins, caffeine, gallic acids, tannins, volatile flavor and aromatic compounds which result in the

Sampling and Isolation of Bacillus spp.
Miang samples were collected from 16 local markets of various locations in upper northern Thailand including Chiang Mai (15 samples), Chiang Rai (8 samples), Lampang (4 samples), Phayao (2 samples), Phrae (3 samples), and Nan Province (7 samples) ( Table 1). All samples were transported to the laboratory in sterile polyethylene bags on ice and analyzed immediately. A sample of 10 g each was mixed well with 90 mL of sterile 0.85% (w/v) NaCl solution using Masticator Homogenizer (Basic/Panoramic, IUL, S.A., Barcelona, Spain) for 10 min. The sample was further diluted in a 10-fold dilution series and heated at 80 • C for 12 min. The suitable dilutions were spread plated onto nutrient agar (NA) medium (Merck, Darmstadt, Germany) and incubated aerobically at 37 • C for 24 h.
The colonies showing different morphological characteristics were picked from the plates and purified by repeated streaking onto the same nutrient agar medium for further characterization. Stock cultures were kept in nutrient broth containing 25% (v/v) glycerol and stored at −80 • C. The tannin tolerance of isolated Bacillus spp. in tea leaves extract (TE) was evaluated on NA supplemented with 20% (v/v) crude TE. Crude TE was prepared from local Miang prepared from Assam tea sourced from Chiang Mai as described by Chaikaew et al. [16]. Briefly, fresh young tea leaves (1000 g) were washed with tap water and then steamed for 30 min. The steamed tea leaves were mixed with 3000 mL sterile water and the mixture was homogenized in a Masticator blender (Basic/Panoramic, IUL, S.A., Barcelona, Spain) for 5 min. The supernatant was collected after centrifugation at 6000 rpm for 20 min, and concentrated by EYELA N-1000 rotary evaporator (Tokyo Rika-kikai, Co. Ltd., Tokyo, Japan) at 40 • C for 12 h to obtain a final volume of 200 mL. The TE solution was estimated for total tannins content using analysis method for tannin in tea infusion with some modification based on method of Tabasum et al. [19]. A single colony of each Bacillus isolate was transferred to NA supplemented with TE to obtain the final concentrations of 0.5% (w/v) total tannins. The growth of bacterial isolates was observed after incubation at 37 • C for 48 h.

Test for Extracellular Tannase and Polysaccharide Degrading Enzymes Production
All tannin-tolerant Bacillus isolates were investigated for their capability for production of extracellular tannase and polysaccharide degrading enzymes. All isolates were transferred into NA media containing 0.5% (w/v) tannic acid and incubated at 37 • C for 48 h. Growth and clear zone surrounding colonies represented the extracellular tannase activity and were observed after adding 1% (v/v) FeCl 3 solution [20]. Similar to tannase activity, 0.5% (w/v) of carboxymethyl cellulose (CMC), locust bean gum (LBG), soluble starch and pectin supplemented with 0.01% (w/v) trypan blue were independently used to detect extracellular activities of cellulase, β-mannanase, amylase and pectinase, respectively. The isolates producing extracellular tannase, distinguished from the clear zone formed surrounding the colony, were selected to determine enzyme activity in culture broth. This was done by taking 1% (v/v) overnight inoculum of each isolate and transferring into 100 mL of nutrient broth containing 0.5% (w/v) tannic acid, pH 6.5, and shaking at 100 rpm at 37 • C for 48 h. The culture broth sample was collected at 24 and 48 h and cell free supernatant (CFS) was separated by centrifugation 10,000× g at 4 • C for 10 min and used for determination of extracellular tannase activity using a modified method of Sharma et al. [21]. Briefly, 0.125 mL of proper dilution of crude enzyme was mixed with 0.125 mL of substrate (12.5 mM of methyl gallate in 0.05 M of citrate buffer, pH 5.0) and incubated at 37 • C for 10 min. The reaction mixture was then mixed with 0.15 mL of 0.67% (w/v) methanolic rhodanine and incubated at 37 • C for 5 min. Next, 0.1 mL of 0.5 N potassium hydroxide was added and incubated at 37 • C for 5 min. The reaction mixture was filled with 2.0 mL distilled water and absorbance was measured at 520 nm. One unit of tannase activity was defined as the amount of enzyme that liberated 1 µmole of gallic acid per minute under the assay condition.

Identification of Extracellular Tannase Producing Bacillus spp. and Phylogenetic Analysis
All extracellular tannase producing Bacillus spp. isolates were identified using molecular identification via 16S rRNA gene analysis. The genomic DNA of each isolate was extracted from the bacterial cell following the standard protocol described by Sambrook and Russell [22]. The 16S rDNA fragment was amplified by polymerase chain reaction (PCR) using genomic DNA as a template with bacterial universal primers, 27F (5 -AGA GTT TGA TCC TGG CTC AG-3 ) and 1525R (5 -AAG GAG GTG WTC CAR CC-3 ) [23]. The amplification reactions were carried out in a thermal cycler (MyCycler DNA thermal cycler; Bio-Rad, Hercules, CA, USA) following the method of Kim and Chun [24]. The 16S rRNA gene sequences were compared to other genes in the GenBank and EzBioCloud databases, and the phylogenetic tree was created based on the neighbor-joining method by MEGA version 4.0 software [25]. All 16S rRNA gene sequences generated in this study have been deposited in the NCBI GenBank database under accession number MH889120 to MH889140.

Acid and Bile Salt Tolerances
All 21 extracellular tannase producing Bacillus spp. were tested for their tolerance to acidic condition and bile salts. Tolerance to acidic condition was determined using the method of Argyri et al. [26] with some modification. Briefly, 5 mL of bacterial cells from overnight (18 h) cultures were harvested by centrifugation 10,000× g at 4 • C for 10 min, washed twice with PBS buffer (pH 7.2), and 5resuspended in 5 mL of the same buffer solution. This suspension was inoculated (1%, v/v) into 5 mL of PBS solution pH 7.2 (control) and the PBS solution was adjusted to pH 2.0 and 3.0 with hydrochloric acid (1 M). These treated suspensions were incubated at 37 • C for 3 h. Viable cell count was determined by plating on NA and incubated at 37 • C for 12 h. The viable cell count was expressed as log value of colony-forming units per mL (logCFU/mL). The survival percentage was calculated as follows: survival (%) = [final (logCFU/mL)/control (logCFU/mL)] × 100.
Bile salts tolerance of bacterial strain was determined according to the method of García-Hernández et al. [27]. Briefly, 5 mL of bacterial cell from overnight (18 h) cultures were harvested by centrifugation at 10,000× g at 4 • C for 10 min, washed twice with PBS buffer (pH 7.2), and resuspended in 5 mL of the same buffer solution. The cell suspension was inoculated (1%, v/v) into 5 mL of PBS solution supplemented with 0.3% (w/v) bile salts (HiMedia, Mumbai, India) and PBS without bile salts served as control, after which all tubes were incubated at 37 • C. Following 3 h of incubation, the viable cell count was determined, and survival rate was calculated.

Cell Surface Hydrophobicity
The cell surface hydrophobicity of selected bacilli was determined in terms of the bacterial cell ability in adhering to hydrocarbons (MATS: Microbial Adhesion to Solvents), according to the methodology described by García-Hernández et al. [27]. Bacterial cultures in stationary phase were harvested by centrifugation 10,000× g at 4 • C for 10 min, washed twice with PBS buffer (pH 7.2), and resuspended in 5 mL of the same buffer solution. The bacterial concentration was adjusted with PBS to OD600 = 1 (A0), and then an equal volume of toluene (BDH Chemicals, Ltd., Poole, England) was added. Toluene was chosen as a nonpolar solvent because it reflects cell surface hydrophobicity and hydrophilicity [28]. The two-phase system was completely mixed for 5 min. After 1 h of incubation at 37 • C, the aqueous phase was measured again (A1). MATS percentage was calculated according to the following equation: MATS (%) = [(A0 − A1)/A0] × 100. Isolates with MATS above 50% were considered to be hydrophobic.

Antibiotic Susceptibility Test
Antibiotic susceptibility of 21 extracellular tannase producing strains was determined by disk diffusion method according to the guidelines of the Clinical and Laboratory Standards Institute [29]. Antibiotic resistance of the isolates was tested against four selected antibiotics, including gentamycin (10 µg), erythromycin (15 µg), vancomycin (30 µg) and kanamycin (30 µg). Fifty microliters of the active bacterial suspension (10 5 -10 6 CFU/mL) was spread evenly on the NA plate and antibiotics discs were placed on the plates. After 24 h of incubation at 37 • C, the inhibition zone diameters were measured including the diameter of the discs. Breakpoints for the interpretation of inhibition zone were expressed as sensitive, S; intermediate, I; and resistant, R as described by CLSI [29].

Antimicrobial Activity
The antimicrobial activity of the isolated Bacillus strains was assessed against Salmonella enterica serovar Typhimurium TISTR 292 using the well diffusion assay according to Abid et al. [30] with slight modification. Briefly, 50 µL of pathogen cell (10 6 -10 7 CFU/mL) were spread onto agar plates. Cell-free culture supernatants (CFCS) were collected by centrifugation (10,000× g at 4 • C for 15 min) and filtered through 0.22 µm membrane filter (Millipore, Bedford, MA, USA). To demonstrate the antimicrobial activity, 75 µL of pH neutralized CFCS (pH 6.5) was added to each well (cut with sterile 6 mm Cork borer) of the pathogen cells agar plates. The agar plates were incubated at 37 • C for 18 h. Growth inhibition was read by measuring the diameter of the inhibition zones.

Isolation of Bacillus spp.
A total of 117 presumptive Bacillus isolates were obtained from the 39 samples of Miang collected from 16 sampling sites within six provinces of northern Thailand (Table 1). All Bacillus isolates clearly showed the properties of bacteria in the genus Bacillus such as Gram-positive, rod shape, endospore-forming ability and catalase positive. The largest numbers of 51 Bacillus isolates were confirmed from Chiang Mai province, whereas the smallest numbers of 7 isolates were confirmed in Miang collected from Phayao province. The high number of samples obtained from Chiang Mai province aligns with the high number of Miang plantation areas which are also well distributed in various districts [1]. Furthermore, the previous reports related to this fermented tea over recent decades mostly investigated samples from Chiang Mai area [5,11,31]. Nan and Phrae provinces also have a long history of Miang production and provide Miang product with unique characteristics that differs from Chiang Mai and Chiang Rai areas [1]. Until now, there have been few published reports on relevance of endospore-forming bacteria from Miang or other fermented tea leaves products. However, the number of endospore-forming bacteria which accounted for approximately 40-45% of total bacterial counts in 40 Miang samples collected from twenty producing locations were detected and therefore suggested that they play specific roles in Miang fermentation [18].

Tannin Tolerance of Bacillus spp.
Tannins concentration in TE prepared for this study was determined after product preparation and also prior to using in medium preparation. The TE from this experiment composed of approximately 2.5% (w/v) total tannins. All 117 Bacillus isolates were tested for their tolerance to tannins in TE at the final concentration of 0.5% (w/v) total tannins and it was found that 95 isolates or approximately 81% were tannin-tolerant based on their growth ability in NA supplemented with 0.5% (w/v) total tannins ( Figure 1a). However, only 21 isolates formed clear zones surrounding the colonies in varying size implying that they were able to produce extracellular tannase. Among 95 isolates of tannin-tolerant Bacillus, 22% (21 of 95) exhibited the extracellular tannase producing ability. The widest clear zone (Figure 2c) was compared to the smallest ( Figure 2b) and control or non-clear zone (Figure 2a). It is known that tea leaves contain significant amounts of phenolic compounds, particularly tannins and other tea phenolics, which have been reported to have inhibitory effect on growth of many microorganisms [32]. Therefore, the microorganisms capable of growth on this substrate potentially harbor the special metabolic mechanisms that allow tolerance to survive the toxicity of tea tannins via the induction of tannin degradation pathways [17]. Likewise, Zhao and Shah [33] also confirmed that the microorganisms originating from tannin-rich environments may have additional response mechanism to overcome the adverse effects of tannin stress to allow their metabolic activity and/or survival. However, 22 of the 117 Bacillus spp. isolated from Miang (18.8%) did not show any growth on NA supplemented with 0.5% (w/v) total tannins. This suggested that these 22 tannin-tolerant negative strains may harbor tannin-tolerant properties, but at concentrations lower than 0.5% (w/v) total tannins. Field and Lettinga [34] reported the toxicity of tannin against various microorganisms was approximately 0.3-0.7% (w/v) and showed 100% inhibition of B. subtilis. Tannins concentration in TE prepared for this study was determined after product preparation and also prior to using in medium preparation. The TE from this experiment composed of approximately 2.5% (w/v) total tannins. All 117 Bacillus isolates were tested for their tolerance to tannins in TE at the final concentration of 0.5% (w/v) total tannins and it was found that 95 isolates or approximately 81% were tannin-tolerant based on their growth ability in NA supplemented with 0.5% (w/v) total tannins (Figure 1a). However, only 21 isolates formed clear zones surrounding the colonies in varying size implying that they were able to produce extracellular tannase. Among 95 isolates of tannin-tolerant Bacillus, 22% (21 of 95) exhibited the extracellular tannase producing ability. The widest clear zone (Figure 2c) was compared to the smallest (Figure 2b) and control or non-clear zone (Figure 2a). It is known that tea leaves contain significant amounts of phenolic compounds, particularly tannins and other tea phenolics, which have been reported to have inhibitory effect on growth of many microorganisms [32]. Therefore, the microorganisms capable of growth on this substrate potentially harbor the special metabolic mechanisms that allow tolerance to survive the toxicity of tea tannins via the induction of tannin degradation pathways [17]. Likewise, Zhao and Shah [33] also confirmed that the microorganisms originating from tannin-rich environments may have additional response mechanism to overcome the adverse effects of tannin stress to allow their metabolic activity and/or survival. However, 22 of the 117 Bacillus spp. isolated from Miang (18.8%) did not show any growth on NA supplemented with 0.5% (w/v) total tannins. This suggested that these 22 tannin-tolerant negative strains may harbor tannin-tolerant properties, but at concentrations lower than 0.5% (w/v) total tannins. Field and Lettinga [34] reported the toxicity of tannin against various microorganisms was approximately 0.3-0.7% (w/v) and showed 100% inhibition of B. subtilis.

Extracellular Polysaccharide Degrading Enzyme Production Test
In addition to the capability for survival in high-tannin condition, 95 tannin-tolerant Bacillus spp. were tested for their ability to produce extracellular polysaccharide degrading enzymes including amylase, pectinase, xylanase, cellulase and β-mannanase by observation of the clear zone formation in NA supplemented with the specific substrates as described previously. We have found that all tannin-tolerant Bacillus isolates were positive for pectinase, amylase and xylanase (Figure 1b). However, only 91 isolates (96%) were positive for cellulase production based on the visualization of the clear zone surrounding colonies on NA supplemented with CMC, while 4 isolates were negative. Similar to cellulase production, only 86 isolates (91%) were positive for β-mannanase, while 9 isolates were negative (Figure 1b). Furthermore, among the 21 extracellular tannase producers, 19 isolates  were also found to form clear zones both on CMC and LBG and only 2 isolates (K22.1 and K23.1) were positive for β-mannanase. Based on the results from this study, most of tannin-tolerant Bacillus spp. were highly associated with the ability to produce extracellular polysaccharide degrading enzymes particularly pectinase, xylanase and amylase (100% positive) and 96 and 91% positive for cellulase and β-mannanase production, respectively. This indicates that the capability to produce extracellular polysaccharide degrading enzymes reflects the effort of the bacteria to find carbon source for their survival and extracellular cellulose, β-mannanase, pectinase, amylase and xylanase producing Bacillus strains have been well documented in several studies on fermented products [35][36][37]. Some of the extracellular polysaccharide degrading enzymes produced by tannin-tolerant Bacillus spp. mentioned in this study may be involved in the Miang fermentation process.

Identification of Tannase Producing Bacillus spp. and Phylogenetic Analysis
Molecular identification techniques present an alternative to other conventional methods because the independent genome for the physiological characteristics may vary among the species. Moreover, the techniques are more reproducible, and the results are achieved more rapidly than with traditional methods. All tannase producing Bacillus isolates were identified using 16S rRNA gene analysis and the data are presented in Table 2. The majority of Bacillus (11 of 21 isolates) were

Extracellular Polysaccharide Degrading Enzyme Production Test
In addition to the capability for survival in high-tannin condition, 95 tannin-tolerant Bacillus spp. were tested for their ability to produce extracellular polysaccharide degrading enzymes including amylase, pectinase, xylanase, cellulase and β-mannanase by observation of the clear zone formation in NA supplemented with the specific substrates as described previously. We have found that all tannin-tolerant Bacillus isolates were positive for pectinase, amylase and xylanase (Figure 1b). However, only 91 isolates (96%) were positive for cellulase production based on the visualization of the clear zone surrounding colonies on NA supplemented with CMC, while 4 isolates were negative. Similar to cellulase production, only 86 isolates (91%) were positive for β-mannanase, while 9 isolates were negative (Figure 1b). Furthermore, among the 21 extracellular tannase producers, 19 isolates were also found to form clear zones both on CMC and LBG and only 2 isolates (K22.1 and K23.1) were positive for β-mannanase. Based on the results from this study, most of tannin-tolerant Bacillus spp. were highly associated with the ability to produce extracellular polysaccharide degrading enzymes particularly pectinase, xylanase and amylase (100% positive) and 96 and 91% positive for cellulase and β-mannanase production, respectively. This indicates that the capability to produce extracellular polysaccharide degrading enzymes reflects the effort of the bacteria to find carbon source for their survival and extracellular cellulose, β-mannanase, pectinase, amylase and xylanase producing Bacillus strains have been well documented in several studies on fermented products [35][36][37]. Some of the extracellular polysaccharide degrading enzymes produced by tannin-tolerant Bacillus spp. mentioned in this study may be involved in the Miang fermentation process.

Identification of Tannase Producing Bacillus spp. and Phylogenetic Analysis
Molecular identification techniques present an alternative to other conventional methods because the independent genome for the physiological characteristics may vary among the species. Moreover, the techniques are more reproducible, and the results are achieved more rapidly than with traditional methods. All tannase producing Bacillus isolates were identified using 16S rRNA gene analysis and the data are presented in Table 2. The majority of Bacillus (11 of 21 isolates) were identified as Bacillus tequilensis while the remaining as B. siamensis (3 isolates), B. megaterium (3 isolates), B. aryabhattai (3 isolates) and B. toyonensis (1 isolate), which showed similarities in 16S rRNA gene sequence related to their closest type strain in the narrow range between 99.8% and 100.0%. The phylogenetic trees based on the sequences of the 16S rRNA gene were constructed by the neighbor-joining method comparing between each Bacillus strain ( Figure 3). From the results, most tannase producing Bacillus spp. isolated from Miang belong to species B. tequilensis which is closely related to B. subtilis [38]. A previous report found that B. siamensis and B. licheniformis have been isolated from tea leaves (raw material for Miang fermentation) and suggested involvement in Miang fermentation process [39].  Note: "T " is indicate the type strain of bacteria.

Extracellular Tannase Production
The tannase activity of all 21 isolates of extracellular tannase producing strains were determined from cell-free supernatant fraction (CFSF) separated from 24 and 48 h old culture broth containing 0.5% (w/v) tannic acid and the results are presented in Figure 4. Extracellular tannase were detected from CFSF of all isolates with variation in activity. B. tequilensis K34.2 showed the highest activity of 0.53 U/mL from 24 h to until the end of the experiment at 48 h (0.60 U/mL), while the lowest activity of 0.25 U/mL was observed in B. megaterium K21.4 and B. siamensis K23.3 at 48 h growth phase. Most of the reported bacterial tannase producers correspond to the presence of extracellular tannase [40]. Until now, several tannase producing Bacillus spp. have been identified such as B. pumilus, B. polymyxa [41], B. licheniformis [20,42], B. cereus [43], B. sphaericus [44], B. massieliensis [45], B. subtilis [46] and B. gotthelii [47]. This study is the first report on extracellular tannase produced by B. tequilensis and B. siamensis.
Foods 2020, 9,  The tannase activity of all 21 isolates of extracellular tannase producing strains were determined from cell-free supernatant fraction (CFSF) separated from 24 and 48 h old culture broth containing 0.5% (w/v) tannic acid and the results are presented in Figure 4. Extracellular tannase were detected from CFSF of all isolates with variation in activity. B. tequilensis K34.2 showed the highest activity of 0.53 U/mL from 24 h to until the end of the experiment at 48 h (0.60 U/mL), while the lowest activity of 0.25 U/mL was observed in B. megaterium K21.4 and B. siamensis K23.3 at 48 h growth phase. Most of the reported bacterial tannase producers correspond to the presence of extracellular tannase [40]. Until now, several tannase producing Bacillus spp. have been identified such as B. pumilus, B. polymyxa [41], B. licheniformis [20,42], B. cereus [43], B. sphaericus [44], B. massieliensis [45], B. subtilis [46] and B. gotthelii [47]. This study is the first report on extracellular tannase produced by B. tequilensis and B. siamensis.

Assessment of Probiotic Potentiality of Tannin-Tolerant Bacillus spp.
A total of 21 tannin-tolerant Bacillus isolates were selected to determine probiotic potential and the results are shown in Table 3. Acid tolerance is generally considered as an essential assessment criterion for probiotic evaluation, since the strains have to survive the acidic condition of gastrointestinal tract environment [48,49]. The tolerances of all 21 isolates against low pH were examined by exposing the strains to pH 2.0 and 3.0 for 3 h. Most of the isolates were able to survive after being exposed to pH 3.0 for 3 h with varying degree of survival rate in the range of 51

Assessment of Probiotic Potentiality of Tannin-Tolerant Bacillus spp.
A total of 21 tannin-tolerant Bacillus isolates were selected to determine probiotic potential and the results are shown in Table 3. Acid tolerance is generally considered as an essential assessment criterion for probiotic evaluation, since the strains have to survive the acidic condition of gastrointestinal tract environment [48,49]. The tolerances of all 21 isolates against low pH were examined by exposing the strains to pH 2.0 and 3.0 for 3 h. Most of the isolates were able to survive after being exposed to pH 3.0 for 3 h with varying degree of survival rate in the range of 51  It should be noted that the sensitivity found in our study may be related to the concentration of each antibiotic and hence, several concentrations should be tested to confirm the results. The capability to produce antimicrobial compounds is related to the metabolic products of bacteria such as organic acids, hydrogen peroxide, bacteriocins and short chain fatty acids, which are among the properties generally used to evaluate the probiotic potential of bacteria [30]. In this study, S. enterica were used as pathogenic indicator, where most of Bacillus isolates were able to show inhibition towards this pathogenic bacterium which included B. tequilensis (isolates K7. 2

Conclusions
In conclusion, Bacillus spp. diversity from traditional Miang, a fermented tea leaves product of northern Thailand was for the first time identified and characterized in this study. Among 117 Bacillus spp. isolated from Miang, 21 isolates showed the ability to produce extracellular tannase. The results of current study also showed that some tannin-tolerant Bacillus strains are potentially useful as probiotic bacteria. Some B. tequilensis and B. siamensis isolates showed high acid tolerance, bile salt tolerance, hydrophobicity and antimicrobial activity. Miang may also be considered a traditional fermented product with health-relevant functional benefits with antioxidant compounds such as high levels of phenolic acid and flavonoids [18], which are potentially biotransformed by tannase producing Bacillus spp. Moreover, due to the high numbers of Bacillus spp. in Miang, these bacteria in themselves may be considered as a possible probiotic. From the promising findings of this study more detailed analysis of probiotic potential of specific strains isolated from Miang should be undertaken. Further understanding of their health effects, including wider functional benefits of biotransformed tannin products should be investigated.

Funding:
The authors are grateful to the Chiang Mai University for financial support via postdoctoral fellowship.