Bacillus siamensis I7B strain as heavy metal quencher and probiotics; isolated from gut of Stolepherous commersonni

Probiotic products have been administered in food, medicine, dairy and fermentation industry to enhance the benecial functions in the host. In the present study gut bacteria of stolepherous commersonnii have been isolated and subjected to screening for heavy metal resistance. A potent heavy metal resistant bacterial strain of gut microbiota was subjected to assessment for its probiotic and antioxidant potential. Four bacterial strains namely I7A, I7B, I7C, and I8 were isolated and the strain I7B showed heavy metal resistant potential (upto 20 mg/ml of Cr, Cr, and Pb). The strain I7B showed high sensitivity to Streptomycin (10 μg), low sensitivity to Rifampin (5 μg) and resistant to pencillin (2 μg, 10 μg). Atomic absorption spectrometry studies showed that the I7B strain possess maximum heavy metal (Cu) removal ability of 81.64 % in textile dye euent and the strain was identied as Bacillus siamensis by 16S rRNA gene sequencing studies. Hence I7B strain was named as Bacillus siamensis I7B and has been submitted in NCBI with accession number MN404539. Bacillus siamensis 17B strain possess high acid tolerance (90.9 %), high bile tolerance (96.9 %) and nonhemolytic. The17B strain showed auto aggregation (28±0.011%) and coaggregation potential (27±0.16 %) which could be a feasible characteristic of potent probiotic strain. Cell free Supernatant of Bacillus siamensis I7B strain possess good DPPH free radical scavenging ability (59.43±0.002 %) and moderate hydroxyl radical scavenging ability (27.6±0.010 %). Biopreservation effect of Bacillus siamensis I7B strain on tomato puree showed 72 % growth inhibition against A. niger for 15 days.


Introduction
Food contamination is the global concern worldwide. Heavy metal de le is caused by 3 main routes via ingestion, inhalation and dermal contact (Abtahi et al. 2017). Still, beyond 90% of heavy metal de le in human exposure is through food contamination. These heavy metals nd accumulated in rice, vegetables, and sh. The consumption of contaminated sh or substances may enter the food chain pathway resulting in adverse effects in biota (Al-Saleh and Abduljabbar 2017). Among several crops, rice can predominantly accumulate heavy metals leading to critical human health risks (Djahed et al. 2018).
Cd, As, Cr, Ni, pollution in food is owing to administration of contaminated water (Omar et al. 2015), metal-based pesticides, and chemical fertilizer (Djahed et al. 2018) in agriculture. Approximately 90% of total metal intake in humans is because of consumption of heavy metal befouled vegetables (Ametepey et al. 2018;Pajevic et al. 2018). Cd, Al, Cr, and As contamination was found in cereals and vegetables. Accumulation of heavy metals in human organs and tissues end up in the kidney, cardiovascular system, and nervous system toxicity (Owolabi and Hekeu 2010).
Studies con rmed administering probiotics can protect animals and humans against adverse health effects caused by food contamination by maintaining the gut microbiota (Abdel-Megeed 2020), which aid in reducing oxidative stress (Chattopadhyay et al. 2019), stimulation of gene expression in host, increasing the intestinal barrier (Duan et al. 2020), and at last enhancing the host ability to assimilate xenobiotics . Lactobacillus strains were proved to reduce chronic and acute heavy metals poisoning (Reddivari et al. 2017).
Probiotics are live microorganisms with bene cial activity to living organism when administered in adequate quantity (Hill et al. 2014). In trend, probiotics and their metabolic compounds have become a global research hotspot and various probiotic products have been administered in food, medicine, dairy and fermentation industry to enhance the bene cial functions in the host (Abatenh et al. 2018; Day et al. 2019). Probiotics have been isolated from fruit juices, grains, honeycomb, and soil, sh, beef, pork (Siripornadulsil et al., 2014), salted crab (Senthong et al. 2012), and seafood (Nanasombat et al. 2014). In dairy industry probiotics are the sources from cheese, yogurt (Mahmoudi et (Martin et al. 2017). LAB is found to be the major group of probiotics and among the LAB group acidophilus, paracasei, casei, plantarum, crispatus, reuteri, rhamnosus, gasseri and bulgaricus were widely used. Bi dobacterium and Lactobacillus are most familiar probiotics among LAB. Pediococcus pentosaceous is predominant in thai fermented foods containing sh and pork (Siripornadulsil et al. 2014). Probiotics are found to possess the following protective functions like Antipathogenic, anti-diabetic, anti-obesity, anti-in ammatory, anti-cancer, anti-allergic, and also have potential in maintaining urogenital health care (Kerry et al. 2017). Administration of probiotics by food and pharmaceutical industry is just adaptable due to supplementation through drugs, foods, dietary supplements, infant formulas and animal feed (T'elessy 2019).
Lactic acid bacteria (LAB) are known to produce a range of antifungal agents such as alcohols, lactic acid, acetic acid, hydrogen peroxide, carbon dioxide, diacetyl, acetaldehyde, phenyl derivates, hydroxy fatty acids, peptides and bacteriocins against pathogenic infections (Ortiz-Rivera et al. 2017). Among the LAB isolates, Lactobacillus kunkeei in giant honey bees showing antagonist effect against yeast spoilage in honey. Moreover, administration of probiotic in bio preservative perspective not only preserve the food but also enhance the additional health bene ts to the host. Probiotics resident in gut and food matrix, have the capacity to produce vitamins with good antimicrobial activity. Vitamin C produced by probiotics elevate the lipid acidity in the bacterial cell membrane, causing cleavage in the cell membrane and bacterial cell wall (Pedros-Garrido et al. 2019), signi cant in food industry for food preservation to increase the shelf life of the food samples.
The rich nutrient of the sh digestive tract gives a nourishing environment for the growth of bacterial cultures. In the digestive tract, the resident bacteria exhibit the wide and enzymatic potential which plays a major role in sh digestion (Kavitha et al. 2018). The gut bacteria can produce various enzymes, like proteolytic, amylolytic, cellulolytic, lipolytic, and chitinolytic enzymes, which are responsible for the digestion of proteins, carbohydrates, cellulose, lipids, and chitin in the host (Ray et al. 2012) and provide the nutritional bene ts to the host (Dutta et al. 2015).
In the aquatic system, the functioning of gut microbiota is similar to the terrestrial mammals (Talwar et al. 2018). The main aim to isolate the gut bacteria of sh is to ease the selection of probiotics, prebiotics and their compounds to improve the gut balance, nutritional performance and host health (Wang et al.

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2018). The production of various chemical like caustic soda and fertilizers by various industries in the coastal area discharge the wastewater without proper treatment into the sea through canals. The external conditions like poor water quality create stress in sh and modi es the composition of gut microbes (Hossain et al. 2013;Sihag et al. 2012). Heavy metal resistant bacteria from marine environment have showed good resistance to heavy metal and many antibiotics (Nithya et al. 2011). For more than a decade, gut microbiota had gained interest of many researchers worldwide, which gives the biological interactions of harbored microbes and the intestinal function (Jandhyala et al. 2015). Hence, many studies focus on isolating indigenous and exogenous microbiota of aquatic animals with probiotic functionality (Reddy 2015).
However, it is apparent that attempt has not been made so far to isolate bacteria from the gut of edible sh (Stolepherous commersonnii). To make use of the valuable quantity of gut microbes of Stolepherous commersonnii, this study has been designed to isolate and characterize the gut bacteria of Stolepherous commersonnii as probiotics. Four indigenous bacterial strains namely I7A, I7B, I7C and I8 have been isolated from the gut of edible marine sh (Stolepherous commersonnii), and their probiotic properties were characterized.

Isolation of gut bacteria
The sh is washed with sterile water and then carefully placed aseptically within laminar air ow chamber on ice slabs. The sh gut was dissected out and homogenized with 5 ml of normal saline under sterile condition. Fungal contaminants were removed by placing the homogenate in the boiling water bath at 80°C for 20 min. The homogenate was serially diluted with PBS to 10-2, 10-3, 10-4, 10-5, 10-6 and 10-7 dilution, and spread plate using L rod in the nutrient agar media. Plates were incubated at 37°C for 24 -48 h. Individual colonies were picked out and puri ed using quadrant streaking method in the nutrient agar media. The pure isolates were revived using nutrient broth and stored at -80°C in 40% glycerol (Kavitha et al. 2018).

Screening for heavy metal resistance
For the selective screening of heavy metal resistant bacteria, the isolates were screened for heavy metal resistance using the agar diffusion method using Marzan et al. (2016). Brie y, the puri ed bacteria were streaked in the plates containing 20 ml of minimal agar medium supplemented with different concentrations (10,20,30,40,50 and 60 mg/ml) of chromium, cadmium, copper and lead. The plates were incubated at 37°C for 48h. The potent isolate is chosen for further studies.
Bioremediation of heavy metals in textile dye e uent Heavy metals present in solution were identi ed by ame atomic absorption spectrometer (AAS, model 220 AA B6; Varian). The bioremediation of Heavy metals was analysed in textile dye e uent collected from Arulpuram Common E uent treatment plant, Tirupur. Four isolates I7A, I7B, I7c and I8 were inoculated in 20 ml of nutrient broth at 37ºC for 24 h. 5 ml of culture was inoculated in 100 ml of sterilized textile dye e uents and incubated for 32 h and the sample was centrifuged at 9500 rpm for 10 min. The supernatant was analysed for heavy metal removal by atomic absorption spectrometry. The heavy metal concentrations were evaluated using appropriate blanks and standards for calibration (Jafarian and Ghaffari 2017). The result was interpreted using the formula as follows: The heavy metal utilized = Heavy metals present in the e uent before treatment (ppm) -Heavy metal present in the e uent after treatment (ppm)

Morphological characterization of potent isolate
Morphological characteristics of potent isolate I7B was assed using gram staining and FESEM analysis.
The potent isolate I7B was grown in nutrient broth at 37°C overnight in a rotary shaker. Gram-staining was performed using the method described by Ajayi et al. (2017). FESEM analysis is performed using Ste et al. 2021. In brief, the potent isolate I7B grown mid-log phase is pelleted by centrifugation using 10,000 rpm for 10 min. The cells were xed using 2.5% glutaraldehyde for 1 h, air dried and dehydrated using ethanol gradient. The cells were visualized in QUANTA -250 FEG electron microscope.

Identi cation of potent isolate
The genomic DNA of the potent isolate I7B were isolated by HiPurA® Bacterial Genomic DNA puri cation kit. The purity of the DNA was checked using nanodrop and agarose gel electrophoresis. The isolated DNA were ampli ed by PCR using PCR master mix (Qiagen) and universal primers forward (518F) 5ʼ CCAGCAGCCGCGGTAATACG-3ʼ and reverse primer (800R) 5ʼ 34 TACCAGGTATCTAATCC-3ʼ, under the following conditions; initial denaturation 94°C for 5 min, followed by 25 cycles each consisting of 94°C of 2 min, 55°C for 1 min and 72°C for 2 min, following a nal extension step at 72°C for 10 min. The PCR products were analyzed using agarose gel electrophoresis. The ampli ed DNA were sent for 16s rRNA sequencing. The obtained nucleotide sequences were compared with the available sequences in the National Center for Biotechnology Information (NCBI) database using the Basic Local Alignment Search Tool (BLAST). The phylogenetic tree was constructed with MEGA 7 software by the neighbor-joining method. The sequences were deposited in the NCBI GenBank (Adeoti et al. 2021).

Antibiotic susceptibility assay
The selected potent strain was tested for the antibiotic susceptibility against the selected antibiotics using the disc diffusion method. Six different antibiotic discs were used, namely Erythromycin: 15 µg/disc, tetracycline (30 µg), streptomycin (10 µg), kanamycin (30 µg), penicillin (10µg and 2 µg), and Rifampin(5µg). Antibiotic discs were placed on the Muller-Hinton agar plates and incubated at 37°C for 24 h. The antibiotic sensitivity of bacteria towards antibiotics was recorded by measuring the Zone of inhibition (mm) (Kavitha et al. 2018).

Screening of probiotic potential
The acid tolerance test The tolerance of the isolate to acid were estimated by previous report by R.P Shastry et al. 2021. The potent strain grown overnight (approximately 1 × 10 8 CFU/ml) is inoculated into 5 ml of tryptone soya broth, and the pH was adjusted to 1.5 and 3.0 using hydrochloric acid. The culture was then incubated at 37°C and the viable bacterial counts were measured and depicted as bacterial growth count at 0, 1, 2, and 3 h time interval on nutrient agar plates.

Cellviability% = logCFUofviablecellssurvived logCFUofinitialcells × 100
Assay of Bile salt tolerance The bile tolerance test was carried out according to previous report by Chen Pei et al. 2014. Firstly, aliquot (approximately 1 × 10 8 CFU/ml) of potent strain grown overnight culture was inoculated into 10 ml nutrient broth which is supplemented with and without 0.3% and 0.7% bile salt and cultures were then incubated at 37°C. The growth medium with 0% bile salt served as control. The absorbance was measured at OD 600 for the time intervals 0h, 2 h, 4h and 24 h and was estimated against the corresponding uninoculated blank samples. Survivability of the isolates was represented by the following formula: Aggregation activity Aggregation activity consisting of both auto-aggregation and co-aggregation was estimated in accordance with the method reported by Li Y et al. (2019). Pathogenic strain, Staphylococcus aureus and the potent strain was incubated separately at 37°C for 18 h and then the supernatant was harvested by centrifugation at 10,000 rpm for 10 mins. The cells were washed twice and resuspended in PBS of approximately 1 × 10 8 CFU/ml was used for the experiment. For determining auto-aggregation ability, 4 ml of the isolate suspension was individually added into sterile tubes and suspended thoroughly. The tubes were placed at room temperature with no agitation and a 150 µl aliquot of the upper suspension was taken after an incubation of 5 hours. The absorbance was determined at 600nm by using a microplate reader. Percentage Auto aggregation was calculated using the formula: Where A0 corresponds to the absorbance at 0 h, and At corresponds to the absorbance of upper suspension after 5h.
For determining the co-aggregation of potent strains with the pathogens, equal amounts (2 ml) of isolates and pathogen cultures were mixed in sterile tubes and vortexed thoroughly. These tubes were placed at room temperature with no agitation and a 150 µL aliquots of upper suspension was taken at a time interval of 0 and 4 h and the absorbance was determined at 600nm absorbance by using a microplate reader. Coaggregation percentage was calculated as follows Where A pat is the absorbance of the pathogens and A probio are the absorbance of the isolate at 0 h and A mix is the absorbance of the mixed suspensions at 4 h.

Hemolytic activity assay
Hemolytic activity assay was conducted for the potent strain following the method reported by Samson J S (2020). Blood agar plates supplemented with 5% goat blood were streaked with the isolates and incubated for 48 h at 37˚C. After incubation the hemolytic zones were observed. A presence of clear halo around the colonies indicates positive hemolysis (Beta-hemolysis) and a greenish zone around the colonies indicate negative hemolysis (Alpha-hemolysis) or the absence of clear zones around the colonies indicate no hemolysis (Gamma-hemolysis).
Free radical scavenging ability of selected strain DPPH radical scavenging assay Assessment of bio-preservation potential of selected strain Fungal biomass growth inhibition Fungal biomass growth inhibition by CFS of potent strain was determined following the method by Abouloifa et al. 2021 with some modi cations. Into a ask containing 10 ml of sterile nutrient broth, 1 ml of the sterile CFS of the isolate was aseptically inoculated and then 1 ml of the fungal spore of Aspergillus niger were inoculated into the broth. Fungal culture without CFS was used as control. The fungal biomass was collected by using Whatman grade I lter after an incubation of 5 days at 25°C. The fungal biomass was then dried in an oven at 100°C for 18 h and the percentage biomass growth inhibition was calculated by the formula: Biomass growth inhibition (BI %) =\frac{ \text{T}\text{c}-\text{T}\text{t}}{\text{T}\text{c}}× 100 Tc = The total fungal biomass obtained without CFS in grams (control) Tt = The total fungal biomass obtained with CFS in grams.

Bio-preservation of tomato puree
The bio-preservation of potent strain was evaluated on tomato puree, using the method of Abouloifa et al. 2021. Brie y twenty grams of tomato puree was prepared from fresh tomato, having an initial pH of 3.8, were introduced in sterile Petri dishes. An overnight grown I7B strain was centrifuged and 10 5 CFU/ml was spread on the surface of tomato puree, and then drop inoculated, in the centre of Petri dishes, with 100 µl of spore suspension (10 5 CFU/ml) of Aspergillus niger. The control sample was added with 1 ml of sterile distilled water and inoculates in the same condition of the other assay. The plates were incubated at 25℃ for 15 days, and the diameter of fungal colonies was measured. The percentage of fungal growth inhibition (I%) was calculated by the formula: Fungal growth inhibiton (I %) =\frac{ \text{T}\text{c}-\text{T}\text{t}}{\text{T}\text{c}} × 100 Tc = The total fungal colony diameter (mm) obtained in control Tt = The total fungal colony diameter obtained with tested isolates

Statistical analysis
All the experiments were performed in triplicates. The results attained were expressed as mean ±SD.

Isolation of gut bacteria
Totally four bacterial strains were isolated from the gut of the Stolipherous commersonnii and they were labelled as I7A, I7B, I7C, and I8. Colony morphology showed that all the four isolates were circular, medium-sized, white color, at, opaque and smooth.

Screening for heavy metal resistant bacteria
Observation of bacterial colonies in heavy metals (Cr, Pb, and Cu) loaded media indicated the capacity of bacterial tolerance against different concentrations (10, 20, 30 and 40 mg/ml) of heavy metals. Out of 4 isolates only 1 isolate (I7B) can grow in 10 mg/ml and 20 mg/ml of copper, chromium, and lead. The presence of bacterial colonies in the media containing 10 mg/ml and 20 mg/ml of copper, chromium, and lead exhibits the heavy metal resistant ability of isolate I7B after 48 h of incubation ( Fig. 4.1).

Assessment of biosorption ability of heavy metal resistant bacteria
The textile dye e uent was treated with I7B strain and incubated for 32 hours for heavy metal biosorption studies. Tests were carried out to evaluate the residual Cu, Cd, Zn, and Pb in the samples. The concentration of Cu was observed in the textile dye e uent. Heavy metal removal ability of the gut bacterial strains was assessed and represented in Fig. 4

Morphological characterization
Microscopic analysis of gram staining result revealed that the Isolate I7B is gram positive, rod shaped. Further, the FESEM analysis con rms the rod-shaped morphology of isolate I7B (Fig. 4.3).

Molecular characterization studies
To intensify the identi cation of selected bacterial isolate after preliminary morphological analysis, molecular identi cation of 16S rRNA gene is proceeded. The concentration of DNA isolated from I7B was 18.15 ng/µl and the ratio of absorbance was found to be 1.9. Hence, the isolated DNA can be selected for ampli cation using PCR. The amplicon of 1312 bp length were obtained after PCR ampli cation of the 16S rRNA gene and subjected to BLAST analysis. For the taxonomical identi cation of the selected isolate I7B, the phylogenetic tree was constructed indicated a considerable genetic homogeneity. The sequence showed 100% similarity with Bacillus siamensis ML121-1. Hence, it was named as Bacillus siamensis I7B and has been deposited in the database of National Center for Biotechnology Information (NCBI) as Bacillus siamensis strain I7B with accession no MW404539. The evolutionary relationship was inferred with the Neighbor-Joining method using MEGA-X software is shown in Fig. 4 Rifampin (5 µg), Streptomycin (10 µg) and Tetracycline (30 µg). I7B is more sensitive to Streptomycin (10 µg) and less sensitive to Rifampin (5 µg). Values are mean ± standard deviation

Aggregation activity
Auto-aggregation is a process which demonstrates the interrelation of probiotic strain forming colonization in the gastrointestinal tract through adhesion of probiotic strain to epithelial cells. Autoaggregation property of I7B strain after 5 h of incubation was found to be 28±0.011 %.
The results of auto-aggregation give the potential of self-aggregate competence of probiotic strain in adhesion to epithelial cells and mucus membrane.
The coaggregation potential of I7B strain with staphylococcus aureus was studied and in vitro studies serve as a primary screening of the nest probiotic strain. Results shows that I7B could coaggregate up to 27±0.16% with Staphylococcus aureus. Coaggregation of I7B strain with staphylococcus aureus enables them to form the barrier that facilitate in colonization of pathogen in the gastrointestinal tract.

Hemolytic activity assay
The hemolytic activity of the I7B strain was recorded by observing no clear zone or greenish zone around their colonies on the blood agar plate (Fig. 4.7). Hence, it illustrated the Gamma-hemolysis property of the I7B strain. It refers to the non-hemolytic property of the I7B strain. Hence it is safe and does not lyse red blood cells during probiotic administration.
Free radical scavenging ability of selected strain DPPH radical scavenging ability In order to investigate the antioxidant capacity of I7B strain, in vitro antioxidant assay has been performed. The scavenging DPPH capacity of intact cells and CFS were evaluated. The percentage of DPPH scavenging of intact cell and CFS was found to be 55.58±0.003% and 59.43±0.002% respectively (Table 4.3). Results shows good DPPH scavenging activity of I7B strain.

Assessment of bio-preservation potential of selected strain
Bio-preservation potential of Bacillus siamensis I7B strain was assessed with fungal biomass assay. Food bio-preservation potential of the Bacillus siamensis I7B strain was evaluated on tomato puree.

Fungal biomass growth inhibition assay
The fungal biomass growth inhibition was evaluated with the strain I7B. The obtained values for the cellfree supernatant of the I7B strain against fungus, Aspergillus avus are reported in Table 4.4. The I7B strain inhibits the growth of A. niger up to 42%. Fungal biomass growth inhibition assay was performed in triplicates. The percentage of survival was expressed as an average from three experiments ± standard deviation.

Bio-preservation effect of Bacillus siamensis I7B on tomato puree
Bio-preservation effect of I7B on tomato puree was tested. Test were performed in a petridish containing tomato puree and A. niger. The selected strain I7B was spread on the surface of the tomato puree. After 15 days of incubation at 25 ℃, the growth of A. niger was observed and showed in Fig. 4.8 and Table  4.5. Inhibition percentage results that the I7B strain has the potential to control the growth of A. niger in tomato puree up to 72%. From this result, it is evident that our I7B strain possesses good bio-preservative potential.

Discussion
Fish gut act as a reservoir of exclusive microorganisms which aid in maintaining the health of the host.
To study the bene cial activity of the gut microbes, the bacteria must be isolated and further characterized. Nowadays, probiotics were gained focus for their heavy metal detoxi cation In our study, we have isolated four bacterial isolates I7A, I7B, I7c and I8, and out of four isolates only I7B showed resistance to 10 mg/ml and 20 mg/ml of copper, chromium, and lead. Atomic absorption spectrometry results clearly demonstrated the removal of Cu on treatment with I7B strain in the textile dye e uent. Hence, we have chosen isolate I7B for morphological, phenotypic and probiotic characterization.
Morphological observation of gram staining and FESEM analysis proved the gram-positive nature of I7B isolate. Phenotypic characterization using 16s rRNA sequencing revealed that the strain in Bacillus siamensis and has been submitted in NCBI as Bacillus siamensis I7B strain with accession no MW404539.
Antibiotic sensitivity is an important parameter to safety assessment as the antibiotic resistant nature of the probiotic is not desirable (European Food Safety Authority, 2012). Bacillus siamensis I7B strain was resistant to Penicillin, Whereas sensitive to Erythromycin, Kanamycin, Rifampin, Streptomycin and Tetracycline. The antibiotic-resistant trait also stipulates that the isolates would survive in the condition by resisting the dreadful situation that occurred because of occasional high antibiotic concentrations. Aggregation activity assay is a preliminary screening test used for selecting potent probiotic strain, which demonstrates the colony forming ability of the microorganism's causing precipitation in the suspended medium. The aggregation ability is interrelated to the adhesion of microbes to host cells, i.e., A strain with good aggregation property can adhere to different types of host cells effectively (Malik et al. 2013). Autoaggregation (which takes place between microorganisms of the same strain) is a process in which the interrelation of probiotic strain forming colonization in the gastrointestinal tract through adhesion of probiotic strain to epithelial cells. Whereas Co-aggregation test reveals the interrelationship of probiotic adhesion to pathogenic strain. The co-aggregation property of probiotic strain prevents the pathogenic colonization in the host intestine (Balakrishna 2013). Auto-aggregation of Bacillus siamensis I7B strain was 28±0.011% after 5h of incubation and co-aggregation with S. aureus showed up to 27±0.16%. The auto-aggregation result portrays the self-aggregate competence of Bacillus siamensis I7B strain in adhesion to mucus membrane and epithelial cells. Sahoo  Hemolysis is a virulent factor, which causes infection in the resident living organisms via skin and mucous membranes (Ramesh et al. 2015;Nandi et al. 2017). Assessment of hemolytic activity of the strain is an important test to assess the safety of probiotic strain (Argyri et al. 2013). Hemolytic activity is important to assess the virulence factor of strain to lyse blood cells in the host. Hence, absents of hemolytic activity strain was characterized as an important property to assess a safe probiotic strain (Argyri et al. 2013). Bacillus siamensis I7B strain showed non-hemolytic property.
Oxidative stress is a major problem during heavy metal toxicity which causes elevated lipid peroxidation and reduction of antioxidant enzyme activity. This assay was performed in both intact cells and cell-free supernatant of the selected bacterial strains I7B. Intact cells act more effectively protecting intestinal epithelial cells against oxidative stress due to heavy metal ingestion. Cell-free supernatant was noticed to be absorbed by the small intestine and enters the bloodstream, involved in defense mechanism to prevent damage to other organs like kidney and liver due to oxidative stress (Ojekunle et al. 2017 Hydroxyl radical seems to be the most reactive oxygen radical causing damage to living cells. Hence, it is essential to scavenge the hydroxyl radical to inhibit oxidative damage (Zhang et al. 2011). Antioxidant molecules produced by the probiotics can remove and neutralize free radicals, protecting them from cellular damage (Yadav et al. 2009). These antioxidant metabolites produced by probiotics work with free radicals by donating a hydrogen atom to free radicals (Adnan et al. 2009). The hydroxyl radical is the most reactive oxygen radical and cause cell damage. And therefore, it is very important to reduce the amount of reactive oxygen by scavenging it. Upon Bacillus siamensis I7B analysis, the scavenging rate of CFS was higher than that of the intact cells, i.e., 25.15±0.009% of hydroxyl radical scavenging activity upon intact cells treatment and 27.6±0.010% in CFS. The hydroxyl radical scavenging activity of lactobacillus LM07 and LM19 was 7.83% and 86.64% (Delgado et al. 2021). Weisella confuse exhibited 24.65% of hydroxyl radical scavenging in CFE and 16.95% in IC (Sharma et al. 2018). Previous reports confer the higher hydroxyl radical scavenging activity of the Bacillus siamensis I7B strain might be due to production of metabolic compounds by the bacteria.
To study the bio-preservative potential of Bacillus siamensis I7B strain they are subjected for fungal biomass assay. The I7B strain inhibits the growth of A. niger up to 42%. Previous report also supports the results of the present study.

Conclusion
In this present study four bacterial strains namely I7A, I7B, I7C, and I8 were isolated from the gut of stolepherous commersonnii. Among the four isolates, isolate I7B showed more heavy metal resistant potential and atomic absorption spectrometry studies showed the maximum heavy metal (Cu) removal ability of 81.64% of I7B strain in textile dye e uent. The I7B strain is found to be gram-positive rodshaped bacterium and it was identi ed as Bacillus siamensis by 16S rRNA gene sequencing studies. The potent heavy metal resistant strain was named as Bacillus siamensis I7B and has been submitted in NCBI GenBank as Bacillus siamensis strain I7B with accession number MN404539. Antibiotic susceptibility test of bacillus siamensis I7B showed high sensitivity to Streptomycin (10 µg) and less sensitive to Rifampin (5 µg) and resistant to pencillin (2 µg, 10 µg). The study con rmed that Bacillus siamensis I7B strain possess signi cant probiotic potential with high acid and bile tolerance, good aggregation ability, effective antibiotic susceptibility and non-hemolytic activity. Bacillus siamensis I7B strain possess good DPPH free radical scavenging ability and moderate hydroxyl radical scavenging ability. Bacillus siamensis I7B strain inhibits the growth of A. niger up to 42%. Biopreservation effect of Bacillus siamensis I7B strain on tomato puree showed 72% growth inhibition against A. niger for 15 days. However, further in vivo studies are essential to assess the probiotic strain in animal models.

Conflict of interest
All authors declare that they have no conflict of interest.

Funding
The requirement for the project was not supported by any funding agency.
Author's contribution AUTHOR NAME

Data availability
The data that support the ndings of this study are available from the corresponding author, upon reasonable request. Figure 1 Screening the heavy metal resistant bacteria   Acid tolerance test of Bacillus siamensis I7B strain

Figures
The acid tolerance test was performed in triplicates. Values are mean ± standard deviation.

Figure 6
Bile tolerance assay of Bacillus siamensis I7B strain The bile tolerance assay was performed in triplicates. Values are mean ± standard deviation. Figure 7