Vol 14, Issue 12, 2021 Online - 2455-3891 Print - 0974-2441 PROBIOTIC CHARACTERIZATION OF BACILLUS SUBTILIS STRAIN ISOLATED FROM INFANT FECAL MATTER REVEALED BY 16S rRNA GENE AND PHYLOGENETIC ANALYSIS DEVARANJAN DAS 1 , CHANDI CHARAN RATH 2 , NAKULANANDA MOHANTY 1 , SMITA HASINI PANDA 1 * 1 Department of Zoology, Maharaja Sri Ram Chandra Bhanja Deo University, Baripada, Odisha, India. 2 Department of Life Sciences, Rama Devi Women’s University, Bhubaneswar, Odisha, India. Email: panda.smita@gmail.com Received: 20 September 2021, Revised and Accepted: 03 November 2021 ABSTRACT Objective: The rationale of our study was to isolate and identify the putative probiotic strain from infant fecal matter exhibiting a broad range of antimicrobial activity and to analyze the effect of different culturing conditions on its probiotic properties and the production of antimicrobial metabolites. Methods: In the present study, bacterial strains were screened for probiotic properties and antimicrobial activity from infant fecal matter (6 months–2 years). The effect of varying culture conditions such as tolerance to acid, bile salt, phenol, NaCl, pH, incubation period, and temperature along with autoaggregation assay, hydrophobicity, and hemolysis was studied. The characterization of the potent strain was studied by morphological, biochemical, and 16S rRNA gene sequencing along the phylogenetic affiliation of the strain was studied. Results: Two putative probiotic bacteria (DAM and IFM) were isolated, identified, characterized, and predicted at pH 2.0, 3.0, and 4.0, the isolate IFM had 50%, 60%, and 70% survivability, while isolate DAM had 55%, 63%, and 75% survivability, respectively. At a bile salt concentration of 0.5%, both isolates had a 75% survival rate. The isolates exhibited a high percentage of hydrophobicity and autoaggregation. The isolates also had non- hemolytic activity and were susceptible to many clinical tested antibiotics (tetracycline, erythromycin, ampicillin, gentamycin, penicillin, etc.). The isolate showed antimicrobial activity against enteric pathogens such as Staphylococcus aureus, Escherichia coli, and Shigella dysenteriae. The accession number of Bacillus subtilis MT279753 and MK453362 was submitted to NCBI. Conclusion: The result revealed that isolates have potent probiotic properties and possess a direct influence on the production of antimicrobial metabolites. These parameters can be modified for the improvement of the potentiality of the isolates. Keywords: Probiotics, Antimicrobial activity, Phylogenetic analysis, Bacillus spp., 16s rRNA Secondary structure. INTRODUCTION Probiotics are considered to be live microbial feed supplements. It is known to exert beneficial effects on the host by improving its intestinal microbial balance [1,2]. Most of the commercially available probiotic strains are Lactobacillus sp. and Bifidobacterium sp [3,4]. However, Bacillus sp. have been considered as potential probiotic strains in various dietary supplements [5,6]. The advantage of Bacillus sp. over other probiotic strains is they can survive in foods that require harsh processing conditions such as high temperature and pressure [7]. Bacillus sp. which are commonly used in different feed supplements as probiotics are Bacillus coagulans, Bacillus subtilis, Bacillus licheniformics, Bacillus cereus, Bacillusnatto (subtilis), Bacillus clause, Bacillus pumilus, Bacillus amyloliquefaciens, and Bacillus polyfermenticus [8,7]. Bacillus sp. are considered to be a highly potential probiotic due to its beneficial effects i.e. secretion of antimicrobials, stimulation of immune system, resistance to high acid, bile salt, non-hemolytic activity, and improvement of the gut microbiome [4,8]. In general, B. subtilis as a probiotics strain has shown outstanding health-boosting records [9-12]. Their application with certain immune- deficient populations especially for critically ill, neonates, and elderly groups should be evaluated and regulated carefully since reports related to bacteria in an immune-compromised patient treated with spore former and other probiotics have been recorded, repetitively [13]. Food additives and therapeutic supplements are lately seeing extensive use of probiotics, especially as digestion enhancers. Ubiquitously found endospore-forming Gram-positive bacterium of the Bacillus group produces several vitamins and digestive enzymes and B. subtilis are used as probiotics. In the present study, we undertook the task of screening and examining potential probiotic bacteria, particularly those with antimicrobial and bacteriocin-producing properties from the feces of healthy newborn babies collected from Mayurbhanj District Hospital. The probiotic products being marketed as functional food, dietary supplements, or drug are also elucidated by the state of probiotic strains. The quality of commercial probiotic products is an important issue to be considered for regulation. In our research, the findings proved the probiotic potency of known probiotic B. subtilis is used as the clear implication for future research in the field and to develop our isolates as probiotics for human and animal nutrition. Studies and screening of lactic acid bacteria (LAB) from fermented vegetables, steamed buns, yogurt, and other materials have previously been pursued by many researchers but a few attempts is initiated to screen antibacterial LAB from the feces of newborn infants. METHODS Isolation Samples were collected from infant fecal matter (6 months–2 year old), from Mayurbhanj district hospital, Mayurbhanj, Odisha, India. They were diluted by serial dilution method and plated using MRS agar media by spread plate and pour plate method. Plates were then incubated at 37°C for 48 h. Colonies were randomly selected based on morphological differences. The isolates were maintained on MRS agar slants at 30°C and preserved as frozen glycerol stocks. From the stock, propagation of isolates was done to MRS broth medium which was considered as working culture. © 2021 The Authors. Published by Innovare Academic Sciences Pvt Ltd. This is an open access article under the CC BY license (http://creativecommons.org/ licenses/by/4.0/) DOI: http://dx.doi.org/10.22159/ajpcr.2021v14i12.43204. Journal homepage: https://innovareacademics.in/journals/index.php/ajpcr Research Article