Abstract
This study focuses on isolation, screening, and characterization of novel probiotics from gastrointestinal tract of free-range chicken (Gallus gallus domesticus). Fifty seven colonies were isolated and three isolates (FR4, FR9, and FR12) were selected and identified as Lactobacillus gasseri FR4, Bacillus tequilensis FR9, and L. animalis FR12 by 16S rRNA sequencing. Three strains were able to survive in stimulated acidic and bile conditions and inhibit the growth of pathogens. Especially, FR9 exhibited maximum inhibition against Listeria monocytogenes and none of them exhibited hemolytic activity. Native-PAGE revealed the presence of low molecular weight (3.4–5.0 KDa) antimicrobial peptide. The peptide was further purified by Sephadex G-50 column and RP-HPLC using C18 column. N-terminal amino acid sequencing of antimicrobial peptide showed 100% consensus to antilisterial peptide Subtilosin A and SboA gene was amplified from FR9 genome. FR9 showed maximum aggregation activity, exopolysaccharide production (85.46 mg/L) and cholesterol assimilation (63.12 ± 0.05 μg/mL). Strong adhesion property (12.6%) and pathogen invasion protection ability was revealed by B. tequilensis FR9 towards HCT-116 human colon carcinoma cell line. This is the first study to demonstrate antilisterial Subtilosin A production of B. tequilensis. Our results indicate that B. tequilensis FR9 strain furnish the essential characteristics of a potential probiotics and might be incorporated into human and animal food supplements.
Highlights
Probiotic microorganisms are live bacteria, which modulates the host immune system and maintain the intestinal microbial balance (Joint FAO/WHO, 2002)
The present study has revealed that L. gasseri FR4, B. tequilensis FR9, and L. animalis FR12 strains isolated from healthy free-range chicken’s gastrointestinal tract (GIT) displayed better performance in all in vitro assays, which is essential to be considered as probiotics
B. tequilensis FR9 was recognized as a reliable probiotic candidate, since they produce antilisterial
Summary
Probiotic microorganisms are live bacteria, which modulates the host immune system and maintain the intestinal microbial balance (Joint FAO/WHO, 2002). Subtilosin A from Bacillus tequilensis FR9 the essential probiotic characteristic and produce group of antimicrobial peptides with broader inhibition spectrum (Khochamit et al, 2015). Probiotic Lactobacillus and Bacillus strains isolated from diverse sources are used as probiotic candidates and it is unlikely that each species/strain possesses all the preferred characteristics that will make it a felicitous probiotic (Swain et al, 2014). A potential probiotic bacteria must fulfill certain fundamental criteria, such as ability to adhere on intestinal epithelium, to overcome potential obstruction, including low pH of the stomach, presence of bile acids in intestines, compete/antagonize enteric pathogens, susceptibility to commonly used antibiotics, cholesterol lowering effect and colonize the intestinal cell wall (Garriga et al, 1998; Chiang and Pan, 2012; Anandharaj et al, 2015). Probiotic microbes synthesis various biologically active compounds including enzymes, bacteriocins, exopolysaccharides (EPS), vitamins, and organic acids and these compounds has immense industrial applications (Nel et al, 2001; Walling et al, 2005)
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