Purification of Antilisterial Peptide (Subtilosin A) from Novel Bacillus tequilensis FR9 and Demonstrate Their Pathogen Invasion Protection Ability Using Human Carcinoma Cell Line

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.

Screening of cattle gut associated Bacillus strains for their potential use as animal probiotic

“…In present study, all Bacillus strains showed no hemolysis. Parveen et al, (2016) had worked on probiotic potential of Bacillus strains and their results were in accordance with present findings in which B. tequilensis FR9 showed no hemolysis. Similar results were given in the one of the experiment of Luis-Villasenor et al (2011), who noted that Bacillus species showed alpha hemolysis.…”

“…The strains B. tequilensis was the most significantly (p<0.05) resistant strain (Table 4). Similar results were given by Parveen et al (2016). A vital property of the probiotic strains is the antipathogenic activity.…”

“…Over all pathogenicity results revels that NCCP-2004a showed maximum (p<0.05) inhibition against all the tested pathogenic strains. This might be due to the antimicrobial protein produced from the B. tequilensis (Parveen et al, 2016). Bacillus species can spread from its habitat to feed and food particles due to its endospore production and its long term survival ability.…”

“…This means the Bacillus species did not show any risk to host. Recently, it has been noted that, B. tequilensis could be used as human probiotic because it produces the novel silver nanoparticles and showed significant cytotoxic effects against cancer (Parveen et al, 2016).…”

“…The tree was constructed from unambiguously arranged nucleotides sequence using an algorism by using MEGA 7 (Saitou and Nei, 1987). The acid tolerance was determine by methods given by Parveen et al, (2016). Briefly, Bacillus strains were growth in MRS broth (pH; 1.0, 2.0, 3.0 and 7.0) and culture plates were incubated at 37 C for 20 h aerobically.…”