Unlocking the Gut’s Treasure: Lipase-Producing Bacillus subtilis Probiotic from the Intestine of Microstomus kitt (Lemon sole)

• Published in: Applied biochemistry and biotechnology 2024-07, Vol.196 (7), p.4273-4286
• Main Authors: Saravanakumar, Sivaneshwaran, Prabakaran, Naresh Narayanan, Ashokkumar, Rathinavel, Jamuna, S.
• Format: Article
• Language: English
• Subjects: agar; Animals; Bacillus; Bacillus subtilis; Bacillus subtilis - enzymology; Bacillus subtilis - isolation & purification; Bacterial Proteins - genetics; Bacterial Proteins - metabolism; Bile; Bile Acids and Salts - metabolism; Bile salts; Biochemistry; Biotechnology; carboxylic ester hydrolases; Chemistry; Chemistry and Materials Science; Enzymatic activity; Enzyme activity; Enzymes; Hydrogen-Ion Concentration; Industrial applications; Intestine; intestines; Intestines - microbiology; Isolation media; Lipase; Lipase - metabolism; Marine fish; Microstomus kitt; Nucleotide sequence; Olive oil; Original Article; Probiotics; RNA; Salinity tolerance; Salt tolerance; Selective media; Sequence analysis; Sodium chloride; Sole; species; Strain analysis; Substrates; viability
• ISSN: 0273-2289; 1559-0291; 1559-0291
• DOI: 10.1007/s12010-023-04749-7

The main objective of this research was to identify potential probiotic candidates belonging to the Bacillus species that could demonstrate tolerance to bile salt and acidic conditions. The study focused on isolating Bacillus strains from the intestine of marine fish— Microstomus kitt . The isolation process involved the use of selective MRS media through the pour plate method. After 24 h, one particular isolate was identified based on its morphological and biochemical traits as Bacillus species. To confirm the identity, molecular characterization of the 16S RNA from the isolated strain was performed, and the sequence analysis verified it as Bacillus subtilis strain ACL_BS 001. With the molecular confirmation, the next step was to assess the probiotic characteristics of this B. subtilis strain. Various tests were conducted to evaluate its acid/pH tolerance, NaCl tolerance, and bile salt tolerance. The results indicated that B. subtilis exhibited high viability percentages even under acidic pH, in the presence of 1.5% bile salt, and at high salt concentrations. Subsequently, we investigated the strain’s ability to produce lipase, an important enzyme with potential industrial applications. B. subtilis was grown in MRS agar amended with olive oil as a lipase substrate. After incubation, the presence of lipase activity was confirmed, and the enzymatic assay revealed a significant lipase enzyme activity of 100.23 µmoles/ml of the sample. In conclusion, the study successfully isolated and identified B. subtilis from the intestine of Microstomus kitt , and the strain exhibited promising probiotic characteristics, including resistance to bile salt and acidic conditions. Furthermore, the strain was found to produce lipase, which opens up possibilities for future research focusing on isolating and purifying the lipase from this potential probiotic B. subtilis strain.