In vitro screening for potential probiotic properties of Ligilactobacillus salivarius isolated from cattle calves

Abstract

• 41 lactic acid bacteria (LAB) strains, isolated from newborn calves' feces were identified as Ligilactobacillus salivarius based on biochemical tests and a molecular approach. • 9 isolates were screened and evaluated for probiotic attributes, biosafety aspects, prebiotic activity scores, and adhesion ability to the HT29 cell line. • Among the evaluated LAB isolates, RBL38 and RBL68 emerged as promising probiotic candidates based on heat-map and principal component analysis (PCA). This research was aimed at the development of a probiotic Lactobacillus culture for administration to cattle calves to treat and/or prevent gastrointestinal disorders, as an alternative to antibiotics. A total of 41 lactic acid bacteria (LAB) strains were isolated from the feces of newborn calves and categorized as Lactobacillus on the basis of biochemical tests and genus-specific PCR. Further, nine strains were selected for in vitro probiotic tests and were confirmed to be Ligilactobacillus salivarius through 16S rRNA sequencing. Among these nine strains, the highest cell surface hydrophobicity was 64.45% for RBL76 and 66.45% for RBL62 in n-hexadecane and xylene, respectively, and auto-aggregation results in RBL76 were satisfactory (60.26%). All nine isolates showed optimal production of amylase, protease, and β-galactosidase enzymes. The antimicrobial and co-aggregation activities of all isolates were also determined against the pathogenic microorganisms E. coli ATCC-25922 and Salmonella arizonae ATCC 13314. The biosafety assessment of all selected isolates revealed that they were non-hemolytic and did not exhibit mucin degradation, showed sensitivity to nine antibiotics (ampicillin, chloramphenicol, clindamycin, erythromycin, gentamicin, kanamycin, streptomycin, trimethoprim, and tylosin) and showed resistant to two antibiotics (nalidixic acid and oxytetracycline). The isolates exhibited substantial antioxidant activity, as tested via the DPPH (2,2-diphenyl-1-picryl-hydrazyl-hydrate) and ferric ion reducing antioxidant power methods. RBL40 and RBL50 showed a prebiotic index of more than 1 for fructo-oligosaccharide, and RBL40 and RBL38 displayed the highest prebiotic activity scores with inulin (1.58±0.04 and 1.46±0.02, respectively), followed by RBL40 (1.44±0.02), with fructose, RBL68 (1.36±0.02), and RBL50 (1.33±0.02) with inulin. In addition, the highest adhesion ability to the HT29 cell line was observed for the isolate RBL50 (13.86±0.43%). Among the evaluated LAB isolates, RBL38 and RBL68 emerged as promising probiotic candidates on the basis of heat maps and principal component analysis.