Probiotic potential of β‑galactosidase‑producing lactic acid bacteria from fermented milk and their molecular characterization

Abstract

Probiotics have attained significant interest in recent years as a result of their gut microbiome modulation and gastrointestinal health benefits. Numerous fermented foods contain lactic acid bacteria (LAB) which are considered as GRAS and probiotic bacteria. The present study aimed to investigate indigenous LAB from homemade fermented milk samples collected in remote areas of Karnataka (India), in order to isolate the most potent and well-adapted to local environmental conditions bacteria, which were then evaluated using a step-by-step approach focused on the evaluation of probiotic traits and β-galactosidase-producing ability. LAB were screened using 5-bromo-4-chloro-3-indole-D-galactopyranoside (X-Gal) and O-nitrophenyl-β-D-galactopyranoside (ONPG) as substrate, and exhibited β-galactosidase activity ranging from 728.25 to 1,203.32 Miller units. The most promising isolates were selected for 16S rRNA gene sequence analysis and identified as Lactiplantibacillus plantarum, Limosilactobacillus fermentum, Lactiplantibacillus pentosus and Lactiplantibacillus sp. Furthermore, these isolates were evaluated by in vitro, viz., survival in gastrointestinal tract, antibiotic susceptibility, antimicrobial activity, cell surface characteristics, and haemolytic activity. All eight isolates demonstrated strong adherence and prevented pathogen penetration into HT-29 cells, indicating potential of the bacteria to scale up industrial level production of milk products for lactose intolerants.

Keywords: GenBank; HT-29 cells; LAB; ONPG; X-Gal; lactose intolerance; probiotic; β-galactosidase.

Figure 1

(A) Bacterial colonies (deep grey colour) were identified on a de Man, Rogosa and Sharpe agar plate containing 5-bromo-4-chloro-3-indole-D-galactopyranoside, for β-galactosidase production. (B) β-Galactosidase production and bar graph represents β-galactosidase activity of Lactiplantibacillus isolates in Miller units. Bar graph was plotted incorporating the sample mean (n=3) and error bar (standard deviation of individual isolate).

Figure 2

Carbohydrate fermentation profile of Lactiplantibacillus isolates.

Figure 3

Phylogenetic neighbor-joining tree based on the 16S rDNA sequences (~1,300-1,500 bp) of the eight selected lactic acid bacteria strains of curd samples based on the results of alignment. The scale bar represents 0.10 nucleotide substitutes per position.

Figure 4

Gastric, bile, and pancreatic juice tolerance tests of Lactiplantibacillus isolates, expressed as percentages [the bar graph was plotted incorporating the sample mean (n=3) and error bar of individual isolates].

Figure 5

(A) Hydrophobicity of Lactiplantibacillus isolates with xylene and chloroform. (B) Auto-aggregation expressed as a percentage after each hour of incubation. (C) Co-aggregation expressed as a percentage after 5 h of incubation with E. coli and L. monocytogenes. (D) HT-29 cell adhesion of Lactiplantibacillus isolates expressed as a percentage after 3 h of incubation. E. coli, Escherichia coli; L. monocytogenes, Listeria monocytogenes.

Figure 6

(Aa) Control HT-29 cell line monolayer and (Ab) arrow showing adhered Gram-positive Lactiplantibacillus isolate to HT-29 cells (magnification, x1,000). (B) HT-29 cell invasion of human pathogens E. coli and L. monocytogenes expressed as a percentage after 3 h of incubation with Lactiplantibacillus spp. E. coli, Escherichia coli; L. monocytogenes, Listeria monocytogenes.