In vitro screening and characterization of lactic acid bacteria from Lithuanian fermented food with potential probiotic properties

Abstract

The present work aimed to identify probiotic candidates from Lithuanian homemade fermented food samples. A total of 23 lactic acid bacteria were isolated from different fermented food samples. Among these, only 12 showed resistance to low pH, tolerance to pepsin, bile salts, and pancreatin. The 12 strains also exhibited antimicrobial activity against Staphylococcus aureus ATCC 29213, Salmonella Typhimurium ATCC 14028, Streptococcus pyogenes ATCC 12384, Streptococcus pyogenes ATCC 19615, and Klebsiella pneumoniae ATCC 13883. Cell-free supernatants of isolate 3A and 55w showed the strongest antioxidant activity of 26.37 μg/mL and 26.06 μg/mL, respectively. Isolate 11w exhibited the strongest auto-aggregation ability of 79.96% as well as the strongest adhesion to HCT116 colon cells (25.671 ± 0.43%). The selected strains were tested for their synbiotic relation in the presence of a prebiotic. The selected candidates showed high proliferation in the presence of 4% as compared to 2% galactooligosaccharides. Among the strains tested for tryptophan production ability, isolate 11w produced the highest L-tryptophan levels of 16.63 ± 2.25 μm, exhibiting psychobiotic ability in the presence of a prebiotic. The safety of these strains was studied by ascertaining their antibiotic susceptibility, mucin degradation, gelatin hydrolysis, and hemolytic activity. In all, isolates 40C and 11w demonstrated the most desirable probiotic potentials and were identified by 16S RNA and later confirmed by whole genome sequencing as Lacticaseibacillus paracasei 11w, and Lactiplantibacillus plantarum 40C: following with the harboring plasmid investigation. Out of all the 23 selected strains, only Lacticaseibacillus paracasei 11w showed the potential and desirable probiotic properties.

Keywords: GIT-gastrointestinal tract; fermented food; in vitro screening; lactic acid bacteria; potential probiotics; probiotics LAB-lactic acid bacteria; safety assessment; tryptophan.

Figure 1

Acid resistance of lactic acid bacteria in phosphate-saline buffer (pH 2). Values are expressed as mean ± standard deviation (n = 3). Bars with the same lower-case letters are not significantly different, whereas those with different lower-case letters are significantly different (p < 0.05). The dotted line represents the minimum percentage survival requirement of the individual isolates.

Figure 2

Resistance of lactic acid bacteria to pepsin (pH = 2). Values are expressed as mean ± standard deviation (n = 3). Bars with the same lower-case letters are not significant, whereas those with different lower-case letters are significantly different (p < 0.05). The dotted line represents the minimal requirement of survival of the individual isolates.

Figure 3

Viability of lactic acid bacteria in the presence of simulated intestinal fluid (0.3% (w/v) bile salt and 1 mg/mL pancreatin in peptone water, pH 7.2). Values are expressed in mean ± standard deviation (n = 3). Bars with the same lower-case letters are not significantly different, whereas those with different lower-case letters are significantly different (p < 0.05). The dotted line represents the minimal requirement of survival of the individual isolates.

Figure 4

Trolox equivalent antioxidant concentration of 13 LAB CFS. Results are expressed as mean of triplicate values ± standard deviation (n = 3). Bars with the same lower-case letters are not significantly different, whereas those with different lower-case letters are significantly different (p < 0.05).

Figure 5

Auto-aggregation abilities of probiotic candidates after 20 h incubation at 37°C. Each value represents the mean ± standard deviation of three independent readings (n = 3). ^*Significant differences at p < 0.05, and ^***significant difference at p < 0.001.

Figure 6

The ability of LAB to adhere to the HCT-116 cell lines. Results are expressed as mean of triplicate values ± standard deviation (n = 3). Bars with the same lower-case letters are not significant, whereas those with different lower-case letters are significantly different (p < 0.05).

Figure 7

The growth curves of LAB isolates were measured at 600 nm with 2% GOS and 4% GOS. Growth curve of (A) 11w supplemented with 2% GOS compared with 11w with 4% GOS, (B) growth curve of 33E supplemented with 2% GOS compared with 33E 4% GOS, (C) growth curve of 40C supplemented with 2% GOS compared with 40C 4% GOS, (D) growth curve of 48C supplemented with 2% GOS compared with 4% GOS, and (E) the growth curve of 66 W supplemented with 2% GOS compared with 66 W 4% GOS. Each value represents the mean ± standard deviation of three independent readings (n = 3 ± SD). ^*Significant differences at p < 0.05, ^**significant difference at p < 0.01, and ^***significant difference at p < 0.001.

Figure 8

Neighbor-joining phylogenetic tree of LAB isolates based on 16S rRNA gene sequences. Bootstrap values based on 1,000 replications are listed as percentages at branch points. The scale bar represents a 0.02% divergence.

Figure 9

Mucin degradation ability of probiotic candidates. (A) growth curve of 11w (Lacticaseibacillus paracasei 11w) supplemented with 0.5% mucin compared with 11w without mucin, (B) growth curve of 33E (Lactiplantibacillus paraplantarum 33E) supplemented with 0.5% mucin compared with 33E without mucin, (C) growth curve of 40C (Lactiplantibacillus plantarum 40C) supplemented with 0.5% mucin compared with 40C without mucin, and (D) the growth curve of 66 W (Lactiplantibacillus paraplantarum 66 W) supplemented with 0.5% mucin compared with 66 W without mucin, (E) growth curve of E. coli ATCC 3515 supplemented with 0.5% mucin compared with E. coli ATCC 3515 without mucin. ^*Significant differences at p < 0.05, ^**significant difference at p < 0.01, and ^***significant difference at p < 0.001.

Figure 10

Gelatin degradation ability of LAB. Bacteria with gelatinase activity exhibited by positive control; Staphylococcus aureus ATCC 29213 shows clear zones around the bacteria colony, whereas the negative control Lactobacillus casei has no zone formation. 11w = Lacticaseibacillus paracasei 11w, and 40C = Lactiplantibacillus plantarum 40C. Staphylococcus aureus ATCC 6538 was used as a positive control. (+) indicates positive results and (−) indicates negative results.