Metagenomic evaluation, antimicrobial activities, and immune stimulation of probiotics from dietary supplements and dairy products

Abstract

Probiotics are widely marketed as dietary supplements and dairy products for their purported antimicrobial and immunomodulatory activities, often with limited supporting evidence. We identified and isolated probiotics from commercial dietary supplements and dairy products using metagenomics and cultured-based methods. We assessed their anti-bacterial activity against diverse pathogens and investigated their immunomodulatory effects on phagocytes and natural killer (NK) cells. Metagenomic analysis revealed that Lactobacillus and Bifidobacterium were the predominant genera in dietary supplements, while Streptococcus spp. was dominated in dairy products. However, only 37% of the predominant microorganisms identified by metagenomics were accurately listed on product labels. Among 70 representative probiotic strains, 4.3-17.1% probiotic strains demonstrated strong antibacterial-effects against pathogenic bacteria. Notably, specific strains of Bifidobacterium longum and Lactobacillus plantarum exhibited strong antagonistic activity against extended-spectrum beta-lactamase-producing and carbapenem-resistant Escherichia coli. Some strains of Lactobacillus spp. significantly enhanced phagocytic activity in monocytes and increased IFN-γ production in NK cells, while members of Lactobacillus rhamnosus significantly suppressed TNF-α, IL-6, and IL-8 production in lipopolysaccharide-stimulated macrophages. In contrast, Bifidobacterium animalis stimulated the production of anti-inflammatory cytokines. This study highlights discrepancies in probiotic labeling and demonstrates the antimicrobial and immunomodulatory potential of specific probiotic strains, suggesting their utility in enhancing health and wellness.

Keywords: Antimicrobial activity; Dairy products; Immune response; Metagenomic; Probiotics; Supplements.

Fig. 1

Metagenomic profiles of probiotics in dietary supplements (A) and dairy products (B).

Fig. 2

The comparison between the microorganism species declared on the labels of probiotic supplement products and those identified through metagenomic sequencing using bioinformatic analyses and cultivation methods. The comparison was performed from six perspectives. First, microbes present in the supplement that were declared on the label and detected through analysis (green). Second, microbes were declared on the label but detected at a very low abundance in the analysis (less than 1% of the reads) (yellow). Third, microbes were declared on the label but not detected via metagenomic analyses (red). Fourth, microbes were detected within the supplements’ metagenomic but were not declared on the label (purple). The numbers in the table indicated the % abundance of microbe in the metagenomic analysis. Fifth, microbes indicate with a star were detected in the supplement’s metagenomics and grown as a single colony and identified by MALDI-TOF. Finally, microbes indicate with a G were grown as a single colony and identified by MALDI-TOF but were not declared on the label and were undetectable by metagenomics.

Fig. 3

Antibacterial activity of probiotic isolates against antibiotic resistance bacteria by agar overlay method. (A) Agar overlay method demonstrated the antibacterial activity of live L. rhamnosus Y03.2 against five bacterial pathogens used as target strains, including E. coli ATCC25922, E. coli PB1 (ESBL), E. coli PB231 (CRE), S. aureus ATCC25923 and S. aureus DMST20654 (MRSA). The inhibition zones were observed surrounding probiotic spots (arrows). The diameter of the inhibition zone (mm), including probiotic spots (approximately 5 mm in diameter) was measured, and reported as means of two independent experiments. (B) The zone of inhibition of 70 tested probiotic strains was summarized in a heatmap chart. The diameter of the inhibition zone was interpreted as follows: >20 mm as a strong activity (red), 10–20 mm as a moderate activity (yellow), and < 10 mm as a weak inhibition (bright yellow).

Fig. 4

Effect of probiotics on phagocytic activity. The phagocytic index is defined as % phagocytosis of monocyte cells × mean fluorescence intensity. Monocyte with RPMI medium was used as the negative control (No treatment), and monocyte treated with PMA was the positive control. Each color indicates probiotic species. Data were presented as bar graphs with mean ± standard deviation from three independent experiments. One-way ANOVA was used to test the difference between probiotic bacteria affecting phagocytic activity. (****, P < 0.0001, ***, p < 0.001, **, p < 0.01).

Fig. 5

Effect of probiotics on cytokine production. Effect of probiotics on cytokine profiles from THP-1-derived macrophages or stimulated with LPS 1 µg/ml for 18 h. The supernatant was collected to measure cytokine profiles using Luminex assay. Cells with RPMI medium was used as a negative control and cells treated with LPS 1 µg/ml alone was used as a positive control. (A) IL-6 production from THP-1-derived macrophages after stimulating with probiotic strains. (B–F) IL-6, IL-8, IL-12p70 and TNF-α levels from cells treated with LPS and probiotic strains for 18 h. (G–H) IL-1RA and IL-10 from cells treated with LPS and probiotic strains for 18 h. (Mann-Whitney test; *, p < 0.05, **p < 0.01). (Ban; B. animalis, Blo; B. longum, Lpa; L. paracacei, L. plantarum, Lrh; L. rhamnosus, Lre; L. reuteri, Lsa; L. salivarius, Lfe; L. fermentum, Lga; L. gasseri, Lde; L. delbrueckii, Lac; L. acidophilus, Sth; S. thermophilus, Efa; E. faecium, Ega; E. gallinarum, Sce; Saccharomyces cerevisiae)

Fig. 6

IFN-γ production by NK-92 MI cells activated with different probiotics. NK-92 MI cells were incubated with 70 isolates of probiotic bacteria from 16 species for 24 h. Mixture of PMA and ionomycin was a positive control and medium was a negative control. The supernatant was collected to measure IFN-γ concentration using ELISA assay. Data were represented as a bar graph with mean ± standard deviation from three independent experiments. One-way ANOVA was used to test the difference between probiotic bacteria affecting NK cell-derived IFN-γ production. ***, p < 0.0001.