Functional characterization and immunomodulatory properties of Lactobacillus helveticus strains isolated from Italian hard cheeses

Abstract

Lactobacillus helveticus carries many properties such as the ability to survive gastrointestinal transit, modulate the host immune response, accumulate biopeptides in milk, and adhere to the epithelial cells that could contribute to improving host health. In this study, the applicability as functional cultures of four L. helveticus strains isolated from Italian hard cheeses was investigated. A preliminary strain characterization showed that the ability to produce folate was generally low while antioxidant, proteolytic, peptidase, and β-galactosidase activities resulted high, although very variable, between strains. When stimulated moDCs were incubated in the presence of live cells, a dose-dependent release of both the pro-inflammatory cytokine IL-12p70 and the anti-inflammatory cytokine IL-10, was shown for all the four strains. In the presence of cell-free culture supernatants (postbiotics), a dose-dependent, decrease of IL-12p70 and an increase of IL-10 was generally observed. The immunomodulatory effect took place also in Caciotta-like cheese made with strains SIM12 and SIS16 as bifunctional (i.e., immunomodulant and acidifying) starter cultures, thus confirming tests in culture media. Given that the growth of bacteria in the cheese was not necessary (they were killed by pasteurization), the results indicated that some constituents of non-viable bacteria had immunomodulatory properties. This study adds additional evidence for the positive role of L. helveticus on human health and suggests cheese as a suitable food for delivering candidate strains and modulating their anti-inflammatory properties.

Fig 1. Production of IL-12p70 and IL-10 cytokines in co-culture experiments between moDCs and bacterial cells.

The production of IL-12p70 (a, b, c) and IL-10 (d, e, f) cytokines, in co-culture experiments between moDCs and bacterial cells of Lactobacillus helveticus SIM12, Lh43, SIS16, 1734 in the presence of lipopolysaccharides (LPS) (b, e), Salmonella FB62 (c, f) or in absence of stimulus (a, d) were displayed. Controls: non stimulated (panels a, d) or stimulated (panels b, c, e, f) moDCs without bacterial cells. Statistical significance of single bacteria strain was evaluated by using two-way ANOVA followed by multiple comparisons (Tukey) against the control. Values represent the means ± SD, 95% CI, (*P<0.05, **P<0.01, ***P<0.001, ****P<0.0001).

Fig 2. Production of IL-10 and IL-12p70 cytokines in co-culture experiments between moDCs and LPS and Salmonella strain in presence of postbiotics.

The production of IL-10 and IL-12p70 cytokines, in co-culture experiments between moDCs and lipopolysaccharides (LPS) (a, b) or Salmonella FB62 (d, e) in presence of postbiotics (expressed as % added supernatants, % SN -25%, 12,5%,6,25%, 3,12%, 1,56% -) of Lactobacillus helveticus SIM12, Lh43, SIS16, and 1734, were displayed. The anti-inflammatory index (IL-10/IL-12p70) is reported for each strain (c, f). Statistical significance of single bacteria strain was evaluated by using two-way ANOVA followed by multiple comparisons (Tukey) against the control and the serial dilution values (b, e). Values represent the means ± SD, 95% CI, (*P<0.05, **P<0.01, ***P<0.001, ****P<0.0001).

Fig 3. Production of IL-12p40 and IL-10 cytokines in co-culture experiments between PBMCs and LPS in presence of homogenized cheese or homogenized and pasteurized cheese.

The production of IL-12p40 (panels a, d) and IL-10 (panels b, e) cytokines in co-culture experiments between PBMCs and lipopolysaccharides (LPS) in presence of homogenized cheese (TQ-CS), or homogenized and pasteurized cheese (P-CS) inoculated with Lactobacillus helveticus strains SIM12 and SIS16 and expressed as % of added supernatants (% SN—12,5%, 6,25%, 3,12%, 1,56%, 0,78% -) were showed. Unfermented milk samples acidified at pH 5.3 with lactic acid were used as controls (CNTR). The anti-inflammatory index (IL-10/IL-12p40, panels c, f) is indicated. Statistical significance of single bacteria strain was evaluated by using two-way ANOVA followed by multiple comparisons (Tukey) against the control. Values represent the means ± SD, 95% CI, (*P<0.05, **P<0.01).