Beneficial Propionibacteria within a Probiotic Emmental Cheese: Impact on Dextran Sodium Sulphate-Induced Colitis in Mice

Abstract

Backgrounds and aims: Inflammatory Bowel Diseases (IBD), including Ulcerative Colitis (UC), coincide with alterations in the gut microbiota. Consumption of immunomodulatory strains of probiotic bacteria may induce or prolong remission in UC patients. Fermented foods, including cheeses, constitute major vectors for bacteria consumption. New evidences revealed anti-inflammatory effects in selected strains of Propionibacterium freudenreichii. We thus hypothesized that consumption of a functional cheese, fermented by such a strain, may exert a positive effect on IBD.

Methods: We investigated the impact of cheese fermented by P. freudenreichii on gut inflammation. We developed an experimental single-strain cheese solely fermented by a selected immunomodulatory strain of P. freudenreichii, CIRM-BIA 129. We moreover produced, in industrial conditions, an Emmental cheese using the same strain, in combination with Lactobacillus delbrueckii CNRZ327 and Streptococcus thermophilus LMD-9, as starters. Consumption of both cheeses was investigated with respect to prevention of Dextran Sodium Sulphate (DSS)-induced colitis in mice.

Results: Consumption of the single-strain experimental cheese, or of the industrial Emmental, both fermented by P. freudenreichii CIRM-BIA 129, reduced severity of subsequent DSS-induced colitis, weight loss, disease activity index and histological score. Both treatments, in a preventive way, reduced small bowel Immunoglobulin A (IgA) secretion, restored occludin gene expression and prevented induction of Tumor Necrosis Factor α (TNFα), Interferon γ (IFNγ) and Interleukin-17 (IL-17).

Conclusions: A combination of immunomodulatory strains of starter bacteria can be used to manufacture an anti-inflammatory cheese, as revealed in an animal model of colitis. This opens new perspectives for personalised nutrition in the context of IBD.

Keywords: Emmental; cheese; colitis; inflammation; inflammatory bowel disease; intestine; probiotic; propionibacteria.

Figure 1

Experimental design of the evaluation of anti-inflammatory effects of a preventive intervention implementing P. freudenreichii-fermented cheeses, in the context of DSS-colitis. C57BL6 mice were divided into 5 groups, receiving different pre-treatments for 5 days, prior to induction of colitis. Colitis was then induced using 3% DSS in drinking water for 7 days prior to euthanasia. Different disease parameters were monitored to study the severity of colitis.

Figure 2

Impact of cheese matrix, single-strain cheese and Emmental cheese on colitis-induced body weight loss. (A) Time-course of mice body weight monitoring, and differences across groups. (B) Body weight loss observed at the 7th day of DSS colitis induction, and differences across groups. Groups were as follows. PBS: healthy group gavaged using PBS buffer as a sham. PBS-DSS: DSS-treated-colitis control group gavaged using PBS buffer as a sham. Cheese matrix-DSS: DSS-treated group gavaged using a germ-free dairy matrix. Single-strain cheese-DSS: DSS-treated group gavaged using an experimental single-strain cheese containing P. freudenreichii CIRM-BIA 129 as a sole bacterium. Emmental-DSS: DSS-treated group gavaged using an industrial Emmental cheese produced using P. freudenreichii CIRM-BIA 129 as a ripening starter. The data represent the mean ± SD of 18 mice per group. Multiple comparisons were performed, * p < 0.05, ** p < 0.01, *** p < 0.001 **** p < 0.0001.

Figure 3

Impact of cheese matrix, single-strain cheese and Emmental cheese on the severity of DSS-induced colitis. Seven days after colitis induction, disease activity index (DAI) (A) and Colon length (B) were determined. Groups were as follows. PBS: healthy group gavaged using PBS buffer as a sham. PBS-DSS: DSS-treated-colitis control group gavaged using PBS buffer as a sham. Cheese matrix-DSS: DSS-treated group gavaged using a germ-free dairy matrix. Single-strain cheese-DSS: DSS-treated group gavaged using an experimental single-strain cheese containing P. freudenreichii CIRM-BIA 129 as a sole bacterium. Emmental-DSS: DSS-treated group gavaged using an industrial Emmental cheese produced using P. freudenreichii CIRM-BIA 129 as a ripening starter. The data represent the mean ± SD of 18 mice per group. Multiple comparisons were performed, * p < 0.05, ** p < 0.01, *** p < 0.001 **** p < 0.0001.

Figure 4

Impact of cheese matrix, single-strain cheese and Emmental cheese on DSS-induced histopathological damages. Representative images of mice colon mucosa sections, stained with haematoxylin, are shown. Image acquisition phase was done with a 20× magnification objective. Scale bar = 50 μm. Histopathological scores were determined. Groups were as follows. PBS: healthy group gavaged using PBS buffer as a sham. PBS-DSS: DSS-treated-colitis control group gavaged using PBS buffer as a sham. Cheese matrix-DSS: DSS-treated group gavaged using a germ-free dairy matrix. Single-strain cheese-DSS: DSS-treated group gavaged using an experimental single-strain cheese containing P. freudenreichii CIRM-BIA 129 as a sole bacterium. Emmental-DSS: DSS-treated group gavaged using an industrial Emmental cheese produced using P. freudenreichii CIRM-BIA 129 as a ripening starter. The data represent the mean ± SD of 6 mice per group. Multiple comparisons were performed, * p < 0.05, ** p < 0.01, *** p < 0.001 **** p < 0.0001.

Figure 5

Impact of cheese matrix, single-strain cheese and Emmental cheese intake on small bowel IgA secretion. Secretory IgA concentration in the small bowel content was determined by ELISA quantification. The data represent the mean ± SD of 18 mice per group. Groups were as follows. PBS: healthy group gavaged using PBS buffer as a sham. PBS-DSS: DSS-treated-colitis control group gavaged using PBS buffer as a sham. Cheese matrix-DSS: DSS-treated group gavaged using a germ-free dairy matrix. Single-strain cheese-DSS: DSS-treated group gavaged using an experimental single-strain cheese containing P. freudenreichii CIRM-BIA 129 as a sole bacterium. Emmental-DSS: DSS-treated group gavaged using an industrial Emmental cheese produced using P. freudenreichii CIRM-BIA 129 as a ripening starter. Multiple comparisons were performed, * p < 0.05, ** p < 0.01, *** p < 0.001 **** p < 0.0001.

Figure 6

Impact of cheese matrix, single-strain cheese and Emmental cheese on colonic expression of markers of cell barrier and oxidative stress. Colonic mRNA expression levels of (A) Ocln and (B) iNOS genes were analysed. The data represent the mean ± SD of 6 mice per group. Groups were as follows. PBS: healthy group gavaged using PBS buffer as a sham. PBS-DSS: DSS-treated-colitis control group gavaged using PBS buffer as a sham. Cheese matrix-DSS: DSS-treated group gavaged using a germ-free dairy matrix. Single-strain cheese-DSS: DSS-treated group gavaged using an experimental single-strain cheese containing P. freudenreichii CIRM-BIA 129 as a sole bacterium. Emmental-DSS: DSS-treated group gavaged using an industrial Emmental cheese produced using P. freudenreichii CIRM-BIA 129 as a ripening starter. Multiple comparisons were performed, * p < 0.05, ** p < 0.01, *** p < 0.001 **** p < 0.0001.

Figure 7

Impact of cheese matrix, single-strain cheese and Emmental cheese on colonic expression of cytokines genes during DSS-induced colitis. Colonic mRNA expression levels of (A) IL-10, (B) IL-1β, (C) TNFα and (D) IFNγ were determined. Data represent the mean ± SD of 6 mice per group. Groups were as follows. PBS: healthy group gavaged using PBS buffer as a sham. PBS-DSS: DSS-treated-colitis control group gavaged using PBS buffer as a sham. Cheese matrix-DSS: DSS-treated group gavaged using a germ-free dairy matrix. Single-strain cheese-DSS: DSS-treated group gavaged using an experimental single-strain cheese containing P. freudenreichii CIRM-BIA 129 as a sole bacterium. Emmental-DSS: DSS-treated group gavaged using an industrial Emmental cheese produced using P. freudenreichii CIRM-BIA 129 as a ripening starter. Multiple comparisons were performed, * p < 0.05, ** p < 0.01, *** p < 0.001 **** p < 0.0001.

Figure 8

Impact of cheese matrix, single-strain cheese and Emmental cheese on colonic secretion of cytokines during DSS-induced colitis. Cytokines concentration of (A) IL-10, (B) TGFβ, (C) IL-6, (D) IFNγ, (E) IL-17, (F) TNFα, (G) IL-12 and (H) IL-1β were quantified by ELISA. Data represent the mean ± SD of 6 mice per group. Groups were as follows. PBS: healthy group gavaged using PBS buffer as a sham. PBS-DSS: DSS-treated-colitis control group gavaged using PBS buffer as a sham. Cheese matrix-DSS: DSS-treated group gavaged using a germ-free dairy matrix. Single-strain cheese-DSS: DSS-treated group gavaged using an experimental single-strain cheese containing P. freudenreichii CIRM-BIA 129 as a sole bacterium. Emmental-DSS: DSS-treated group gavaged using an industrial Emmental cheese produced using P. freudenreichii CIRM-BIA 129 as a ripening starter. Multiple comparisons were performed, * p < 0.05, ** p < 0.01, *** p < 0.001 **** p < 0.0001.