Starter SURFace against INFlammation of the Gut: Role of Lactobacillus delbrueckii and Propionibacterium freudenreichii surface proteins in the modulation of gut inflammation – SURFING : Starter SURFace against INFlam

The strategy followed in the SURFING projects relies on an integrative approach, from the molecule to the functional food. Immunomodulatory properties of strains belonging to both species will be quantified and classified in vitro on 2 models, human leukocytes (PBMC) and colic epithelial cells (HT-29), by quantification of immunoregulatory cytokines and by monitoring of NFkB activation, respectively. For each species, 8 genomes have been fully sequenced and annotated. The genes potentially responsible for immunomodulatory effects are identified by a comparative genomic, transcriptomique and proteomic approach (number, nature, abundance). In particular, the surface proteins of these strains are identified by approaches combining proteomics and bioinformatics. Surface proteins potentially involved are analyzed by comparing in vitro immunomodulatory properties of enriched fractions, gene inactivation mutants and gene over expression mutants of the corresponding genes. Immunomodulatory properties of strains are validated in the context of experimental colitis in mice. Expression of key genes is checked within model cheeses and fermented milks by a transcriptomics and proteomic approach developed by the partners on different diary matrices. Anti-inflammatory properties of strains, provided within cheese or fermented milk, studied in vivo, will confirm the proof of concept in a complex matrix.

First in vitro results show a great variability between strains of both species. Some strains having an anti-inflammatory effect equal or above that of recognized probiotic bacteria. In vitro immunomodulatory properties, nature and amount of surface proteins, but also genes encoding these proteins, are variable. In Pf, strains exhibiting a surface polysaccharide are poorly anti-inflammatory. An ongoing statistical analysis seeks correlations between the presence of key surface proteins and the most anti-inflammatory profiles. First results showed that 13 genes are correlated, positively or negatively, with the phenotype and 6 genes are correlated with the presence/absence of surface proteins. A Pangenomic DNA chip was designed for the project. It will allow identification of the genes differentially expressed according to the immunomodulatory profile of Pf and Ld stains. Following part of the project will aim at confirming the link between these key proteins and the immunomodulatory potential, in vitro and in vivo. In that aim, the protective effect of 5starins of Pf and of 5 strains of Ld, selected on the basis of their immunological diversity, is analyzed on 2 distinct experimental models of colitis in mice. Finally, fermented dairy products will be developed and their anti-inflammatory effects sought in vivo in these colitis contexts, thus combining real dairy matrices with intrinsic properties of strains. A web site is being developed to build an interface between research, industry and public and to communicate the key results.

Long-term perspectives are preclinical and clinical studies aiming at the evidence of immunomodulatory effects, not of dried probiotic preparations, but of fermented dairy products made with bacterial strains with bioactive properties that we will have demonstrated.
For scientists, the awaited consequences are better knowledge of bacterial molecules involved in interaction with the host. For stakeholders of the dairy field, it is the selection of bioactive strains and better knowledge of their starters. For the public, communication about the SURFING project will aim at reinforcing the awareness of the benefits linked to fermented airy products consumption.

The SURFING project was advertised and described at international symposia.
International Probiotic Conference 2011, Kosice, Slovaquie, Juin 2011 ; Probiotech 2011, Milano, Italia ; FEMS 2011, 4th Congress of European microbiologists, Geneva, Switzerland, 26-30 June 2011 ; Microbes for health symposium, Paris, France, 1-2 December 2011 ; INRA-Rowett Symposium on Gut Microbiology, Clermont-Ferrand, France, 17-20 June 2012 ; JOBIM 2012, Rennes, Le Maréchal et al. Combination of in silico and proteomic approaches to identify candidate genes responsible for the immunomodulatory properties of Propionibacterium freudenreichii.
First results were published in international journals.
Deutsch et al. 2012. Appl. Environ. Microbiol. ; Cousin et al. 2012. Food Microbiol. ; Van de Guchte et al. 2012 Curr. Opin. Microbiol.

Inflammatory bowel diseases (IBD) constitute a main health concern in industrialized countries. Their incidence is currently about 1% of the population in these countries. In addition to some genetic predisposition, they are linked to lifestyle and diet. They are caused by a disregulation of the immune system leading to disequilibrium between protection towards pathogens and tolerance towards gut commensal bacteria.
Intestinal inflammation, key factor in IBD, can be partly treated by consumption of bacterial strains selected for their immunomodulatory and anti-inflammatory properties. In this context, the effect of some bacteria commercialized in this aim, mainly belonging to the genus Bifidobacterium or Lactobacillus, was thoroughly studied.
Propionibacterium freudenreichii (Pf) and Lactobacillus delbrueckii (Ld) are two bacterial species massively used as starters in fermented dairy products. Despite this massive use, the anti-inflammatory potential of these two species is surprisingly rarely studied. Pf is massively ingested via the consumption of Swiss type cheeses and of a growing number of other cheeses in which this starter is more and more used to improve organoleptic properties. Ld is very abundant in various fermented dairy products. The subspecies lactis is more and more used in Swiss type cheeses because of its acidifying abilities. As an example, one gram of Emmental cheese contains 108 to 109 cells of Pf and 103 to 109 cells of Ld, which are potentially immunomodulatory. The Ld subspecies bulgaricus is also highly consumed in various fermented milks, including yogurt.
In the SURFING project, we will focus on the immunomodulatory properties of these two dairy starters. Indeed, cheeses and fermented milks already on the market contain high amounts of Pf and Ld. These two bacteria can thus help in treating IBD, via specific surface components showing variable anti-inflammatory properties which are crucial and should be characterized. Our preliminary results revealed anti-inflammatory properties which are highly strain-dependent in both Pf and Ld. One selected strain of each species was tested in an experimental model of colitis in mice and revealed a promising protective effect. The aims of the SURFING project are:
1- Show that the ingestion of Pf or of Ld can have an anti-inflammatory impact in the context of IBD.
2- Identify the surface proteins responsible for the two species immunomodulatory properties
3- Elucidate the molecular mechanisms of the immune response triggered in vitro and in vivo in epithelial cells
4- Confirm the presence of the key surface immunomodulatory proteins in real conditions during starter preparation and dairy products making using these Pf and Ld starters.