Modulating poultry microbiota to promote gut function by enzymatic tailoring of feed

The meat industry, which is already responsible for about 15% of the total anthropogenic greenhouse gas (GHG) emission, is expected to continue expanding due to population and economic growth. Notably, the demand for chicken meat has the highest projected increase (17.8% by 2030) relative to other meat sources. Life-cycle assessment studies implicate feed as responsible for more than two thirds of both the financial cost and GHG emission of chicken production, and soybean meal (SBM) as a common ingredient with major environmental impact. Exogenous feed enzymes are attractive as tools that allow optimisation of the efficacy of established feed ingredients and enable the use of alternative counterparts. Besides these obvious gains, enzymes are expected to be instrumental in modulating the feed-gut microbiota interplay to overcome the challenges imposed by the ban on antibiotic use as growth promoters. Overall, feed enzymes have the potential to markedly improve the sustainability of poultry production, which will be crucial during the likely lengthy process until other protein sources become viable.

Interestingly, SBM and rapeseed meal (RSM), that are major protein-rich feed ingredients, contain 15-30% non-starch polysaccharide (NSP) per weight, especially pectin and xyloglucan. The microbiota that colonise the caeca-pair in chicken are equipped with the machinery needed to ferment distinct NSP in feed into short-chain fatty-acids (SCFAs), which are beneficial to animal nutrition and protection against pathogens. However, the efficacy of this bacterial community in utilising feed NSP is limited due to the insolubility and the complexity of the NSP compounds as well as the short transit time. Recently, multiple ex vivo studies showed promising effects of supplementing SBM- or RSM-containing diets with multi-enzyme products to degrade the pectin matrix.

In this project, the aim was to identify a minimal blend of carbohydrate active enzymes (CAZymes) able to break down the NSP content of these plant meals into fragments that are fermentable by the chicken caeca microbiota. For that, two approaches were explored: 1) testing enzymes obtained from the collection of enzymes available at Novozymes for their effect in solubilising NSP from SBM and RSM, and 2) a rational substrate-oriented strategy for feed CAZymes discovery. After an initial screening, we pursued the enzyme discovery approach that involved the secretomic analyses of 11 pectinolytic fungal strains, which guided the selection of four endo-acting CAZymes. The selected enzymes targeting pectin and xyloglucan were heterologously expressed, purified, and their ability to solubilise NSP from SBM and RSM was demonstrated. The combination of mainchain/sidechain and of pectin/xyloglucan enzyme pairs synergistically increased the solubilisation of NSP from the target plant meals, and increased short-chain fatty-acid (SCFA) concentrations, notably butyrate, in chicken caecal microbiota fermentations. The preferential increase in butyrate is likely to confer immuno modulatory effects and provide a higher caloric yield from the pectin-rich feed ingredients, which remains to be corroborated in vivo. Taxonomic profiling using 16S sequencing showed a decrease in Proteobacteria that harbours potential pathogens, and an increase in Firmicutes and Bacteroidetes, which are both associated with glycan fermentation and SCFA production. This thesis offers a methodological platform for discovery of Prebiozymes tailored to transform less accessible feed ingredients into precision prebiotics.