Glycerol and reuterin-producing Limosilactobacillus reuteri enhance butyrate production and inhibit Enterobacteriaceae in broiler chicken cecal microbiota PolyFermS model

Abstract

Background: Administering probiotic strains of Limosilactobacillus reuteri to poultry has been shown to improve poultry performance and health. Some strains of L. reuteri taxa can produce reuterin, a broad-spectrum antimicrobial compound from glycerol conversion, with high inhibitory activity against enterobacteria. However, little is known about the metabolism of glycerol in the complex chicken cecal microbiota nor the effect of glycerol, either alone or combined with L. reuteri on the microbiota. In this study, we investigated the effect of L. reuteri PTA5_F13, a high-reuterin-producing chicken strain and glycerol, alone or combined, on broiler chicken cecal microbiota composition and activity using the continuous PolyFermS model recently developed to mimic chicken cecal fermentation.

Methods: Three independent PolyFermS chicken cecal microbiota models were inoculated with immobilized cecal microbiota from different animals and operated continuously. The effects of two additional levels of glycerol (50 and 100 mM) with or without daily supplementation of chicken-derived L. reuteri PTA5_F13 (10⁷ CFU/mL final concentration) were tested in parallel second-stage reactors continuously inoculated with the same microbiota. We analyzed the complex chicken gut microbiota structure and dynamics upon treatment using 16S rRNA metabarcoding and qPCR. Microbiota metabolites, short-chain and branched-chain fatty acids, and glycerol and reuterin products were analyzed by HPLC in effluent samples from stabilized reactors.

Results: Supplementation with 100 mM glycerol alone and combined with L. reuteri PTA5_F13 resulted in a reproducible increase in butyrate production in the three modelled microbiota (increases of 18 to 25%). Glycerol alone resulted also in a reduction of Enterobacteriaceae in two of the three microbiota, but no effect was detected for L. reuteri alone. When both treatments were combined, all microbiota quantitatively inhibited Enterobacteriaceae, including in the last model that had very high initial concentrations of Enterobacteriaceae. Furthermore, a significant 1,3-PDO accumulation was measured in the effluent of the combined treatment, confirming the conversion of glycerol via the reuterin pathway. Glycerol supplementation, independent of L. reuteri addition, did not affect the microbial community diversity.

Conclusions: Glycerol induced a stable and reproducible butyrogenic activity for all tested microbiota and induced an inhibitory effect against Enterobacteriaceae that was strengthened when reuterin-producing L. reuteri was spiked daily. Our in vitro study suggests that co-application of L. reuteri PTA5_F13 and glycerol could be a useful approach to promote chicken gut health by enhancing metabolism and protection against Enterobacteriaceae.

Keywords: Butyrate; Chicken cecal microbiota; Glycerol; In vitro model; L. reuteri; Reuterin.

Fig. 1

Glycerol metabolism by reuterin-producing L. reuteri. Anaerobic metabolism of glycerol by reuterin-producing L. reuteri to 3-hydroxypropionaldehyde (3-HPA) and further to 1,3-propanediol (1,3-PDO). In an aqueous environment, 3-HPA is quickly dimerised and hydrated to form HPA-dimer and HPA-hydrate and also spontaneously dehydrates to acrolein together form the reuterin system. PduQ, 1,3-PDO dehydrogenase

Fig. 2

Alpha diversity measured by Shannon index in microbiota from three independent in vitro fermentations under different conditions. F1 (A), F2 (B) and F3 (C). Values are mean of results ± standard deviation of the last 3 days of fermentation. CR, Control reactor; 50G, 50 mM glycerol; 100G, 100 mM glycerol; Lbr, L. reuteri PTA5_F13; Lbr-100G, L. reuteri PTA5_F13 and 100 mM glycerol

Fig. 3

Experimental set-up of the PolyFermS chicken cecal in vitro models. A Experimental set-up and conditions tested in the PolyFermS model mimicking the chicken cecal microbiota. Control reactor (CR) and test reactors (TR) were continuously inoculated with 5% fermentation effluent from the inoculum reactor (IR) and 95% nutritive media. B Experimental conditions for three independent fermentations (F1, F2 and F3) during the pre-treatment and treatment periods. L. reuteri PTA5_F13 (Lbr) was added daily for 8 days in F2 and F3 to reach a concentration of 10⁷ CFU/mL. 50G, 50 mM glycerol; 100G 100 mM glycerol