Compatibility and safety of five lectin-binding putative probiotic strains for the development of a multi-strain protective culture for poultry.

The ban on the use of antibiotics as feed additives for animal growth promotion in the European Union and United States and the expectation of this trend to further expand to other countries in the short term have prompted a surge in probiotic research. Multi-species probiotics including safe and compatible strains with the ability to bind different nutritional lectins with detrimental effects on poultry nutrition could replace antibiotics as feed additives. Lactobacillus salivarius LET201, Lactobacillus reuteri LET210, Enterococcus faecium LET301, Propionibacterium acidipropionici LET103 and Bifidobacterium infantis CRL1395 have proved to be compatible as evaluated through three different approaches: the production and excretion of antimicrobial compounds, growth inhibition by competition for essential nutrients and physical contact, and a combination of both. The safety of P. acidipropionici LET103 was confirmed, since no expression of virulence factors or antibiotic resistance was detected. The innocuity of E. faecium LET301 should be further evaluated, since the presence of genes coding for certain virulence factors (gelE, efaAfm and efaAfs) was observed, albeit no expression of gelE was previously detected for this strain and there are no reports of involvement of efaAfm in animal pathogenicity. Finally, a combination of the five strains effectively protected intestinal epithelial cells of broilers from the cytotoxicity of mixtures of soybean agglutinin, wheat germ agglutinin and concanavalin A. To our knowledge, this is the first time that a combination of strains is evaluated for their protection against lectins that might be simultaneously present in poultry feeds.

Feed supplementation with avian Propionibacterium acidipropionici contributes to mucosa development in early stages of rearing broiler chickens.

Different studies in animal rearing claim the probiotic potential of species of the genus Propionibacterium. The effects of strains of Propionibacterium acidipropionici isolated from poultry intestine on microbiota activity and intestinal mucosa development were investigated in the early stage of rearing chicks and the safety of the dose used was investigated. The strains P. acidipropionici LET105 and LET107, administered as monoculture to chicks from the 1st to 14th day of life in a daily dose of 106 cfu/ml administered in the drinking water resulted harmless. The animals arrived at the expected weight for age and no differences were observed with respect to the food intake and water consumption related to control without bacteria administration. The analysis of microbiota composition revealed the presence of propionibacteria at the middle and end of the trial only in treated groups. Normal development of lactic acid bacteria and bifidobacteria, and slow colonisation by Bacteroides at the 7th day of the study was observed in the same groups. Analysis of the organic acids concentrations in the caecal content of birds revealed higher lactic acid and lower butyric acid production. Lower short chain fatty acids total concentration than expected during treatment was related to a better development of the gut mucosa. Increase in length of villus-crypt units, goblet cells counts and neutral mucins production were evidenced. Higher mucus secretion produced by dietary supplementation with propionibacteria could provide increased protection against pathogens.

Feruloyl esterase activity is influenced by bile, probiotic intestinal adhesion and milk fat.

Cinnamoyl esterases (CE) are microbial and mammalian intestinal enzymes able to release antioxidant hydroxycinnamic acids from their non-digestible ester-linked forms naturally present in vegetable foods. Previous findings showed that oral administration of Lactobacillus fermentum CRL1446 increased intestinal CE activity and improved oxidative status in mice. The aim of this work was to evaluate the in vitro CE activity of L. fermentum CRL1446 and the effect of bile on this activity, as well as strain resistance to simulated gastrointestinal tract (GIT) conditions and its ability to adhere to intestinal epithelium and influence its basal CE activity. L. fermentum CRL1446 and L. fermentum ATCC14932 (positive control for CE activity) were able to hydrolyse different synthetic hydroxycinnamates, with higher specificity toward methyl ferulate (3,853.73 and 899.19 U/g, respectively). Feruloyl esterase (FE) activity was mainly intracellular in L. fermentum CRL1446 and cell-surface associated in L. fermentum ATCC14932. Both strains tolerated simulated GIT conditions and were able to adhere ex vivo to intestinal epithelium. Pre-incubation of L. fermentum strains with bile increased FE activity in both whole cells and supernatants (~2-fold), compared to controls, suggesting that cells were permeabilised by bile, allowing more substrate to enter the cell and/or leakage of FE enzymes. Three-fold higher FE activities were detected in intestinal tissue fragments with adhered L. fermentum CRL1446 cells compared to control fragments (without bacteria), indicating that this strain provides exogenous FE activity and could stimulate esterase activity in the intestinal mucosa. Finally, we found that milk fat had a negative effect on FE activity of intestinal tissue, in absence or presence of adhered L. fermentum. These results help explaining the increase in intestinal FE activity previously observed in mice fed with L. fermentum CRL1446, and support the potential use of this strain for the development of new functional foods directed to oxidative stress-related ailments.

Dairy bacteria remove in vitro dietary lectins with toxic effects on colonic cells.

AIMS: To assess in vitro the ability of some dairy bacteria to bind concanavalin A (Con A), peanut agglutinin (PNA) and jacalin (AIL), preventing their toxicity on mouse intestinal epithelial cells (IEC). METHODS AND RESULTS: Con A and AIL reduced significantly IEC viability in vitro, as determined by Trypan Blue dye exclusion or by propidium iodide/fluorescein diacetate/Hoescht staining. Different strains of dairy bacteria were able to remove lectins from the media. Two strains were subjected to treatments used to remove S-layer, cell wall proteins, polysaccharides and lectin-like adhesins. They were then assayed for the ability to bind dietary lectins and reduce toxicity against IEC and to adhere to IEC after interaction with lectins. Con A and AIL were removed by Propionibacterium acidipropionici and Propionibacterium freudenreichii by binding with specific sugar moieties on the bacterial surface. Removal of lectins by bacteria impaired IEC protection. Adhesion of P. acidipropionici to IEC was reduced but not abolished after binding Con A or AIL. CONCLUSIONS: Removal of Con A or AIL by dairy propionibacteria was effective to avoid the toxic effect against colonic cells in vitro. SIGNIFICANCE AND IMPACT OF THE STUDY: Consumption of foods containing these bacteria would be a tool to protect the intestinal epithelia.

Competitive Inhibition of Propionibacterium acidipropionici by Mixed Culturing with Lactobacillus helveticus.

Lactobacillus helveticus and Propionibacterium acidipropionici were grown in pure and mixed cultures in a complex medium to assess the associative interaction. The specific growth rates, substrate consumption coefficient, substrate utilization and product formation rates were determined in each case. Propionibacterium acidipropionici utilized glucose preferably when it grew in a medium containing a mixture of glucose and lactate. Its growth rate was higher on glucose than on lactate in pure culture. However, lactic acid was the substrate utilized by propionibacteria in the associative growth. The fast pH reduction produced by the growth of lactobacilli and the slow lactate utilization by propionibacteria in mixed culture determined the inhibition of propionic acid bacteria in associative growth.