Cytotoxicity in Vero cells and cytokines analyses in Balb/c mice as safety assessments of the probiotic mixture Saccharomyces cerevisiae RC016 and Lactobacillus rhamnosus RC007 for use as a feed additive.

The objective was to carry out cytotoxicity assays in Vero cells and cytokines analyses in Balb/c mice as safety assessments to evaluate the probiotic mixture (M) Saccharomyces cerevisiae RC016 (Sc) and Lactobacillus rhamnosus RC007 (Lr) for use as feed additive. Vero cells (104 cells per well) were exposed to Sc (2·08 × 107 , 2·08 × 106 ; 2·08 × 105 cells per ml), Lr (8·33 × 107 ; 8·33 × 106 ; 8·33 × 105 cells per ml) and their M (1 : 1). Sc concentrations did not affect the Vero cells viability; in contrast, they were lower when exposed to Lr (P Ë‚ 0·0001). Vero cells showed increasing viability with M decreasing concentrations (91% viability with M2). Control BALB/c mice received only phosphate buffer saline and the others received the M. The IL-10, IL-6 and TNFα concentrations from intestinal fluid were analysed and no significant differences were observed among treatments. The same occurred with the ratio between IL-10/TNF-α. Beneficial effects of probiotics are associated with the regulation of the excessive inflammatory response; it is desirable they can modulate the cytokines production only under pathological conditions. Here, M administration to healthy mice did not induce negative side effects and expands the knowledge about beneficial effects of using probiotic microorganisms in mixture for feed additives development.

Aflatoxin B1 adsorption/desorption dynamics in the presence of Lactobacillus rhamnosus RC007 in a gastrointestinal tract-simulated model.

AIMS: (i) To determine the aflatoxin B1 (AFB1 ) adsorption and desorption dynamics in the presence of Lactobacillus rhamnosus RC007 under simulated transit of AFB1 at each gastrointestinal tract (GIT-saliva, stomach and intestine) stage consecutively and then, separately, (ii) to study the ability of L. rhamnosus RC007 to biotransform AFB1 as a strategy that complements the adsorption process. METHODS AND RESULTS: The AFB1 adsorption and desorption assay simulating the GIT passage of AFB1 (93·89 ng g-1 ) in the presence of L. rhamnosus RC007 (108 CFU per ml) was conducted. Moreover, lactic acid production was determined. Results demonstrated that predominant environmental conditions in salivary solution induced a low AFB1 adsorption, while the transit through the gastric solution and intestinal solution allowed high percentages of adsorption and did not generate significant AFB1 desorption. CONCLUSIONS: The AFB1 adsorption and desorption dynamics in the presence of L. rhamnosus RC007 was favoured by gastric and intestinal environment. SIGNIFICANCE AND IMPACT OF THE STUDY: The knowledge of the adsorption dynamics of AFB1 with a micro-organism of interest will allow predicting its behaviour at each stage of the GIT.

Comparison of toxicogenic and immunosuppressive capacity of Aspergillus fumigatus strains isolated from clinical and corn silage samples.

AIMS: To compare clinical and environmental Aspergillus fumigatus strains on their toxicogenic and immunosuppressive capacity. METHODS AND RESULTS: A total of 51 strains of A. fumigatus isolated from clinical and corn silage samples were assayed. All A. fumigatus strains were assayed for gliotoxin production, therefore strains with different gliotoxin capacities and isolated from different sources were selected and assayed for their effects on bovine macrophages and lymphocytes. Spore diffusates (SDs) obtained from all A. fumigatus strains were able to inhibit macrophage phagocytosys, regardless of their gliotoxin production capacity. However, most but not all strains were able to inhibit bactericidal activity. SDs from all A. fumigatus strains reduced lymphocytes viability. The heat treatment was not always able to inhibit the negative effect on immune cells. CONCLUSIONS: There was no difference between clinical and environmental isolates in their toxicogenic and immunosuppressive capacity. Gliotoxin would not be responsible for the immunosuppressive activity observed by the assayed A. fumigatus strains. However, gliotoxin could be present in the SD, together with some other substances. SIGNIFICANCE AND IMPACT OF THE STUDY: The results obtained suggest that any environmental strain of A. fumigatus is a putative infectious strain. Prevention measures should be applied to control environmental Aspergillus conidia.

Selection of lactic acid bacteria to promote an efficient silage fermentation capable of inhibiting the activity of Aspergillus parasiticus and Fusarium gramineraum and mycotoxin production.

AIMS: To select lactic acid bacteria with potential silage inoculant properties. The bio-control activity against mycotoxicogenic fungi and the presence of antibiotics resistance gene were also evaluated. METHODS AND RESULTS: Lactobacillus rhamnosus RC007 and Lactobacillus plantarum RC009 were selected on the basis of growth rate and efficacy in reducing the pH of maize extract medium; therefore, they were evaluated for their bio-control ability against Fusarium graminearum and Aspergillus parasiticus. Studies on lag phase, growth rate and aflatoxin B1 (AFB1) and zearalenone (ZEA) production were carried out in vitro under different regimes of aw (0·95 and 0·99); pH (4 and 6); temperature (25 and 37°C); and oxygen availability (normal and reduced). Lactobacillus rhamnosus RC007 was able to completely inhibit the F. graminearum growth at all assayed conditions, while Lact. plantarum RC009 only did it at pH 4. Both Lactobacillus strains were able to significantly reduce the A. parasiticus growth rate mainly at 0·99 aw . A decrease in ZEA production was observed as result of Lactobacillus strains -F. graminearum interaction; however, the A. parasiticus- Lact. plantarum interaction resulted in an increased AFB1 production. Lactobacillus rhamnosus RC007 proved to have no genes for resistance to the tested antibiotics. CONCLUSIONS: The ability of Lact. rhamnosus RC007 to rapidly drop the pH and to inhibit fungal growth and mycotoxin production and the absence of antibiotic resistance genes shows the potential of its application as inoculant and bio-control agent in animal feed. SIGNIFICANCE AND IMPACT OF THE STUDY: This study demonstrated the importance of selecting bacteria for silage inoculants not only for the improvement of silage fermentation but also for their effects on mycotoxicogenic fungi and the resulting mycotoxin production due to the risk that they may involve.

Mutagenicity evaluation with Ames test of hydro-alcoholic solution of terpenes.

Mutagenic properties of terpenes (both synthesis and plant derived) have been tested, up to now, as a single molecule. A terpenes containing hydro-alcoholic solution deriving from frankincense and myrrh resins and hyssop essential oil was assayed for mutagenicity by means of ames test. Extraction technique conducted with electromagnetic fields at room temperature enabled to obtain a solution of free active molecules that did not undergo thermal degradation and characterized by biocidal activity. In order to verify lack of mutagenic hazard in coming into contact with human, the solution was appropriately diluted and tested with Salmonella typhimurium TA98, TA1535 and YG1024 strains, both in absence and in presence of metabolic system S9. For none of the tested conditions a 2-fold increase of induced revertants, as regards to spontaneous, was registered. The ratio between induced and spontaneous His+ revertants (Mutagenic Index) was around 1.00 in all the determinations and no statistically significant differences have been identified comparing the sample and the negative control. A similar result has been obtained for the dose-response curve. In conclusion, we verified that tested terpenes solution lacks of mutagenicity on Salmonella typhimurium with and without metabolic activator so this plant extract can be safely used as biocide.

Beneficial effects of Saccharomyces cerevisiae RC016 in weaned piglets: in vivo and ex vivo analysis.

Probiotics represents an alternative to replace antibiotics as growth promoters in animal feed and are able to control enteric bacterial diseases and to improve gut immunity. Saccharomyces cerevisiae RC016 showed previously inhibition/coagregation of pathogens) and mycotoxins adsorbent ability (aflatoxin B1, ochratoxin A and zearalenone). The aim of this work was to evaluate beneficial properties of S. cerevisiae RC016 in a non-inflammatory in vivo model in weaned piglets and in an intestinal inflammation ex vivo model induced by the mycotoxin deoxynivalenol (DON). Secretory immunoglobulin A (s-IgA) levels, intestinal cytokines, goblet cells and production parameters were evaluated in a pig model. For the in vivo assays, twelve pigs were weaned at 21 days and assigned to two groups: Control (n=6) and Yeast (n=6). Animals received yeast strain for three weeks. After 22 days the small intestine was recovered for determination of goblet cells and s-IgA. For the ex vivo assay, jejunal explants were obtained from 5 weeks old crossbred piglets and treated as follow: (1) control; (2) treated for 3 h with 10 µM DON used as an inflammatory stressor; (3) incubated with 107 cfu/ml yeast strain; (4) pre-incubated 1 h with 107 cfu/ml yeast strain and then treated for 3 h with 10 µM DON. CCL20, interleukin (IL)-1ß, IL-8 and IL-22 gene expression was determined by qPCR. Oral administration of S. cerevisiae RC016 increased s-IgA, the number of goblet cells in small intestine and all the growth parameters measured. In the ex vivo model, the cytokine profile studied showed a potential anti-inflammatory effect of the administration of the yeast. In conclusion, S. cerevisiae RC016 is a promising candidate for feed additives formulation to improve animal growth and gut immune system. This yeast strain could be able to improve the gut health through counteracting the weaning-associated intestinal inflammation in piglets.