Enterococci as probiotics and their implications in food safety

Supplementation of broiler chicken diets with probiotics may improve carcass characteristics and meat quality. However, the underlying molecular mechanism remains unclear. In the present study, 2D-DIGE-based proteomics was employed to investigate the proteome changes associated with improved carcass traits and meat quality of Arbor Acres broilers (Gallus gallus) fed the probiotic Enterococcus faecium. The probiotic significantly increased meat colour, water holding capacity and pH of pectoral muscle but decreased abdominal fat content. These meat quality changes were related to the altered abundance of 22 proteins in the pectoral muscle following E. faecium feeding. Of these, 17 proteins have central roles in regulating meat quality due to their biological interaction network. Altered cytoskeletal and chaperon protein expression also contribute to improved water holding capacity and colour of meat, which suggests that upregulation of chaperon proteins maintains cell integrity and prevents moisture loss by enhancing folding and recovery of the membrane and cytoskeletal proteins. The down-regulation of β-enolase and pyruvate kinase muscle isozymes suggests roles in increasing the pH of meat by decreasing the production of lactic acid. The validity of the proteomics results was further confirmed by qPCR. This study reveals that improved meat quality of broilers fed probiotics is triggered by proteome alterations (especially the glycolytic proteins), and provides a new insight into the mechanism by which probiotics improve poultry production.

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Proteome changes underpin improved meat quality and yield of chickens (Gallus gallus) fed the probiotic Enterococcus faecium

The aim of the current study was to evaluate and compare two representative samples of different classes of adsorbents intended for use as feed additives in the prevention or reduction of the adverse effects exerted by mycotoxins, specifically ochratoxin A (OTA) and zearalenone (ZEN). The adsorbents, an organically activated bentonite (OAB) and a humic acid polymer (HAP), were tested in a common in vitro model with a pH course comparing the maximum pH changes that can be expected in the digestive system of a monogastric animal, i.e. pH 7.4 for the oral cavity, pH 3.0 for the stomach, and pH 8.4 for the intestines. In the first experiment, the concentration-dependent adsorbent capacity of OAB and HAB were tested using a fixed concentration of either mycotoxin. Thereafter, adsorption was evaluated applying different isotherms models, such as Freundlich, Langmuir, Brunauer-Emmett-Teller (BET) and Redlich-Peterson, to characterize the adsorption process as being either homo- or heterogeneous and representing either mono- or multilayer binding. At the recommended statutory level for the mycotoxins of 0.1 mg kg(-1) OTA and 0.5 mg kg(-1) ZEN, OAB showed an adsorbed capacity of >96% towards both mycotoxins, regardless of the pH. The HAP product was also able to absorb >96% of both mycotoxins at pH 3.0, but extensive desorption occurred at pH 8.4. Based on χ-square (χ(2)) values, Langmuir and Redlich-Peterson equations proved to be the best models to predict monolayer equilibrium sorption of OTA and ZEN onto the organically activated bentonite and the humic acid polymer. The applied methodology has a sufficient robustness to facilitate further comparative studies with different mycotoxin-adsorbing agents.

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Isotherm modeling of organic activated bentonite and humic acid polymer used as mycotoxin adsorbents

Humic acids are known for their overall positive health and productivity effects in animal feeding trials and, controversially, as an aetiological factor of cancer. We tried to assess the in vitro effect of humic acids from a selected source in Slovakia when used at recommended prophylactic dosage. We investigated antioxidant properties, enzymatic and non-enzymatic antioxidant defence system in liver mitochondria and cultured cancer cell lines in vitro. We observed a significant decrease in superoxide dismutase activity after humic acids treatment irrespective of dissolving in dimethyl sulphoxide or direct addition to mitochondria suspension in a respiration medium. Activities of other antioxidant enzymes measured, such as glutathione peroxidase and glutathione reductase, showed no significant differences from the control as well as the reduced glutathione content. Percentage of inhibition by humic acids of superoxide radical indicated lower efficacy compared with that of hydroxyl radical. Survival of six different cancer cells lines indicated that only the acute T lymphoblastic leukaemia cell line was sensitive to the tested humic acids. Despite relatively low solubility in aqueous solutions, humic acids from the selected source participated in redox regulation. By recapturing the radicals, humic acids reloaded the antioxidant defensive mechanism. Results from in vitro study conducted with humic acids from the natural source showed potential of these substances as promising immunity enhancing agents.

Effects of humic acids in vitro.

The high prophylactic doses of ZnO commonly used to control post-weaning diarrhoea (PWD) in piglets have become an environmental problem. The possibility of partial replacement of ZnO by sodium humate (HNa) in PWD treatment was investigated in this study. Weaned piglets (32) were challenged with two enterotoxigenic E. coli strains (ETEC/O149/F4/LT and ETEC/O147/F18/LT) and allocated into four treatments maintained for three weeks: C (no supplementation); ZnO2.5 (2.5 g ZnO/kg diet); HNa + ZnO1.0; HNa + ZnO1.5; HNa + ZnO1.7 (20 g HNa and 1.0, 1.5 and 1.7 g ZnO/kg diet, respectively). The effects on incidence, severity and duration of diarrhoea, faecal shedding of total E. coli and both ETEC strains, growth rate and selected blood parameters were investigated. In contrast with ZnO2.5 and HNa + ZnO1.7, high daily diarrhoea scores, incidence and duration and mortality due to severe dehydra - tion were seen in C, HNa + ZnO1.0 and HNa + ZnO1.5 groups. The administration of ZnO and HNa did not affect the faecal shedding of the challenged ETEC strains for eght days, even in clinically healthy piglets in ZnO2.5 and HNa + ZnO1.7 groups. Signs of growth depression were found in C; HNa + ZnO1.0 and HNa + ZnO1.5 groups during the first week. No difference in growth performance was observed in ZnO2.5 and HNa + ZnO1.7 piglets. Most of the selected biochemical and haematological parameters did not differ significantly among the treatments. However, a significantly higher serum Zn as a result of high dietary ZnO intake in the ZnO2.5 group compared to the control and HNa groups was detected. Significantly lower serum P in ZnO2.5; HNa + ZnO1.7 and HNa + ZnO1.0 groups compared to the control group was most likely induced by the increased Zn in serum. The results indicate the possibility of reducing the high pharmacological levels of ZnO in the prophylaxis of PWD through partial replacement with HNa. Such a treatment maintains the favourable prophylactic effect while lowering the Zn content in faeces.

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Prophylaxis of post-weaning diarrhoea in piglets by zinc oxide and sodium humate

The present study investigated the effect of the probiotic strain Enterococcus faecium DSM 7134 (2 x 10(7) cfu per g of feed) (probiotic group), natural humic substances (7g x kg(-1) feed) (HS group), and the combination of both (probiotic + HS group) on the performance, serum biochemistry, and biological activity of leucocytes of broiler chickens. Average body weight of chickens in different phases as well as throughout the observation period under different treatments did not differ significantly. Final body weight and feed conversion ratio (FCR) reached in 41 days of trial were 2542.5; 2568.9; 2610.5, 2516.6 g and 1.93; 1.81; 1.78; 1.88 kg for HS, probiotic, probiotic + HS and control group respectively. The best performance variables including the European efficiency index (EEI) were observed in the probiotic + HS group, where the effect of the probiotic supplementation was improved by HS. There were no significant differences among groups in the serum levels of total protein, glucose, cholesterol, aminotransferase and phosphorus. A significant decrease of total lipids in the probiotic group (P < 0.01) and probiotic + HS group (P < 0.001) groups, triglycerides levels in the HS (P < 0.01), probiotic and probiotic + HS (P < 0.001) groups on day 35, calcium level of the probiotic (P < 0.05) and probiotic + HS (P < 0.01) groups on day 41 and alkaline phosphatase levels in the probiotic group (P < 0.01) on day 14 were observed in comparison to the control group. Significantly higher values of indices of metabolic activity of phagocytes (IMA) after 14, 35 and 41 days of the experiment were observed in the probiotic + HS group (P < 0.01, P < 0.05) when compared with the control group. No adverse health effects were observed in experimental groups The results of the present study indicate that HS and E. faecium DSM 7134 had no significant effect on performance. However an improvement in FCR and EEI was observed which is believed to be due to the significant increase of indices of metabolic activity of phagocytes, caused by HS when combined with. E. faecium DSM 7134.

The effect of the probiotic strain Enterococcus faecium DSM 7134 in combination with natural humic substances on performance and health of broiler chickens.

Objective of this study was to investigate changes in biological activity of blood leukocytes in Japanese quails, caused by zinc and cadmium administration. Four groups of Japanese quails were used. Three experimental groups of quails were exposed either to Cd (0.12 mg Cd/quail), Zn (4 mg Zn/quail) or a combination of Cd and Zn (0.12 mg Cd, 4 mg Zn/quail), which were added daily to the drinking water. The fourth group was the control group. The metabolic activity of phagocytes and mitogenic activation of lymphocytes to phytohemaglutinine (PHA) were determined on day 37, 58 and 118 of exposure. The numbers of peripheral blood leukocytes of Japanese quail after cadmium and zinc addition in all groups of birds were without significant differences, however, the functional activities of phagocytes and lymphocytes in the Cd-group of quails were significantly decreased. The metabolic activity of phagocytes decreased significantly at all time points analysed (P < 0.05-0.001) in comparison to the control group and the Cd-Zn group. Similarly, the response of lymphocytes to PHA activation in the Cd exposed group of quails decreased significantly on day 58 and 118 of exposure when compared to the control group (P < 0.05) and Zn-group of quails (P < 0.01). Zn in combination with Cd eliminated the immunotoxic effect of Cd on metabolic activity of phagocytes and improved lymphocyte answer to PHA, when compared to the Cd-group on day 58 and 118 of metal administration. The results of the present study indicate that cadmium caused a significant decrease of metabolic activity of phagocytes and mitogenic activation of lymphocytes in peripheral blood of Japanese quails. Simultaneous administration of Zn and Cd eliminated the immunotoxic effect of Cd on functional activity of phagocytes and lymphocytes, and zinc improved both investigated functional parameters of immune cells.

Juraj Pistl

Papers

The effect of the probiotic strain Enterococcus faecium DSM 7134 in combination with natural humic substances on performance and health of broiler chickens.

Effect of oral cadmium and zinc application on leukocytes biological activity in Japanese quails (Coturnix coturnix japonica)