Saccharomyces cerevisiae strains with AFB1 binding ability and beneficial effects on the ruminal ecosystem

DOGI C.A; ARMANDO, MR; LUDUEÑA, RICARDO; DALCERO A.M.; CAVAGLIERI L.R.

Congreso; VI Latin American Congress of Mycotoxicology. II International Symposium of fungal and algal toxins in industry; 2010

Mycotoxins are metabolites produced by fungi growing on foods or feeds and represent a serious hazard to humans and animals. They can be produced preharvest or during storage, transport, processing or feeding. Aflatoxin B1 (AFB1) is one of the most harmful mycotoxins in animal production and food industry (1). Among the developed strategies to eliminate mycotoxin, biological decontamination using microorganism is highly promising. Saccharomyces cerevisiae strains are among promising candidates that can be applied in animal feed promising candidates that can be applied in animal feed towards improved robustness of animals in the production environment (2,3). Aim: Evaluate if the yeast strains (RC008; RC009; RC016) isolated from animal environmental and with AFB1 binding ability previously in vitro demonstrated are able to survive and binding AFB1 under rumiants gastrointestinal conditions. This study was conducted to the further selection of potentially probiotic strains to be included in animal feed. Matherials and Methods: Rumen fluid (RF) was obtained by ruminocentesis from 3 healthy cows. The RF was strained through 4 layers of gauze and used as strained RF (SRF), or it was set for 1 h in cones tubes after which the middle layer was centrifugated for 30 minutes at 15000 rpm and the supernatant autoclaved (ARF). To evaluate the viability of yeast strains under gastrointestinal conditions, ARF was inoculated with each yeast strain (1x106 CFU/ml) for 24 h at 39°C in anaerobiosis (a). Yeast cells were washed and resuspended in simulated gastric juice and incubated for 60 min at 37º C under agitation (b). After centrifugation, cells were added to artificial intestinal fluid and incubated for 60 min at 37ºC under agitation (c). Aliquots of yeast cells after a; b and c conditions were taken and serial dilutions were carried out and spread on YPD agar. In order to study AFB1 binding ability under physic-chemical conditions of gastrointestinal tract, yeast strains were someted to the same conditions described above. After that, yeast cells were washed twice with PBS and incubated with 1 ml of AFB1 (50 ng/ml in PBS) for 1 h at 37ºC in a shaking bath. Cells were centrifugated and the supernatant containing unbound mycotoxin was collected and stored at -20ºC for HPLC analysis. Yeast cells without gastrointestinal passage were included as control group. To determine the effect of yeast strains on ruminal cellulolytic bacteria, SRF was used as inoculum and incubated in medium 10 of Caldwell and Bryant (4) with filter paper strip as only carbon source. The incubation was carried out in anaerobic conditions during 10 days at 39°C. The activity of cellulolytic bacteria was evaluated by the paper strip degradation and ruminal pH was measured at the end of incubation. SRF without yeast addition were included as control. Results and Discusion: All yeast strains were able to survive under gastrointestinal conditions, but they decrese one log after rumen passage. However, cell viability was maintained after stomach and intestinal conditions. The AFB1 binding percentages varied among yeast strains: RC008 38.4%; RC009 48% and RC016 39%. After passage in simulated gastrointestinal conditions, the strains RC008 and RC016 increased their AFB1 binding ability to 58 and 46% respectively. Aflatoxin adsorption is influenced by pH and phosphate concentration in an aqueous environment (5). The results obtained suggest that the conditions in the gastrointestinal tract would enhance adsorption and not decrease the mycotoxin-adsorbent interactions. However, the strain RC009 decreased the percentage of AFB1 binding to 36% after passge to simulated gastrointestinal tract. The number or activity of cellulolytic bacteria in SRF was increased in the presence of RC008 and RC016 yeast strains. This was assumed by the observation of total degradation of filter paper in comparision with control group (SRF without yeast strain addition) in which the paper strip was still present after the incubation period. These results were not obsserved with the strain RC009. Conclusions: the strains RC008 and RC016 are promisory probiotic candidates to be include in animal feed. They are able to survive and binding AFB1 under gastrointestinal conditions. They also affect beneficially the rumen microbiota.