EFFECT OF BILE AND LOW pH ON OCHRATOXIN A BINDING BY Saccharomyces cerevisiae STRAINS

ARMANDO M.R.; DOGI C; ESCOBAR F; DALCERO A.M. ; CAVAGLIERI L.R.

Congreso; VI Congreso Latinoamericano de Micotoxicología; 2010

Background. Ochratoxin A (OTA) is a secondary metabolite produced by two genera of fungi Aspergillus and Penicillium. A big concern is about its occurrence in many commodities (feeds, foods and beverages), because it is suspected to be nephrotoxic, teratogenic, hepatotoxic and carcinogenic (Marquardt and Frohlich 1992). Several technologies in food processing may play an important role for reducing the content of OTA in foods and beverages like physical, chemical, and microbiological methods. However, few of them have practical applications (Bata et al., 1999; Heilmann et al., 1999, Belajová et al., 2007; Ringot et al., 2007). Saccharomyces cerevisiae strains, due to their GRAS status and their use as probiotics, are of particular interest on reducing the bioavailability of mycotoxins (Shetty et al, 2006; Var et al., 2009). The use of Saccharomyces cerevisiae strains to reduce the animal exposure to OTA has a great promise; however additional studies are required as well as GI relevant conditions that effect OTA binding.   Aim. To examine four strains of Saccharomyces cerevisiae from animal ecological niches for the ability to bind OTA using adsorption isotherms and the effect of bile and low pH on OTA binding.   Materials and Methods. To determine the effect of exposure to bile salts and low pH on OTA binding of Saccharomyces cerevisiae strains, 1 ml of each strain (RC008; RC009; RC012 and RC016) were incubated for 4 h at 37 ºC in YPD broth (30 mL) containing 0.5 % (w/v) bile salts and YPD pH 3 modified with HCl (1M). After incubation, cells were harvested, used in the AFB1-binding assay. .The OTA binding assay was performed according to Bueno et al. (2007) with modifications. Solution stock of OTA was suspended in PBS to obtain concentrations of 1; 5; 10; 40 and 100 µg ml-1. Yeasts (107cells ml-1) were washed twice with PBS and incubated for 1 h at 37ºC in a shaking bath with 1 ml of PBS OTA. Then, cells were pelleted by centrifugation at 5,000 rpm at room temperature for 15 min, and the supernatant containing unbound mycotoxin was collected and stored at -20ºC for high-performance liquid chromatography (HPLC) analysis using the methodology described by Ringot et al. (2007). Each adsorption test was performed in duplicate and controls were performed. Curves representing the amount of bound OTA as a function of the amount of added OTA were plotted according to the mathematical expressions proposed by Hill’s theoretical model, selected according to the isotherms form. Results and Discussion. The amount of OTA bound was strain specific with a percentage ranging from 56.7% to 74.24%. The amount of OTA bound by Saccharomyces cerevisiae RC0016 was higher than the other examined strains. Growth in the presence of 0.5% bile salts (except RC016) increased OTA binding (71.33% to 78.66%) by Saccharomyces cerevisiae strains. Moreover, an increase in the OTA binding when all strains were subjected to pH 3 was observed (75.88% to 83.32%). Therefore, to screen strains of Saccharomyces cerevisiae for the ability to bind OTA during passage through the gastrointestinal tract, it is important to evaluate the influence of bile and low pH on OTA binding. The results presented in this study demonstrate that exposure of yeast strains to gastrointestinal conditions have a significant impact on OTA binding. In general, all strains bound more amount of OTA when they were exposed to bile and low pH. This finding could have an important biological significance, since probiotic yeast are normally exposed to bile secreted in the small intestine, which suggest that probiotics may be more capable of binding OTA in the intestine, and thus reduce OTA bioavailability in the gut.   Conclusion. 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