Patulin degradation by yeast protein extract

Abstract

The mycotoxin patulin, produced by a number of fungi, most prominently Penicillium expansum, has proven problematic for the apple industry due to contamination of apple juice and apple cider. Presently, techniques to control patulin accumulation have proven increasingly ineffective due to the presence of antifungal resistant strains of mold, stability of patulin during thermal processing, and conflicting data on the efficacy of other treatments. However, fermented apple products such as hard ciders and apple cider vinegars are devoid of patulin. Fermentation with yeast resulted in complete degradation of patulin, possibly due to enzymatic degradation by yeast enzymes. Patulin has also been shown to be susceptible to adduct formation with free thiol containing molecules such as glutathione, which is naturally present in yeast cells. Limited studies have also looked at patulin adsorptivity onto the call walls of yeast. Degradation of patulin is, therefore, hypothesized to be caused by multiple mechanisms mainly caused by yeast proteins/enzymes.To assess the loss of patulin by protein extracted from yeast (Rhodosporidium kratochvilovae strain 62-121), patulin extraction methods were compared to determine the optimal method for patulin extraction from protein rich environments. The effect of boiling to halt any possible enzymatic degradation on total patulin loss was assessed by comparing patulin recovery to that of samples placed on ice after the assay. Yeast growth was optimized for the production of patulin-degrading protein extracts by surveying days of growth and subsequent storage at 4°C. Additionally, free thiol group reactivity with patulin was assessed upon incubation with protein extract, cysteine, and glutathione. Liquid chromatography and mass spectrometry (LC/MS) was used to detect patulin degradation products. Potential enzymatic activity was assessed by comparing the degradation activity of different protein extracts from yeast. Finally, patulin loss due to adsorption to inactivated yeast cell walls was determined. The use of acid and salt to precipitate the protein before patulin extraction resulted in the best patulin recovery from protein rich media, and an additional extraction following a modified AOAC method allowed for removal of excess salt without sacrificing patulin recovery. The use of boiling to denature the protein after the assay resulted in 10% higher patulin loss than when the samples were placed on ice, presumably due to adduct formation with thiol groups. Growing yeast for 6 days at room temperature was deemed adequate to obtain optimal patulin degradation; and subsequent incubation of the yeast at 4°C did not impair the patulin degradation activity. Yeast protein extracts were found to be inconsistent with respect to patulin degradation activity, nevertheless patulin degradation activity (up to 100% patulin) was observed in several batches. Patulin incubated with cysteine showed signs of free thiol blockage in both samples of protein extract and pure cysteine. Patulin incubated with glutathione was degraded at both pH 7 and 3.7, and one patulin-glutathione adduct (462 m/z) was identified via LC/MS. Lyophilized yeast cells demonstrated patulin adsorption capabilities after incubation at 30°C for 20 min. Observed results confirm that patulin can be degraded by the protein extract from yeast. The exact mechanism of patulin degradation by protein extracts remains unclear, yet it appears to be either enzymatic or chemical through thiol adduct formation. Our results indicated that the mechanism is a combination of the two. This research offers insight into possible patulin degradation mechanisms, and can give direction in applying this new method of patulin control in an industrial setting.