Patulin Detoxification By Saccharomyces Cerevisiae And Its Mechanism

Patulin is a mycotoxin that is mainly produced by Penicillium expansum,the causal agent of apple blue mold.This fungal disease often occurs in the postharvest storage of damaged apples and can remain in apple-based products jeopardizing human health.Longterm exposure to PAT may result in immunotoxicity,cytotoxicity,neurotoxicity,genotoxicity,and severe damage to mammalian organs,especially kidneys and liver.Accordingly,many jurisdictions have implemented the regulation which the maximum level of PAT in apple juice and apple cider should be no more than 50 ?g/L.The PAT contamination in apple products continues to be a worldwide problem largely because of the complicated relationships of pathogenic Penicillium,apple resistance and environmental conditions.In addition,PAT is stable in acidic condition,even pasteurization only marginally reduces PAT level in apple-based products.Because of this,the development of strategies to effectively control PAT levels is of great importance.With safety and high efficiency,the biocontrol agents have become the promising way to control patulin contamination in apple products.Saccharomyces cerevisiae has a GRAS(Generally Recognized as Safe)status,which the PAT was disappeared in the fermentation of contaminated apple juice to apple cider.However,the degradation mechanism,the degradation products and its toxicity are not clear,which adversely affect its development and its application in food industry.In this study,the S.cerevisiae S288C,with high efficacy in PAT degradation,was screened from the preserved yeast strains in our lab.The fermentation conditions for patulin degradation by S.cerevisiae S288C were studied.Based on that,patulin-degrading enzymes were extracted,followed by the analysis of the enzymatic properties and degradation products,as well as the RNA-seq analysis,to uncover the enzymatic degradation of PAT.With the identification of several glutathione-patulin(GSH-PAT)conjugates from the cells of S.cerevisiae S288C and from the reaction mixture of PAT-degrading enzymes,the gene knockout strategy was used to demonstrate the role of glutathione in the non-enzymatic detoxification of patulin.The protection of GSH on the hepatic and renal damage caused by PAT was furthered evaluated by animal experiments.The main findings are as follows:1.The study of conditions of Saccharomyces cerevisiae on patulin detoxification.The pH stability and temperature stability of PAT was first analyzed.The results indicated that PAT was stable in the buffer of pH from 4.0 to 6.0.The PAT solution was hardly degraded under 4? and room temperature,indicating its stability at low temperature.Moreover,PAT was stable in the use of MM,PDB or YPD medium.With high efficiency in degrading PAT,the S.cerevisiae S288C strain was screened and the degradation conditions was further analyzed.After static fermentation at 30? for 96.h,the S.cerevisiae S288C could completely degrade 5 mg/L of PAT from the MM medium.LC-MS analysis was used to identify the degradation products.The E-ascladiol and Z-ascladiol were found in the yeast culture,as well as the GSH-PAT conjugates,especially c-GSH-PAT(m/z 434.1225)and lGSH-PAT(m/z 452.1329).These results indicate that S.cerevisiae S288C could effectively degrade patulin from the medium and produce the ascladiol and GSH-PAT conjugates,which provide the basis for the further identification of its degradation mechanism.2.The enzymatic degradation of patulin by S.cerevisiae.The mechanism of patulin degradation by S.cerevisiae was first analyzed by separation the different components of S.cerevisiae.The results showed that the cell lysate for yeast has a superior ability in degrading PAT when compared with its culture supernatant or cell debris.The cell lysate,especially from 96 h fermentation culture,could reduce 10 mg/L of PAT in 8 h.The remarkable effect was disabled after heat treatment,which indicates that enzymes may responsible for the degradation.Moreover,the PAT-degrading enzymes was induced by PAT.The PAT degrading-enzymes was extracted by ammonium sulfate precipitation.The 60%ammonium sulfate could precipitate the majority of enzymes,which were able to degrade 98.2%of PAT from the reaction mixture.In the analysis of its enzymatic properties,the optimal reaction temperature and the optimal pH for PAT-degrading enzymes are 40? and 6.0,respectively.These enzymes were NADH-or NADPH-dependent and free from metal ion.In the analysis of the degradation products,a new metabolite(C7H10O5,(4.6-dihydroxy3-(hydroxymethyl)hexa-2,4-dienoic acie))with RT at 6.21 min,m/z 175.1193 at[M+H]+was identified by LC-MS chromatography.It is highly possible that the new metabolite was a redox product of ascladiol.Moreover,several GSH-PAT conjugates were also found after PAT degradation,especially c-GSH-PAT(m/z 434.1225)and l.c-GSH2-PAT(m/z 723.1944).Therefore,it is highly possible the enzymatic degradation was responsible for the PAT degradation by S.cerevisiae S288C.With low concentration of the new metabolite,it is possible other mechanism was exist for PAT detoxification by S.cerevisiae S288C.The RNA-seq analysis indicates.1 86 of DGEs are up-regulated and 207 of DGEs are downregulated.The up-regulated DGEs are enriched in metabolic pathway,biosynthesis of amino acids,Glutathione metabolism and drug metabolism.The down-regulated DGEs are enriched in carbon metabolism,glyoxylate and dicarboxylate metabolism,AMPK signaling pathway and fatty acid metabolism.In addition,a tight protein-protein interaction was found among SAH1,GLR1,TKL1 and GND1.3.The non-enzymatic detoxification of patulin by S.cerevisiae.The conjugation of GSH and PAT was studied in vitro.The results indicate that the molar concentration of GSH and PAT played an important role in the conjugation of GSH and PAT.The reaction speed happened fast when the ratio of GSH to PAT was 5:1.The concentration and conformation of GSH was not affected by the heat treatment,and thus,the conjugation of GSH and PAT was thermal stable.Moreover,the role of GSH in PAT detoxification by yeast was analyzed by constructing the gene knockout and claw back strains of y-glutamyl cysteine synthetase(GSH1)and glutathione synthetase(GSH2),respectively.Results showed that the gene knockout GSH1 or GSH2 yeast was unable to reduce PAT from the medium;the ability was recovered and reinforced when GSH1 or GSH2 was complemented and overexpressed in the gene knockout yeasts.The addition of glutathione(1 mg/L)could speed up the reduction of PAT in the used of PAT-degrading enzymes.Both the in vivo and in vitro experiments showed that GSH was the main factor in the non-enzymatic detoxification of PAT.4.The toxicity study of glutathione-patulin conjugates on experimental mouse.The protection of GSH on the hepatic and renal damage caused by PAT was evaluated by animal experiments.After 7 days of administration,no significant difference was found in the feed intake and the body weight of mice in the group of saline,different dosage of PAT,GSH and different dosage of GSH-PAT conjugates.The analysis of ALT and AST indicates that the treatment of PAT could significantly cause the increase of ALT and AST in serum,while the difference treatment of GSH-PAT conjugates and the saline was insignificant.The antioxidant index of liver and kidney showed that,the high dosage of PAT could significantly reduce the level of T-AOC,SOD,GSH-PX.LDH and GSH,but significantly increase the level of MDA when compared with the NC group.Moreover,no significant difference was found in the treatment of GSH-PAT conjugates or saline.The HE-stained pathological slices of liver and kidney indicated that,high dosage of PAT could cause severe hepatotoxicity,including the necrosis and dilated sinusoids.The liver was in a healthy status in the treatment of GSH-PAT conjugates or saline.The high dosage of PAT also cause the nephrotoxicity,like the degeneration of renal corpuscles and hemorrhage of tubules.The kidney was in a healthy status in the treatment of GSH-PAT conjugates or saline.Therefore,the toxicity of PAT to laboratory mice was smoothed after the conjugation with GSH.