Preliminary Study On Degradation Of Alternariol By Saccharomyces Cerevisiae A20

Alternariol(AOH) is a group of toxic secondary metabolites predominantly produced by Alternaria alternata.There are several studies have confirmed that AOH is highly genotoxic,mutagenic,carcinogenic,cytotoxic,etc.,which can cause cell cycle arrest and apoptosis,affect the normal proliferation of cells,inhibit the activity of topoisomerase I and II.AOH has extensively polluted agricultural and sideline products and often synergistically enhanced with other Alternaria toxins.The superposition of the two makes the toxicity significantly enhanced.As a highly hazardous mycotoxin,AOH has seriously affected the health of humans and animals.It is urgent to find a method for biodegradation of AOH to reduce its harm and the risk of human contact,so as to improve the safety of food and feed.In this article,one Saccharomyces cerevisiae strain with high AOH degradation efficiency was isolated,and the efficiency and mechanism of AOH degradation by a yeast was studied.Besides,the structure and properties of the co-culture product P₁was analyzed.The main achievements and conclusions are as follows:(1)In this study,with extracted AOH as the sole carbon source for isolation and screening through enrichment and culture of microorganisms in grape epidermis and its contents,orchard soil and pickle during storage.Results:a strain with high AOH degradation efficiency was screened,named as A20,and the degradation efficiency reached more than 50%.Through combining with morphological observation,physiological and biochemical characteristics and 26S r DNA gene sequence determination analysis,strain A20 was preliminarily identified as Saccharomyces cerevisiae.At the same time,the paper diffusion method showed that strain A20 had no antagonism to Alternaria alternata.In addition,the effect of AOH on the growth of strain A20 under the optimal degradation conditions was further evaluated.The results of PI fluorescence staining showed that the cell membrane of a small amount of bacteria was damaged after strain A20 was cultured for 4 days under the stress caused by the addition of AOH.(2)The effects of different culture medium,p H value,initial concentration of AOH,inoculation amount and other factors on the degradation efficiency of AOH by S.cerevisiae A20 were studied.The results showed that S.cerevisiae A20 degrades65.89%AOH(4?g/m L)in YEPD medium incubated for 4 days with 28°C,2%inoculation amount,4?g/m L initial concentration of AOH and p H 6,whose degradation efficiency reach the peak.(3)Preliminary study on the mechanism of AOH removal by A20 strain.Through the use of ultrasonic instrument and repeated extraction experiments,it was shown that the removal process of AOH by A20 strain was mainly degradation,and a single stable substance P₁ was newly produced.In addition,the degradation kinetics of AOH showed that strain A20 could rapidly degrade AOH in YEPD within 4 days of incubation time,and the degradation of AOH was almost stable after 4 days incubation.(4)The catalytic properties of S.cerevisiae A20 for the degradation of AOH was preliminarily determined.The active substances of extracellular extract,bacterial suspension and intracellular extract of S.cerevisiae A20 for the degradation of AOH were located and identified.The results showed that the extracellular extract of S.cerevisiae A20 had good degradation effect on AOH,and the degradation efficiency was 58.47%,while the suspension of S.cerevisiae A20 had little degradation effect on AOH,and the degradation rate was 3.33%.The intracellular extract of S.cerevisiae A20 cultured in YEPD medium had poor degradation effect on AOH,and the degradation rate was only 13.35%.It was preliminarily speculated that the removal of AOH by S.cerevisiae A20 was mainly degradation rather than adsorption.According to the degradation rate of AOH decreased significantly after heat treatment and protease K treatment on extracellular extract.Therefore,it was speculated that extracellular protease secreted by S.cerevisiae A20 may degrade AOH and reduce its concentration.(5)The single component P₁ produced by the co-culture of S.cerevisiae A20 and AOH was isolated and purified by organic solvent extraction,silica gel column chromatography and high performance liquid chromatography.The structure of P₁was analyzed by liquid chromatography—mass spectrometry and nuclear magnetic resonance spectroscopy.The molecular weight of P₁ was 161,and the molecular formula was C₁₀H₁₁NO.Pale brown crystal,comprehensively identified as tryptophol.