Structural Identification And Mechanisms Of Lentinus Edodes Cellulose Derivative Against Aflatoxin Production By Aspergillus Flavus

Aflatoxins (AFTs) are highly toxic and careinogenic secondary metabolites produced mainly by Aspergillus flavus and Aspergillus parasiticus, recognized as the Class I carcinogens by Food and Agriculture Organization of the United Nations (FAO) and Word Health Organization (WHO).They severely contaminate agricultural commodities and thus pose serious health hazards to both humans and domestic animals. Control of aflatoxin contamination has been a worldwide problem.Beta-glucan has been proved associated with the inhibition of aflatoxin B1production by A. flavus, which provides a new perspective to control the aflatoxin contamination. In this paper, the inhibitory effects of several polysaccharides on the growth and toxin production of A. flavus were investigated, including lentinan, laminaria, pustulan and etc. We found a derivative (glycoprotein, GPX) from Lentinus edodes cellulose, which inhibited the aflatoxin B1(AFB1) production by A. flavus effectively. The following studies were carried out on the structural identification and the mechanisms of GPX inhibit AFB, production. The results showed as follows:1. The inhibitory rate of10、50μg/ml GPX on AFB1production were70%、95%respectively and AFB1can be completely inhibited by GPX at concentrations ranging from100μg/ml to1000μg/ml。The peanut seeds treated with100μg/ml GPX showed good resistance to A. flavus infection. According to the single-factor experiment and central composite experimental design principles by Response Surface Analysis, the optimizing extraction technology parameters of GPX were determined as follows:the extraction temperature130℃, the ratio of raw material to water1:200(w/v), the time20min. The extraction rate of GPX under the given conditions was1.66%.2. GPX were mainly composed of glucose, and their molecular weights of GPX were approximately240 000Da. Neither the typical absorption peak of nucleic acid at260nm nor the typical absorption peak of protein at280nm was observed by UV spectrum. GPX were pyranose containing β-glucesidal bonds according to the analysis of Infrared ray spectra、1H NMR and electrophoresis. Alkaline β-elimination reaction showed that GPX had O-glycopeptide key, and periodate oxidation and Smith degradation reaction indicated that GPX contained only1→4glycosidic bonds. In addition, GPX were determined containing14kinds of amino acid, accounting for17.969%of whole molecular weights with higher levels of glutamic acid (3.114%), aspartic acid (2.527%), leucine (1.603%), phenylalanine (1.297%), threonine (1.284%), and serine (1.238%), respectively.3. GPX could hardly be determined in the antioxidant activity in vitro, whereas could modify the oxidative stress level of A. flams. By microscope, numerous vacuoles were found in the mycelieum of A. flavus treated with GPX, and the fusion of vesicles to large vacuoles has been proved against the aflatoxin production by A. flavus. By RT-PCR analysis, the modulatory genes of aflatoxin synthesis, aflS and aflR, in the mycelium of A. flavus treated with GPX was found to be down-regulated to an undetectable level in the transcription.Through structural analysis, GPX were finally identified as soluble β-1,4-glucan-peptide complexes. The mechanism involved in the aflatoxin production inhibition by GPX was hyperthesiszed that GPX could alleviate oxidative stress via specific receptors, inhibit the transcription of aflR and aflS, and promote premature vacuolization of vesicles of A. flavus resulting in the inhition of AFB. production.