Key Factors Of Dried Fish Matrix And Cysteine Hydrochloride Regulation Mechanism Of Aflatoxin Production Of Aspergillus Flavus

Aspergillus flavus and aflatoxin（AFT）are widely present in nature and easily enriched through the food chain,posing a threat to public health.In light of aflatoxins’physiological and toxicological effects,many countries have set regulatory limits for high-carbon agriculture products,but not for high-protein meat and dried aquatic products.This study aims to identify key regulatory factors for the growth and aflatoxin B1（AFB₁）production of A.flavus,specifically focusing on the fatty acid oxidation products and protein decomposition products derived from dried fish products.This work investigated the regulatory mechanism of key products of fat oxidation-oxylipins,cysteine（Cys,a protein decomposition product）and its derivatives on the synthesis of AFB₁ by A.flavus,and elucidated the antioxidant effect of L-cysteine hydrochloride（L-CH）in a simulated fish dry environment,providing scientific insights and a theoretical framework for solving mycotoxin contamination in meat products.Specific research methods and conclusions are summarized as follows:1.The water content,cure fat content,cure protein content,salt content,oxidation level（MDA）,fatty acid concentration of 6 common aquatic dried products were detected by classical analysis methods.Subsequently,the correlation between these parameters and the sporulation of A.flavus as well as the concentration of AFB₁ was examined.The study revealed that unsaturated fatty acids exerted a significantly inhibitory effect on the mycelial growth,sclerotinia formation and AFB₁ synthesis of A.flavus.A comprehensive analysis suggested that this effect could be attributed to the presence of the key products of fat oxidation,namely oxylipin and protein decomposition products.2.The spore development and sporulation of A.flavus were assessed using conventional morphological techniques,while AFB₁ synthesis was quantified through LC-MS/MS method.Additionally,the expression of genes involved in G Protein-Coupled Receptors（GPCR）pathway was determined by RT-q PCR.Through analysis of dose-effect relationship between oxylipins and detection indices,it was found that the production of spore was sensitive to oxylipins,with 9S-HODE exerting a stronger effect than 13S-HODE.As 9S-HODE dose increased,spore germination rate first inhibition and then increase,while AFB₁ synthesis showed the opposite trend.The results indicated that gpr C may be the key molecules responding to 9S-HODE signal,while the response molecule of13S-HODE may be gpr D.Oxylipin 9S-HODE promoted the expression of gpa B and fad A gene in GPCR pathway,and inhibits AFB₁ synthesis by inhibiting the expression of afl R and afl K.3.The potential mechanisms of Cys,acetylcysteine（NAC）and L-CH on the development and AFB₁ synthesis of A.flavus were explored by observing the changes of mycelium morphology and cell structure,as well as detecting the changes of mycelium antioxidant enzyme activities and related gene expression.The findings indicate that the inhibition of spore germination and germ tube formation by Cys and its derivatives contributes to their antifungal activity.Cys significantly inhibited mycelia growth,conidia and sclerotium formation.However,it did not affect the biosynthesis of ergosterol in mycelia.NAC had a detrimental effect on the development of conidial heads,while L-CH hindered mycelial growth and development by disrupting chitin metabolism within cells.Cys,NAC,and L-CH regulate growth and AFB₁ synthesis by promoting Glutathione（GSH）synthesis and inhibiting the expression of Blr A,afl R,afl S,and afl K.4.On the basis of transcriptome analysis,the expression of target genes was analyzed by RT-q PCR.The results showed that the anti-fungal and anti-aflatoxin mechanism of L-CH was mainly as follows:L-CH reduced intracellular ROS accumulation by activating the GSH metabolic pathway,there by delaying cell wall remodeling,leading to hyphal dwarfing,hypobranching,and decreased sporulation and number of sclerotia.Furthermore,AFT cluster genes,afl S and afl R,as well as AFLA＿091090 transcription factors were also down-regulated on L-CH treatment.AFLA＿091090 gene could be a potential indicator of L-CH anti-aflatoxin in A.flavus.These findings contribute to further understanding the molecular and gene expression mechanisms of L-CH’s antifungal and anti-mycotoxin activities,and further supports that L-CH can be used as a food additive to control AFB₁ in food.5.The effects of L-CH on the lipid oxidation of traditional dried fish were evaluated by lipid hydrolysis and lipid oxidase changes,and the succession of the dried fish microbial community during storage was analyzed by high-throughput sequencing technology.Meanwhile,the relationship between microbial flora and lipid oxidation was also analyzed.The results showed that L-CH exerted its ability to control fatty hydrolysis in dried fish at low temperatures（4℃）.L-CH inhibited lipid hydrolysis and lipid oxidation by suppressing the activities of lipid hydrolase and lipoxysynthase,thus inhibiting the deterioration of dried fish.Bacteroides,Faecalibacterium and Prevotella were the dominant microflora in dried fish.Storage at 4℃and L-CH treatment reduced the diversity and abundance of microorganisms in dried fish.LEf Se analysis showed that Prevotella,Veillonella and Clostridia＿UCG＿014 were the main microbial biomarkers of L-CH-induced microbial community differences.Correlation analysis of microorganisms and lipid oxidation indicators indicated that Enterobacter might be a key bacterial group leading to lipid oxidation and quality deterioration of red fish.A reduction in Enterobacter abundance may be a protective effect of L-CH on dried fish.