Enviromental Factors Mediate The Regulatory Mechanism Of Ochratoxin A In Dry Cured Ham Filamentous Fungi

Chinese dry cured ham,one of the most famous traditional meat product ripened for a long time.The environmental conditions throughout this ripening time,favor growth of a fungal population on the surface of hams,which could include a number of potentially toxigenic molds.Some fungal species can produce mycotoxins in dry cured ham which can produce carcinogenic and mutagenic effects in humans or animals.The natural processing methods of low temperature curing,medium temperature dehydration and high temperature fermentation have been used up to now.Therefore,it is important to identify and reduce the contamination of dry cured ham,minimizing the consumption and deleterious effects caused by mycotoxins.Mycotoxins are products of fungal secondary metabolism,which are regulated in response to the e.cophysiological factors.In this paper,to detect mycotoxin of dry cured ham during ripening as the starting point,through colonization,Hiseq,metagenomics and RNA-Seq,we research filamentous fungi OTA biosynthesis mechanism and potential osmotic stress regulation factor in NaCl rich dry cured ham simulation.It will provide theoretical basis for scientific guidance of traditional ham production,elimination or reduction of mycotoxin accumulation.The main contents and conclusions were as follows:(1)A simple and efficient QuEChERS sample preparation method was modified to provide good analytical results for 9 mycotoxins in dry cured ham.The analysis was performed using QTrap liquid chromatography tandem mass chromatography.Under optimized conditions,the calibration curves displayed good linear relationships with all coefficients of determination higher than 0.994.Throughout the validation experiments,74.5?103.4%spiked recoveries were achieved with RSDs 3.2?14.3%.The limits of detection were 0.1?2.5?g/kg.The limits of quantification were 0.5?10.0?g/kg.7 of 25 dry cured hams were contaminated with OTA ranged from 3.73 to 128.32?g/kg.And 3 hams were detected OTA in the deep section.A few hams contaminated with CIT and CPA,but AFB1,AFB2,AFG1,AFG2,DON were not detected.Spearman rank correlations between physicochemical property and OTA in dry cured ham had also been investigated.(2)Metagenomic approaches and ITS high-throughput sequencing were used to monitor changes in fungal populations,secondary metabolites pathway and function of related genes.Phylogenetic analysis based on ITS genes indicated that dry cured ham fungus was composed of 2 phyla,7 class,11 order,16 family and 17 genera.The predominant fungal genera were Penicillium and Debaryomyces during pre-ripening and then change to Aspergillus during ripening of dry cured ham.The relationships between fungal populations environmental factors were analysed by RDA.The mycotoxin biosynthesis pathway was selective analysis and presented 112 unigenes,14 enzymes,33 proteins,20 pathway entry,10 KO entry by KEGG database.(3)A total of 215 filamentous fungal colonies were isolated from 25 dry cured hams.The potential ability to produce OTA was studied by isolating the culture followed by ELISA and HPLC analysis.The results indicated that 22(10.23%)of them produced OTA.Seven highly toxigenic strains were selected and identified by morphology and ITS sequences which were A.ochraceus H1905,A.steynii H1611,A.subramanianii H2307,A.westerdijkiae H1409,P.brevicompactum H1016,P.chrysogenum H0808,P.verruculosum H1118.(4)The temperature and NaCl content(which affects water activity,aw)affect sporulation and OTA production of fungal species.The optimum temperature of sporulation is 20?30 0C and the optimum aw is 0.95?0.97.Sporulation was positively affected by the highest temperature and aw values indicating that Aspergillus species might be expected in warm climates.The sporulation was negatively correlated with aw and there was a significant difference between groups(P<0.05).The tolerance of NaCl was quite different between stains and P.verruculosum H1118 produced high level concentration of OTA(320.7 ?g/kg)at NaCl 8%,aw 0.85.But other Aspergillus species produced low level concentration of OTA at that situation.(5)To better understand the mechanism of different adaptation of P.verruculosum H1118 and A.subramanianii H2307 in rich NaCl,RNA-Seq analysis was used to profile the transcriptome changes associated with OTA production.By comparing high NaCl(80 g/L)to low NaCl(8g/L)cultural conditions,a total of 1120(H1118)and 1074(H2307)differentially expressed genes(DEGs)were identified.Analysised by GO enrichment,genes involved in secondary metabolite synthesis,carbohydrate metabolism,amino acid metabolism,MAP kinase phosphorylation were up regulated in H1118 Pv H group,which will benefit to OTA biosynthesis.By KEGG analysis,there were 15 up regulated genes and 3 down regulated genes in the MAPK signaling pathway of H1118 Pv H group.However,only 2 and 8 DEGs were found in H2307 As H group.The differentially expressed genes were concentrated in Sln1-Ypd-Ssk1 pathway,and there were no DEGs in other three branches Sho1-Stel1,Hkr/Msb-Cdc42 and Opy2-Ste50.OTA synthesis genes pks?nrps?chl?cyp?pal were up regulated in H1118 Pv.However,three key OTA synthesis genes pks?nrps?chl were obviously down upregulated in H2307 As by NaCl stress.Based on RNA-Seq transcriptome analysis,the coding genes of H1118 Pv histidine/aspartate kinase Sln1,histidine kinase Ypdl,aspartate kinase Sskl,MAPK kinase Pbs2 and Hogl were up regulated,which activates Hog1 phosphorylation and then regulated the expression of toxin producing genes.Hog1 is a potential transcription factor,which is closely related to the mechanism of H1118 Pv tolerance to NaCl stress.