The Distribution Of Ochratoxin A In Daqu And Its Producing Microorganisms

In this paper,the distribution of ochratoxin A?OTA?in Chinese liquor of different types and geographical origins was studied as well as that in Daqu by using new methods for the quantitative determination of OTA developed.And,the dynamic changes of OTA in the production of Daqu and its producing microorganisms were also studied by using the combination of the traditional culture technology and the molecular ecology technology.The main results of this paper are as follows:?1?A method for the quantitation of OTA in Chinese liquor was developed.Chinese liquor with high content of alcohol and esters is a kind of distilled liquor.The matrix of Chinese liquor is very complex and special.In addition,there was no method for the quantitative detection of OTA in Chinese liquor before this study began.In view of the above problems,the method development was conducted by a new solid-phase extraction clean-up?SPE?combined with high performance liquid chromatography-fluorescence detection?HPLC-FLD?.Firstly,a SPE column,a reversed phase/strong anion-exchange mixed-mode column(ProElut^TM PXA),was selected topurify OTA in Chinese liquor.The ProElut^TM PXA column loaded with polystyrene/divinylbenzene copolymer was first used to purify mycotoxin.Then,the conditions of OTA purification were optimized based on ProElut^TM PXA column,and the analysis conditions of HPLC were improved.Finally,the method was verified by using the three aroma-types of Chinese liquor in the concentration range of 0.05⁰.2?g/L,including strong aroma-,soy sauce aroma-,and light aroma-type.The recovery rates were between 81.6 and 100.8%.The relative standard deviations?RSDs?were 3.6⁷.3%for the intra-day test and 5.1¹2.8%for the inter-day test.The signal suppression/enhancement?SSE?values were in the range of 6¹4%.The limit of detection?LOD?and limit of quantitation?LOQ?were respectively 0.006 and 0.02?g/L.The method is comply fully with the Directive2002/26/CE of European Union States,and,therefore,considered appropriate for the quantitative determination of OTA in Chinese liquor.?2?The OTA was detectable in Chinese liquor,and the distribution of OTA is different in Chinese liquor of different types and geographical origins.The results showed that¹2%of tested samples were detected with OTA at a maximum concentration of 0.17?g/L.Therefore,the safety risk of OTA probably exists in Chinese liquor.Meanwhile,the contents and detection rates of OTA in different aroma-types of Chinese liquor were distinct,and 15%and10%of strong aroma-and light aroma-type raw liquors were detected with OTA at mean contents of 0.09 and 0.05?g/L,respectively.But OTA was not detected in soy sauce aroma-type raw liquor.The risk of OTA in strong aroma-type was highest,and then was light aroma-type,and doesn't seem to exist in soy sauce aroma-type.In addition,the contents and detection rates of OTA in Chinese liquor from southwest region were a little more than that from east region.?3?A method for the quantitation of OTA in Daqu was developed.Daqu is a complex and special matrix.Daqu is spontaneously fermented under thermophilic condition,which contains proteins,carbohydrates,pigments,amino acids,organic acids,and other unknown compounds.In the meantime,texture of Daqu is very firming,and its hydrophilicity is very poor.In addition,the content of OTA in Daqu is in trace levels.Therefore,the quantification of OTA in Daqu is very difficult,especially the pretreatment process.Meanwhile,the specific method to detect OTAin Daqu has not been reported before this study began.In view of the above problems,therefore,the method development was carried out by ultrasound-assisted extraction?UAE?combined with ultra-high performance liquid chromatography-tandem mass spectrometry?UPLC-MS/MS?.Firstly,the UAE conditions of OTA in Daqu were optimized using Plackett-Burmandesign coupled with Box-Behnkendesign.And then,the conditions of OTA purification and the analysis conditions of UPLC-MS/MS were improved.Finally,the method was verified by using three types of Daqu in the concentration range of 0.5¹5.0?g/Kg,including high temperature-,medium temperature-,and low temperature-type.Under the optimized conditions,the recovery rates were between 87.1 and 105.7%,and the LOD and LOQ were respectively 0.33 and 0.41?g/Kg.The RSDs were 2.6¹3.6%for the intra-day test and 5.1¹4.6%for the inter-day test.The SSE values were in the range of 10¹8%and the external standard method can be used to determine OTA in Daqu.The method is comply fully with the Directive 2002/26/CE of the European Union States,and,therefore,considered appropriate for the the quantitative determination of OTA in Daqu.?4?The distribution of OTA in Daqu of different types is remarkable,and their fermentation temperature is the key factor affecting the OTA distribution.The results showed that⁵0%of tested samples were detected out OTA with a maximum content of 28.87?g/Kg.OTA was detected out in the three types of Daqu samples including high temperature,and not detected out in all raw materials for Daqu.Therefore,it was speculated that OTA was produced in the production of Daqu,and that the spontaneously fermented products under high temperature?>45 ^oC?may be at risk of OTA.It showed that the detectable rates in low temperature-,medium temperature-,and high temperature Daqu were respectively 100%,90.91%and 16.22%.And,it was also found that the maximum contents in low temperature-,medium temperature-,and high temperature Daqu were respectively 28.87?g/Kg,14.84?g/Kg and 2.08?g/Kg.Statistical differences in OTA occurrence were found among the three types of Daqu samples?p<0.05?.It was revealed that the OTA occurrencein Daqu decreased in the order of low temperature-,medium temperature and high temperature-type Daqu based on their frequency and levels of OTA.This implied that fermentation temperature is key factor influencing OTA occurrence in Daqu of different types.?5?The OTA occurrencein Daqu may be related to geographical origins.The results showed that there was significant difference on OTA occurrence in medium temperature Daqu between the samples from northern Jiangsu province and southern Sichuan province?p<0.05?,and the frequency and levels of OTA in Daqu from the southern Sichuan province is more than that from northern Jiangsu province.Therefore,OTA occurrence in Daqu?at least in medium temperature-type?is related to geographical origins,and the relative humidity and rainfall maybebe the key factor influencing the OTA occurrence in Daqu.?6?It was revealed by using high throughput technology and multivariate statistical analysis that the influence of seasons on the fungal community of Daqu is very significant,and it mainly affects the early stage of Daqu fermentation.The results showed that fungal species in the production of Daqu in the autumn were more than that in the summer,but the uniformity of fungal community in the production of Daqu in the summer was higher than that in the autumn.It was known that the microbial community is more stable when the distribution of microbial community is more uniform,and which may be a reason for the better quality of Daqu produced in the summer.These genera including Wickerhamomyces,Pichia and Thermoascus,were the main differential microorganisms in the production of Daqu in the autumn and summer.It was showed by?diversity analysis that the production of Daqu can be divided into two stages with the boundary about at 8¹2 d.It was showed by?diversity analysis that the difference of the Shannon diversity index in the first stage of production of Daqu in the autumn and summer is very significant?p<0.05?,and that was not significant in the late stage of production of Daqu?p>0.05?.It can be seen that the influence of seasons on the fungal community of Daqu is mainly in the early stage of Daqu fermentation.In addition,the difference on the relative abundance of Penicillium in the production of Daqu in the summer and the autumn is quite large?p=0.058?,but that for Aspergillus is very small?p=0.46?.It is well known that the main difference between different seasons is in the climate conditions.Therefore,it was preliminary speculated that the difference on the contents and positive rates of OTA in Daqu of different geographical origins may be that the difference on penicillium in Daqu produced in different seasons.?7?The dynamic changes of OTA in the production of Daqu were that the content of OTA was increased firstly,then decreased,and finally tended to be stable.Specifically,the OTA content was increase quickly from zero in the early stage of Daqu production?10¹2 d?,then decreased continuously in the middle period of Daqu production?14²5d?,and finally the content of OTA remained stablein the later period of Daqu production as well as in the storage period of Daqu.The dynamic trend of OTA is basically consistent with the growth and metabolism law of molds in the production of Daqu.Meanwhile,it was confirmed that OTA in Daqu is produced by microorganisms in its production.Therefore,ochratoxigenic fungi were isolated,screened and identified from Daqu by using the traditional microbial culture technology,and 14 strains of OTA producer were obtained.Among them,there were 11strains of Aspergillus and 3 strains of Penicillium,including A.nidulans?A.tubingensis?A.niger?A.flavus and P.chrysogennm.Through the combination of traditional microbial culture technology and DGGE molecular ecology technology,it was finally confirmed that P.chrysogenum is the major ochratoxigenic fungi in the production of Daqu.