Research On Analysis And Confirmed System Of Typical Toxins During The Production Of Chinese Rice Wine

Chinese rice wine,a traditional alcoholic beverage,has been well-known for the long history,unique flavor and enjoyable taste in China.It always plays essential roles in fermented alcoholic beverage industry around the world.The variety and complexity of raw materials,the traditional and unique processing techniques,and the various microorganisms involved in the production,can easily lead to the generation of specific toxins in the production procedures.The production mode of Chinese rice wine can also hinder the effective control of these toxins,and invariably,increase the food safety risk.Therefore,considering the critical steps that can generate and accumulate the toxins during the production of Chinese rice wine,the establishment of an analytical system,which includes the rapid detection and confirmatory techniques,for reliable and feasible detection of the by-products,will be an urgent subject with important application value.Ethyl carbamate,cyanide（a key precursor of ethyl carbamate）,aflatoxin B₁,ochratoxin A,and citrinin,have been involved in the study due to their widespread occurrence and adverse impacts on human beings and animals.This study aims to explore the new strategies for the identification,detection,and monitoring of these toxins during the production process of Chinese rice wine.The by-products from two major Chinese rice wines,Mai Qu rice wine and Hong Qu rice wine,were collected in this study.Taking the chemical structure and characteristics of the contaminants into account,a comprehensive analytical system,which included fast,on-site immunoassays（LFIA and ELISA）and the confirmatory mass spectrum methods（UPLC-MS/MS and GC-MS）was developed and validated.These techniques can greatly facilitate the monitoring of the toxins,and provide solid support for the development of the mitigation proposals.To be specific,the main content and results of the research are as follows:1.LFIA for detection of AFB₁,OTA and CIT in the by-products of Chinese rice wineDifferent types of fluorescent microspheres（FMs）were used as signal materials,and the preparation of the probes,the formulation of the pre-treatment solution for sample pad had been optimized in the construction of LFIA.（1）Concretely,the FMs were covalently coupled with AFB₁,OTA,CIT specific antibodies to produce the signal probes.During the preparation of the probes,the activated p H value,the amount of antibody,and the coupling condition were optimized,respectively.（2）Then,the test strip was fabricated basing on the sensitive signal probes.The formulation of the solution for sample pad pretreatment was also evaluated to improve the performance parameters of LFIA.（3）The LFIA presented satisfactory sensitivity and specificity.The IC₅₀ values were from 9.31μg/kg to 11.59μg/kg for AFB₁,8.34μg/kg to 9.74μg/kg for OTA,and 12.03μg/kg to15.28μg/kg for CIT.This developed method was applicable in real samples for rapid,on-site analysis,and the results can be achieved in 10 min.2.UPLC-MS/MS for confirmatory of AFB₁,OTA and CIT in the by-products of Chinese rice wineTo effectively improve the efficiency of the analysis and confirm the LFIA,a UPLC-MS/MS was developed to simultaneously detect three mycotoxins,AFB₁,OTA,and CIT.（1）According to the EU Council Directive of 96/23/EC,the retention time and characteristic fragment ions for each mycotoxin were identified.（2）The sample preparation procedures were optimized,and the methanol-water solution（80+20,v/v）,the ethyl acetate solution（0.1%formic acid）were used for efficient extraction.（3）Then,the C₁₈ SPE column was selected for better purification and concentration of the target analytes.（4）Under the optimized conditions,the performance parameters of the UPLC-MS/MS were evaluated.The method showed good sensitivity,for instance,the LODs for AFB₁were from 0.07μg/kg to 0.21μg/kg,and the LOQs were from 0.25μg/kg to 1.61μg/kg.Under two level of fortified concentrations,the accuracy and precision were studied,and the results were acceptable:the recovery rates were from 85.9%to 115.8%,the coefficients of variation were all<10%.To verify the application of the proposed method,the established UPLC-MS/MS was used to test AFB₁,OTA,and CIT in real sample.The results exhibited good correlation with LFIA,and the correlation coefficients were acceptable（R²>0.99）.3.ELISA for detection of EC in the by-products of Chinese rice wineBasing on the principle of indirect competitive ELISA and the construction of the high-quality antibodies,A rapid,reliable immunoassay for EC detection has been established and validated.（1）Firstly,the haptens and artificial antigens against EC were synthesized and identified,respectively.（2）The critical analytical parameters for ELISA,such as the dilution ratio of coating antigen,the volume of antibody,and the reaction time,were optimized and validated.（4）For better extraction of target analyte,the extraction solvents were optimized,and acetonitrile was finally selected.The proposed ELISA represented appreciable specificity towards other EC analogues.（5）After evaluation of performance parameters,the ELISA presented satisfactory sensitivity,the IC₅₀values were from 7.04μg/kg to 11.59μg/kg.The results of recovery studies supported acceptable accuracy and precision of the ELISA:the recovery rates were from 80.0%to 112.7%,and the CVs were<10%.The established method was used to quantify EC levels in real samples,and was applicable for rapid,in-field analysis in by-products from Chinese rice wine.4.UPLC-MS/MS for confirmatory of EC in the by-products of Chinese rice wineA UPLC-MS/MS technique was developed as the confirmatory method for EC detection in real samples.（1）To decrease the interference from the complex matrix and achieve the reliable results,an isotopically internal standard,d₅-EC,was selected and added into the samples before the pretreatment of the samples.（2）To improve the response signal of target analytes and boost the extraction efficiency,the alkaline silica column was involved in the sample re-preparation,and the formulation of liquid phase of LC chromatography was also evaluated.（3）Under the optimized conditions,the accuracy,sensitivity and precision of the method were satisfactory.When the sample size was set at 5.00 g,the LODs were from 1.2μg/kg to 1.5μg/kg,the LOQs were from 3.5μg/kg to 4.0μg/kg.The excellent correlation was observed when real samples were detected by UPLC-MS/MS and ELISA（R²>0.99）.The content of EC was from 3～10μg/kg in the by-products of Chinese rice wine,and increased during the fermentation steps.5.GC-MS for detection of cyanide in the by-products of Chinese rice wineA GC-MS method for detection of cyanide,a key precursor of EC,in the by-products of alcoholic beverage industry was developed and applicated in real samples.（1）To effectively decrease the matrix effect,the headspace technique was select for the extraction of target analyte,and the experimental conditions were also optimized.（2）The sensitivity,accuracy,and precision of the GC-MS were systematically assessed.The LOQs of the method can completely meet the requirements of existing maximal limits of cyanide.The recovery rates were from 86.2%to115.0%,and the CVs were<10%,completely meeting the corresponding requirements.