Research On The Rapid Detection Of Citrinin In Food Products

Background Citrinin is usually found in the red yeast rice products, a kind ofChinese traditional product with a long history. Red yeast can be used as antiseptic,flavoring agent, hametz, ingredient for wine making and other many traditional foods.Citrinin is a secondary metabolite of fungal with significant renal toxicity andteratogenicity. It also causes animal death at high doses. Enzyme-linkedimmunosorbent assays (ELISA), high-performance liquid chromatography (HPLC)and high performance capillary electrophoresis (HPCE) are mostly used detectionmethods for citrinin. However, the complex pre-processing steps of these methods aretime consuming and cannot fulfill the purpose of trace detection. Immunoaffinitychromatography column is a preconcentration device. Citrinin can be rapidly enrichedand purified from samples through this effective system with high reproducibility andspecificity. This research prepared the citrinin immunoaffinity chromatographycolumn and detected the purified product through high performance capillaryelectrophoresis system. This rapid detection method is suitable for the normalregulations check and supervision of citrinin.Proposal To produce the anti-citrinin antibody, rabbits were immunized withimmunogen synthesized by linking citrinin to protein carrier. IAC column wasprepared by coupling the obtained antibody to the filling material synthesized usingCuⅡthrough bulk polymerization method. The citrinin of two different red yeastrice products were purified and concentrated by this effective column and detected byHPCE. This rapid detection method of citrinin can be established accordingly in foodproducts.Methods Synthesize the immunogen and identify its structures. Extract the polyclonalantibody from the experiment rabbits immunized by this immunogen. The titer of antiserum was detected by indirect non-competitive enzyme-linked immunosorbentmethod and the specificity of the antibody was determined by HPCE system. The CuⅡfilling material of ICA column was synthesized through bulk polymerizationmethod with acrylamide as the functional monomer and Ethyleneglycoldimethacrylate as the cross linker. The polyclonal antibody can be easily adsorbed tothe column due to the chelating power of CuⅡfor protein. The complete ICAcolumn was prepared after fill the filling agent into the column and the adsorptioncapacity and fortified recovery were investigated under the optimal conditions. Finally,citrinin was determined by HPCE system at 25℃under the optimized examinationcondition with borate buffer at concentration of 20 mmol/L (pH 9.28) and segregationvoltage of 20 kV. The interior diameter of capillary was 75μm with its action lengthat 45 cm and the wave length of UV detector was at 319 nm.Result The amount of substance ratio, determined by UV detector, of citrinin toprotein is 26.38:1 and of citrinin to OVA is 17.58:1. The production process and tilterwere analyzed after the immune of experiment rabbit using the couplet of citrinin andBSA as the immunogen. 35 days after immunization, antibody was detected in No.1rabbit. 14 days later, antibody was produced similarly in No.2 rabbit. The quantity ofantibody reached its peak 77 days after immunization. However, No.3 rabbit did notproduce antibody from the beginning of the experiment to the end. The titers ofantiserum from No.1 to No.3 were 1:135000, 1:16000 and 1:4000, respectively. Thesynthesize condition of filler was optimized through the experiment. The maximumadsorption of filler to antibody was 196 mg/g in water solution with 30 mg cupricacetate. The optimal adsorption time was 10 hours. The best combination stability ofantibody to polymer was at the condition when solution was methanol at theconcentration of 60% with pH 7. This research investigated influence of type,concentration and pH of elution to the separation effect. Citrinin could be completelyelution from the column and the stability of the column was intact under the optimalcondition. The best extraction solution was methanol (80% V/V). The optimalwashing agent was PBS solution with methanol (60% V/V) as the elution agent. Theresult shows that the maximum adsorption capacity of ICA is 597 ng and the recovery rate of citrinin in red yeast rice and Monascus color 83.7% to 93.2% with theconcentration range is 50～150μg/kg and the RSD is 0.84%～2.06%。Conclusion This research prepared the immunogen and obtained the polyclonalantibody of citrinin. ICA column, which was developed independently, was used topurify the citrinin in food sample and HPCE system with UV detector was used todetect the citrinin. Eventually, the optimal conditions were summarized and the ICAdepuration and HPCE detection method to citrinin in leavened food were established.