Immunoassay Based Simultaneous Detection Of Capsaicinoids And Aflatoxins In Edible Plant Oils

The quality and safety of edible oil is not only essential to the national economy,but also to the health of consumers.Edible oils are parts of the indispensable nutrition components and widly used in food industry.It has been estimated that 10 million tons of waste cooking oil is generated over the world every year.How to deal with these waste oils is a seriously problem for the industry.It would be a good resource if these waste oils are used as the raw material of biodiesel.However,recently the cooking waste oil becomes one of the threats on people's health because it was added into or directly used as the qualified vegetable edible oils by some unscrupulous traders to reduce costs and pursuit of high profits.After frying,boiling and other cooking process,the nutritional value of edible oil is greatly reduced becaused of the high peroxide value,trans-fatty acids and other harmful substances.And because of mixing with food debris and animal tissues,the edible oil also easily polluted by bacteria or fungi which may secreted toxins,such as aflatoxin,a strong carcinogen which is seriously threat consumer health and difficult to be removed in the refining process.According to the reported results of the content of capsaicinoids in edible vegetable oils and waste cooking oils,the capsaicinoids could be used as an external marker for waste cooking oils.For the lack of rapid method for identification of edible vegetable oil,an immunoaffinity-ultra performance liquid chromatography-mass spectrometry?HPLC-MS/MS?and on-site immunoassay methods were developed for the determination of capsaicinoids.At last,the technology of simultaneous detection of capsaicinoids and aflatoxins in edible vegetable oil was established,which provide the key technical support for the discrimination of waste cooking oil.The main contents and innovations of this study are as follows:1.The hapten design principle of capsaicinoids was studied and the most effective antigen structure was proved,and the high affinity monoclonal antibody of capsaicinoids was successfully developed.Four kinds of capsaicinoids haptens were designed and synthesized for the first time.These haptens were conjugated to bovine serum albumin to synthesize immune antigens,and conjugated ovalbumin to synthesize coating antigens.The Japanese big-eared rabbits were immunized with four capsaicinoids immune antigens,and the effects of different antigenic structures on immunological were examined.Balb/c mice were immunized with the most immunogenic antigens.The mouse that produced the highest titer and most sensitive antibodies against capsaicin was chosen for cell fusion.Finally,an ultra-sensitive anti-capsaicinoids mAb YQQD8 was screened out and the sensitivities(expressed by IC₅₀)to capsaicin,dihydrocapsaicin and N-vanillylnonanamide were 8.5,5.0,13.5 ng/mL,respectively.The result indicated that YQQD8 can be used as capsaicinoids generic monoclonal antibody.2.A competitive indirect enzyme-linked immunosorbent assay?icELISA?was established for rapid detection of capsaicinoids in multiple samples.Based on the mAb YQQD8 and coating antigen OVA-hapten C,a competitive indirect enzyme-linked immunosorbent assay?icELISA?was established for capsaicinoids detection in waste cooking oils,the IC₅₀ values of blank standard curve,dilution matrix standard curve and concentrated matrix standard curve were 5.93,6.64 and 8.36 ng/mL,respectively.This method is more suitable for the analysis of a large number of samples.3.The fluorescence polarization immunoassay?FPIA?for capsaicinoids detection was established.By using the best capsaicinoids tracer CPC-3-EDF?Rf=0.9?and YQQD8,the fluorescence polarization immunoassay?FPIA?for capsaicinoids detection was established,the IC₅₀ values of FPIA was 0.0356?g/mL,and the linear range(IC₂₀^IC₈₀)was 0.0126⁰.1009?g/mL.Compared with the ELISA method,the fluorescence polarization detection technique is more operate,and the result can be read once the sample is added.4.The nanogold probe-based immunochromatographic assay?NGICA?for qualitative detection of capsaicinoids was developed.By combining immunoassay with chromatographic technique,the nanogold probe-based immunochromatographic assay?NGICA?for capsaicinoids detection was developed,the Visual detection limit?VDL?to capsaicinoids was 2.00 ng/mL.Because of the concentrated extract in the pretreatment process,the VDL of this method reached 0.40?g/kg in real oil samples.The results anayted by NGICA were not only visible by the naked eye but also with small matrix effect,and greatly improving the detection sensitivity.5.A time-resolved fluorescent immunochromatographic assay?TRFICA?for rapidly monitoring capsaicinoids in oil samples was development.The TRFICA for capsaicinoids detection in oil samples was development by combining fluorescence immunoassay with the chromatographic technique,the linear range of the method was 1.95⁶2.5 ng/mL.Because of the concentrated extract in the pretreatment process,the minimum linear range of TRFICA for capsaicinoids was 0.39¹2.5?g/kg in oil samples.The TRFICA can be used to distinguish the peanut oil,waste cooking oil or qualified oil by quantitative determination of the capsaicinoids in oil samples.6.A method for the determination of capsaicin and dihydrocapsaicin was established by combining immunoaffinity columns with high performance liquid chromatography-tandem mass spectrometry HPLC-MS/MS based on a quality anti-capsaicinoids antibody.The limits of detection?LOD?for capsaicin and dihydrocapsaicin were 0.02 and 0.03?g/kg,and the limits of quantification?LOQ?were0.08 and 0.10?g/kg,respectively.This method can be used to detect the content of capsaicinoids in oils,so as to discriminate the waste oils and ensure the quality and safety of edible vegetable oils.8.The nanogold probe-based immunochromatographic assay for simultaneous detection of capsaicinoids and aflatoxins was developed by using anti-capsaicinoids YQQD8 mAb and anti-aflatoxns 1C11 mAb.The Visual detection limit to capsaicinoids and alatoxins were 2.00 and 0.10ng/mL.This method is successfully using in discrimination waste cooking oils by determination of capsaicinoids and aflatoxins.The advantages of this method are operate simply,low cost,and working well without large-scale equipment and professional operators,which provides the relevant departments with a important technical support for supervision on the quality and safety of edible oil.