Study On The Formation Mechanism Of Contaminant Mepiquat In Thermal-processed Food

This research was supported by the National Natural Science Foundation of China(31772092).Mepiquat(Mep),also known as roborant,auxiliaries,etc.,is the trade name of N,N-dimethylpiperidinium chloride.It is a commonly used quaternary ammonium compound plant growth regulator in China.In 2014,research first indicated that Mep was formed at mg/kg levels in roasted coffee and soluble coffee,up to 1.4 mg/kg.Mep was a process-induced compound formed from natural ingredients in the food material during the baking process.Mep generated during thermal processing of certain foods has become an important potential factor affecting food safety.And it has also led to continued attention to its formation mechanism.This study intended to start from the model systems and food systems to explore the multiple reaction pathways of Mep.Using the model reaction systems,the kinetics and mechanisms of chemical reactions based on decarboxylation and methylation reactions were elucidated.And in the food systems represented by plant-based and animal-based foods,the effects of Mep formation reaction pathways,processing methods and conditions on Mep formation were clarified.Finally,the multi-reaction pathways and mechanisms of the formation of Mep,a new contaminant in food thermal processing,were comprehensively revealed from the model systems and food systems.The results of this study will provide an important theoretical and scientific basis for expanding the frontier theories of food safety and ensuring food safety.The main results of this study are as follows:1.The detection method of Mep was established by HPLC-MS/MS technology in different food matrices,and the methodological evaluation was carried out.The results showed that the limit of detection(LOD)and limit of quantification(LOQ)of Mep in food samples were 0.45 and 1.5?g/kg,respectively,and the sample recoveries were in the range of 80%-116%.Precision,expressed as RSD,was<9%within days and<5%among days.The ME values were all less than 1%,which further verified the effectiveness of the method for Mep detection.It could meet the detection needs of trace Mep in food.At the same time,some common thermally processed commodities in the market such as coffee and tea were selected to investigate Mep contents.Results showed that the Mep contents in coffee were relatively high,and different varieties of coffee had a greater impact on the Mep content.The Mep content in Yunnan Arabica coffee was 892±49?g/kg,and the Mep content in Vietnam Robusta was 1037±5?g/kg.The contents of Mep in different varieties of tea,potato chips and oat flakes were quite different,and the highest Mep content in potato chips was 9005.6±54.4?g/kg.The distribution of Mep in commercial products was clarified,which will provide certain theoretical data for formulating more scientific and reasonable Mep limit standards.It also lays the foundation for the subsequent study of Mep generation mechanism and reaction pathways.2.Five model systems were established with representative amino acids and pipecolic acid(Pip Ac)as precursors(alanine(Ala)/Pip Ac,methionine(Met)/Pip Ac,valine(Val)/Pip Ac,leucine Acid(Leu)/Pip Ac and isoleucine(Ile)/Pip Ac).The generation law of Mep was studied by heating in the oil bath.With the increase of temperature,the Mep content first increased and then decreased,and a temperature-dependent trend was observed.In Met/Pip Ac model system,Mep levels were up to 1.25%at 240°C for 30 min.Likewise,the Mep content in Ala/Pip Ac model system was 1.24%at 260°C.The Ala/Pip Ac and Met/Pip Ac model systems formed Mep more readily than the Val/Pip Ac,Leu/Pip Ac and Ile/Pip Ac model systems.At the same time,the time-dependent reaction kinetics of Ala/Pip Ac,Met/Pip Ac,Val/Pip Ac,Leu/Pip Ac and Ile/Pip Ac model systems were discussed.The kinetic model showed that the highest k value of the Met/Pip Ac system was10.68(×10^-4)at 260°C,and the Ea value of the Met/Pip Ac system was 21.12 k J/mol.Met had a greater contribution to Mep production than other amino acids.3.Accurate mass spectra of related compounds(such as Mep and reaction intermediates)were acquired by SIM mode of HPLC-MS/MS to verify the formation mechanism of Mep.The results showed that Mep,piperidine and N-methylpiperidine could all be formed in model systems containing Ala/Pip Ac,Met/Pip Ac,Val/Pip Ac,Leu/Pip Ac and Ile/Pip Ac(260°C for 30 min).Among them,piperidine and N-methylpiperidine of the Met/Pip Ac model system showed slightly higher responses than other model systems.At the same time,isotope tracing technique was applied to further confirm the specific role of amino acid methyl donors in Mep formation.When Pip Ac was thermally treated with ¹³C-Met,a Mep signal with accurate mass was observed at m/z 116.12,which meant two ¹²C atoms were replaced by two ¹³C atoms.The results showed that Met provided the methyl donor of N-methylpiperidine and Mep.Similar results were obtained using ¹³C-Ala,¹³C-Val,¹³C-Ile and ²H-Leu as methylating reagents.It was further demonstrated that amino acids could provide methyl groups as important precursors for Mep generation.In addition,the thermal characteristics of the amino acid/Pip Ac model systems were investigated by DSC analysis.In the amino acid/Pip Ac systems,there was only a broad endothermic peak between 240.15 and 255.25°C,which was consistent with the denaturation temperature of Mep.Pip Ac affected the amino acid denaturation process.Pip Ac could form Mep by decarboxylation in the presence of amino acids.The Met/Pip Ac model system detected a larger peak area with a higher enthalpy(?H=94.03 J/g)to complete the denaturation.The thermal stability of the Met/Pip Ac system was lower,less energy was required to complete the reaction,and the reaction was more likely to occur.4.Plant-based and animal-based food systems were used as typical food systems.Common food processing methods such as pan-cooking,deep-frying,oven-baking,and microwave were selected to study the effects of processing methods,processing time and processing temperature on the formation of Mep in different food systems.Results showed that different cooking methods affect the formation of Mep in food matrices.Deep frying required more oil compared with pan cooking.Oil was absorbed toward the inner portions of foods,which helped food ingredients to react at a much higher speed,producing higher mepiquat content.The heat transfer during microwaving was not obvious;therefore,lower levels of mepiquat were formed in contrast.The highest mepiquat level was detected upon oven baking because of the rapid and direct heat transfer on the food surface at a high temperature.In particular,the Mep content in the potato samples reached 1064±40?g/kg after heat treatment at240°C for 20 min.After treatment at 260°C for 20 min,the highest Mep content in broccoli was 293±1?g/kg.However,the Mep contents under several heat treatment conditions in the animal food system were significantly lower than the Mep contents after vegetable processing.5.Among the above vegetable systems,the processed potatoes led to a high level of Mep,which was significantly related to its rich precursor contents.The Mep content after thermal processing was closely related to potato varieties due to the differences in precursor substances of different potato varieties.The Mep contents first increased and then decreased with the increase of temperature and time.The Mep contents fluctuated obviously in different potato varieties after different processing.The Mep content in Chunshu No.5 sample was the highest at 800±1?g/kg when oven baked at 220°C for 30 min.The highest contents of Mep in Chunshu No.5,V7and golden potato samples after frying were higher than that of pan-cooked potato samples but lower than that of oven-baked potato samples.The Mep contents of Chunshu No.3,Holland No.7 and Eugene samples after frying treatment were lower than that of pan-cooked samples and oven-baked samples.At the same time,Chunshu5 variety with the highest Mep content was selected to analyze the content of precursor substances after heat treatment.The results showed that different precursor substances worked together to promote the formation of Mep during heat treatment.Pip Ac formed Mep through decarboxylation in the presence of methyl reagents(mainly betaine,choline,trigonelline,and Met).The precursor substances contained in Chunshu 5 cultivar significantly promoted the formation of Mep during heat treatment.It was further verified that the high Mep content formed by Chunshu 5 was due to its abundant Mep-forming precursors.In conclusion,amino acids,as newly discovered precursors,could utilize decarboxylation and transmethylation reactions to promote the formation of Mep in thermally processed foods.Mep-forming precursors in food systems and different processing methods would affect the formation of Mep.The study on the formation law of Mep,a contaminant of thermally processed food,will provide a theoretical basis and application basis for finally revealing the multiple reaction pathways and mechanisms of Mep formation.