浸泡消毒对蔬菜中5-甲基四氢叶酸结构影响的理论研究

Theoretical investigation of the effect of immersion disinfection on the structure of 5-methyltetrahydrofolate in vegetables

新鲜绿叶蔬菜携带的致病性微生物已经成为引发食源性疾病的一大诱因,为减少绿叶蔬菜携带的病原体,次氯酸的浸泡消毒工艺已广泛应用在很多生鲜蔬菜的生产、加工及包装过程中.然而,次氯酸在杀死病原体的同时,也很可能对蔬菜中的营养成分产生影响.叶酸作为细胞生长和繁殖不可或缺的营养成分,与人类机体重要的生化过程密切相关,而浸泡消毒对叶酸影响方面的研究甚少且影响机制尚不清楚.本文运用量子化学密度泛函理论方法,在B3LYP/6-31G(d)水平上以蔬菜中最普遍存在的5-甲基四氢叶酸为研究对象,系统探究其与次氯酸反应的可能反应位点、机制及活性.研究发现,次氯酸能够与叶酸上的富电基团——(亚)氨基、酰胺基和苯环发生亲电取代反应,有趣的是不同于以前发现的氨基N是活性最强的位点,本研究发现与亚氨基直接相连的苯环的α-位的C具有最强的反应活性(ΔG^(≠)分别为41和61 kJ·mol^(-1)),这可能与其能形成亚胺共轭结构有关;(亚)氨基N的活性仅次之(ΔG^(≠)为76~79 kJ·mol^(-1));酰胺基N和与亚氨基直接相连的苯环β-位的C的活性远低于前两者(ΔG^(≠)为149~157 kJ·mol^(-1)).有关发现的5-甲基四氢叶酸氯化产物的毒性还有待进一步研究.本研究结果可为后续深入探讨次氯酸对叶酸结构产生的影响奠定理论基础,也为扩展了解次氯酸的浸泡消毒对蔬菜中其他营养成分的影响提供参考依据。

Pathogenic microorganisms carried by fresh green leafy vegetables have become a major inducement of food borne diseases. In order to reduce the pathogens, the disinfection technology of immersing with hypochlorous acid has been widely applied into the production, processing and packaging processes of fresh vegetables. However, hypochlorous acid not only can kill pathogens, but also might have some influences on the nutritional composition of vegetables. Folic acid as an indispensable nutrient for the growth and reproduction of cells in green leafy vegetables, plays a major role in important biochemical processes of the human body, however, few investigations about the effects of the immersion disinfection technology on the folic acid nutrient composition have been performed and little knowledge regarding the reaction mechanisms has been known to date. Therefore, 5-methyltetrahydrofolate as the most common form of folic acid in vegetables was chosen as model precursor, and its potential reaction sites, reaction mechanisms, and reactivity of various sites in the electrophilic substitution reaction with hypochlorous acid were systematically investigated by using density functional theory method in this study. There are two kinds of electron-rich sites as potential reaction sites in 5-methyltetrahydrofolate for its electrophilic substitution reaction with hypochlorous acid. One is the N site with lone pair electrons and the other is the C site of benzene ring with big π bond. The N sites studied here include the pyrimidine-linked amino and imino N, the benzene-linked imino N, and the amide N, and the C sites contain the α-C sites and the β-C sites of the imine-linked benzene ring. Our results indicate that all the above reactive sites could undergo concerted reactions. Although from the viewpoint of thermodynamics, the reactions of all the above sites with hypochlorous acid are spontaneous and favorable, there are quite large differences between them in kinetics. Interestingly, in contrast to previous results that amino N being the most reactive site, the α-C site of the benzene ring directly connected with imino is the most reactive site(ΔG^(≠)is about 41 and 61 kJ·mol^(-1), respectively), which may be attributed to the formation of imine conjugated structure during the reaction,(imino)amino is the second(ΔG^(≠)is between 76~79 kJ·mol^(-1)) reactive site, amide N and the β-C site of the benzene ring directly connected with imino have dramatically lower reactivity than the above(ΔG^(≠)is between 149~157 kJ·mol^(-1)). The toxicity of the chlorinated 5-methyltetrahydrofolate products found in this study remains to be further investigated. The results of this investigation can lay a theoretical foundation for further study on the impact of hypochlorous acid on folic acid structure, and also provide a reference for expanding the understanding of the influence of hypochlorite immersion disinfection on other nutrients in vegetables.