Study On Glucosinolate Metabolism And Antioxidant Activity Of Broccoli Treated With Different Cutting Methods During Storage

Objective:Brassica(Brassica oleracea var.italica),also known as broccoli,is a variety of cabbage vegetable,belonging to Brassicaceae,annual or biennial plant.In recent years,broccoli has attracted much attention because it is rich in glucosinolate,polyphenols and other antioxidants and anticancer active substances.The analysis of glucosinolate biosynthesis pathway has also become a hot research spot.Glucosinolates,also known as glucosinolates oil,is widely found in dicotyledonous angiosperms of 11 species and genera,among which genussica has been found to have the most abundant glucosinolates,of which more than 200 compounds were isolated so far.When plants are subjected to mechanical damage,the degradation products such as sulforaphane and indole-3-methanol produced by the reaction of glucosinolate and myrosinase show good anticancer activity,among which sulforaphane is the strongest phase II enzyme inducer discovered so far,and it is the most effective active ingredient in vegetable with anti-cancer effect.As one of the vegetables with the most abundant glucosinolate and economical availablity,the demand on broccoli is increasing nowadays,so it is a material of great research significance and value.However,mechanical damage caused by processing in post-harvest transport of broccoli induces a series of physiological and defensive responses to prevent further tissue damage.Traumatic stress has been shown to induce the accumulation of secondary metabolites in broccoli,such as phenylpropanes and glucosinolates.In addition,the content of secondary metabolites in broccoli was affected by different injury styles,such as mechanical injury intensity,etc.Therefore,the metabolomics and physiological analysis methods were used in this study to explore the dynamic changes of bioactive metabolites of broccoli.Based on the above considerations,this study will be carried out from the following three aspects:1.Based on UHPLC-Quadripole-Orbitrap MS/MS technology combined with the metabolomics method of multivariate statistical analysis,the composition differences and distribution rules of metabolites in the storage process of broccoli treated with different cutting styles were analyzed;2.The glycosides in the broccoli samples were quantitatively analyzed by using HPLC-MS/MS method,and the activity of myrosinase was evaluated to further clarify the influence of different cutting styles on the content of glycosides;3.The changes of antioxidant activity of fresh-cut broccoli were assessed,and the effects of different cutting styles on the biological activity of fresh-cut broccoli were preliminarily explored from the aspect of antioxidant activity.Methods:1.The untargeted metabolomics method based on UHPLC-ESI-Orbitrap MS was applied to discriminate the composition changes of metabolites in fresh-cut broccoli with different cutting styles.After data preprocessing,the precise mass number was measured by comparing the compounds.Combined with the retention time,isotopic abundance and the secondary spectrum characteristic of compounds such as qualitative,significant difference of treatment between each components in single factor variance analysis and orthogonal partial least-squares discriminant analysis,screening of VIP group>1 were divided into groups of metabolites,the main differences between cluster analysis on differences between the groups of metabolites.2.HPLC-MS was used for quantitative analysis of seven glucosinolate components including SIN,NAP,PRO,RAA,GBC,4ME and NEO in broccoli with different cutting style during storage,and the activity of myrosinase was measured.Correlation analysis and cluster heat map analysis were conducted based on glucosinolate data.To compare the effects of different cutting styles on the content of glucosinolate in broccoli during storage.3.The content of antiscorbic acid was determined by 2,6-dichlorophenolindigophenol titration.The activity of L-phenylalanin ammo-nialyase was determined by enzyme plate analyzer,and the antioxidant activity of the samples was measured by DPPH free radical scavenging ability.Combining with the targeted phenolic acids compounds in metabonomics heat map clustering analysis in first chapter,we could compare in vitro antioxidant activity between broccoli with different cutting styles during storage,so that to understand the influence of different cutting styles on its quality.Results:1.In this study,non-targeted metabolomics method based on UHPLC-Quadrupole-Orbitrap-MS/MS technology was used to comprehensively evaluate the changes of metabolite profile in fresh-cut broccoli during storage.The more obvious the changes of metabolite profile were the acid substances increased significantly with the increase of mechanical strength.Among them,abscisic acid,N-ethyl maleimido-S-glutathione,2-hydroxy-3-?-D-glucopyranosylbenzoic acid(4-O-glucoside of procatechuic acid),1(2)-methylpropyl(butyl)glucoside,and 3(4)-O-Feruloylquinic acid were the compounds with the greatest difference in broccoli under different cutting styles.They were also the compounds that contributed the most to differentiating the differences between different storage stages.2.Different cutting styles were closely related to the content and components of glucosinolate in broccoli.With the increase of mechanical strength,the content of indole glucosinolate in the sample also increased,but RAA and GBC were still the main components.It was note worthy that mild and moderate mechanical damage resulted in significant decreases in the contents of PRO?RAA and GBC on the day of treatment,while severe mechanical damage had little effect.In addition,mechanical damage intensity could also affect the activity of myrosinase in the sample,the decrease of activity of myrosinase delayed when samples subjected to slightly damaged,while the degradation of myrosinase accelerated when the damage degree was serious.3.Different cutting styles could affect the phenylalanine ammonia-lyase activity and ascorbic acid content during the storage of broccoli.Except for severe mechanical damage,the change trend of enzyme activity in all groups was similar to that of the myrosinase activity.Slightly mechanical damage would increase the content of ascorbic acid,while moderate and severe mechanical damage would accelerate the metabolism of ascorbic acid during storage.However,different cutting styles had little effect on the antioxidant capacity of broccoli during storage.Except for severe mechanical damage to accelerate the oxidation of samples,the antioxidant capacity of other groups showed a trend of first decreasing and then increasing.Non-targeted metabolomics was used to comprehensively characterize the secondary metabolites produced in the storage process of broccoli treated with different cutting styles.Then,combined with the quantitative analysis of glucosides,enzyme activity and antioxidant activity determination,the intrinsic chemical base and biological activity of fresh-cut broccoli were described.This study provided research ideas and basic data for the research on the quality and biological activity of broccoli that processed and transported after picking,and provided methodological support and theoretical basis for its quality improvement.