| Broccoli(Brassica oleracea var.italica),belonging to the Brassicaceae genus,is an annual or biennial herbaceous plant,which is native to Italy.Known for its nutritional value,broccoli is popular worldwide and is a good source of many nutrients and bioactive substances.In recent years,broccoli and its derivatives have been reported to have various health benefits,such as antioxidant,anti-inflammatory,anti-cancer,antibacterial,neuroprotective,metabolic syndrome regulatory,and kidney protective activities.In addition,selenium is an essential trace element for human body,but most of the crop production areas in China are in low selenium regions,so it is important to supplement selenium-enriched foods in daily diet,and cruciferous crops generally have strong selenium enrichment ability.Based on this,this project explored the effect of broccoli on the antioxidant activity and metabolite composition and content of sprouts during selenium enrichment based on in vitro antioxidant activity using metabolomics technology to provide a theoretical basis for breeding broccoli sprouts with high antioxidant activity.The relevant study results are as follows:(1)Six cultivars of broccoli seeds(LH,MTL,FZLG,YX,WX and BL)were selected for the study and their antioxidant activities and phenolic contents were determined.The results showed that the antioxidant activity and total phenolic content of LH were significantly higher than those of other varieties,while the antioxidant activity and the total phenolic content of BL were the lowest,but there was no significant difference between its total flavonoid content and that of LH.The metabolites of six broccoli seeds were analyzed based on UPLC-MS/MS,and a total of 988 metabolites were identified,317 of which were differential metabolites,among which the top 20 differential metabolites in VIP value were mainly in "amino acids and derivatives" and "organic acids",and the most differential metabolite was γ-aminobutyric acid.(2)Three cultivars with high,medium and low antioxidant activity in seeds were selected and germination treatments were performed under the same conditions to analyze the antioxidant activity and phenolic content of the sprouts.The results showed that the antioxidant activity of the broccoli sprouts was significantly enhanced after germination,while the phenolic substances were enriched in large amounts.Among them,LH had the highest antioxidant activity and phenolic content.The analysis of metabolite changes before and after germination of the three cultivars of broccoli was compared and it was found that there were 612,602 and 621 differential metabolites before and after germination for LH,YX and BL,respectively.There were 404 differential metabolites present among all three cultivars.Among the "phenolic acids",the differential metabolites with the highest abundance values were all disinapoyl glucoside.Among the "flavonoids",kaempferol-3-O-(2’’-sinapoyl)glucosyl-(1→2)-(6’’-malonyl)glucoside and isorhamnetin-3-O-sophoroside were the differential metabolites with high abundance values in all three broccoli germination processes.In "alkaloids",the top five different metabolites in abundance between the three cultivars were the same: N-(2-hydroxy-4-methoxyphenyl)acetamide,3-amino-2-naphthoic acid,3-indoleacrylic acid,putrescine,6-deoxyfagomine,and the largest difference multiplier substances are p-coumaroyl spermidine.Among the "amino acids and their derivatives" metabolites,L-histidine was the most abundant differential metabolite in all three species,L-tyrosine and L-phenylalanine were also in the top 5 in terms of abundance among the three cultivars,while the substance with the highest differential multiplicity was glycyl phenylalanine.(3)LH was selected as the cultivar for subsequent studies.First,they were subjected to ordinary culture for different days.It was found that the phenolic accumulation was mainly concentrated in the middle of germination,showing an increasing trend,followed by a decreasing trend,while both antioxidant activity and phenolic content peaked on the day 4 of light culture.On this basis,two selenium sources,nano-selenium and sodium selenite,were used for selenium enrichment of broccoli sprouts,and the results showed that the suitable concentrations of both selenium sources could enhance the basic indexes of sprouts,but the nano-selenium solution with 75 μmol/L had better antioxidant activity and phenolic content than sodium selenite.A total of 1974 metabolites were detected in normal sprouts and selenium-enriched sprouts,173 were differential metabolites and 129 were up-regulated in selenium-enriched sprouts,and mainly in "phenolic acids" and "flavonoids".The top 5differential metabolites in "phenolic acids" were phenyl acetate,4-hydroxyacetophenone,propyl 4-hydroxybenzoate,vanillin acetate,5’-glucosyloxyjasmanic acid,in addition to1-O-p-cumaroylglycerol,2-hydroxy-3-phenylpropionic acid and trihydroxycinnamoylquinic acid,which were only present in selenium-enriched sprouts,and trihydroxycinnamoylquinic acid had the highest FC value after selenium enrichment.Among the "flavonoids",3,5,3’-Trihydroxy-7,4’-dimethoxyflavone(ombuin),quercetin-3-O-galactoside(hyperin),azaleatin,isorhamnetin-3,7-O-diglucoside,and quercetin-3-O-glucoside(isoquercitrin)were among the top 5 metabolites in terms of abundance.Isosalipurposide and cyanidin-3-O-(6’’-O-malonylglucoside)-5-O-(6’’’-O-caffeoylglucoside)-3’-O-glucoside were detected only in selenium-enriched sprouts,and isosalipurposide was the most significantly different substance in selenium enrichment.(4)Further analysis of the KEGG enrichment pathway between normal and selenium-enriched sprouts was most significant for "flavone and flavonol biosynthesis".A total of four metabolites were upregulated in this pathway,isovitexin,isoquercitrin(quercetin-3-O-glucoside),rutin,and syringetin,with associated enzymes EC 2.4.1.91,EC2.4.1.159,and EC 2.1.1.267,while vitexin and luteoloside were downregulated,with associated enzymes EC 2.4.1.81.The "flavonoid biosynthesis" was associated with this significant pathway,and the analysis revealed that only naringin and isosalipurposide were upregulated in this pathway,while isosalipurposide was the significantly altered the substance in "flavonoids",the related enzyme EC 2.4.1.-.These results suggest that isosalipurposide and the related enzyme EC 2.4.1.-may be the key substances in the regulation of antioxidant activity during selenium enrichment.In this study,we used broccoli as the research object,antioxidant activity and phenolic content as indicators,and high-throughput metabolomics technology to explore the metabolite changes between the different cultivars of broccoli seeds and sprouts,based on which key metabolites and related enzymes of selenium-enriched sprouts were identified.The results of the study provide a reference for the selection of broccoli cultivars and the germination of broccoli,and also provide a theoretical basis for the cultivation of selenium-enriched broccoli sprouts with high antioxidant activity. |
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