Effects Of Exogenous Additives On Main Active Components And Flavonoid Metabolomics Of Tartary Buckwheat Sprouts

Tartary buckwheat（Fagopyrum tataricum（L.）Gaertn.）is a kind of medicine and food homologous crop.Because it is rich in flavonoids,proteins,trace elements and various essential amino acids with antioxidant activity,its research value is much higher than that of other buckwheat varieties.Many studies have shown that flavonoids and other secondary metabolites increase after Tartary buckwheat germination,and the effects of reducing blood sugar,blood lipids,antioxidation and scavenging free radicals are significantly enhanced.Therefore,it is very important to explore the enrichment mechanism of flavonoids after Tartary buckwheat germination.Stress affects plant growth status and metabolic balance.In recent years,more and more researchers have turned their attention to the stress adaptability of plants.This paper attempts to explore the reasons why plants can adapt to environmental stress,change their metabolic pathway and achieve dynamic balance.In this study,Tartary buckwheat was taken as the research object,caffeic acid,L-Phenylalanine（L-Phe）and Na Cl were selected as exogenous additives to make the stress environment for Tartary buckwheat germination.The best conditions for flavonoids enrichment and germination were chosen after studying the Tartary buckwheat germination characteristics,the primary bioactive components,the antioxidant capacity before and after in vitro digestion,and the differences of their metabolites.It provides an innovative concept for how stress can enhance the germination and flavonoid enrichment of Tartary buckwheat and provides a theoretical basis for the investigation of functional foods containing flavonoids from Tartary buckwheat.The main results of the study are as follows:1.The germination characteristics of Tartary buckwheat under the treatment of caffeic acid,L-Phe and Na Cl were investigated using a single factor test and response surface optimization experiment,and the optimal concentration ratio was determined based on the synergistic action of the three solutions.The results showed that when the three solutions were treated alone,the germination rate,germination characteristics,sprout length and seed vigor index of Tartary buckwheat were effectively improved,and the content of total flavonoids was increased.The concentration ratio of caffeic acid 17mg/L,L-Phe 2.7 mmol/L and Na Cl 2.7 mmol/L（T₄）was selected by response surface optimization experiment.Total flavonoids content in Tartary buckwheat sprouts under the treatment was 6.94 g/100 g DW,21.97%more than in the control group（CK）,and 6.12%more than predicted by the response surface.And under T₄treatment,the germination potential,germination index,promotion rate,stem length and sprout length of Tartary buckwheat sprouts were the highest,which were 97.33%,112.40,40.44%,5.80 cm and 15.83 cm,respectively.2.The effects of caffeic acid,L-Phe and Na Cl on the contents of main nutrients in Tartary buckwheat during germination were studied.The results showed that seed germination enhanced the content of total flavonoids in Tartary buckwheat.The content of total flavonoids in the T₄group was the highest after3,5 and 7 days of germination,reaching in at 5.24,6.30 and 6.82 g/100 g,respectively.The synergistic treatment of caffeic acid,L-Phe and Na Cl on the increase of total phenolics content of germinated Tartary buckwheat was better than that of single treatment.And the total phenolics content of T₄group reached17.04 mg/g after 7 days of germination.In Tartary buckwheat sprouts,exogenous additives can stimulate an increase in soluble protein to protect cells from damage caused by stress,and the content of soluble protein increases as the concentration of exogenous compounds rises.The content of soluble sugar in T₄group was significantly lower than that in CK group at 7 days after seed germination（P<0.05）.On the 7th day after germination,the content of reducing sugar in sprouts reached the highest,and 19.12 g/100 g could be reached in T₄group,which was 416.78%and 37.09%higher than that of seed and CK group,respectively（P<0.05）.Seed germination and exogenous additive stimulation can aggravate membrane lipid per-oxidation and increase the content of MDA,which increases with the increase of exogenous additive concentration,while L-Phe can alleviate the degree of membrane lipid per-oxidation.3.In vitro digestion of germinated Tartary buckwheat with caffeic acid,L-Phe,and Na Cl was investigated,as well as the concentrations of total flavonoids,total phenolics,and antioxidant capacity.The results exhibited that the oral,stomach,and intestinal stages at T₄had the highest contents of total flavonoids,measured 1.32,2.25 and 3.51 g/100 g,respectively,which were significantly greater than those in the CK group（P<0.05）.The total phenolics content in T₄group decreased by 73.26%after intestinal digestion,and the bioaccessibility was only 26.74%.The scavenging ability on DPPH free radicals increased significantly with the progress of germination,and the highest scavenging ability on DPPH free radicals was 174.74μmol TE/g at 7 days after germination.After intestinal digestion,the scavenging ability on DPPH free radicals was 155.21μmol TE/g,and the bioaccessibility was 88.82%.The scavenging ability on ABTS free radical in Tartary buckwheat sprouts increased significantly at 5-7 days after germination.Among the sprouts germinated for 7days,the scavenging ability on ABTS free radicals in sprouts treated with T₂and T₄was the strongest,and there was no significant difference between them.In T₄group,after intestinal digestion,the scavenging ability on ABTS free radicals decreased by 29.65%,and the bioaccessibility was 70.35%.After 7 days of Tartary buckwheat germination,the total antioxidant activity（FRAP）of T₄group was 37.78%higher than that of CK group（P<0.05）.With the progress of digestion,the FRAP in sprouts increased gradually.The FRAP of intestinal CK group was 21.24μmol TE/g,which was 224.52%higher than that of seeds,while the bioaccessibility of Tartary buckwheat sprouts treated with T₄was62.19%after intestinal digestion,which was much lower than that on DPPH free radical scavenging ability.It was indicating that a single evaluation standard of total antioxidant activity could not be used to evaluate Tartary buckwheat antioxidant capacity.4.The effects of caffeic acid,L-Phe and Na Cl and their synergistic treatment on differential metabolites and their KEGG metabolic pathways during Tartary buckwheat germination were studied by non-targeted metabolomics.Studies have shown that after Tartary buckwheat germination,a variety of metabolic pathways are awakened,and the body stimulates a variety of metabolites to maintain system homeostasis and biological needs in order to adapt to the effects of growth and stress.A total of 1449 metabolites were identified and annotated by metabolomics,including 163 flavonoids,140organic oxygen compounds,22 isoflavonoids and so on.Compared with seed group,differentially expressed metabolites（DEMs）in germinated Tartary buckwheat sprouts was mainly concentrated in metabolic pathways such as biosynthesis of unsaturated fatty acids,cutin,suberine and wax biosynthesis,fatty acid biosynthesis,phenylalanine and tyrosine and tryptophan biosynthesis.However,the DEMs of T₄is mainly concentrated in phenylalanine metabolism and flavonoid biosynthesis metabolic pathways.Furthermore,Tartary buckwheat germinated by T₄treatment up-regulated the metabolites of phenylpropanoid biosynthesis and flavonoid biosynthesis,brought about the synthesis and transformation of related compounds in the metabolic pathway,helped the body adapt to stress,and simultaneously encouraged the enrichment of flavonoids.This study revealed the effects of three exogenous additives under T₄treatment on Tartary buckwheat germination,body adaptation to stress and flavonoid enrichment.It offers a theoretical foundation for the enrichment of flavonoids and the safe and efficient manufacture of Tartary buckwheat flavonoid-based functional foods and other items.