| Barley is a whole grain raw material with high nutrition and functional activity.It has been reported that lactic acid bacteria fermentation can significantly improve the nutritional and quality characteristics of grain raw materials.In our previous study,Lactobacillus plantarum dy-1 was used to ferment the whole barley powder,and the results showed that the physiological activity and function of barley fermented extract were significantly improved.On this basis,this paper further analyzes the characteristic small molecule active components of Lactobacillus plantarum dy-1 fermented barley extract(LFBE),studies the effect of fermentation on the antioxidant activity of the main characteristic components in LFBE,and explores its possible antioxidant activity mechanism.The main research contents and results are as follows.1.Ultra-performance liquid chromatography-high resolution mass spectrometry(UPLC-HRMS)was used to study the influence of fermentation on the main components and characteristic components of LFBE,and initially revealed the substance change of LFBE during the fermentation process.A a total of 124 compounds in LFBE were identified by high-resolution mass spectrometry data collection and analysis of 8 groups of LFBE samples prepared within 0-28 h fermentation time;Principal component analysis(PCA)was used to analyze LFBE compound in different fermentation time groups.The results showed that materials in the barley samples with fermentation time of 4 h,8 h and 12 h were gradually biotransformed to produce fermentation products.The compounds whose content decreased significantly with the fermentation time,included sugars,amino acids,nucleosides,organic acids,etc.During the fermentation process,metabolites such as glucose-6-phosphate and phenyllactic acid were accumulated,as well as lipid components and some biologically active molecules such as homovanillic acid,indole-3-lactic acid etc were released.Partial least squares discriminant analysis(PLS-DA)Obtain the main characteristic components of LFBE.The results showed that some metabolites and active substances such as indole-3-lactic acid,homovanillic acid and cafestol were the main characteristic components of LFBE increased significantly during the fermentation process.2.Comprehensive use of chemical antioxidant evaluation methods and HepG2 cell model,the antioxidant activity of LFBE and its 9 main characteristic components in LFBE were analyzed.The results showed that the total polyphenols content in LFBE increased significantly with the increase of fermentation time,and the polyphenols content continued to slowly increase with the increase of fermentation time after 8 h,and reached the peak at 24 h,and the trend of change was basically consistent with the antioxidant activity of LBFE.The chemical antioxidant activity analysis results showed that most of the compounds in the main characteristic components of LFBE have high antioxidant activity,among which coumaric acid and quercetin have relatively high DPPH clearance rates,respectively 95 %,89 %,docosahexaenoic acid methyl ester,cinnamaldehyde,6-hydroxycaproic acid are relatively low,respectively 58 %,53 %,49 %;ABTS analysis results showed that quercetin,coumaric acid these phenolic compounds have higher ABTS clearance rates,78 % and 52 %,respectively,while cinnamaldehyde,6-hydroxycaproic acid,and docosahexaenoic acid methyl ester are relatively low,17 %,15 % and 19 % respectively.Compared with the model group,most of the characteristic components such as cinnamaldehyde,homovanillic acid,cafestol,etc.can significantly increase the superoxide dismutase in HepG2 cells active,docosahexaenoic acid methyl ester has no significant effect;Except 6-hydroxyhexanoic acid and indole-3-lactic acid,most of the characteristic components can significantly reduce the level of reactive oxygen level;The results of cell antioxidant activity(CAA)analysis showed that the CAA value of coumaric acid was the highest,followed by 6-hydroxyhexanoic acid,homovanillic acid,cinnamaldehyde,docosahexaenoic acid methyl ester,indole-3-lactic acid,etc.Real-time fluorescent quantitative PCR analysis of the expression of genes related to cell antioxidant activity showed that compared with blank control cells,the main characteristic components of LFBE,homovanillic acid and coumaric acid,mainly up-regulated the expression of CAT and HOMX-1genes,cafestol and indole-3-lactic acid mainly up-regulates the expression of Keap1 and NQO1 genes,indicated that there may be differences action in the pathways of different substances in the cellular antioxidant process.3.The main characteristic components of LFBE,included homovanillic acid,coumaric acid,cafestol and indole-3-lactic acid as the analysis objects,and use metabonomics technology to study the effects of different characteristic components on HepG2 intracellular metabolites and their metabolic characteristics,revealed its possible influence on the metabolic pathway in the cellular antioxidant process.The results showed that the metabolites of HepG2 cells treat with characteristic components mainly include organic acids,amino acids,lipids and other substances.The results of principal component analysis(PCA)showed that there was no significant difference in the metabolites of HepG2 cells treat with the main characteristic components of different LFBE,indicated that the effects of different treatment groups on cell antioxidation are generally similar.Orthogonal partial least squares discriminant analysis(OPLS-DA)is used to obtain significant differences in metabolites between different treatment groups.Combined with metabolic pathway analysis,compared with the blank group,the characteristic component treatment group had a significant effect on 17 metabolic pathways included intracellular pantothenic acid and coenzyme A biosynthesis,glutathione metabolism.Compared with the quercetin treated group,the metabolites in the intracellular amino acid biosynthesis,glycolysis and gluconeogenesis related to antioxidant were significantly up-regulated by homovanillic acid and indole-3-lactic acid,and there are differences in other metabolic pathways between different treatment groups.The analysis of the correlation between cellular metabolic pathways and antioxidant activity showed that the DPPH and ABTS scavenging ability of the compound and the SOD activity change trend were negatively correlated with changes in the content of most metabolites,and the level of ROS was positively correlated with the expression of most metabolites.These results indicated that metabolic pathways,especially the biosynthesis of pantothenate and Co A biosynthesis,TCA cycle,glutathione metabolism,glycolysis and gluconeogenesis,are the main pathways for the main characteristic components of LFBE to exert antioxidant activity in HepG2. |
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