Study On Carbonyl Scavenging Capacity Of Insoluble Dietary Fiber-bound Polyphenols From Black Highland Barley In Gastrointestinal Tract Based On In Vitro Simulation

Reactive carbonyl species(RCS)are highly reactive electrophilic compounds with one or more carbonyl groups.Among the RCS,methylglyoxal(MGO),glyoxal(GO),acrolein(ACR)and malondialdehyde(MDA)are extensively characterized markers.The human digestive tract is repeatedly attacked by carbonyls from exogenous intake,gastrointestinal digestion and gut microbial metabolism.Abnormal accumulation of carbonyls in the digestive tract can lead to carbonyl stress,which is an important factor in the progression of gastrointestinal inflammatory and colitis-associated colorectal cancer.Free polyphenols and insoluble bound polyphenols have been proven to be effective carbonyl scavengers.Most of polyphenols(approximately 95%)within cereal bran are covalently conjugated to insoluble dietary fiber(IDF)via ester bond,which is rich source for preparing IDF-bound polyphenols(IDF-BP).Compared to free polyphenols,IDF-BP can remain intact during gastrointestinal digestion and reach the colon,and maintain a considerable concentration for more than 24 h.Scavenging carbonyls in the digestive tract could be an efficient strategy to minimize its toxicity on human body.However,the capacity of IDF-BP from cereals to scavenge RCS in the digestive tract remain unknow.In this dissertation,carbonyl scavenging capacity of IDF-BP from black highland barley and underlying mechanism of the IDF-BP actions were studied.And the change in content,structure and carbonyl scavenging capacity of IDF-BP from black highland barley during in vitro gastrointestinal digestion and colonic fermentation were studied.Meanwhile,the influence of black highland barley IDF-BP on the generation of RCS during in vitro gastrointestinal digestion of different foods were evaluated,as well as the effect of MGO,GO,ACR and MDA on human gut microbiota was explored with or without black highland barley IDF-BP.This study will provide theoretical foundation for the research on protective role of bound polyphenols on carbonyl threat in human digestive tract.The main research contents and results are as follows:The polyphenols composition and content,as well as scavenging capacity for MGO、GO、ACR and MDA of IDF-BP from black/blue highland barley,black/red quinoa,rye and wheat were compared with each other.The results showed that black highland barley IDF exerted the highest bound polyphenols content,especially flavonoids.The dominant bound polyphenols in black highland barley IDF-BP were ferulic acid,followed by myricetin,luteolin and quercetin.All tested cereal IDF-BP could effectively scavenge RCS under p H 7.0.The black highland barley IDF-BP showed the most pronounced effect in scavenging carbonyls by trapping 75.8%,39.6%,82.9% and 18.3% for MGO,GO,ACR and MDA during incubation,respectively.However,the wheat IDF-BP only scavenged 35.5%,21.6%,53.8%and 5.4% for MGO,GO,ACR and MDA during incubation,respectively.Time courses of RCS scavenging capacity of black highland barley IDF-BP was further studied,the results indicated that the amounts of RCS scavenged by black highland barley IDF-BP increased continuously within 24 h,and the scavenging rate of ACR and MGO reached its highest within the first 8 and 12 h,respectively.Black highland barley IDF-BP which show the highest scavenging capacity was selected to further elucidate its scavenging mechanism.Firstly,continuous alkaline and acidic hydrolysis were used to remove polyphenols bound to black highland barley IDF,and carbonyl scavenging ability of black highland barley IDF containing or removing bound polyphenols was compared.The results showed that the scavenging rate of black highland barley IDF reduced more than 57.6% of MGO,27.1% of GO,61.5% of ACR and 13.7% of MDA after alkaline hydrolysis and nearly no scavenging activities on four carbonyls were observed after further acidic hydrolysis,indicating that IDF-BP play a key role in scavenging carbonyls.Moreover,the formation of adducts between black highland barley IDF-BP and RCS were detected in chemical model systems.Mono-luteolin-MGO adduct,mono/diquercetin-MGO adducts,mono-quercetin-GO adduct,mono-luteolin-ACR adduct and monoquercetin-ACR adduct were identified,suggesting that black highland barley IDF-BP could exert scavenging efficacy through trapping RCS.After investigating the release and carbonyl scavenging capacity of black highland barley IDF-BP during in vitro gastrointestinal digestion,it was found that residual black barley IDFBP accounted for 97.9% of the total bound polyphenols content,and considerable antioxidant activities were still retained.Almost no carbonyls scavenging capacity of black highland barley IDF-BP at p H 3.0(gastric),while it was showed effective scavenging activity at p H 7.0(intestinal).Compared with the non-digested black highland barley IDF-BP,carbonyl scavenging capacity of digested samples for MGO,GO,ACR and MDA only decreased by1.3%,1.7%,1.1% and 1.6% of the total,respectively.These results suggested that gastrointestinal digestion had almost no influence on carbonyl scavenging capacity of black highland barley IDF-BP.Moreover,the influence of black highland barley IDF-BP on the generation of RCS during in vitro gastrointestinal digestion of cookies and cooked high-fat pork were further explored.The results showed that the concentration of MGO and GO in digestive juice decreased by 63.4% and 54.3% after simulated gastric digestion when black highland barley IDF-BP co-digestion with cookies,while their concentration decreased by60.5% and 46.8% after simulated gastrointestinal digestion,respectively.Meanwhile,when black highland barley IDF-BP co-digestion with cooked high-fat pork,the concentration of MDA,4-hydroxy-2-nonenel and 4-hydroxy-2-hexenal in digestive juice decreased by 52.3%,46.5% and 48.7% after simulated gastric digestion,while their concentration decreased by39.1%,34.4% and 37.3% after simulated gastrointestinal digestion,respectively.These results suggested that black highland barley IDF-BP could reduce the concentration of RCS during in vitro gastrointestinal digestion of different foods as antioxidant and carbonyl scavenger.After investigating the release and carbonyl scavenging capacity of black highland barley IDF-BP during colonic fermentation(6-24 h),the results showed that the cell wall structure of black highland barley IDF-BP was coarse and disintegrated into a honeycomb shape during fermentation processes.Meanwhile,the polyphenols bound to black highland barley IDF were released due to the decomposition of the rigid structure,and the release amount was significantly higher than that in the simulated gastric and small intestine digestion stages.Ferulic acid and quercetin were the dominant released polyphenols from black highland barley IDF-BP during colonic fermentation,and they could be metabolized and converted into p-hydroxybenzoic acid under the action of gut microbiota.Remarkedly,more than 74.2% of black highland barley IDF-BP was still retained in fermentation residues after 24 h of colonic fermentation.During fermentation processes,the released and transformed polyphenols showed limited scavenging capacity for MGO,GO and ACR,nearly no scavenging activity were observed for MDA.However,residual black highland barley IDF-BP in fermented residues exerted more effective scavenging capacity by trapping from 45.5% to 56.5% for MGO,from 19.0% to 25.6% for GO,from 54.8% to 64.5% for ACR and from 6.6% to 11.2%for MDA.Finally,the effect of MGO,GO,ACR and MDA on human gut microbiota was evaluated with or without black highland barley IDF-BP.The results showed that MGO,GO,ACR,and MDA increased the relative abundance of total pathogenic bacteria from 14.1% to 37.5%,39.0%,34.1% and 39.6% without the addition of black highland barley IDF-BP,respectively.However,the relative abundance of total pathogenic bacteria decreased to 8.2%,27.5%,8.0%and 32.6% when black highland barley IDF-BP was added,respectively.Meanwhile,the proliferation of probiotic bacteria,including Megaspaera,Bifidobacterium and Lactobacillus,was effectively promoted by black highland barley IDF-BP.Moreover,the four RCS inhibited the production of short-chain fatty acids,MGO,GO,ACR,and MDA decreased the concentration of acetate from 5.7 m M to 2.0,3.5,4.8 and 1.5 m M without the addition of black highland barley IDF-BP,respectively.The concentration of propionate decreased from3.3 m M to 2.1,2.0,2.9 and 2.8 m M,while the concentration of butyrate decreased from 2.5m M to 1.6,1.0,0.5 and 0.8 m M without the addition of black highland barley IDF-BP,respectively.In the presence of black highland barley IDF-BP,the concentration of acetate increased to 8.9,5.3,8.2 and 5.5 m M,while the concentration of butyrate increased to 7.1,4.1,10.3 and 1.8 m M,respectively.However,black highland barley IDF-BP could not effectively simulate the production of propionate.These results suggested that the four RCS negatively affected the human gut microbiota.The addition of black highland barley IDF-BP could improve RCS-induced detrimental consequences on gut microbiota.The beneficial role of black highland barley IDF-BP was achieved by inhibiting the proliferation of pathogenic bacteria,promoting the increase of probiotic bacteria and stimulating the production of acetate and butyrate.Black highland barley IDF-BP showed more effectively modulation capacity on MGO and ACR-induced detrimental effects on gut microbiota,which was consistent with the trend of its scavenging capacity towards the four RCS,indicating that black highland barley IDF-BP could modulate gut microbiota by scavenging carbonyls.