| There is a close relation between intestinal flora and human health.The intestinal flora plays essential roles in intaking nutrient,protecting mucosal barrier,and regulating immune system.However,If the flora dysbacteriosis occurred,inflammatory cytokines,inflammatory mediators,proteases and oxygen free radicals will be released largely,resulting in the injury of intestinal mucosal mechanical barrier,causing bacterial translocation and the out of control of intestinal immunoregulation,which poses a serious threat to human health.Therefore,it is necessary to exploit an effective prebiotics to regulate the balance of intestinal flora and therefore maintain the intestinal health.The preparation of Semen coicis rsistant starch has opened up an important approach for developing a type of new prebiotic.Semen coicis(Coix lachryma-jobi),belongs to the Gramineae family,Coix,which are rich in starch and is an important specialty economic resources in China.Semen coicis was chosen as raw material in this paper,the autoclaving treatment,ultrasonic-autoclaving treatment,enzyme-autoclaving treatment,and microwave treatment were used to prepare Semen coicis resistant starch.Then some modern analytical testing technologies were taken to analysize the structural characteristics and physicochemical property of Semen coicis resistant starch.On this basis,the in vitro experiment was conducted to investigate the effect of Semen coicis resistant starch on the proliferation of some probiotics,and the survival rate of probiotics in simulated gastrointestinal tract conditions were also researched to demonstrate the relationship between structure and prebiotics efficacy.After that,the effect of Semen coicis resistant starch prepared by different methods on the intestinal flora microecology was investigated to prove the results of in vitro experiment.Base on the in vitro and in vivo experiment,a Semen coicis resistant starch preparation mothod with best prebiotics efficacy was selected to study its role in the regulation of intestinal morphology.Eventally,the metabonomics technology was employed to study the pathway and mechanism.The results were concluded as follows:(1)The structural characteristics and physicochemical property of Semen coicis resistant starch prepared by different methods and traditional prebiotics high alymose maize starch(HAMS)were different.HAMS had an oval appearance,while the surfaces of GP-SRS3,UP-SRS3,EP-SRS3,and MP-SRS3 were rougher compared to HAMS.Some shallow and deep layered strips could be found on the particle surfaces of GP-SRS3,UP-SRS3,EP-SRS3,and MP-SRS3.There were many cavities on the particle surface of UP-SRS3,and the MP-SRS3 showed an irregular structure,with large numbers of deeply layered strips.The results of FT-IR showed that the DO and DD value of MP-SRS3 was 1 · 118 and 1.114,compared to GP-SRS3?UP-SRS3?EP-SRS3 and HAMS,the molecular chain of MP-SRS3 displayed higher degree of ordered structure and double helix structure.The results of 13C NMR showed that compared to GP-SRS3?UP-SRS3?EP-SRS3 and HAMS,MP-SRS3 exhibited a higher relative crystallinity and a lower amorphous component,which means the its crystalline structure was more stable.The results of DSC showed that the crystalline structure was tighter than other samples.Besides,MP-SRS3 displayed a lower solubility and swelling power than other samples,which means the lager particle density,degree of double helix structure,and an order molecular arrangement.The results of iodine absorption characteristics indicated that molecular weight distribution of Semen coicis resistant starch was concentrated.(2)The Bifidobacterium bifidum?B.longum?B.breve?B.adolescentti?B.animalis?B.infantis were selected to investigate the prebiotics efficacy of Semen coicis resistant starch and HAMS,the results showed that compared with HAMS,GP-SRS3,UP-SRS3,EP-SRS3,MP-SRS3 could better promote the fermentation of Bifidobacteria,and MP-SRS3 displayed the best effect prebiotics efficacy.Moreover,Bifidobacteria exhibited a higher survival rate under simulated gastrointestinal tract conditions,such as low pH,bile acid,pepsin and trypsin,in resistant starch media,especially in MP-SRS3 madium.(3)In order to investigate the regulation effect of Semen coicis resistant starch on intestinal flora in mice,and traditional prebiotic HAMS was selected as a positive control.The results showed that Semen coicis resistant starch could control the weight of mice,and the intake of Semen coicis resistant starch would not cause any side effect to mice.The thymus and spleen index of the GP-SRS3,UP-SRS3,EP-SRS3,MP-SRS3 group increased,which indicated that Semen coicis resistant starch had the capability of improving the immunity of the mice.Semen coicis resistant starch could effectively reduce the pH of colon and cecal contents.Specifically,the pH value of ascending colon,transverse colon,descending colon,sigmoid colon and cecum contents in MP-SRS3 group were lower than that of other groups.The amounts of Bifidobacteria in the feces of mice were increased.In contrast,and the number of E.coli was decreased.After 6 days,the number of Bifidobacteria in the feces of MP-SRS3 group was larger than that of the other groups.Moreover,Semen coicis resistant starch could remarkable improve the short chain fatty acid(SCFAs)concerntration in intestine of mice.To be more specific,the concentration of acetic acid,propionic acid and butyric acid in the feces of MP-SRS3 group was higher than that of other groups.Eventually,Semen coicis resistant starch could reduce the number of pathogenic or potentially pathogenic flora such as Porphyromonadaceae,Clostridium and Rikenellaceae in the intestine of mice,as well as increase the number of beneficial intestinal microflora such as Butyrivibrio,Bacteroidaceae,and Prevention,which exhibited satisfactory flora regulating effect.(4)On the basis of the previous in vitro and in vivo experiment,MP-SRS3 was selected as the best prebiotics to investigate the effect of Semen coicis resistant starch on the intestinal morphology.The results indicated that MP-SRS3 could significantly reduced the body weight and feed efficiency of rat(p<0.05),and it did not cause any side effect to rat.In addition,MP-SRS3 could increase fecal wet weight,improve fecal moisture content,promote intestinal peristalsis,and accelerate intestinal toxic substances discharge.What is more,MP-SRS3 could increase the total mass of cecum,cecal wall quality and cecal contents,promote the metabolism,growth and differentiation of epithelial cell.The results of morphology showed that MP-SRS3 could not only increase the length of duodenum,jejunum and ileal villus,reduce the depth of crypt,and improve the duodenum,but also increase the mucosal thickness and muscle thickness of duodenum,jejunum,ileum,cecum and colonic,which plays an important role in intestinal growth and metabolism.(5)The metabonomics method was utilized to analysize the change of metabolic phenotype of rat,the results showed that the metabonomics in each group were significantly different,and all groups could be distinguished in PCA score plot,some biomarkers were acquired by PLS-DA,the levels of which changed dynamically between different time.The metabolites of succinic acid,citric acid,acetoacetic acid,tryptophan,phenylalanine,tyrosine,2-phosphoglyceric acid,phosphoenolpyruvic acid,linolenic acid,linoleic acid,oleic acid and hippuric acid has been adjusted by MP-SRS3,suggesting MP-SRS3 could adjust the metabolism of glycolysis,amino acid,fatty acid related to intestinal microflora,and then affect the whole metabolism of rat. |
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