| Corn gluten meal(CGM)is the main by-product of wet production of corn starch.Its pH is about 4.0.It has no emulsification activity,is insoluble in water and its processing characteristics is poor.It is mainly used as animal feed,so its added value is low.CGM contains 62-74% protein,which is an ideal raw material for the preparation of bioactive peptides.Therefore,the use of CGM to prepare active peptides can greatly increase the added value of corn.CGM was disposed by heating and ?-amylase,and high purity corn peptides(CPs)were prepared by controlled enzymatic hydrolysis and activated carbon decolorization.The mechanism of anti-alcoholism and hepatoprotection of(CPs)was further explored through acute alcoholic liver injury in mice.Gel chromatography was applied on the separation and Mass-spectrometric technique was used to characterize the peptide sequence.Furthermore,the identified peptides were chemically synthesized and their mechanism of activating alcohol dehydrogenase was studied by molecular docking technology.(1)By comparing the different pretreatment methods of CGM,it was found that the heating and ?-amylase pretreatment can increase the protein content and improve enzymatic hydrolysis efficiency.Therefore,heating and ?-amylase was selected for the pretreatment of CGM.The pretreated corn gluten meal was hydrolyzed by papain and pancreatin to obtain CGMHs.The optimum enzymatic conditions are as follows: the material-to-liquid ratio was 10:1,the quantity of enzyme was 2%,reaction time was 12 h.Under such condition,the protein recovery rate was 69.89%,the ADH activation rate was 65.72%,the DPPH clearance rate was 86.11%,and the Trolox equivalent(TE)values in ABTS and ORAC assays with 1318.97 and 1192.39 ?mol TE/g,respectively.After decolorization of CGMHs by activated carbon,the decolorization rate was 89.92%;the total amount of amino acids of CPs was 94.21 mg/100 mg,and the contents of Leu and Ala were 15.94 mg/100 mg and 12.13 mg/100 mg,respectively.(2)The mechanism of anti-alcoholism and hepatoprotection of CPs was further explored through acute alcoholic liver injury in mice.Results showed that CPs can significantly reduce alcoholic liver damage in mice.Compared with model group,early administration of different doses of CPs can increased the time of loss of righting reflex(LORR),and shorten the duration of LORR;inhibited the increase of liver index in mice,reduced the degree of hepatocyte degeneration and inflammatory cell infiltration,reduced serum ALT activity,AST activity and MDA content,increased liver SOD activity,GSH-PX activity and SOD content;inhibited CYP2E1 mRNA levels and its protein expression,increased liver ADH activity and ALDH activity;inhibited the content of serum TG,TC and liver TG;decreased the expression of SREBP-1c and improved expression of PPAR?.The above studies found that CPs may alleviate alcoholic liver damage in mice through three pathways: anti-oxidation,regulating of alcohol metabolism-related enzymes and regulating fatty acid metabolism.(3)Sephadex G-15 chromatography was applied on the separation and UPLC-ESI-Q-TOF-MS/MS was applied to identify peptide sequences with high ADH activating activity of CPs.Furthermore,the identified peptides were chemically synthesized and their mechanism of activating ADH was studied by molecular docking technology.Results showed that CPs was isolated to four fractions(P1,P2,P3 and P4).The fraction P4 of small molecule showed the highest ADH activation rate;a total of 66 peptides were identified,among them,LH at a concentration of 0.5 mM exhibited the highest ADH activation rate with 28.34%,followed by AF?YL and LLP;Furthermore,AF? LLP?YL and LH displayed different affinities(-6.5 kcal/mol?-6.3 kcal/mol?-7.6 kcal/mol?-6.7 kcal/mol)to ADH.Obviously,many hydrogen bonds were observed between AF?LLP?YL?LH and ADH,which implied that these polypeptides can activate ADH activity may be related to the affinity and the number of hydrogen bonds. |
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