| In order to use corn protein resources and increase the addition value of corn,corn gluten meal which is by-product of starch industry,was used to produce corn peptides (CP) with effects of antialcoholism and hepatoprotection.The hydrolysis methods by one or two kinds of enzymes(Neutrase and Alcalase) were compared.The orthogonality test was designed to discover optimum hydrolysis conditions of preparing CP with two kinds of enzymes.Desalination,adsorption and fractionation with macroporous resins for CP were researched.The antialcoholism bioactivitiy and mechanism of CP were studied by mensuration of the alcohol concentration in mice blood and the activity of alcohol dehydrogenase(ADH) in mice liver after given alcohol.The hepatoprotective effects and mechanism of CP were investigated by model mice induced liver damage by D-galactosamine(D-GalN)/Bacille- Calmette- Gu’erin and lipopolysaccharide(BCG+ LPS).CP was separated by Sephadex G-15,then the amino acid sequence of peptides was analyzed by RP-HPLC and HPLC-MS/MS.The structure-effect relationship of CP was discussed.The main results were shown as follows:1.Study on preparation methods and inhibitory·OH activity of CPThe optimum hydrolysis condition of CP preparation methods was as follow:First corn proteins were hydrolyzed by Neutrase,when[E]/[S]=1.5%,temperature=50℃,pH=7.3, 2h.Then they were further hydrolyzed by Alcalase,when[E]/[S]=1.0%,temperature= 60℃,pH=8.0,4h.The rate of inhibiting·OH can reach 75.54%,and the rate of recovery of peptides was 95.56%.The activity of CP from two kinds of enzyme hydrolysis was higher than that from one kind of enzyme hydrolysis.The inhibitory·OH activity of CP following digestion by gastrointestinal proteases had little change.2.Study on desalination and adsorption capability of macroporous resins for CPThe effects of desalination and adsorption for CP treated by 7 kinds of macroporous resins were compared.The desorption rate of D4006 was highest with 90%ethanol as desorpting agent,however DA201-C had the best abilities of desalination and adsorption for CP with 85%ethanol as desorpting agent.The dynamic adsorption orthogonality tests of DA201-C resin was done,and the optimum adsorbing condition was 2.0 mL/min of flowing velocity,8.5 of pH and 10.0mg/mL of sample concentration.The CP was fractionated by different concentrations ethanol grads elution sequently with 15%,50% and 85%concentration ethanol.Amino acid composition of each fraction was different. The best inhibitory·OH activity fraction was eluted by 85%concentration ethanol.3.Study on antialcoholism bioactivitiy and mechanism of CPCP could activate ADH in liver.A positive correlation between dose and rate of activating ADH was observed.CP at the dose of 600 mg/kg·bw could significantly increase the activity of ADH after given alcohol(p<0.01),the rate of activating ADH was 30.1%;it could remarkably reduce the alchol concentration in blood of mice(p<0.01).The scavenging rate of alchol concentration and CP dose had positive correlation.The scavenging rate of alcohol concentration in blood and the activity of ADH of CP group’s were remarkably higher than that of model group’s in 20-200min after given alcohol.Compared to model group,the alcohol concentration of CP group’s in blood was significantly decrease in 20-120min after given alcohol(p<0.05～p<0.01);the activity of ADH of CP group’s in liver were manifested by a significant increase in 50-120min after given alcohol(p<0.05).The antialcoholism mechanism of CP owes to its effective reduction of ethanol concentration in blood after drinking,promotion of the liver ADH activity and its high contents of alanine,leucine and glutamate.4.The hepatoprotective activity and mechanism of CP(1) Compared to model mice of D-GalN-induced liver injury,pretreatment of mice with CP at doses of 720mg/kg·bw was manifested by a significant decrease in the activities of marker enzymes(AST,ALT) in serum and MDA level in liver(p<0.01),and by a significant increase in SOD activity and GSH level in liver(p<0.01).Its hepatoprotective effect was near to positive control(bifendate) groups’(200mg/kg·bw), and its liver histopathological characters were close to normal groups’.CP possesses potent hepatoprotective effect against D-GalN -induced liver damage in mice.(2) Compared to model mice of BCG+LPS-induced hepatic injury,pretreatment of mice with CP at doses of 600 mg/kg·bw was manifested by a significant decrease in the activities of AST/ALT in serum and in content of MDA/NO in liver(p<0.01),and by a significant increase in activities of SOD,GSH-Px and GSH level.The biochemical results were supplemented by histopathological examination of liver sections.The hepatoprotective effect was close to positive control(Silymarin) groups’(50mg/kg·bw) and their liver histopathological characters were close to normal groups’.CP possesses potent hepatoprotective effect against BCG+LPS-induced liver damage in mice.(3) The hepatoprotection mechanism of CP is related to its antioxidation, immunomodulatory effect and high F value.5.Study on the primary structure of CPAfter ultrafiltration treatment,the CP was with higher inhibitory activity on·OH than it before treatment.Then the CP were separated by Sephadex G-15 column and finally divided into two peaks,the CP of peak 1 had higher inhibitory activity on·OH than that of peak 2.By means of HPLC,MS,HPLC-MS/MS,with m/z 388.5 and 443.3 from high peak in HPLC,was determined,which was Lys/Gln-Leu/Ile-Lys/Gln and Ala-Asn-Ala-Ala -Pro. |
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