Preparation, Activities And Structures Of Neotype Potent Bioactive Corn Peptides As Alcohol Metabolism

Bioactive peptides have beneficial effects upon human health. The byproduct of corn industry---corn gluten meal (CGM) was used as the material in present study. Corn protein was hydrolyzed by Alcalase, then the hydrolyzate, which showed capabilities to facilitate alcohol metabolism and to protect liver against injury, was fractionationed by the ultrafiltration(UF). The potent bioactive CP fractions as alcohol metabolism stimulator were sieved and its mechanism of action was discussed according to their activities in vitro/vivo. Furthermore hepatoprotective effect of potent fraction with anti-alcoholism activity was investigated by carbon tetrachloride acute liver injury in mice. The primary structure of peptides in the most effective fraction was evaluated by the HPLC-MS/MS. In addition, preparation and purification of CP by the UF and nanofiltration(NF) was investigated. The aims of present study were to give guidance to mass preparation corn peptides, which could be used as alcohol metabolism stimulator and hepatoprotective agent. The major results of present study were showed as follow.1. Preparation of crude corn peptides and its’molecular weight distributionThe optimal condition of hydrolysis:Concentrated corn protein powder was reconstituted with water at 1:25 ratio (w/v) and was then hydrolyzed by Alcalase at an enzyme to substrate ratio of 0.1536 U/g for 5 h. Molecular weight (Mw) distribution of unfractionationed hydrolyzate and permeation of 10 kDa ultrafiltration membrane was concentrated upon about 4540 Da and 3320 Da, respectively.2. Fractionation of hydrolyzate and mechanism of CP as alcohol metabolism stimulator(1) Mw 3-5 kDa CP fraction, which exhibited the highest·OH radical scavenging activity (IC50= 1.89 mg/mL) in vitro, the highest Q value(6.01 kj/mol), and the lowest Ficsher’s ratio; The Mw< 1 kDa CP fraction had the strongest capability to activate ADH in vitro (170.83% activating rate), and highest percentage of Ala and Leu residues in the total amino acids residue (27.50%) and in hydrophobic amino acid residue (52.16%); Mw< 5 kDa CP fraction showed the highest Ficsher’s ratio and maximum ability to eliminate blood alcohol concentration (BAC) in mice, a 45.1% decrease of BAC was observed at the dose of 200 mg/kg.bw, compared to that of control group. There was a correlativity between Ficsher’s ratio and capability to eliminate BAC, with correlation coefficient 0.8067.(2) Mechanism of CP as alcohol metabolism stimulator:CP’ capability to facilitate alcohol metabolism and alleviate alcohol consumption related oxidative stress to liver could be mainly correlated with the activation to alcohol dehydrogenase (ADH) by the hydrophobic CP with low Mw. The OH scavenging activity of CP may also have synergistic contribution to the oxidative stress release.3. Hepatoprotective effects and mechanism of CP against CCl4-induced liver injury in miceThe pretreatment of CP at the dose of 200 mg/kg.bw showed a significant protection effect to alleviate CCl4-induced hepatocellular injury, which was close to that of positive control drug--Silymarin (50 mg/kg.bw). The hepatoprotective effect of CP may be due to its excellent antioxidant capability, reducing power and higher Fischer’s ratio.4. The separation and purification of corn peptides by UF/NF(1) Ultrafiltration (molecular weight cut off, MWCO 5 kDa):The transmission (Tr) and flux decreased, while volume concentrated factors (VCF) increased at all test pH with the increase of time during ultrafiltration. The flux, VCF and yield increased with the increase of pH at the same time. The pH value controlled at 8-9, would be the best choice during the ultrafiltration of corn protein hydrolyzate.(2) Nanofiltration:NF membrane with MWCO 360 Da exhibited higher transmission to peptides, amine acids and Na+, compared to 160 Da NF membrane. The rejection rate of peptides by 160 Da NF membrane was above 88.80%。160 Da NF membrane effectively retained the OH inhibiting activity and activation to ADH of corn peptides, and showed similar desalination in diafiltration process, compared with 360 Da NF membrane. Keeping the activity retain, desalinization and energy consumption in consider,160 Da NF membrane is more suitable for the concentration, purification and diafiltration of corn peptides, the frequency of diafiltration process properly controlled under 3 times.5. The primary structure of peptides from Mw< 5 kDa CP fractionsThe primary structure of CP were evaluated by HPLC-MS/MS, and the primary results were retrieved in the MS-MS database. Primary structure of six peptides was definited, which were peptidel:KPFIT or KPFLT,7:QLLPF,9:HSIPF or SHLPF,10: EEIPF. They were all hydrophobic short peptides. The hydrophobic and basic amino acids, such as L, I, P, F, K and H, could play an important role in anti-alcoholism and hepatoprotective activities.