The Preparation Of Rice Protein ACE Inhibitory Peptides And The Research On Its Effective Separation By Membrane Technology

Compared to other plant protein, rice protein is more nutritional, but for chronic kidney patients it is still a major burden. TU(Tianjin) Pharmaceutical Technology Co.,Ltd. intends to develop a deproteinized rice to meet the kidney patients demand and reduce their burden on kidney. The aim of this paper is retrieving the removal rice protein, and establishing a highly efficient method of ultrafiltration membrane for recovering rice protein peptide with angiotensin converting enzyme(ACE) inhibition.Firstly, we Compared two bacillus with lactobacillus,which was original used as a protein removal production process. Results show that Bacillus Y, Bacillus Y4-2and Lactobacillus 2-18 could all multiply to 108 CFU/m L, and produce protease.However, as to the ability of deproteinization, Lactobacillus 2-18 is much better than two bacillus. The experiments of the combination of three strains with pepsin,bromelain, alkaline protease showed that Lactobacillus 2-18 combined with pepsin and bromelain had the highest removal rate of 91%, so the experiment was still using the original process to remove protein.Having obtained the recycling rice protein, we filtered it to remove the solid particles and used microfiltration to remove impurities such as bacteria, fungi. Then,we determined its basic components: total protein, peptide, mineral content was0.66%±0.026%, 0.63%±0.035%, 7.3±0.096 mg/10 m L respectively, and after dialysis,the content of mineral decreased to 1±0.035 mg/10 m L. According to amino acid analysis, the essential amino acid was 39.62% of total amino acid content, which was close to the FAO/WHO recommended protein pattern, and the value of SRC reached to 91.05, which is much higher than pork and beef. On the basis of the distribution of molecular analysis, rice protein peptides mainly between 1000 Da~1500 Da,accounting for 41.66 percent of the total peptides, which may had ACE inhibitory activity of that molecular weight range; ACE inhibition rate of rice protein peptide increased as the increasing of the degree of hydrolysis. Lactobacillus 2-18 combined with pepsin and bromelain had highest ACE inhibition rate reached 91.95%±6.45%;In the light of simulated gastrointestinal digestion(SGID), we obtained peptides will be partially hydrolyzed in the gastrointestinal, and ACE inhibition rate declined partially, and dropped to 41.05%±7.28% at last.According to molecular weight distribution, we preselection 1 k Da, 3 k Da ultrafiltration membrane, the retention rate of 1 k Da was 89.29, while 3 k Da 65.69.Based on the investing of the effect of pressure, temperature, concentration on membrane flux, we practiced a Box-Behnken design, ultrafiltration optimization model was identified as: Y = 30.92+2.56·A+4.7·B-0.58 ·C+0.9·A·B+0.0025·A·C+0.11·B·C-1.42·A2-1.22·B2-0.54·C2, ANOVA and response surface analysis showed that: temperature, pressure affected the membrane flux very significantly,while concentration, the interaction of temperature and pressure on membrane flux significantly. Based on the actual production cost considerations, through design expert software, we optimized ultrafiltration conditions as 50℃, 0.4 MPa and 0.63%,which membrane flux value was 35.43 L/(h·m2) theoretically, while the actual was35.54±0.70 L/(h·m2); studies had shown that after every 150 min we should clean membrane at 0.2MPa for 10 min, to maintain the permeability of membrane.Through cation exchange column chromatography,we initially separated peptides into three components: AⅠ, AⅡ, AⅢ; through the Superdex peptide 10/300 GL gel filtration chromatography, we purified AI into seven groups: BⅠ, BⅡ, BⅢ, BⅣ,BⅤ, BⅥ, B Ⅶ, and then purified by RE-HPLC, BⅤ was further separated into CⅠ,CⅡ, CⅠ were analysised by LC/MS and amino acid analyzer, and compared with the rice glutelin amino acid sequences, we preliminarily determined the sequence of the peptide: Val-Val- Phe-Phe-Ala-Ala-Ala- Leu.