Study On The Technology Of Depolymerization Of Proanthocyanidins From Grape Seeds

Proanthocyanidin (PC) is a kind of flavanols, which had been proved of strong antioxidant properties. It is obviously benefit for prevention and treatment of aging caused by free radicals, heart disease and cancer. However, snice the PC has a large number of phenolic hydroxyl, extremely easy oxidation by oxygen in the air as anthocyanins. At the same time, the PC is composed of different monomer polymerization of the mixture, which contains a lot of high polymers (degree of polymerization is greater than 4), it is difficult to through the biological membrane, so they cannot be absorbed by human body. Thus obtained can be used by human body of oligomeric proanthocyanidin (degree of polymerization is less than 4) and to prevent the oxidation is becoming a hot spot of current research. Degradation of high polymeric proanthocyanidin methods at home and abroad mainly include two categories:acid hydrolysis and rare metal catalysis. However, the former is not easy for recycle of acid, which not only wastes energy but also cause environmental pollution; besides, the instrument is higher of the latter, and rare metal belongs to the precious metal, which is expensive and not conducive to industrial production.Based on the research background, this study started from the structure and the rational medicine of proanthocyanidin, and discussed the degradation of high polymeric proanthocyanidin as a absorbable oligomeric proanthocyanidin new method. First, the inspection method and established the conditions of proanthocyanidin was founded up. On basis of those, the method of alkaline degradation of proanthocyanidin to explore, to make a study of alkaline degradation proanthocyanidin by optimizing the four groups of degradation for the degradation of the optimal conditions. Explores the enzyme hydrolysis proanthocyanidin, pick out the N-acetylneuraminic lyase, and achieved some results. In this study, explores the enzyme hydrolysis proanthocyanidin, pick out the N-acetylneuraminic lyase, and achieved some results.The main research conclusions are as follows:In order to determine the degradation effect, high polymeric proanthocyanidin monitoring and its degradation products, set up and optimize the measuring method of proanthocyanidin. First of all, the vanillin-hydrochloric acid method for determining the content of total proanthocyanidin. Then by reversed-phase high performance liquid chromatography (HPLC) method in the preliminary determination of monomer (+)-catechin and (-)-epicatechin, dimers proanthocyanidin B1 and B2 content. Finally with reversed phase high performance liquid chromatography and mass spectrometry determination of monomer (m/z=289) and dimer content (m/z=577). By these methods and conditions optimization, simplify the measuring steps, improve the detection accuracy.Second, the selection of sodium hydroxide, sodium carbonate, potassium hydrogen carbonate and sodium bicarbonate four lye to study the effect on the degradation of proanthocyanidin:sodium hydroxide> sodium carbonate> sodium carbonate or potassium bicarbonate sodium bicarbonate. The study found that PC degradation rate is proportional to the solution of basicity. Compared with acid hydrolysis and metal catalytic degradation, alkali degradation generated 1.5% of monomer and 2.0% of dimer, and acidolysis generated 2.0% of dimer, carbon palladium catalyst to generate 1.5% of monomer and 3.2% of dimer. Although the product yield was only slightly lower, but as a kind of new method of degradation of proanthocyanidin, through the regulation of experimental conditions are expected to further improve the biodegradation efficiency.Enzyme is a field of high yield, no pollution and high catalytic efficiency. In the paper, using N-acetylneuraminic lyase to degradate high polymeric proanthocyanidin. This chapter selected the Staphylococcus aureus and Escherichia coli nanA genes, respectively connected with plasmid pET duet-I, pMAL and pZE, and insert into E.coli BL21 (DE3), building 6 strains of recombinant bacteria, fermentation and SDS-PAGE validated, screening for gene expression of S.aureus nanA, use high copy plasmids pET duet-I as a carrier of E.coli BL21 SananA pETduet-I work best. At the same time, fermentation and purification SananA pETduet-I expression of N-acetylneuraminic lyase, optimal degradation conditions shows that, at 50?, the pH of 10,8 h reaction conditions of the enzymatic degradation of high polymeric proanthocyanidin optimal, the monomer is 35.67 mg·g-1, dimers is 14.49 mg·g-1All in all, this study explores the alkali degradation and enzyme hydrolysis degradation characteristics of high polymeric proanthocyanidin. Alkali degradation method is simple, cheap, which can further enhance the degradation rate are optimized by the conditions; N-acetylneuraminic lyase degradation enzyme degradation method is a new kind of green, although production is low, it has good effect on environmental protection, energy conservation and the development potential extremely.