Study On The Enzymatic Preparation And Bioactivities Of Neoagaro-oligosaccharides

As a functional oligosaccharide,neoagaro-oligosaccharides have lots of biological activities such as anti-oxidation,lipid reduction,treatment of diabetes,anti-inflammatory and anti-tumor,etc,which is one of focus on natural product development in this century.In this project,?-agarase produced by Vibrio Natriegens?HJPHYXJ-1?fermentation could degrade agar to obtain neoagaro-oligosaccharides.At the same time,antioxidant activity in vitro,Hypoglycemia and regulation of intestinal flora in vivo of high purity neoagaro-oligosaccharides were researched.The specific results are as follows:?1?Investigation of fermentation conditions during?-agarase production and preparation of neoagaro-oligosaccharides using enzymatic hydrolysis:in order to obtain?-agarase with high enzyme activity and high yield,5 L fermentation tanks were used.The result showed that the optimized fermentation condition was at 30°C,7.5 pH,8 h inoculation age,10%inoculation amount and 30%dissolved oxygen amount and the maximum enzyme activity was 376.7 U/mg.Both of 75 L and 200 L fermentation tanks were used to produce enzyme and enzyme activity were 366.1U/mg and 362.6 U/mg,respectively,which had almost no amplification effect.Used?-agarase to degrade agar and then prepared neoagaro-oligosaccharides with a polymerization degree of 2-12 was proved by HPLC.Simultaneously,the enzymatic products with different degree of polymerization could be controlled by the enzymatic time.?2?Isolation,purification and structural analysis of neoagaro-oligosaccharides:isolation and purification of neoagaro-oligosaccharides with ultrafiltration,nanofiltration and Bio-Gel P2,six monomers with a polymerization degree of 2-12consists of 99.3%,98.9%,98.0%,97.6%,97.3%and 97.4%purity analyzing by TLC and HPLC,respectively.Furthermore,FTIR,MS,and NMR were used to analyze the functional group,molecular weight and structure of each monomer.The result showed that the six monomers were Neoagarobiose?NA2?,neoagaroteraose?NA4?,neoagarohexaose?NA6?,neoagarooctaose?NA8?,neoagarodecaose?NA10?and neoagarododecaose?NA12?.?3?Study on the antioxidative activity of neoagaroteraose in vitro:the positive effect of neoagaro-oligosaccharides on the damage of HepG2 cells induced by H2O2showed that NA4 had the best protective effect of on HepG2 cells with MTT.Then,the antioxidant experiment revealed that NA4 had a significant effect on the scavenging active oxygen caused by H2O2 and dose-dependent.Studying the mechanism of HepG2 cell injury through Nrf2-ARE known that NA4 could enhance the expression of Nrf2,HO-1 and NQO1 proteins in the nucleus.These results indicated that NA4 has a certain protective effect on HepG2 cell injury with H₂O₂-induced.?4?Hypoglycemic effect of neoagaro-oligosaccharides on diabetic mice:administer gavage of neoagaro-oligosaccharides were applied for a mouse model of type 2 diabetes built by high-fat diet and tetraoxopyrimidine.The experimental results showed that neoagaro-oligosaccharides reduced the blood glucose concentration of diabetic mice and improved serum lipid levels.Simultaneously,neoagaro-oligosaccharides reduced MDA levels in the liver and increased GSH-Px and SOD levels.According to the result of HE staining,neoagaro-oligosaccharides could improve the bullous steatosis and hepatocellular swelling induced by high-fat diet and increase the number of islet cells.Additionally,neoagaro-oligosaccharides protected liver by regulating the expression of p-ERK1/2,p-JNK,NQO1,HO-1 and PPAR?.?5?The effect of neoagaro-oligosaccharides on the glycolipid metabolism and intestinal flora of mice with a high-fat diet:in order to study the mechanism of how neoagaro-oligosaccharides improve the intestinal flora,neoagaro-oligosaccharides were used as a dietary intervention in high-fat diet mice.The results showed that neoagaro-oligosaccharides improved serum lipids and enhanced lipid metabolism in the liver and adipose tissue.The increase of antioxidant enzymes such as SOD,CAT and GSH-Px and reduction of MDA could protect the liver.Simultaneously,the gene expression levels of PPAR?,PPAR?,CPT-1,FAS,SREBP-1c and LXR?were regulated in the liver.The reduction of the levels of pro-inflammatory factors LPS,TNF-?,IL-6 and IL-1?change the intestinal flora proportion in mice and reshaped the intestinal flora homeostasis to varying degrees.