Efficient Preparation Of Linear β-1,3-glucooligosaccharides By Directed Hydrolysis Of Curdlan And Characterization Of Its Biological Activity

Linear β-1,3-glucooligosaccharides is a new kind of functional oligosaccharides, which also is a kind of efficient biological response modifier. Linear β-1,3-glucooligosaccharides have many kinds of functions such as inducing plant disease resistance, stimulating the production of cytokines, anti-tumor, enhancing immunity. Curdlan is the main raw material for the preparation of linear β-1,3-glucooligosaccharides. This research explored the method and optimized the conditions to efficiently preparae linear β-1,3-glucooligosaccharides by directed hydrolysis of Curdlan. Oligosaccharin bioactivity was also studied of linear β-1,3-glucooligosaccharides. The main content and results are as follows:1. Methods to prepare linear β-1,3-glucooligosaccharides by degradation of Curdlan were investigated. Dimethyl sulfoxide is the best solvent to dissovle Curdlan(150 g·L-1 at 80oC). Various methods were tested to degradate Curdlan, and acid treatment of Curdlan proved to be efficient method for preparing linear β-1,3-glucooligosaccharides.2. Linear β-1,3-glucooligosaccharides was separated with fractional precipitation by organic solvents. Nine kinds of commen organic solvents can precipitat Curdlan hydrolysate and methylene chloride and ethyl acetate have higher application feasibility. For 10 g·L-1 Curdlan hydrolysis solution, 2.0-fold and 4.7-fold volume of methylene chloride or 2.2-fold and 3.4-fold volume of ethyl acetate can separate linear β-1,3-glucooligosaccharides by fractional precipitation. Methylene chloride had higher precipitation selectivity than ethyl acetate. Curdlan concentration, water content and ionic intensity have different influences on fractional precipitation, but degree of hydrolysis has no influence.3. Quantification of linear β-1,3-glucooligosaccharides by thin layer chromatography and Image Analysis were established. The developing reagent acetic acid, ethyl acetate, H2 O in the ratio of 2:2:4:1.4(v/v/v) can separate Curdlan hydrolysate from degree of polymerization of 1 to 13 on 20 cm Silica gel 60 F254 plate. The method had been established to quantitatively analyze Curdlan β-1,3-glucooligosaccharides by Image J software. Good linear relationship(R2>0.99) can be obtained between saccharides concentration(0.02-0.1 g·L-1, 0.1-0.8 g·L-1, 0.8-5.0 g·L-1 respectively) and grayscale integration response. The detection limit of this method is 0.02 g·L-1, and the relative standard deviation is 1-5% with good stability.4. The technologies to efficiently prepare linear β-1,3-glucooligosaccharides by directed hydrolysis of Curdlan were optimized. With the yield of linear β-1,3-glucooligosaccharides of degree of polymerization from 4 to 10 which have high bioactivity and composite score for the main index, one-factor-at-a-time experiments and response surface analysis experiment were used to obatain the best hydrolysis conditions: 100 g·L-1 Curdlan, 1.0 mol·L-1 hydrochloric acid concentration, 92oC and 20 min. Under this condition, the yield is 38.03 g·L-1, the composite score is 26.64, the production intensity reaches 114.09 g·L-1·h-1 with conversion rate of 38.03%. Fruther, 300 m L scale-up experiment verified the optimal conditions.5. Oligosaccharin activity of linear β-1,3-glucooligosaccharides was investigated. Three kinds of methods including irrigating, spraying and irrigating plus spraying were applied to strawberry with 50, 100, 150 mg·L-1 linear β-1,3-glucooligosaccharides. Results showed that linear β-1,3-glucooligosaccharides can not only induce disease resistance, promote the growth but also improve the quality of strawberry fruits and keep fruits fresh by physiological detection. Better result could be observed by using higher concentration of oligosaccharin. Irrigating plus spraying was the best way. Spraying treatment was applied to lettuce also. Results showed linear β-1,3-glucooligosaccharides can improve β-1,3-glucanase activity, decrease malondialdehyde content and increase chlorophyll content of lettuce leaves. Therefore, linear β-1,3-glucooligosaccharides can induce disease resistance, improve resistance ability and promote growth of lettuce. Better result could be got by higher concentration treatment and oligosaccharides solution containing 0.5-1.5% DMSO can improve the effect by 1-3%.