Study On Isolation,Purification And Stability Of Chitooligosaccharides

Chitooligosaccharide(COS)is a kind of oligosaccharides with a degree of polymerization of 2~20 obtained by degradation of chitosan.It is the only positively charged alkaline cationic oligosaccharide in nature.Chitooligosaccharide has good water solubility and is easily to be used by organisms.It has many biological activities due to its special amino structure.Preparation of COS by enzymatic hydrolysis is one of the main ways to prepare COS.However,the commercial COS limits its application because of its wide molecular weight distribution.This project used membrane separation technology to separate and purify COS,increased the content of COS with a degree of polymerization of 3~6,improved the purification efficiency by using the cationic property of COS solution and optimized parameters based on the industrial production,providing theoretical guidance for the industrial separation and purification of high quality COS.The factors affecting the browning of COS during storage were investigated in order to achieve stable preservation of COS.The main contents are as follows:1.Analysis of crude COS showed: the content of COS in crude COS was89.11%(the content of COS with a degree of polymerization of 3~6 was 52.56%),the content aminoglucoe was 8.36%,and the content of protein was 1.33%.The average molecular mass of COS was 3021,the content of free amino group was 7.96%,and the isoelectric point was pH 9.36.2.The separation and purification of crude COS were studied by ultrafiltration.The ultrafiltration membrane of 10 kDa was selected.The optimize operation parameters: the operating pressure was 0.25 MPa,the operating temperature was 35°C,the concentration of hydrolyzate was 2%,the pH of hydrolyzate was 3.0,and the amount of water was 3 times of the original volume of hydrolyzate.After ultrafiltration,the average molecular weight of the sample was 2547,the content of protein was 0.22%,the content of COS with a degree of polymerization of 3~6 was61.86% and the product yield reached 78.58%.The cleaning effect of different mass fraction of HCl solution on the ultrafiltration membrane was investigated.It was found that 0.50% HCl solution and cyclic cleaning 30 min.The recovery rate of membrane flux was 99.19%.3.The separation and purification of crude COS was further studied by nanofiltration.The nanofiltration membrane of 500 Da was selected.The optimizeoperation parameters: the operating pressure was 0.50 MPa,the operating temperature was 35°C,the pH of hydrolyzate was 3.0,and the amount water was 4 times of the original volume of hydrolyzate.After nanofiltration,the content of glucosamine was1.21%,the content of ash was 0.15%,the content of COS with a degree of polymerization of 3~6 was 70.25%,the product yield reached 70.12%.The cleaning effect of different mass fraction of HCl solution on the nanofiltration membrane was investigated.It was found that 0.75% HCl solution and cyclic cleaning 30 min.The recovery rate of membrane flux was 99.01%.4.Stability tests were performed on the COS obtained after separation and purification.The effects of temperature,relative humidity,pH on the browning of COS products were investigated.The results showed that the browning rate of COS increased with the increase of temperature and relative humidity,and the longer the time,the worse the browning;The pH had a significant effect on the browning of COS.With the increase of pH,the absorbance value first increased and then decreased,and the highest absorption value was at pH 9.0.oxygen accelerates the browning of COS.Therefore,the COS should be stored in a low temperature,dry and acidic environment.