Fabrication Of Chitosan Functional Carriers And Its Application In Lysozyme And Protease Immobilization

The egg resource in our country is very abundant,and a large amount of egg white have been used in food industry.However,the relatively traditional processing technology resulted in the low utilization efficiency of egg white.It is significant to improve the comprehensive utilization value of egg white by using the scientific and advanced processing technology.In this study,we put forward an idea that the utilization efficiency of egg white can be increased by using enzyme immobilization technology.Chitosan,a widely used polysaccharide in food field,was chosen as a basic material to fabricate the functional carrier for enzyme immobilization.Currently,the processing and utilization of egg white can be mainly divided into two aspects.Therefore,this study is going to improve the water resistance and mechanical properties of chitosan film,which will be used in lysozyme immobilization for preparing an excellent antibacterial food packaging.Furthermore,based on the traditional chitosan microsphere,we fabricated a novel porous chitosan microsphere for enhancing the activity of immobilized protease,which was suitable for the egg white hydrolysis system with high viscosity.The main results are as follows:(1)According to the organic-inorganic hybrid method,the nanosilica with different content was added into chitosan film,and the composite films were soaked in sodium hydroxide solution according to the traditional neutralization method.The physical interaction between nanosilica and chitosan molecules increased the degree of crosslinking of the molecular network in film,which resulted in the decline in the swelling rate of composite film.During the content of nanosilica was 25%,the composite film possessed the maximum tensile strength,which was equivalent to 212.88% of the pure chitosan film.However,the traditional neutralization method gave rise to about 90% decrease in activity of lysozyme immobilized in chitosan/nanosilica composite films.After treatment with sodium hydroxide solution,the lysozyme loaded composite films appeared little inhibition to E.coli and S.aureus.(2)The p H value of chitosan film forming solution was adjusted by disodium hydrogen phosphate solution.With the increase in p H value of film forming solution,the charge of chitosan molecular chain decreased and the intermolecular interaction increased,resulting in the significant decline in swelling behavior of obtained chitosan film.The maximum mechanical properties of chitosan films were observed at CS-6.5,its elongation at break and swelling rate were equivalent to 122.98% and 36.64% of chitosan film obtained with original p H value.During the p H value of film forming solution higher than 6.5,the chitosan molecules aggregated due to the higher intramolecular and intermolecular interactions.This destroyed the uniformity and continuity of the internal structure in obtained films,resulting in the performance of the films decreased.(3)In order to prepare the CSNSL composite films with immobilized lysozyme,the lysozyme with different content was introduced into the chitosan/nanosilica mixed solution,which was pre-adjusted to p H 6.5.The addition of lysozyme could increase the water resistance,transmittance,and smoothness of CSNSL.Also,a certain amount of lysozyme could improve the mechanical properties of CSNSL.The largest tensile strength and elongation at break of CSNSL were observed at film with 4% lysozyme,which were equivalent to 126.36% and 116.78% of film without lysozyme.CSNSL4 possessed the swelling rate lower than 25%.Importantly,the lysozyme loaded composite films prepared by p H induction method did not destroy the structure of lysozyme.Furthermore,the activity and antibacterial ability enhanced with the increase in lysozyme content.(4)The CSESM composite microspheres were prepared by replacing part of chitosan with ESM powder.The addition of ESM powder significantly increased the amount of immobilized enzyme on composite microspheres.Meanwhile,the ESM adding observed increased the pores density and uniformity,as well as the size of mesopores.The papain immobilized on CSESM2 had the highest ELMA and SA,which were equivalent to 156.37% and 111.95% of that in CSM microsphere without ESM powder.(5)The pore structure of carrier is important for the catalysis activity of immobilized enzyme.In order to improve the pore structure of traditional chitosan microsphere,the ammonium acetate was used as a gas porogen.The addition of ammonium acetate effective improved the porosity and pore size of chitosan microsphere,also increased the thermo thermal stability of chitosan microsphere.However,the mechanical stability and strength of chitosan microsphere decreased significant with the increase of gas porogen.The immobilization rate of papain on chitosan microsphere was mainly limited by covalent binding.The activity of papain immobilized on porous chitosan microsphere prepared via gas porogen was significantly higher than that of CSPM microsphere without gas porogen.The specific activity of immobilized papain on CSPM-AG9 was equivalent to 170% of immobilized papain on CSPM.(6)Based on the porous chitosan microsphere prepared by using ammonium acetate as a gas porogen,the sodium carbonate was used as the second gas porogen to improve its pore structure.The pore structure of outer layer of CSPM-DAGs became looser and larger in pore size with the addition of sodium carbonate.The ELMA and SA of immobilized papain on CSPM-DAG4 were equivalent to 141.70% and 143.22% of that on CSPM-DAG0.The CSPM-DAG4 also possessed a great mechanical stability.Furthermore,the immobilized papain on CSPM-DAG4 had broadened p H and temperature profile than free papain,it also possessed higher thermal and storage stability than free papain.Importantly,the immobilized papain on CSPM-DAG4 could effectively hydrolyze the egg white.After the separation of immobilized enzyme from egg white,the molecular weight of egg white hydrolysis production exhibited a negligible change after storage for 10 h.This indicated that the novel porous chitosan microsphere is a suitable carrier of protease immobilization for achievement of controlled enzymatic hydrolysis of egg white.