Study On Preparation And Antibacterial Activity Of Chitooligosaccharides From Different Raw Materials

Chitin is a natural high molecular polymer that is widely found in nature,and chitosan is a product of chitin after deacetylation.The hydrolyed product of chitosan is chitooligosaccharides（COS）.Both chitosan and COS have been reported to possess diverse bioactivities,such as antibacterial,antitumor,antioxidant,immunity-enhancing,and so on.They are widely used in food,medicine,agriculture and other industries.Chitosan is usually extracted from the exoskeletons of shrimp and crab.However,the preparation of chitosan by shrimps and crabs is limited by seasons and regions.In addition,with the increase of marine pollution in recent years,shrimp and crab-derived chitosan may cause some harm to human health;the application of chitosan is limited.The cell wall of Trichoderma asperellum contains chitin.However,there are few reports about the extraction and preparation of chitosan from Trichoderma asperellum.The residue of Trichoderma asperellum after the extraction and preparation of polysaccharides,proteins,etc.is usually directly buried in the open air,resulting in environmental pollution and waste of biological resources.In this paper,chitooligosaccharides were prepared from shrimp husk powder and Trichoderma asperellum dregs.To compare the structural properties and antibacterial activities of chitooligosaccharides from different raw sources.The main research methods and results are as follows:Chitosan was prepared by alkaline method.The crude chitosan obtained from shrimp shell powder was named CT₁ with a yield of 10.78±0.02%;the crude chitosan product from Trichoderma asperellum dregs was named CT₂ with a yield of 9.53±0.03%.The characteristic absorption peaks of chitosan in both CT₁ and CT₂ were obtained by infrared spectroscopy.Therefore,both CT₁ and CT₂ were chitosan.According to the national standard of food safety GB 29941-2013,the degree of deacetylation of CT₁ and CT₂ was 82.40±0.57%and 87.59±0.42%,respectively,and the viscosity was 23.251±0.02 mPa.s and 5.63±0.01 mPa.s respectively.The chitosan content of CT₁ and CT₂ was 33.90±0.09%and 42.15±0.11%respectively by visible spectrophotometry.HPLC method was used to determine the molecular weight distribution of chitosan.The molecular weight distribution of CT₁ is approximately 800 kDa and 9 kDa;the molecular weight distribution of CT₂ is approximately 18 kDa.Using single factor experiments and orthogonal experiments,chitosanase was used to degrade CT₁ and CT₂,respectively,to obtain chitooligosaccharides,named COS₁ and COS₂.The best preparation process of COS₁ was as follows:temperature 43℃,time 7 h,enzyme dosage 30 U/g chitosan,substrate concentration 6%（w/v）,chitosan hydrolysis rate 91.98±0.12%.The optimal preparation conditions of COS₂ were as follows:temperature 40 ℃,time 6 h,enzyme dosage 30 U/g chitosan,substrate concentration 8%（w/v）,hydrolysis rate of chitosan 91.03±0.57%.The sugar content of COS₁ and COS₂ measured by the phenol-sulfuric acid method was 88.5±1.14%and 90.2±0.97%,respectively.The analysis of chitooligosaccharides by TLC,HPLC and MALDI-TOF showed that COS₁ was a mixture of three sugar,four sugar,five sugar,six sugar and four acetyl six sugar,and COS₂ was a mixture of three sugar,five sugar and six sugar.The antibacterial test results showed that COS₁ and COS₂ had no inhibitory effect on Rhizopus,Mucor,Penicillium,and Aspergillus;they had inhibitory effects on Salmonella,Escherichia coli,Staphylococcus aureus,and Bacillus subtilis;and COS₂ had better inhibitory effect than COS₁.