Study On Preparation And Application Of Marine Polysaccharide Bioactive Gel

In this thesis,ultrasound-assisted enzymatic method was used to prepare antimicrobial peptides from Euphausia superba.Taking the yield of antimicrobial peptides as indicators,response surface methodology was used to optimize enzymolysis conditions;The Marine polysaccharide compound gel was prepared with sodium alginate,chitosan and bioactive glass as the main raw materials,and its properties and structure were determined to determine the optimum preparation conditions;On this basis,the new marine polysaccharide/antibacterial peptide bioactive antibacterial gel was further prepared by loading antibacterial peptide of Euphausia superba.The performance and infrared characterization were carried out,and the antibacterial activity of the gel was studied.The main research contents are as follows:1.Preparation and optimization of antimicrobial peptides from Euphausia superba.The antibacterial peptides were prepared by ultrasound-assisted enzymatic method,and the neutral protease was selected as the best enzyme.In the follow-up experiments,ultrasultrasonic-assisted neutral protease enzymolysis method was used to prepare antibacterial peptides from Euphausia superba.The effects of substrate concentration,enzymolysis temperature,enzymolysis time,ultrasonic time,ultrasonic power and enzyme dosage on antibacterial peptides were investigated using the yield of antibacterial peptides and the diameter of inhibition zone as indicators.The three most significant factors affecting the yield of antimicrobial peptides were selected,and the yield of antimicrobial peptides was taken as the response value,and the process was optimized by response surface methodology.The results showed that the influence of the three factors on the yield was substrate concentration>ultrasonic power>enzymolysis time,and the optimal preparation conditions were as follows:Under the conditions of substrate concentration 21%,enzymolysis temperature 50℃,ultrasonic power 120 W,ultrasonic time 25 min,enzymolysis time 4 h,enzyme dosage 6000 U/g,the yield of antimicrobial peptide was 51.24%,the diameter of inhibition zone was 20.8 mm,which were significantly higher than that of Non-ultrasonic enzymolysis method（P<0.05）.2.Preparation and properties determination of Marine polysaccharide compound gel.The Marine polysaccharide compound gel was prepared by mixing sodium alginate,chitosan and bioactive glass（BG）and crosslinking with 0.3 mol/L calcium chloride solution.The optimum conditions for the preparation of Marine polysaccharide compound gels were determined by gelling time,water content,swelling rate and mechanical properties,and the morphology and structure of the gels were characterized and analyzed.The results show that:Under the conditions of SA/CS volume ratio of 17:3 and BG addition（m/V）of 0.5 g/m L,the gel-forming time of Marine polysaccharide compound gel was about 60 s.Infrared analysis and SEM characterization showed that chitosan,sodium alginate and BG were crosslinked,the network structure was close,the dispersion was good,and the water content was 94.11%.The swelling rate is 44.82%,which has certain swelling capacity and mechanical properties,and the mechanical properties is much higher than that of the gel without BG,which can be used as the optimal preparation conditions for the subsequent preparation of marine polysaccharide/antibacterial peptide bioactive antibacterial gel.3.Preparation and properties determination of marine polysaccharide/antibacterial peptide bioactive antibacterial gel.The marine polysaccharide/antibacterial peptide bioactive antibacterial gel was prepared by loading the antibacterial peptide of Euphausia superba on the basis of Marine polysaccharide compound gel by physical blending method.Taking the content of antimicrobial peptide as variable,the effects on gel gelation time,water content,swelling rate,mechanical properties and other properties were investigated.The gel was analyzed by infrared,and the antibacterial activities of Staphylococcus aureus and streptococcus mutans were compared.The results show that:With the increase of the addition of antimicrobial peptides,the fluidity of the gel was enhanced,the gelation time prolonged,and the water content and swelling rate kept a downward trend.Compared with the gel without antimicrobial peptides,the swelling rate decreased from 44.82%to 28.78%,while the mechanical properties increased to a stable level with the increase of antimicrobial peptides content.When the content of antimicrobial peptide was 40mg/m L,the mechanical properties reached the highest value.FTIR spectrogram analysis showed that the functional groups of Marine polysaccharide/antimicrobial peptide bioactive antibacterial gel were similar to those of Marine polysaccharide compound gel.There are intermolecular force caused by Si-O,hydrogen bond force caused by-OH,hydrophilic and hydrophobic action caused by CH₃and so on.The antibacterial results showed that chitosan as the control,marine polysaccharide/antibacterial peptide bioactive antibacterial gel had antibacterial effect on both STaphylococcus aureus and streptococcus mutans,and the antibacterial activity against Staphylococcus aureus was stronger than that against streptococcus mutans.The maximum antibacterial zone diameter of staphylococcus aureus was 25.12 mm.At this time,the antimicrobial peptide content was 50 mg/m L,and when the antimicrobial peptide content was40 mg/m L,the antibacterial activity against alternation bacteria was the highest,and the antibacterial zone diameter was 22.02 mm.The addition of antimicrobial peptides improved the mechanical properties of Marine polysaccharides/antimicrobial peptides bioactive antibacterial gel,the maximum of 135.80 k Pa,can be adjusted by changing the content of antibacterial peptide gel time,the gel has good swelling property,mechanical properties and bacteriostatic ability.There are 26 figures,12 tables and 165 references in this thesis.