Preparation And Purification Of Antimicrobial Peptides From Crucian Carp Scales And Its Application In Preservation Of Fruits And Vegetables

China is a large country of freshwater fish consumption,but also a country of freshwater fish culture.Fish scales,as by-products of fish,are often discarded as waste.In order to make rational use of abandoned fish scales resource and realize its high value,the optimum preparation process of antimicrobial peptides from crucian carp scales was optimized by single factor and response surface.Then it was isolated and purified,and its antimicrobial and antioxidant activity were determined.Finally,the preservation effect of the crucian carp scale antimicrobial peptides on fruits and vegetables and its products was determined and evaluated by spraying and adding test.The main results are as follows:1.In order to prepare crude enzyme solution of antimicrobial peptides from crucian carp scales,the antimicrobial activity of Escherichia coli and Staphylococcus aureus was selected as the optimum index.Pepsin was selected as enzyme for enzymatic hydrolysis in this experiment.The optimum conditions were determined by single factor test:citric acid concentration was 10%,the liquid-solid ratio of acid to fish scales was 14:1?mL/g?,the extraction temperature was 50?,the extraction time was 12 h,the addition of pepsin was 0.8%?w/w?and the enzymatic hydrolysis time was 2 h.On the basis of this,the optimum extraction process of crude enzyme solution of crucian carp scales was determined by the response surface.The optimum preparation processes were determined as follows:the acid concentration was 11.3%,the liquid-solid ratio of acid to fish scales was 15:1?mL/g?,the addition of pepsin was 0.9%?w/w?,and the enzymatic hydrolysis time was 2.4 h.Under these conditions,the extraction rate of collagen from crucian carp scales reached 79.47%.2.The crude enzyme solution of fish scale antimicrobial peptides was prepared.And then after dialysis,it was separated and purified by Sephadex G-15,Sephadex G-50 gel filtration column and cellulose DEAE-52 anion exchange chromatography column in turn.Finally,a protein component Ac?with strong antimicrobial activity was obtained.The SDS-PAGE electrophoretogram of the component showed a single band,indicating that it was electrophoretically pure,and its molecular mass was about 20.1 kD.The antimicrobial activity of fish scale antimicrobial peptides against a variety of bacteria and fungi showed that fish scale antimicrobial peptides had good broad-spectrum antimicrobial activity against bacteria and fungi.And the determination of its MIC and MBC showed that the MIC of Staphylococcus aureus,Bacillus subtilis,Staphylococcus albus,Vibrio parahaemolyticus,Pseudomonas fluorescens and Shewanella putrefaciens was 16?g/mL.The MIC of Escherichia coli and Salmonella choleraesuis was 32?g/mL.The MBC of Staphylococcus aureus,Bacillus subtilis,Escherichia coli,Pseudomonas fluorescens and Shewanella putrefaciens was 64?g/mL,and the MBC of Staphylococcus albus,Vibrio parahaemolyticus and Shewanella putrefaciens was 128?g/mL.The determination of?·OH?scavenging ability,?DPPH·?scavenging ability and?O₂^-·?scavenging ability showed that it had the same antioxidant capacity as ascorbic acid at a certain concentration.This proved that it also has good antioxidant activity.3.By spraying a certain concentration of crucian carp scale antimicrobial peptides solution on the surface of plum fruit could obviously delay the senescence and decay of fruit.The results showed that it could significantly reduce the weight loss rate and respiratory intensity,delay the formation of malondialdehyde?MDA?content,inhibit the increase of activity of polyphenol oxidase?PPO?and the decrease of activity of peroxidase?POD?.Compared with the positive control group,the results showed that the concentration of crucian carp scale antimicrobial peptides solution with 40 mg/mL could reach or even better than the level of 0.05%potassium sorbate solution.It was proved that the spraying of crucian carp scale antimicrobial peptides solution had a good effect on preservation of fruits and vegetables.4.By adding a certain concentration of crucian carp scale antimicrobial peptides to Fresh Cucumber Juice could obviously prevent Fresh Vegetable Juice from corrupting and turbid.The results showed that the addition of crucian carp scale antimicrobial peptides could maintain the color of Fresh Vegetable Juice,maintain the stability of vegetable juice,prevent rancidity of vegetable juice and the change of pH,and effectively inhibit the growth of microorganisms in Fresh Vegetable Juice.Compared with the positive control group,the results showed that 0.20%antimicrobial peptides of crucian carp scales could reach the same bacteriostatic level as 0.05%potassium sorbate.It was proved that the addition of crucian carp scale antimicrobial peptides had a good antiseptic and antimicrobial ability on fresh squeezed fruit and vegetable juice.