Preparation Of Antimicrobial Peptides Of Crucian Scales And Researches Of Its Antimicrobial Mechanism And Application In Foods

As one of the wastes which has high production but low price,crucian scales is of high research value owing to its high content of protein.The objective of this study was to made antimicrobial peptides from crucian scales by stepwise dual-enzymatic hydrolysis,so that to improve its economic value and solving the waste of high quality protein.Furthermore,optimization of enzymatic hydrolysis conditions of the crucian scales was studied.And the antimicrobial activity,antimicrobial mechanism as well as application in foods preservation of the enzymatic hydrolysates were investigated,too.It was thought that all of the researches are helpful to the further exploration and application of the antimicrobial peptides of crucian scales.1.In order to enhance the antimicrobial antivity of enzymatic hydrolysates of crucian scales,the second enzymolysis protease was selected between five proteases(Pepsin,Trypsin,Papain,Acid protease,Nutrase)on the base that Alcalase was the first enzymolysis protease and the enzymolysis conditions were optimized by single-factor test and response surface method(RSM)based on the antimicrobial activity of enzymatic hydrolysates.Results showed that enzymatic hydrolysates enzymolyzed by Alcalase firstly and by Acid protease secondly had the highest antimicrobial activity.The optimum hudrolysis conditions were determined to be first enzymolysis 62 min,second enzymolysis at 34.4?for 3 h under the precondition of second enzymolysis at pH 3.0 with an Acid protease dosage of 0.05%(w/v)and a substrate concentration of 30%(w/v).Under the optimized conditions,the maximum diameter of inhibition zone against Vibrio parahemolyticus was 27.72 mm,closed to the predicted value of 27.37 mm,which means that the optimization of enzymolysis conditions by RSM allowed accurate theoretical prediction.2.The enzymatic hydrolysates under optimum conditions was separated by Sephadex G-25 gel filtration chromatography so as to abtain effective antibiotic fraction.Consequently,three fractions were obtained,but there was only the second one named G2 showed the antimicrobial activity.And the protein concertration of G2 decreased from 0.50 mg/mL to 0.20 mg/mL.It could be concluded that a part of other peptetides had been removed,and the specific antimicrobial peptides consisted in enzymatic hydrolysates had been roughly separated and purified.3.The stability of temperature and pH of G2 was investigated,so was its antimicrobial activity.Results showed that G2 had thermal stability but did not have pH stability.With the pH increasing,its antimicrobial activity reduced rapidly and even lost when pH reached 6.0.Moreover,G2 was demonstrated to be strong at antibacterial function against both Gram-positive and Gram-negative bacterial.The minimum inhibitory concentration(MIC)of G2 on Pseudomonas and Shewanella putrefaciens was 1.56?g/mL,better than MIC(6.25?g/mL)of G2 on Staphylococcus aureus,Escherichia coli,Bacillus subtilis,Salmonella choleraesuis and Vibrio parahemolyticus.And the maximum inhibitory concentration(MBC)of G2 on Vibrio parahemolyticus,Pseudomonas and Shewanella putrefaciens was 50?g/mL,worse than MBC(6.25?g/mL)of G2 on other tested strains.4.Taking Staphylococcus aureus and Escherichia coli as research objects,determination of bacterial growth curve,cell membrane permeability and morphological observation by optical microscope were utilized to explore the antimicrobial mechanism of G2.The growth curve of Staphylococcus aureus and Escherichia coli showed that hydrolysates could efficaciously slow down or restrain bacterial growth,appearing as lengthen its lag phase and shorten its logarithmic phase.With time going by,the relative conductivity,OD260 value and protein level of the Staphylococcus aureus and Escherichia coli treated with G2 increased significantly while for the control group it kept stable,which obviously indicated that the membrane permeability increased under the addition of G2.In addition,Staphylococcus aureus and Escherichia coli were both observed to lose its original morphological characteristics and come up with severe lysogenesis after treated with G2 for 12 h.All these results indicated that G2 killed cells mainly by disrupting its membrane system and then resulting in the leak of the material in cells.5.There were five indexs,including chromatic aberration,pH,total bacterial count,TVB-N and sensory evaluation,used to estimate the effect of treatment by different concentration(0.2,0.4 mg/mL)of enzymatic hydrolysates of crucian scales on the quality of bighead carp stored at 4? compared with the controlled group without any treatment.The results reflected that the hydrolysates could efficiently inhibit the growth of bacterial in fish,thereby preventing fish protein from decomposition and as much as possible keeping its color and good sensory quality.Additionally,the results showed that the preservation effect of 0.4 mg/mL hydrolysates was better than 0.2 mg/mL hydrolysates.On the whole,the data suggested that the hydrolysates of crucian scales could be applied in food for the purpose of prolonging its quality guarantee period.