Study On Antibacterial Activity And Mechanisms Of Silver Carp Recombinant Cystatin C And Its Hydrolysates

Cysteine proteinase inhibitors（CPIs）are widely distributed in various tissues of fish,and viscera,fish skin and minced meat are often discarded as scraps or wastes in the processing of aquatic products.It was found that the CPIs superfamily member Cystatins（FamilyⅡ）from terrestrial fauna and flora had potential antibacterial activity.The antibacterial activity of recombinant cystatin C（HmCystatin C）from silver carp（Hypophthalmichthys molitrix）and its hydrolysates against 9 common spoilage bacteria in aquatic products was studied.G^-Pseudomonas aeruginosa and G⁺Staphylococcus saprophyticus,G^-Pseudomonas fragariae and G⁺Bacillus subtilis were used as typical test bacteria for HmCystatin C and its hydrolysates,respectively.The possible antibacterial mechanisms were explored from the perspectives of interacting with the cell surface,destroying cell integrity,affecting the synthesis of intracellular protein and the cysteine protease activity of the test bacteria.These results laid an experimental foundation for clarifying the bacteriostatic activity and potential mechanisms of fish Cystatins,and also provide a theoretical reference for the development of efficient natural preservatives of aquatic products by comprehensive utilization of fish Cystatins.The results are as follows:（1）Preparation of HmCystatin C hydrolysatesTaking the Diameter of inhibition zone（DIZ）of Pseudomonas aeruginosa and Pseudomonas fragariae as the index,the types of proteases and enzymatic hydrolysis conditions were screened by single factor test.The results showed that the alkaline protease was used as the enzyme source.When the enzyme:HmCystatin C（w/w）was 1:3,the products obtained by enzymatic hydrolysis for 6 h had the strongest antibacterial activity.The molecular weight of the hydrolysates were mainly distributed around 14.4 and 3.3 KDa,and a high concentration band was presented at 3.18 KDa.（2）Determination of antibacterial activity of HmCystatin C and its hydrolysatesHmCystatin C showed antibacterial effect on 6 strains of Gram-negative bacteria（G^-）:Shewanella putrefaciens,Pseudomonas fluorescens,Pseudomonas aeruginosa,Aeromonas media,Pseudomonas fragi,and Gram-positive bacteria（G⁺）,Staphylococcus saprophyticus.The diameter of inhibition zone（DIZ）was positively correlated with the concentration of HmCystatin C.Pseudomonas aeruginosa,Aeromonas media and Staphylococcus saprophyticus were the most sensitive to HmCystatin C,the minimum inhibitory concentration（MIC）and minimum bactericidal concentration（MBC）were 3.5 mg/m L.At 3/4×MIC of HmCystatin C,the survival rate of the typical test bacteria was only about15%.The growth curve monitoring showed that the growth of typical test bacteria were significantly（P<0.01）lower than that of the control group at 1×MIC of HmCystatin C.After enzymatic hydrolysis of HmCystatin C,part of the antibacterial spectrum changed.it lost inhibitory effect on Shewanella putrefaciens and Staphylococcus saprophyticus,but it showed obvious inhibitory effect on Escherichia coli and Bacillus subtilis which could not be inhibited by intact protein.After enzymatic hydrolysis,the inhibitory activity against Pseudomonas fragi was significantly enhanced,and there was still no inhibitory activity against Staphylococcus aureus.Pseudomonas aeruginosa and Aeromonas media had the same sensitivity to HmCystatin C before and after enzymatic hydrolysis.At 3/4×MIC of HmCystatin C hydrolysates,the survival rates of typical test bacteria were 15.36%and23.98%,respectively.The growth curves of the test bacteria were significantly（P<0.01）lower than those of the control group at 1×MIC of HmCystatin C hydrolysates.（3）The antibacterial mechanisms of HmCystatin C and its hydrolysatesWestern blot and ELISA analysis showed that HmCystatin C could bind to G^-and G⁺typical bacteria,which was the prerequisite for HmCystatin C to play an antibacterial role.In addition,HmCystatin C and its hydrolysates increased the surface electronegativity and hydrophobicity of typical tested bacteria,prompting the tested bacteria to accumulate and die.The tested bacteria with the treatment of HmCystatin C and its hydrolysates,exogenous N-phenyl-1-naphthylamine（NPN）entered the bacteria and alkaline phosphatase（AKP）exuded from the wall membranes,indicating that the bacterial cell wall was destroyed.In addition,the conductivity,OD₂₆₀,OD₂₈₀ and OD₄₂₀ values of the typical test bacterial suspension increased,indicating that the intracellular electrolytes,nucleic acids,proteins andβ-galactosidase were leaked through the cell membrane.Scanning electron microscopy also observed significant changes in the morphology of the tested bacteria.The surface of the bacterial cells appeared concave and damaged,and the contents overflowed.SDS-PAGE was used to detect the effect of HmCystatin C and its hydrolysates on intracellular protein synthesis.The protein patterns of typical tested bacteria in the experimental group were all changed,especially the proteins of Pseudomonas fragi were significantly reduced at about 66 KDa and 45 KDa,and the protein synthesis was blocked.Inhibitory activity of HmCystatin C and its hydrolysates against cysteine protease from typical tested bacteria in vitro were determined.It was found that only HmCystatin C showed58%to 77%inhibitory activity against cysteine proteinase of four tested bacteria at the same concentration（0.005 mg/m L）.The concentration of the hydrolysate needed to reach 700times to show similar inhibitory activity on cysteine protease of the four typical tested bacteria.These results indicated that some components of the hydrolysate retained the inhibitory activity centers of HmCystatin C.In conclusion,In summary,HmCystatin C and its hydrolysates can act on multiple targets of bacteria and exert antibacterial effects together.