| As an essential part of innate immunity,antimicrobial peptides?AMPs?have been receiving increasing attention because of their unique antimicrobial mechanism over recent decades.As a byproduct and even one of the major dissolved pollutants in slaughter house wastewater,appropriate treatment of chicken blood is of great benefit to both environmental protection and economic development.However,antimicrobial peptides from chicken hemoglobin?CHAP?have not been documented yet.In this study,CHAP from chickens was isolated and their biological activities were further detected.These experiments we conducted as follows:1.Isolation and identification of CHAP The crude extracts of CHAP were isolated by papain proteolysis and acetic acid extraction.The enzymatic time,extraction time,enzyme solution temperature,enzyme dosage and extraction temperature were optimized to further obtain AMP with better biological activity.After purified by gel chromatography,detected with Tricine SDS-PAGE and subjected to mass spectrometry,peptides of gallus hemoglobin subunit alpha were confirmed with the molecular weight of 3.3 KDa.2.Antimicrobial and bactericidal activities of CHAP The antimicrobial and bactericidal activities of CHAP were tested by the agarose diffusion assay,minimum inhibitory concentration?MIC?analysis,minimal bactericidal concentration?MBC?analysis,and time-dependent inhibitory and bactericidal assays.The results demonstrated that CHAP had potent and rapid antimicrobial activity against 19 bacterial strains,including 8 species and 9 multidrug-resistant bacterial strains.The diamter of inhibition zone of CHAP against multi-drug resistant?MR?-E.coli?equine?was 33.5 cm,the MIC and MBC of MR-Pseudomonas aeruginosa M140 is 1.56 ?g mL-1 and 5 ?g mL-1 respectively,the inhibitory rates of all six chosen strains reached 100%in 90 min,CHAP demonstrated significant bactericidal effect on both the bacteria grown to early and late exponential phases from 10?240 min?P<0.01?and good bacteriolysis agaisnt bacteria in stationary phase.Bacterial biofilm assay and NaCl permeability assay and electron microscopy observation were further performed to detect the mechanism of its antimicrobial effect.The results showed that CHAP could inhibit the formation of bacterial biofilms,change the permeability of some Gram-negative bacteria and Gram-positive bacteria,and accumulate copious pathogens nearby and punch through their cell surfaces swiftly,suggesting that the target of CHAP is bacterial cell membrane in the antibacterial and bactericidal processes.3.Stability and toxity of CHAP According to thermal and serum stability tests,the antimicrobial activity of CHAP did not decrease in different temperatures even when it was treated in 121 ? for 30 min?P>0.05?and treated with chicken and rabbit serum for 6 h?P>0.05?,suggesting its good stability.After using hemolytic assay and embryotoxicity assay,the CHAP demonstrated lower than 50%hemolysis concentration?HC50?at 360?g mL-1 and no embryotoxicity at rather high concentrations of 6 × MIC against E.coli ATCC25922,which further implied that CHAP had relative high selectivity between eukaryote cells and prokaryote cells,and low hemolysis and embryotoxicity.4.Anti-heat stress effect of CHAP The effects of CHAP on growth performance,blood biochemical indicators,the structure and function of small intestine and immune organs of chicken under chronic and acute heat stress were detceted.The results showed that compared with the heat stress group,the CHAP adding group showed greater average daily gain?P<0.05?and feed efficiency?P>0.05?;more stable concentration of SOD,ATPase and MDA in blood,less histological lesions in small intestines,greater height of villus,thickness of gut mucosa and villus height/crypt depth,greater activity of alkaline phosphatase?P<0.01?in duodenum,more intestine intraepithelial lymphocytes,less goblet cell number,higher ratio of secreting IgA,and lower HSP70 and PCNA in small intestines;higher immune organ indexes and less lession and apoptosis in immune organs.These results indicated that CHAP could effectively resist the adverse effects of heat stress by reducing the injury of intestine and immune organs and maintaining the normal intestinal structure,absorption function,mucosal immunity in chicken under heat stress.5.Immunologic effect of CHAP The preliminary study results of the effects of CHAP on the immune function showed that chicken can get higher body weight,better NDV and AIV immune effect,higher immune organ indexes and lymphocyte conversion rate in spleen after adding CHAP,suggesting CHAP could improve the immunologic function of chicken to some extent.In conclusion,this study firstly reported a practical method of isolating chicken hemosidins?CHAP?from the byproduct and even the pollutant of chicken-slaughtering industries.CHAP has an attractive antimicrobial and bactericidal ability with low hemolysis,low or none embryotoxicity,good temperature resistance and high stability in serum,and chicken could resist the reserve effects of heat stress amd increase the immune function after treated with CHAP,which well accounts for their potential of expanding production and high development value in animal medication,breeding industry and environment protection. |
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