The Crude Extraction And Anti-corrosion, Preservation Efficacy Testing Of Antimicrobial Peptides From Bovine Blood

To extract and study cattle neutrophil peptides, the cattle waiting forslaughter were selected. Neutrophil antimicrobial peptides in Cattle blood would be simple coarseextraction. Then the antimicrobial activities being tested, the minimal inhibition concentrations(MIC)of the extracts were tested and their bactericidal activities were compared. In addition，Itslaid a foundation as a biological preservative for meat preserved. The study of antimicrobialpeptides from Bovine blood is divided into three series.Series I The crude extract of cattle blood leukocyte antimicrobial peptides and theoptimization of extraction process.1. Collect fresh anticoagulant blood of cattle, filter to remove cattle hair. Blood is divided intothree parts. One part of them is water bath standing under37℃,so the anticoagulant blood willnatural stratification. And then four low temperature centrifugal after remove part of the red bloodcells and plasma. Another part of them direct centrifugal, centrifugal separation remove out mostof the plasma and red blood cells. The middle layer white blood cells and a small amount of theplasma and red blood cells were left. Treated the blood left with0.83%ammonium chloride tolyse the red blood cells whereas keep the leukocytes intact. Until observed by microscope withoutintegrity red blood cells. Centrifugal and collected sedimentation was white blood cells. The whiteblood cell is divided into three part, using sonication, cooking machine grinding broken andhomogenizer broken for crushing until without integrity cells. Low-temperature high-speedcentrifugation and collect the precipitate obtained by centrifugation of the group of sonication.Added10%acetic acid solution in the precipitate. Foil seal, Stirring overnight at4℃toextraction antimicrobial peptides. The two groups of Mechanical crushing, after high-speedcentrifugation the supernatant was collected directly foil seal extraction overnight at4℃.Antimicrobial peptides of the extraction from the solution low-temperature high-speedcentrifugal and collected supernatant. Rotary evaporator55℃water bath to remove acetic acidand some water. Low-temperature high-speed centrifugation, the supernatant was collected, placedon the plate,-20℃freezer overnight, then placed in the vacuum freeze-drying machine drying. Todissolve the crude extract antimicrobial peptides with0.01%acetic acid solution. The resultsshowed that: antimicrobial peptides crude extracts obtained by the mechanical crushing relativelyhigh yield with ultrasonication.2. The traditional extraction process is0.83%ammonium chloride solution added directly tolysis red blood cells in the anticoagulant. The two processes improved. First, the initial naturalsedimentation and centrifugation steps are to remove plasma and the vast majority of red bloodcells. Each component is separated, the plasma and red blood cell streams were further processedand in particular has potential to be converted into high-value products. Another advantage wasthat a lot fewer red blood cells needed to be lysed.Since the majority were removed from thewhite blood cell fraction during the centrifugation. Natural sedimentation method can reduce thenumber of centrifugation, thereby reducing some of the waste of resources, responded to the call of low carbon environmental protection. The improved extraction process can also be carried outlarge-scale production, to avoid the waste of resources, can also increase the production rate oflarge-scale production. Comparison of the three process: using the three process crude extractantimicrobial peptide production of the same amount, but improved technology can reduce theconsumption of raw materials costs and on a larger scale, the larger output production.Series II The study of bovine blood leukocyte antimicrobial peptides antibacterial activityand anti-viral activity. Application the trace agarose diffuse method to determination antibacterialactivities of the antibacterial peptides crude extract. The results showed that crude extracts ofantimicrobial peptides have an antibacterial effect on both Gram-positive bacteria andgram-negative bacteria; Compared to some of the commonly used antimicrobial agents, have agood effect on anti-bacterial, and avoid the disadvantages of that many antimicrobial drugs areonly effective against gram-negative bacteria.96well microtiter plate determination minimum inhibitory concentration of the antimicrobialpeptides crude extract. The results show that the antibacterial activity of antimicrobial peptidescrude extract is better than many conventional antimicrobial drug effects.Antiviral experiments use Newcastle disease virus (NDV) virus as subjects. To explore theinfluence of antimicrobial peptides coarse extraction on NDV active, the method of chickenembryos was be used. Three methods are used: Embryo was inoculated by Virus and extracts afterthey react2h in vitro, virus was inoculated before Bovine blood extract for4h, Bovine bloodextract was inoculated before virus for4h. These three groups were arranged testing the controlgroup. Collected allantoic fluid and determined its HI titer. The results show that antimicrobialpeptides crude extract from Bovine blood have strong influence on the activity of NDV at eachgroup.Series III antimicrobial peptides refrigerated meat antiseptic preservation efficacy judgmentThe tested samples is fresh pork. The fresh meat is divided into two parts, one surface withantimicrobial peptides powder, another one don’t do disposal. Two fresh meat is placed in a cleantray into the refrigerator freezer. Every4h smear observed for bacterial count and contrast betweenthe two trends. The results show that: the preserved time of the surface coated with theantimicrobial peptide powder meat preserved longer than the no disposal meat.