Antibacterial Peptides Separated From Goat Milk By Magnetic Microspheres And Its Application In Sauced Beef

Antibacterial peptide is a natural substance with broad-spectrum antibacterial activity.In order to establish a rapid and effective method for the separation of antibacterial peptides from goat milk casein hydrolysate,used to inhibit the main spoilage bacteria of beef and its products to prolong their shelf life.Therefore,the magnetic nanoparticles coated with carboxylic groups were prepared for adsorption of cationic polypeptides in casein enzymatic hydrolysis solution of goat milk,and then desorption to separate antibacterial peptides.The spoilage bacteria of vacuum package sauced beef were stored at 37°C as test strains to detect the activity of the isolated antibacterial peptides.The colonies were cultured and counted at regular intervals,extracted DNA from the single colony liquid culture,and obtaining base sequence was compared with the bacteria Gene Bank to determine the species of the bacteria.Analyzing the characteristics of the spoilage bacteria of beef and Vacuum package sauced beef,the bacteriostatic effect was tested with the isolated antimicrobial peptides or compounding other natural antimicrobial agents in order to provide reliable theoretical data and method for prolonging the shelf life of beef products.The main contents and results of the experiment are as follows:?1?Synthesis of magnetic nanoparticles by co-precipitation in alkali solution,Fe₃O₄particles show a spherical morphology with an average diameter of about 30 nm from picture of scanning electron micrographs.X-ray diffraction pattern proved the inverse spinel structure of the material synthesized.Fourier transform infrared spectroscopy absorption peaks containing Fe-O bonds,amino,the Fe₃O₄ surface is rich in amino nanoparticles were synthesized.The formation of amide bonds between the amino groups on the surface of Fe₃O₄particles and the carbonyl group of the polyacrylic acid with carbodiimide activation,Fe₃O₄-COOH magnetic microspheres with average diameter of 600 nm were successfully prepared?called magnetic adsorbent?,used for the adsorption of cationic peptides.?2?Weighing moderate dried magnetic adsorbents added to appropriate casein hydrolysate of goat milk,mixing by vortex oscillation for two minutes.Magnetic separation and poured of liquid under the action of magnetic field.Then phosphate buffer containing 1 M sodium chloride was added?p H 6.0?,the magnetic adsorbent was removed and the cationic polypeptide was desorbed into the solution after 3 minutes of oscillating mixing,and the concentration of the peptides was 8.09 g/L,which was used in the bacteriostasis test.?3?The fresh beef was cut into small bulk and stored under the condition of ice temperature.The colony number of common spoilage bacteria was measured every 2³ days,and the colony structure of fresh beef was detected.In the initial colony of fresh beef,the lactic acid bacteria were the most.After 7 days of preservation,Pseudomonas and B.thermosphacta became the dominant bacteria,the dominant spoilage bacteria at the end of storage were Pseudomonas,B.thermosphacta,next Enterobacteriaceae and Staphylococcus.?4?Vacuum package sauced beef samples were cultured on a plate counting medium,and the spoilage bacteria isolated.Extraction 16S r DNA determine of base sequence.Twelve strains were identified as Enterobacteriaceae,among them,9 were Enterobacter cloacae.One strain was Staphylococcus epidermidis and two strains were Pseudomonas.Three strains of bacteria Enterobacter cloacae,Staphylococcus epidermidis and Pseudomonas were selected to test the peptides antibacterial activity.The turbidity of each bacterium was adjusted to 0.5Mack's turbidity.The diameter of antibacterial peptide against were 14.20±0.03 mm,17.24±0.45 mm and 11.30±0.11 mm,respectively?aperture was 7.16 mm?.?5?Antibacterial peptide,compounding Nisin and natamycin were used in sauced beef for preservative.After 30 days preservation at 37?,the group with 0.046 g/kg antibacterial peptide and 0.3 g/kg natamycin,its total number of bacteria was 3.0×10⁴ CFU/g,which was2 orders of magnitude less than that in blank group?3.6×10⁶ CFU/g?.In the combination of0.406 g/kg antibacterial peptides and 0.5 g/kg lactic acid streptococcin group and 0.406 g/kg antibacterial peptides coating group,them colony counts decreased by one order of magnitude.The results showed that antimicrobial peptides could inhibit microbial growth to some extent and the effect of antibacterial peptides combined on prolonging the shelf life of sauced beef.