| Bacillus amyloliquefaciens is one of the important strains in the field of biological control.The various bacteriostatic substances secreted by the fermentation have strong antagonistic effects on fungi,bacteria,actinomycetes and viruses.It has broad application prospects in the fields of medicine,health care products,feed,food and cosmetics.Staphylococcus aureus was used as an indicator,and the antibacterial activity and lactic acid concentration of the supernatant were selected as indicators.Among the strains deposited in the laboratory,the B815 strain was selected,the initial lactic acid yield was 2.05 g?L-1,and the inhibition zone diameter was 15.4 mm.Through molecular identification and physiological and biochemical identification,it was identified as a strain of Bacillus amyloliquefaciens.By means of normal temperature and atmospheric pressure plasma mutagenesis?ARTP?,combined with specific high-throughput screening,a mutant strain with a lactic acid yield of 3.92 g?L-1and a zone of inhibition of 20.3 mm and stable inheritance was obtained,the mutagenized strain was numbered B815-1.By optimizing the culture medium and culture conditions of the B815-1 strain,the optimal formula was obtained:maltose 10 g?L-1,peptone 15 g?L-1,MgSO4·7H2O 2 g?L-1,active CaCO310 g?L-1;optimal culture conditions:culture temperature 50°C,initial pH 7,seed age 3 h,inoculum 2%,liquid volume 50 mL.After optimization,the inhibition zone diameter of the strain was 25.6 mm,and the lactic acid yield reached 8.42 g?L-1.The total bacteriostatic substance in the supernatant of strain B815-1 has a tolerance temperature of 60°C,a tolerant pH range of 6 to 9,and is insensitive to neutral protease,alkaline protease,papain,trypsin,soy peptide hydrolase,pepsin pair Its activity has a slight effect.It has inhibitory activity against Salmonella enteritidis,Bacillus licheniformis,Staphylococcus aureus,Enterococcus faecalis,and Listeria monocytogenes.After 3 hours of treatment in the simulated stomach and intestinal juice,the survival rate of the mutagenized strain was 86.14%and 95.14%,respectively.After treated at 100°C for 5 min,the viable count of the mutagenized strain decreased from 7.00×108 CFU?mL-1 to 5.40×108 CFU?mL-1,and the survival rate was77.4%.A solution of acid precipitation,organic solvent extraction,DEAE-Sepharose Fast Flow anion exchange chromatography,RP-HPLC was established,and two antibacterial active substances were obtained from the fermentation supernatant.Two purified products were analyzed by LC-MS,and one of them was found to contain the Surfactin homologue containing C1315;the other amino acid sequence was Leu-Val-Asn-Pro-Pro-Thr,a bacteriopeptide with a molecular weight of 639.75 Da.The Hexapeptide had an isoelectric point of 5.52,a GRAVY of 0.100,and a fat solubility index of 113.33 as analyzed by ProtParam software.Comparing this Hexapeptide with the reported bacteriopeptides in the APD database,the highest homology was only 37.5%.The tolerance temperature of Surfactin in the tolerance experiment was 90°C,and the tolerance range was 29;the tolerance of Hexapeptide was 80°C,and the tolerance range was 38.The antibacterial spectrum found that Surfactin and Hexapeptide have antibacterial activity against Gram-positive bacteria and Gram-negative bacteria.The MIC of Surfactin,Hexapeptide and lactic acid against indicator bacteria were 0.5mg?mL-1,0.8 mg?mL-1,and 1.563 mg?mL-1,respectively.Three kinds of bacteriostatic substances were used to act on the indicator cells separately and in combination.The cell membrane permeability and integrity were determined by the extracellular concentration of K+,OD260 and OD280;The cell morphology was observed by transmission electron microscopy and cold field emission scanning electron microscopy.By using flow cytometry to quantify cell damage,it was found that lactic acid can damage the cell wall,and the Hexapeptide can penetrate the cell wall to act on the cell membrane to further damage the cell.Surfactin not only destroys the cell wall,but also acts through the cell membrane to interfere with cell division.When the three cooperated to indicate the bacterial cells,the degree of damage to the cells was greater.Using the checkerboard dilution method,the synergistic inhibitory effect of Surfactin,Hexapeptide and lactic acid on the indicator bacteria was investigated.It was found that when the concentration of lactic acid was 0.098 mg?mL-1 and the concentration of Surfactin was 0.125mg?mL-1,the FICI value was 0.3125?<0.5?,which had a synergistic effect;When the concentration of lactic acid was 0.195 mg?mL-1 and the concentration of Hexapeptide was 0.20mg?mL-1,the FICI value was 0.375?<0.5?,which had a synergistic effect;When the concentration of Surfactin was 0.125 mg?mL-1,the concentration of Hexapeptide was 0.40mg?mL-1 and the concentration of Hexapeptide was 0.20 mg?mL-1,The concentration of Surfactin was 0.250 mg?mL-1,both FICI values were 0.750?0.5<0.75?1?,which had a additive effect. |
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