Extraction And Identification On Antibacterial Active Substance Of Bacillus Amyloliquefaciens And Its Application In Banana Preservation

Bacillus amyloliquefaciens,as a biocontrol Bacillus,has a wide range of bacteriostatic spectrum,can inhibit many kinds of pathogenic bacteria,and has great potential application in biopesticide,food and feed additives,biomedicine and so on.Different B.amyloliquefaciens have different metabolites and characteristics.In this study,B.amyloliquefaciens?strain No.?was used as the experimental object to optimize the response surface of fermentation medium,and to study the antibacterial components produced by B.amyloliquefaciens in the laboratory.Its physicochemical properties and bacteriostatic activity were studied,and its antibacterial activity was first applied to banana preservation by using the bioantagonistic action of B.amyloliquefaciens.The main findings and conclusions of the study are as follows:1.In the optimization of single colony separation,the antibacterial activity of the strain with the best antibacterial effect was 57.83%higher than that before the optimization.The optimum formula was glucose 10.0 g/L,K₂HPO₄×3H₂O 1.2 g/L,KH₂PO₄ 1.0 g/L,?NH₄?₂SO₄2.00 g/L,trihydrate sodium citrate 0.9 g/L,MgSO₄×7H₂O 0.15 g/L.The optimum culture medium was composed of glucose 6.15 g/L,K₂HPO₄×3H₂O 1.2 g/L,KH₂PO₄ 1.0 g/L,?NH₄?₂SO₄ 2.50 g/L,trihydrate sodium citrate 1.01 g/L,MgSO₄×7H₂O 0.15 g/L.The bacteriostatic activity was increased by 65.79%compared with that before optimization,and the content of bacteriostatic active substance in the fermentation metabolite was obviously increased after the optimization.2.In the determination of acid-base stability,the antibacterial effect of pH=3 was the best,up to 98.08%,and when the UV irradiation was within 180s,the bacteriostatic rate decreased by 6.13%.In the determination of thermal stability,the bacteriostatic activity was70.88%of that of the blank group after being sterilized at 121?for 60 min in a high pressure sterilizing pot,and only the activity of methanol in the determination of the stability of organic solvent.In the determination of stability of ammonium sulfate,50%ammonium sulfate treatment has the highest activity.According to the above results,the antibacterial active substance of B.amyloliquefaciens has good stability in pH?8,ultraviolet irradiation for a few minutes,high temperature less than 60?,methanol and 50%ammonium sulfate.Among the pathogenic bacteria available in the laboratory,including Staphylococcus aureus,Bacillus subtilis,Bacillus pumilus,Micrococcus luteus,Escherichia coli,Salmonella pullorum,Standard Salmonella gallinarum,Mutant Salmonella gallinarum,Pasteurella multocida and Salmonella enteritidis was determined.The results showed that the fermentation broth had inhibitory effect on the above 10 pathogenic bacteria and reached the minimum bactericidal concentration?MBC?,when the fermentation broth concentration was358.50 mg/mL that could kill 99.9%of the tested bacteria.3.After vacuum silica gel column chromatography and high performance liquid chromatography?HPLC?,the antibacterial active substance of B.amyloliquefaciens was separated and the molecular weight of the purified compound was determined by mass spectrometry and NMR.The antibacterial activity of B.amyloliquefaciens CGMCC 1.936 is a small molecule glycoside compound.The molecular weight of the compound is 325.0 and the molecular formula is C₁₂O₁₀NH₉.4.With the prolongation of storage life,compared with other treatments,the weight loss rate of banana was 1.13%,the hardness changed to 41.72%,and titratable acid decreased by36.69%,soluble solid increased by 18.18%on the 5th day after being treated with 400 mg/mL antifungal active substance of B.amyloliquefaciens and preservative film.It can effectively delay the ripening and deterioration of bananas and maintain good quality.