Optimization Of Fermentation System Of Leuconostoc Mesenteroides WZ-44 And Preliminary Study On Antibacterial Substances

Pear fire blight is a devastating bacterial disease on pears,apples and other plants of the Rosaceae family,caused by Erwinia amylovora.At present,the control for pear fire blight mainly relies on chemical pesticides.But the long-term use of chemical pesticides,resulted in the rising of control cost,development of pathogen resistance,ecological environment disorders in pear orchards,increasing of pesticide residues risk,limited improvement of pear fruit quality and other problems.In recent years,biological control has become one of the important development directions in the green pesticide industry by virtue of its advantages of high safety,friendly to the environment,no residue,and not easy to induce pathogen resistance.Lactobacilli are recognized a group of food-grade microorganisms,and some strains possess anti-bacterial and fungal activity and produce a wide range of antimicrobial substances.In this study,Leuconostoc mesenteroides WZ-44 obtained in the laboratory was used as the research object,and the pear fire blight was used as the indicator strain.The optimization of fermentation system and the preliminary exploration of antibacterial substances were carried out.The main results are as follows:1.The best factor combination of Leuconostoc mesenteroides fermentation nutrientsWZ-44 strain was obtained from our laboratory and has a strong antagonistic activity against various pathogens such as Erwinia amylovora.In this study,MRS medium was used as the base medium,and the fermentative bacterial density and antagonistic circle diameter were as evaluation indicators of WZ-44 fermentation optimization,and the medium formulation for high bacterial density of WZ-44 was obtained by single-factor test and orthogonal design test:glucose 25.0 g/L,yeast 25.0 g/L,Ca Cl₂ 0.25 g/L,Mn SO₄·H₂O0.15 g/L,Mg SO₄·7H₂O 0.18 g/L,Na₂HPO₄ 2.0 g/L,KH₂PO₄ 2.0 g/L,sodium acetate 5.0g/L,Tween-80 1.0 m L/L.Bacterial density in the fermentation broth reached 3.47 x 10⁹CFU/m L in 24 h,67.63%higher than that of MRS medium.,but There was no significant difference in antagonistic diameter between before and after optimization,indicating that the optimized medium was more conducive to the growth of WZ-44.2.The best factor combination of Leuconostoc mesenteroides fermentation parameters To explore the antibacterial mechanism,the fermentation parameters such as inoculum,loading volume,temperature and p H were optimized by single-factor tests and orthogonal combination tests,and obtaining the optimum fermentation conditions for WZ-44:inoculum 4%（v/v）,loading volume 40%,temperature 20°C,p H value 7.0.Bacterial density in the fermentation broth reached 4.07 x 10⁹ CFU/m L in 36 h,94.68%higher than that of MRS medium.,but There was no significant difference in antagonistic diameter between before and after optimization,indicating that the optimized medium was more conducive to the growth of WZ-44.3.Antibacterial active substances of Leuconostoc mesenteroidesIn order to further understand the antibacterial activity of WZ-44,the antibacterial substance was initially analyzed using organic solvent extraction,ammonium sulphate precipitation,macroporous resin adsorption and ultrafiltration separation,and then the crude isolate was further separated and purified using HPLC.Based on the results of the initial separation and purification,it was hypothesized that the antibacterial active substance produced by WZ-44 was probably water-soluble substance with a molecular weight of less than 3 kDa.