Preparation Of B. Subtilis Q7 Lipopeptide And Its Inhibitory Effect On Alternaria

Alternaria is the main pathogenic bacteria after apple harvest.At present,more than 500 species of Alternaria are found to be facultatively parasitic in apple cells or tissues.They cause fruit diseases during the storage and transportation of apples and bring huge economic losses.The prevention and control of Alternaria in postharvest apples is mainly at the level of physical control and chemical reagent control.With more and more attention to the problem of pesticide residues,many fungicides commonly used in production were banned,and the cost of physical control is too high.A new way is urgently needed to replace traditional methods.Therefore,biological control has been attached great importance.It has been found that Bacillus can produce some antimicrobial substances in the growth process,which have strong antimicrobial effect and are widely used in agriculture,medicine and light industry.An endophyte Bacillus subtilis Q7 was screened from Ginkgo biloba L.in our laboratory.The active product obtained from preparation after fermentation was preliminarily identified as a lipopeptide compound,which had an inhibitory effect on Alternaria in apples.The research results are as follows:1.Preparation of lipopeptides(1)The active substance lipopeptide in B.subtilis Q7 was prepared,and the active substance contained lipopeptide component was determined by ultraviolet chromatography,high performance liquid chromatography and infrared chromatography,and its purity was 72.86%.(2)The preparation conditions of B.subtilis Q7 producing lipopeptide were optimized,and the optimum medium for B.subtilis Q7 was determined: taking glucose as carbon source,soybean peptone as nitrogen source and NaCl as inorganic salt;the optimum fermentation conditions were: fermentation time was 4 days,fermentation temperature was 28 ℃,initial pH was 5.0,inoculation volume was 3% and the revolving speed of table was 140 r/min;the optimum extraction conditions were that firstly using HCl for precipitation and then using methanol for extraction.The production of lipopeptide after optimization was 0.312 g,which increased by 45.8% in comparison with that before optimization.(3)The temperature and pH stability of lipopeptide were studied.The results found that lipopeptide had good temperature and pH stability,and showed stable bacteriostatic activity when the temperature was 4-100 ℃ and the pH was 3-13.The oil spreading test of lipopeptide showed that it is a good surfactant.2.Antibacterial spectrum of lipopeptide(1)The results of lipopeptide antimicrobial spectrum showed that lipopeptide had a strong inhibitory effect on Gram-negative bacteria such as Salmonella and Escherichia coli.By measuring the diameter of inhibition zone,it was found that the inhibiting effect on Escherichia coli was the largest,and the diameter of inhibition zone was 11.9(±0.26)mm.(2)Lipopeptides have different inhibitory effects on pathomycete genic Alternaria and Botrytis cinerea.Among them,the inhibition effect on Alternaria was the largest.The inhibition rate for Botrytis cinerea and Alternaria was 45.8% and 83.2% respectively when the concentration of lipopeptide was 2 mg/mL.3.Inhibition mechanism of lipopeptides against Alternaria(1)The study on the inhibitory effect of lipopeptide on Alternaria found that the minimum inhibitory concentration of lipopeptide for Alternaria was 1.2 mg/mL.With the increase of lipopeptide concentration,the effect on mycelial growth and sporulation ability increased gradually.When lipopeptide concentration was 2.0 mg/mL,the inhibitory rate reached 83.2%.(2)By detecting the absorbance of protein and nucleic acid,it was found that lipopeptide acted on the mycelium of Alternaria and destroyed the cell membrane of Alternaria,leading to the leakage of internal protein and nucleic acid.By measuring the use ratio of protein and total sugar in the fermentation broth of Alternaria dealt with lipopeptide,it was found that lipopeptide also affected the absorption of nutrients by Alternaria,so as to reach the goal of bacteriostasis.4.The results of apple stab wound experiments showed that lipopeptide can reduce the weight loss of apple and the formation of natural wrinkles of apple during apple storage.