Screening Of Fruit Fresh-keeping Bacterium,Brevibacillus Brevis And Its Culture Conditions Analysis

Brevibacillus brevis can be isolated from soil,water and vivo plants or animals,which plays important role in ecological environment and displays great environmental suitability and competitiveness due to its strong stress resistance and protein secretion ability.In the study,when Lasiodiplodia theobomae acted as an indicator,we built mutant library mainly based on different level of antibacterial activities through three breeding methods including ultraviolet mutagenesis,LiCl chemical mutagen and UV-LiCl combination mutagen.And then screened strains possessed better ability of antibacterial and antifungal from the library.Our result showed the optimum UV mutagenesis condition was bacterium suspension exposed under 30 w UV lamp,50 s irradiation time and distance of 20 cm.In the method of chemical mutagen,the optimum added proportion between LiCl solution and agar of lysogeny broth was 0.3:100.Compared with single UV and LiCl mutagen,UV-LiCl mutagen had the same mutagen condition,except for optimal irradiation time changed to 30 s.In this mutation conditions,we found 80 mutants have better antibacterial abilities than original strain Brevibacillus brevis FJAT-0809-GLX,while 79 mutants with poorer antibacterial abilities had been identified through primary screening,secondary screening and molecular identification.Afterwards,we selected 6 strains of mutant conducted stability test on inhibition activity,strains FJAT-17952,FJAT-17953,FJAT-17954,FJAT-17978,FJAT-17982 and FJAT-17986 have great stability.Three mutants FJAT-17952,FJAT-17953 and FJAT-17954 and original strain FJAT-0809-GLX were selected as the test strains among the six mutants,and carried out further study on their biological characteristic,active substances determination and fruit fresh-keeping detection.The results showed that the four test strains presented similar character of individual form and colony form after cultivated 48 hours on the plate of LB agar.The colony character represent light yellow,similar circular,wetting,slight uplift,smooth surface and exit winkles around colony edge.In addition,four test strains represented similar growth tendency.Furthermore,there were different results in the determination of influence on various culture conditions containing temperature,pH,rotate speed and loaded liquid.Four test strains possess the best antifungal capacities at the optimal temperature 30℃,optimal pH 7.0,optimal rotate speed 150 r/min and 40%liquid broth content in the same other conditions.The final results indicated that three strains of mutant have better tolerance of high temperature,acid-base and dissolved oxygen compared with the original strain.And the mutants FJAT-17953 had the best stress resistance and antifungal ability toward to Lasiodiplodia theobomae among the three mutants.In order to explore the reason of different inhibition activity between original strain and mutants,our study selected ethylparaben act as standard indicator in the HPLC determination of four tests strains’s fermentation broth.The results showed that compared with Brevibacillus brevis original strain FJAT-0809-GLX,ethylparaben secreted from three strains of mutants FJAT-17952,FJAT-17953 and FJAT-17954 were boosted by 419.45%、248.81%and 23.20%,respectively.This results proved that the promotion of antibacterial and antifungal ability of three mutants maybe attributed to the more content of secreted ethylparaben.At last,we determined antimicrobial spectrum of three mutants and detected fresh-keeping effect on wax apple fruit which was the separate origin of Lasiodiplodia theobomae.The result indicated that three mutants had better antimicrobial activity compared with original strain,and the indicator pathogenic microorganism contained Colletotrichum orbiculare,Lasiodiplodia theobomae,Fusarium oxysporum,Escherchia coli and salmonella.Moreover,four test strains represented great fresh-keeping effect,and there were visible difference among four strains treatment groups when storage time was 8 d.Then the fresh-keeping effect of three mutants was better than Brevibacillus brevis original strain,in which mutant FJAT-17954 inclined to the best effect.Besides,mutants had better inhibition activity toward to Lasiodiplodia theobomae compared with the original strain,which showed in the experiment of inoculate Lasiodiplodia theobomae to the skin of wax apple fruit.In all,Brevibacillus brevis mutants FJAT-17952,FJAT-17953 and FJAT-17954 have better function on biological control and fruit fresh-keeping than the original strain Brevibacillus brevis.