Studies On Mass Preparation Of Bacterial Lipopeptide And BM Lipopeptide Toxicological Tests As Well As Its Biocontrol Effects Against Grey Mold

Bacterial lipopeptide was discovered as the metabolite derived from Bacillus subtilis in the late 1960s.In the past half-century,numerous researches about the antibiotic activities against plant pathogen fungi,bacteria,and cancers resulting from bacterial lipopeptides were reported.However,mass extraction and purification for bacterial lipopeptides was not available until now,which severely hampered their related application research.In this research,the lipopeptide surfactant and foaming interference on separation and purification process were overcome to establish the mass preparation technology for bacterial lipopeptides possessing novel molecular structure and good biocontrol activity."Strictly gradual removal of impurities in dimension of polarity" was thought as key research course.Mature scale-up unit technologies of isolation and purification,including acid precipitation,methanol extraction,solvent precipitation,salting out,extraction,silica gel column chromatography and HZ806 macroporous absorption resin column chromatography,were tested and optimized.According to the advantages and disadvantages that they had performed under the surfactivity interference,these unit technologies of separation and purification would proceed in a certain order to remove the impurities and to gain purified bacterial lipopeptides gradually.Finally,novel mass preparation technology was established,which could effectively prepare BM lipopeptides with 87.51%-100%purity in hectograms with more than 81.73%recovery rate from marine-derived Bacillus marinus B-9987 fermentation broth yielding 120 mg/L BM lipopeptides.Therefore,the practical hectogram production for high-purity bacterial lipopeptides came true.This new method performed no strict limitation on fermentation medium and low requirement on raw materials.Therefore,the established large-scale manufacturing of Bacillus-based biocontrol agent using natural media could offer sufficient feedstock to massive lipopeptide purification,which endowed the new method with further practical application.Moreover,no foaming problem and no time-and-energy-consuming water evaporation involved this isolation and purification process.Thus,the new pilot-scale purification technology was practical and economical.Subsequently,the mass preparation technology of BM lipopeptides was applied to bacillomycin D lipopeptides derived from Bacillus amyloliquefaciens TYg 3-2 for general applicability verification.Due to response surface method,bacillomycin D fermentation in 5 L bioreactor could supply raw extracts at 21.31%content.Benefiting from the high-content raw material,subsequent scale-up purification technology for bacillomycin D with 94.72%purity and 64.41%recovery rate was established,which was more efficient because of simplification from 11 to 6 steps after optimization.Based on the high-purity BM lipopeptides derived from the new mass preparation technology,acute toxicity tests for BM lipopeptides,including acute oral toxicity test,acute dermal toxicity test,acute inhalation toxicity test,skin irritation test,skin allergy test,and skin allergy test,were conducted according to the requirement of pesticide registration in China.Then,BM lipopeptides were confirmed as low-toxicity substances after their toxicities were quantified.Field experiment of partially purified samples of BM lipopeptides against grey mold was reported:500 mg/L BM lipopeptides formed 67.53%biocontrol efficacy against Botrytis cinerea,slightly lower than the 74.05%reached by 400 mg/L pyrimethanil and the 76.57%reached by 500 mg/L iprodione,after 3 times of applications and disease severity investigations;And BM lipopeptides could significantly raise the biocontrol efficacy of their producing strain B.marinus B-9987 against cucumber grey mold in pot test in vivo.3×106 CFU/mL B.marinus B-9987 biomass(the spores in BCA)mixed with 500 mg/L BM lipopeptides could form 83.33%biocontrol efficacy,which was higher than the 72.83%and 64.17%reached by BM lipopeptides alone and Bacillus BCA alone,respectively,and even higher than the 73.33%reached by 400 mg/L pyrimethanil as positive control.In conclusion,the first practical hectogram production for high-purity bacterial lipopeptides came true,after the establishment of economical and practical mass preparation for BM lipopeptides.Subsequently,mass preparation of BM lipopeptides was propagable and available to bacillomycin D lipopeptides secreted by B.amyloliquefaciens TYg 3-2,which meant the further propagation and enhancement of scale.Furthermore,the acute toxicological tests compulsorily required by pesticide registration were conducted using high-purity BM lipopeptides,which were definitely identified as low-toxicity substances.Moreover,the field experiment of BM lipopeptides first determined that marine bacterial lipopeptide could biologically control the grey mold.In this paper,scale-up preparation technology for BM lipopeptides possessing novel molecular structure and good biocontrol activity was scientifically optimized.This results would not only offer important reference to the establishments of scale-up preparation technologies for other lipopeptides,but also provide good foundation for the development of BM lipopeptides as biocontrol agents against grey mold.