| Over the years, the development of the novel antimicrobial substances with efficient, safe, stable and broad-spectrum activity has received increasing attention due to their potential use as natural preservatives in food industry, as probiotics in human health, and as therapeutic agents against pathogenic microorganisms. Bacillus species are widely distributed in nature, and possess diverse physiological characteristics. Bacillus is a genus that has been commonly found in soil and plants at the point of harvest. The objective of this work were (1) to isolate and screen antagonistic bacteria from the soil sample;(2) to purify and characterize the antimicrobial compounds from P. polymyxa strain JSa-9, which possessed broad-spectrum antibacterial and antifungal activities towards several pathogens;(3) to optimize the fermentation process for improved LI-F production by JSa-9;(4) to investigate the efficacy of LI-F for controlling Fusarium graminearum in vitro and in vivo. The results are described as follows:1. Screening and identification of the isolate of Bacillus spp. with a wide-spectrum antimicrobial activity. About119Bacillus spp. bacteria were isolated from different farmland and grassland soil samples collected from different six regions of China in terms of the divergence in morphology. Among them,9strains were found to have a broad-spectrum antimicrobial activity using the agar block and shaking flask method. Finally, the isolate JSa-9was determined for the next step of experimental strain because of its strongest and most broad-spectrum antibacterial and antifungal activities. According to the morphological and cultural characteristics, the physiological and biochemical properties, it was indicated that strain JSa-9was belong to the genus of Paenibacillus. From the16S rRNA sequence analysis (GenBank accession NO. EU882855), it was confirmed that strain JSa-9shared99%sequence similarity with that of Paenibacillus polymyxa. Therefore, strain JSa-9was designated as P. polymyxa JSa-9, and stored in China General Microbiology Culture Collection Center (CGMCC accession No:4314).2. Purification and identification of an antimicrobial glycoprotein from the culture supernatant of P. polymyxa JSa-9. A protein of about71.9kDa, isolated from a culture of strain JSa-9, exhibited wide-spectrum antibacterial and antifungal activities against M. luteus, B. cereus, S. aureus, E. coli, P. fluorescens, A. niger, P. expansum, A. oryzae, P. notatum and R. stolonifer. The purification procedure consisted of20～80%ammonium sulfate precipitation, ion-exchange chromatography on DEAE-Sephacel and Sephadex G-100column chromatography. The purified protein was characterized to be a glycoprotein, which contained53%of amino acids and28%of carbohydrate. The results also showed that large amounts of valine (30.2%), tyrosine (14.1%), lysine (12.6%), and threonine (11.5%) existed in the protein. Its N-terminal amino acid sequence was determined as Lys-Cys-Ala-Thr-Ile-Pro-Val-Val-Ile-Lys-His-Leu and the amino acid composition showed no significant homology with any known antagonistic proteins published so far using the BLAST network service in NCBI, so these results suggest that the purified antimicrobial glycoprotein from strain JSa-9may be novel.3. Extraction and identification of LI-F, polymyxin B6and DBP from the culture supernatant and cells of P. polymyxa JSa-9. Another two kinds of antimicrobial compounds were extracted from the fermentation broth of JSa-9using ethyl acetate and subsequently purified by high performance liquid chromatography. Four major distinct peaks were obtained in the elution time period through RP-HPLC. Of these fractions in the chromatogram, the fourth fraction was active against all the tested bacteria and fungi including M. luteus, B. cereus, S. aureus, E. coli, P. fluorescens, A.niger, P. expansum, A. oryzae, P. notatum and R. stolonifer, while three other fractions only exhibited similar inhibitory activity against fungi and Gram-positive bacteria namely M. luteus, B. cereus, S. aureus, A. niger, P. expansum, A. oryzae, P. notatum and R. stolonifer, but there was no activity against Gram-negative bacteria. By means of LC-MS and MS/MS, one of two antagonistic compounds with m/z278.89was determined as di-n-butyl phthalate. And another was characterized as a mixture of related peptides of molecular masses of883,897,911,947, and961Da, with the most likely structure of them determined to be a cyclic depsipeptide with an unusual GHPD bound to a free amino group. The fragmentation patterns for these peptides were consistent with the molecular structures of LI-F03a,03b,04a,04b, and05b, respectively.Additionally, three antagonistic substances were extracted from strain JSa-9cell pellets by methanol. Using HPLC method, two antagonistic fractions (peak A and B) were separated and collected from the methanol extract. One showed inhibition against both Gram-positive and Gram-negative bacteria (including M. luteus, B. cereus, S. aureus, E. coli and P. fluorescens), while the other was active against Gram-positive bacteria and fungi (M. luteus, B. cereus, S. aureus, A. niger, P. expansum, A. oryzae, P. notatum and R. stolonifer). Data from ESI-MS analysis showed that [M+H]+ion peaks were1219.72and901.55and915.51corresponding to peak A and B, respectively. The tentative sequences Dab-Thr-Dab-Dab-Thr-Dab-Dab in m/z1219.72, Val-Val-Thr-Asn-Ala in m/z901.55and Val-Val-Thr-Gln/Lys-Ala in m/z915.51were revealed by tandem mass spectrometry, respectively. They were found to be structurally related to polymyxin B6and the members of the LI-F family:LI-F04a and04b respectively by comparing their partial amino acid sequences with those from the literature. Amino acid analysis of peak A and B showed the presence of Dab, Thr, Phe, and Leu/Ile in a molar ratio of nearly6:2:1:1and Thr, Ala, Val, Asn, and Gln in a molar ratio of4:2:4:1:1, respectively. The data of infrared spectroscopy also illustrated that the two compounds with molecular masses of901and915Da were characterized as the linear lipopeptide analogs of antibiotics LI-F04a and04b respectively, while the other antimicrobial substance from peak A was identified as polymyxin B6.4. Optimization of fermentation process for improved LI-F production by P. polymyxa JSa-9. The medium screening experiment indicated that the strain JSa-9could obtain high yield of LI-F antibiotics in the Landy medium. The further PB design was undertaken to screen the key factors rapidly from the related13factors. By analyzing the statistical regression and the prediction profiler, the concentrations of KC1, FeSO4, MgSO4and CUSO4were found to be the most important factors for the production of LI-F. Based on the results of previous PB design, a four-factor six-level BBD was applied to optimize the LI-F production. The critical factors selected for the investigation were mentioned above. By analysis of the3D plots and their corresponding plots, the optimum values of the concentrations of KCl, FeSO4, MgSO4and CUSO4for obtaining the most production of Ll-F were1.1g/L,0.27mg/L,0g/L,0.64mg/L, respectively. The optimized cultivation conditions allowed LI-F production to be increased to733.9mg/L.5. Determination of the efficacy of LI-F for controlling the Fusarium graminearum in vitro and in vivo. LI-F antibiotics from P. polymyxa JSa-9could significantly inhibit the hypha growth and spore germination of Fusarium graminearum. The50%effective concentration (EC50) against Rhizopus slolonifer hyphae was fengycin13.69μg/mL. The concentration of LI-F antibiotics was30μg/mL when the conidia germination was completely inhibited and the germ tube did not appear. We clarified the application possibility of LI-F on the biological control of Fusaruim head blight of wheat by the test in vivo. The results indicated that the protective effect of LI-F was much better than its curative effect. Besides, LI-F at800μg/mL provided75.19%curative control efficacy against F. graminearum, which was much higher than that of MBC. |
PDF Full Text Request