• Title/Summary/Keyword: GC-1 cell

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Genomic Insights and Its Comparative Analysis with Yersinia enterocolitica Reveals the Potential Virulence Determinants and Further Pathogenicity for Foodborne Outbreaks

  • Gnanasekaran, Gopalsamy;Na, Eun Jung;Chung, Han Young;Kim, Suyeon;Kim, You-Tae;Kwak, Woori;Kim, Heebal;Ryu, Sangryeol;Choi, Sang Ho;Lee, Ju-Hoon
    • Journal of Microbiology and Biotechnology
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    • v.27 no.2
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    • pp.262-270
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    • 2017
  • Yersinia enterocolitica is a well-known foodborne pathogen causing gastrointestinal infections worldwide. The strain Y. enterocolitica FORC_002 was isolated from the gill of flatfish (plaice) and its genome was sequenced. The genomic DNA consists of 4,837,317 bp with a GC content of 47.1%, and is predicted to contain 4,221 open reading frames, 81 tRNA genes, and 26 rRNA genes. Interestingly, genomic analysis revealed pathogenesis and host immune evasion-associated genes encoding guanylate cyclase (Yst), invasin (Ail and Inv), outer membrane protein (Yops), autotransporter adhesin A (YadA), RTX-like toxins, and a type III secretion system. In particular, guanylate cyclase is a heat-stable enterotoxin causing Yersinia-associated diarrhea, and RTX-like toxins are responsible for attachment to integrin on the target cell for cytotoxic action. This genome can be used to identify virulence factors that can be applied for the development of novel biomarkers for the rapid detection of this pathogen in foods.

The Expression of Codon Optimised Hepatitis B Core Antigen (HBcAg) of Subgenotype B3 Open Reading Frame in Lactococcus lactis

  • Mustopa, Apon Zaenal;Wijaya, Sri Kartika;Ningrum, Ratih Asmana;Agustiyanti, Dian Fitria;Triratna, Lita;Alfisyahrin, Wida Nurul
    • Microbiology and Biotechnology Letters
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    • v.47 no.3
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    • pp.449-458
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    • 2019
  • Hepatitis B treatments using immune therapy are gaining interest because of the improvements in dendritic cell performance for antigen presentation, which induces an appropriate immune response and raises patient survival rates. This research aims to produce a significant amount of the HBcAg antigen, which can induce an immune response and have a curative effect on HBV infection. In this study, the HBV subgenotype B3 of the HBcAg gene was used, which is dominant in Indonesia. Further, Lactococcus lactis bacteria was used as the host because of its safety and tightly regulated protein expression. The codon usage for the HBcAg gene was optimized to improve protein expression in L. lactis, which is important because a codon is not random between species. The HBcAg gene is attached to a pNZ8148 plasmid and transformed into the L. lactis NZ3900 expression host. The results confirm that a positive protein band (21 kDa) in two fractions of purified HBcAg was recognized by both western blotting and dot blot hybridization, even if the HBcAg optimized codon has higher GC contents than that suggested for L. lactis expression. Overall, this research strengthens the broad use of L. lactis bacteria for any protein expression, including higher protein expression of codon optimized HBcAg gene compared to non-optimized genes. Furthermore, the improvement in the codon optimization of the HBcAg gene significantly increases the total protein expression by 10-20%, and the expression level of the codon optimized HBcAg increases 1.5 to 3.2-times that of the native HBcAg.

Cheonggukjang Fermented with Bacillus subtilis SCGB574 Ameliorates High Fat Diet-Deteriorated Large Intestinal Health in Rat Model

  • Jae Ho, Choi;Jiyon, Kim;Taekyun, Shin;Myeong Seon, Ryu;Hee-Jong, Yang;Do-Youn, Jeong;Hong-Seok, Son;Tatsuya, Unno
    • Microbiology and Biotechnology Letters
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    • v.50 no.4
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    • pp.522-532
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    • 2022
  • Cheonggukjang is a traditional fermented food in Korea, which is known to exert beneficial effects on health. In this study, we evaluated the effects of cheonggukjang fermented by Bacillus subtilis SCGB 574 (B574) on high fat diet (HFD)-deteriorated large intestinal health. Rats were fed with HFD or HFD supplemented with 10.1% cheonggukjang (B574). Fecal microbiota was analyzed based on 16S rRNA gene sequences, and the fecal and serum metabolome were measured using GC-MS. Our results showed that SCGB574 intake significantly reduced body weight, restored tight junction components, and ameliorated inflammatory cell infiltration. SCGB574 also shifted gut microbiota by increasing the abundance of short chain fatty acid producers such as Alistipes and Flintibacter, although it decreased the abundance of Lactobacillus. Serum and fecal metabolome analyses showed significantly different metabolic profiles between the groups. The top five metabolites increased by SCGB574 were i) arginine biosynthesis, ii) alanine, aspartate, and glutamate metabolism; iii) starch and sucrose metabolism; iv) neomycin, kanamycin, and gentamicin biosynthesis; and v) galactose metabolism. These results showed that cheonggukjang fermented by SCGB574 ameliorates adverse effects of HFD through improving intestinal health.

Composition and Cell Cytotoxicity of Essential Oil from Caryopteris incana Miq. in Korea (층꽃나무(Caryopteris incana Miq) 정유의 성분 분석과 세포 독성 평가)

  • Kim, Song-Mun
    • Applied Biological Chemistry
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    • v.51 no.3
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    • pp.238-244
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    • 2008
  • The essential oil was obtained from the aerial part of Caryopteris incana Miq. by steam distillation, samples were collected by headspace (HS) and solid-phase microextraction (SPME) methods, and the compositions of the essential oil were analyzed by gas chromatography-mass spectrometry (GCMS). The fragrance of the essential oil was fougere and woody. There were sixty-nine constituents in the essential oil: 28 carbohydrates, 22 alcohols, 7 acetates, 7 ketones, 3 aldehydes, and 2 others. Major constituents were 4,6,6-trimethyl [1S-($1{\alpha},2{\beta},5{\alpha}$)]-bicyclo[3.1.1]hept-3-en-2-ol (11.8%), taucadinol (9.4%), myrtenyl acetate (9.2%), pinocarvone (7.0%), 1-hydroxy-1,7-dimethyl-4-isopropyl-2,7-cyclodecadiene (6.3%), ${\delta}$-3-carene (6.2%). By SPME extraction, forty-nine constituents were identified: 22 hydrocarbons, 16 alcohols, 6 acetates, 3 ketones, and 2 ethers. Major constituents of the SPME-extracted sample were ${\delta}$-3-carene (12.6%), (-)-myrtenyl acetate (11.2%), 6,6-dimethyl-2-methylene-bicycol [3.1.1] heptan-3-o1 (10.9%), pinocarvone (9.3%). By HS extraction, ten constituents were identified: 5 hydrocarbons, 2 amines, 1 alcohol, and 2 others. Major constituents of the HS-extracted sample were (Z)-2-fluoro-2-butene (34.9%), ${\delta}$-3-carene (6.9%), 6-(4-chlorophenul)tetrahydro-2-methyl-2H-1,2-oxazine (5.9%). The $IC_{50}$ value (0.011 ${\mu}g/mg$) in MTT assay using HaCaT keratinocyte cell line was lower than those of commercially-selling rosemary and tea tree, suggesting more toxicological studies are needed for commercial use of the essential oil of Caryopteris incana Miq.

Analysis of the Component and Immunological Efficacy of Chamaecyparis obtusa Leaf Extract (편백나무 잎 추출물의 성분분석과 면역효능에 관한 연구)

  • Kim, Joung Hee;Lee, Syng-Ook;Do, Kook Bae;Ji, Won Dae;Kim, Sun Gun;Back, Young Doo;Kim, Keuk-Jun
    • Korean Journal of Clinical Laboratory Science
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    • v.50 no.1
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    • pp.37-43
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    • 2018
  • Chamaecyparis obtusa (CO) has recently been attracting attention because of its beneficial effects on skin allergies, atopic dermatitis, and skin diseases, such as acne and eczema. In the present study, the extract from CO leaf grown in Jangseong gun, Jeollanam-do, Korea was evaluated for its anti-oxidant, anti-inflammatory, and anti-allergic effects in vitro. The total polyphenol content of the CO leaf extract was $25.89{\pm}0.31mg$ gallic acid equivalents (GAE)/g. Gas-chromatography mass-spectrometry (GC-MS) analysis revealed the presence of six compounds in the CO leaf extract: ${\alpha}-terpinene$ (3.03 mg/g), ${\alpha}-terpineol$ (9.48 mg/g), limonene (5.96 mg/g), borneol (59.78 mg/g), myrcene (4.85 mg/g), and sabinene (11.31 mg/g). The $RC_{50}$ values of the CO leaf extract for $H_2O_2$ and ABTS radical were $5.47{\pm}0.13mg/mL$ and $4.00{\pm}0.01mg/mL$, respectively. In addition, the CO leaf extract showed significant inhibitory effects on lipopolysaccharide-induced nitric oxide production in RAW 264.7 cells and IgE-induced release of ${\beta}-hexosaminidase$ (degranulation) in mast-cell like RBL-2H3 cells. The cell viability assay showed that the CO leaf extract ($100{\sim}800{\mu}g/mL$) did not affect the viability of human normal skin fibroblast CCD-986sk cells significantly. Overall, these results suggest that the CO leaf extract is a potential functional cosmetic ingredient that can exert anti-oxidant, anti-inflammatory, and anti-allergic effects.

Association of Single Nucleotide Polymorphisms in the Prostaglandin-endoperoxide Synthase 2 (PTGS2) and Phospholipase A2 Group IIA (PLA2G2A) Genes with Susceptibility to Esophageal Squamous Cell Carcinoma

  • Liu, Fen;Wei, Wen-Qiang;Cormier, Robert T.;Zhang, Shu-Tian;Qiao, You-Lin;Li, Xin-Qing;Zhu, Sheng-Tao;Zhai, Yan-Chun;Peng, Xiao-Xia;Yan, Yu-Xiang;Wu, Li-Juan;He, Dian;He, Yan
    • Asian Pacific Journal of Cancer Prevention
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    • v.15 no.4
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    • pp.1797-1802
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    • 2014
  • Background: The prostaglandin-endoperoxide synthase 2 (PTGS2) and phospholipase A2 group IIA (PLA2G2A) genes encode enzymes that are involved in arachidonic acid and prostaglandin biosynthesis. Dysregulation of both genes is associated with inflammation and carcinogenesis, including esophageal squamous cell carcinoma (ESCC). We therefore hypothesized that there is an association between single nucleotide polymorphisms (SNPs) in these genes and susceptibility to ESCC. Methods: We performed a gene-wide tag SNP-based association study to examine the association of SNPs in PTGS2 and PLA2G2A with ESCC in 269 patients and 269 healthy controls from Taihangshan Mountain, Henan and Hebei Provinces, the rural area of China which has the highest incidence of esophageal cancer in the world. Thirteen tag SNPs in PLA2G2A and 4 functional SNPs in PTGS2 were selected and genotyped using a high-throughput Mass Array genotyping platform. Results: We found a modest increased risk of ESCC in subjects with the PTGS2 rs12042763 AA genotype (OR=1.23; 95% CI, 1.00-3.04) compared with genotype GG. For PLA2G2A, a decreased risk of ESCC was observed in subjects with the rs11677 CT (OR=0.51, 95%CI, 0.29-0.85) or TT genotype (OR=0.51, 95%CI, 0.17-0.96) or the T carriers (CT+TT) (OR=0.52, 95%CI, 0.31-0.85) when compared with the CC genotype. Also for PLA2G2A, rs2236771 C allele carriers were more frequent in the control group (P=0.02). Subjects with the GC (OR=0.55, 95%CI, 0.33-0.93) or CC genotype (OR=0.38, 95% CI, 0.16-0.94) or the C carriers (GC+CC) (OR=0.52, 95%CI, 0.32-0.85) showed a negative association with ESCC susceptibility. Conclusions: Our results suggest that PTGS2 and PLA2G2A gene polymorphisms may modify the risk of ESCC development.

Production of Poly-3-hydroxybutyrate from Xylose by Bacillus megaterium J-65 (Bacillus megaterium J-65에 의한 xylose로부터 poly-3-hydroxybutyrate 생산)

  • Jun, Hong-Ki;Jin, Young-Hi;Kim, Hae-Nam;Kim, Yun-Tae;Kim, Sam-Woong;Baik, Hyung-Suk
    • Journal of Life Science
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    • v.18 no.12
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    • pp.1625-1630
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    • 2008
  • A microorganism capable of producing high level of poly-3-hydoxybutyrate (PHB) from xylose was isolated from soil. The isolated strain J-65 was identified as Bacillus megaterium based on the morphological, biochemical and molecular biological characteristics. The optimum temperature and pH for the growth of B. megaterium J-65 were $37^{\circ}C$ and 8.0, respectively. The optimum medium composition for the cell growth was 2% xylose, 0.25% $(NH_4)_2SO_4$, 0.3% $Na_2HPO_4{\cdot}12H_2O$, and 0.1% $KH_2PO_4$. The optimum condition for PHB accumulation was same to the optimum condition for cell growth. Copolymer of ${\beta}$-hydroxybutyric and ${\beta}$-hydroxyvaleric acid was produced when propionic acid was added to shake flasks containing 20 g/l of xylose. Fermenter culture was carried out to produce the high concentration of PHB. In batch culture, cell mass was 9.82 g/l and PHB content was 35% of dry cell weight. PHB produced by B. megaterium J-65 was identified as homopolymer of 3-hydoxybutyric acid by GC and NMR.

Characteristics of $CO_{2}$ Absorption and Degradation of Aqueous Alkanolamine Solutions in $CO_{2}$ and $CO_{2}-O_{2}$ System ($CO_{2}$$CO_{2}-O_{2}$ 시스템에서 알카놀아민류 흡수제를 이용한 $CO_{2}$ 흡수 및 흡수제 열화 특성)

  • Choi, Won-Joon;Lee, Jong-Seop;Han, Keun-Hee;Min, Byoung-Moo
    • Korean Chemical Engineering Research
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    • v.49 no.2
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    • pp.256-262
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    • 2011
  • Amine can undergo irreversible reactions by $O_{2}$ and high temperature in amine scrubbing process and these phenomena are called "degradation". Degradation causes not only a loss of valuable amine, but also operational problems such as foaming, corrosion and fouling. In this study, using various chemical absorbents(MEA; monoethanolamine, AMP; 2-amino-2-methyl-1-propanol, DAM; 1,8-diamino-p-menthane), we examined the following variable. I) loading ratio of $CO_{2}$ at $50^{\circ}C$ and $120^{\circ}C$, ii) concentration variation and initial degradation rate constant of absorbent in $CO_{2}$ and $CO_{2}/O_{2}$ system, and iii) effect of degradation by $O_{2}$. The $CO_{2}$ loading of 20 wt% DAM was 400% and 270% higher than that of 20 wt% MEA and AMP at 50, respectively and was the largest the difference of $CO_{2}$ loading between absorption $(50^{\circ}C)$ and regeneration $(120^{\circ}C)$ condition. The initial degradation rate constant of 20 wt% DAM was $2.254{\times}10^{-4}cycle^{-1}$ which was slower than that of MEA $(2.761{\times}10^{-4}cycle^{-1})$ and AMP $(2.461{\times}10^{-4}cycle^{-1})$ in $CO_{2}$ system. Also, it was increased 30% by $O_{2}$ that effects on the degradation by $O_{2}$ was less than 100% increased. these degradation reactions was able to identify by formation of new peak in GC and FT-IR spectrum analysis.

Fatty acid analysis and regulatory effects of citron (Citrus junos Sieb. ex TANAKA) seed oil on nitric oxide production, lipid accumulation, and leptin secretion (유자씨유의 지방산분석 및 Nitric Oxide 생성, 지방축적능, 렙틴분비 조절효과)

  • Kim, Tae Woo;Kim, Kyoung Kon;Kang, Yun Hwan;Kim, Dae Jung;Choe, Myeon
    • Journal of Nutrition and Health
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    • v.47 no.4
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    • pp.221-228
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    • 2014
  • Purpose: Citron seed oil (CSO) has been reported to have high antioxidant activity. However, the composition and other biologically activities of CSO have not been reported. In this study, we confirmed the fatty acid composition of CSO, which may be beneficial to vascular disease and obesity. Methods: We investigated the oil composition of CSO using gas chromatography coupled with mass spectrometry (GC-MS) analysis, and cytotoxicity was confirmed by Cell Counting Kit-8 (CCK-8) assay. Nitric oxide (NO) production in human umbilical vein endothelial cells (HUVECs) was measured using Griess reagent, and lipid accumulation and leptin secretion in 3T3-L1 cells were measured by Oil-Red O staining and commercial ELISA kit, respectively. Results: GC-MS analysis indicated that CSO contains several components, including linoleic acid, oleic acid, palmitic acid, stearic acid, linolenic acid, palmitoleic acid, and arachidic acid. In physiological activity analysis, CSO did not induce cytotoxic effects in HUVECs and 3T3-L1 cells. Further, CSO significantly induced nitric oxide and leptin secretion as well as inhibited lipid accumulation. Conclusion: CSO increased NO release, inhibited lipid accumulation, and induced leptin secretion, suggesting it may be useful for the management of vessels and weight gain. Although further studies are required to investigate the safety and mechanism of action of CSO, our results show that the composition and physiological activity of CSO are sufficient for its use as functional edible oil.

Brief Introduction of Research Progresses in Control and Biocontrol of Clubroot Disease in China

  • He, Yueqiu;Wu, Yixin;He, Pengfei;Li, Xinyu
    • 한국균학회소식:학술대회논문집
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    • 2015.05a
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    • pp.45-46
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    • 2015
  • Clubroot disease of crucifers has occurred since 1957. It has spread to the whole China, especially in the southwest and nourtheast where it causes 30-80% loss in some fields. The disease has being expanded in the recent years as seeds are imported and the floating seedling system practices. For its effective control, the Ministry of Agriculture of China set up a program in 2010 and a research team led by Dr. Yueqiu HE, Yunnan Agricultural University. The team includes 20 main reseachers of 11 universities and 5 institutions. After 5 years, the team has made a lot of progresses in disease occurrence regulation, resources collection, resistance identification and breeding, biological agent exploration, formulation, chemicals evaluation, and control strategy. About 1200 collections of local and commercial crucifers were identified in the field and by artificiall inoculation in the laboratories, 10 resistant cultivars were breeded including 7 Chinese cabbages and 3 cabbages. More than 800 antagostic strains were isolated including bacteria, stretomyces and fungi. Around 100 chemicals were evaluated in the field and greenhouse based on its control effect, among them, 6 showed high control effect, especially fluazinam and cyazofamid could control about 80% the disease. However, fluzinam has negative effect on soil microbes. Clubroot disease could not be controlled by bioagents and chemicals once when the pathogen Plasmodiophora brassicae infected its hosts and set up the parasitic relationship. We found the earlier the pathogent infected its host, the severer the disease was. Therefore, early control was the most effective. For Chinese cabbage, all controlling measures should be taken in the early 30 days because the new infection could not cause severe symptom after 30 days of seeding. For example, a biocontrol agent, Bacillus subtilis Strain XF-1 could control the disease 70%-85% averagely when it mixed with seedling substrate and was drenching 3 times after transplanting, i.e. immediately, 7 days, 14 days. XF-1 has been deeply researched in control mechanisms, its genome, and development and application of biocontrol formulate. It could produce antagonistic protein, enzyme, antibiotics and IAA, which promoted rhizogenesis and growth. Its The genome was sequenced by Illumina/Solexa Genome Analyzer to assembled into 20 scaffolds then the gaps between scaffolds were filled by long fragment PCR amplification to obtain complet genmone with 4,061,186 bp in size. The whole genome was found to have 43.8% GC, 108 tandem repeats with an average of 2.65 copies and 84 transposons. The CDSs were predicted as 3,853 in which 112 CDSs were predicted to secondary metabolite biosynthesis, transport and catabolism. Among those, five NRPS/PKS giant gene clusters being responsible for the biosynthesis of polyketide (pksABCDEFHJLMNRS in size 72.9 kb), surfactin(srfABCD, 26.148 kb, bacilysin(bacABCDE 5.903 kb), bacillibactin(dhbABCEF, 11.774 kb) and fengycin(ppsABCDE, 37.799 kb) have high homolgous to fuction confirmed biosynthesis gene in other strain. Moreover, there are many of key regulatory genes for secondary metabolites from XF-1, such as comABPQKX Z, degQ, sfp, yczE, degU, ycxABCD and ywfG. were also predicted. Therefore, XF-1 has potential of biosynthesis for secondary metabolites surfactin, fengycin, bacillibactin, bacilysin and Bacillaene. Thirty two compounds were detected from cell extracts of XF-1 by MALDI-TOF-MS, including one Macrolactin (m/z 441.06), two fusaricidin (m/z 850.493 and 968.515), one circulocin (m/z 852.509), nine surfactin (m/z 1044.656~1102.652), five iturin (m/z 1096.631~1150.57) and forty fengycin (m/z 1449.79~1543.805). The top three compositions types (contening 56.67% of total extract) are surfactin, iturin and fengycin, in which the most abundant is the surfactin type composition 30.37% of total extract and in second place is the fengycin with 23.28% content with rich diversity of chemical structure, and the smallest one is the iturin with 3.02% content. Moreover, the same main compositions were detected in Bacillus sp.355 which is also a good effects biocontol bacterial for controlling the clubroot of crucifer. Wherefore those compounds surfactin, iturin and fengycin maybe the main active compositions of XF-1 against P. brassicae. Twenty one fengycin type compounds were evaluate by LC-ESI-MS/MS with antifungal activities, including fengycin A $C_{16{\sim}C19}$, fengycin B $C_{14{\sim}C17}$, fengycin C $C_{15{\sim}C18}$, fengycin D $C_{15{\sim}C18}$ and fengycin S $C_{15{\sim}C18}$. Furthermore, one novel compound was identified as Dehydroxyfengycin $C_{17}$ according its MS, 1D and 2D NMR spectral data, which molecular weight is 1488.8480 Da and formula $C_{75}H_{116}N_{12}O_{19}$. The fengycin type compounds (FTCPs $250{\mu}g/mL$) were used to treat the resting spores of P. brassicae ($10^7/mL$) by detecting leakage of the cytoplasm components and cell destruction. After 12 h treatment, the absorbencies at 260 nm (A260) and at 280 nm (A280) increased gradually to approaching the maximum of absorbance, accompanying the collapse of P. brassicae resting spores, and nearly no complete cells were observed at 24 h treatment. The results suggested that the cells could be lyzed by the FTCPs of XF-1, and the diversity of FTCPs was mainly attributed to a mechanism of clubroot disease biocontrol. In the five selected medium MOLP, PSA, LB, Landy and LD, the most suitable for growth of strain medium is MOLP, and the least for strains longevity is the Landy sucrose medium. However, the lipopeptide highest yield is in Landy sucrose medium. The lipopeptides in five medium were analyzed with HPLC, and the results showed that lipopeptides component were same, while their contents from B. subtilis XF-1 fermented in five medium were different. We found that it is the lipopeptides content but ingredients of XF-1 could be impacted by medium and lacking of nutrition seems promoting lipopeptides secretion from XF-1. The volatile components with inhibition fungal Cylindrocarpon spp. activity which were collect in sealed vesel were detected with metheds of HS-SPME-GC-MS in eight biocontrol Bacillus species and four positive mutant strains of XF-1 mutagenized with chemical mutagens, respectively. They have same main volatile components including pyrazine, aldehydes, oxazolidinone and sulfide which are composed of 91.62% in XF-1, in which, the most abundant is the pyrazine type composition with 47.03%, and in second place is the aldehydes with 23.84%, and the third place is oxazolidinone with 15.68%, and the smallest ones is the sulfide with 5.07%.

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