[Objective] This study aimed to establish molecular identification methods for Bacillus licheniformis. [Method] Based on clone sequencing and difference analysis for 16S and ITS sequences of B. licheniformis TS-01, sp...[Objective] This study aimed to establish molecular identification methods for Bacillus licheniformis. [Method] Based on clone sequencing and difference analysis for 16S and ITS sequences of B. licheniformis TS-01, specific primers were designed using region sequences as the targets used for amplifying all test strains. [Result] The specific primers of B. licheniformis were designed from the ITS and 16S rDNA regions. The optimal annealing temperature of the specific primers for PCR was 67.2 ℃ with 24 cycles. A 905 bp marker fragment was amplified for B. licheniformis TS-01, while all other test strains showed negative results. This indicated that a specific 16S-ITS marker was obtained, which accurately identified the strain at the species level. [Conclusion] This molecular identification method for B. licheniformis TS-01 has laid the foundation for molecular diagnosis of B. licheniformis.展开更多
Chromatographic separation of the marine-derived bacterium Bacillus licheniformis resulted in the isolation of two new cyclic lipopeptides named ai-Cl6 surfactin （1） and ai-Cl4 surfactin （2）, together with iso-Cm5...Chromatographic separation of the marine-derived bacterium Bacillus licheniformis resulted in the isolation of two new cyclic lipopeptides named ai-Cl6 surfactin （1） and ai-Cl4 surfactin （2）, together with iso-Cm5 surfactin and iso-Cl6 surfactin. The structures of the new cyclic lipopeptides were determined through extensive spectroscopic analysis. The sequences of the amino acids in cyclic nucleus were established by the ESI-MS/MS fragmentation, which provided an efficient method to detect lipopeptides from bacterium extracts without senaration展开更多
Background:Enterotoxigenic Escherichia coli(ETEC)F4 commonly colonizes the small intestine and releases enterotoxins that impair the intestinal barrier function and trigger inflammatory responses.Although Bacillus lic...Background:Enterotoxigenic Escherichia coli(ETEC)F4 commonly colonizes the small intestine and releases enterotoxins that impair the intestinal barrier function and trigger inflammatory responses.Although Bacillus licheniformis(B.licheniformis)has been reported to enhance intestinal health,it remains to be seen whether there is a functional role of B.licheniformis in intestinal inflammatory response in intestinal porcine epithelial cell line(IPEC-J2)when stimulated with ETEC F4.Methods:In the present study,the effects of B.licheniformis PF9 on the release of pro-inflammation cytokines,cell integrity and nuclear factor-κB(NF-κB)activation were evaluated in ETEC F4-induced IPEC-J2 cells.Results:B.licheniformis PF9 treatment was capable of remarkably attenuating the expression levels of inflammation cytokines tumor necrosis factor-α(TNF-α),interleukin(IL)-8,and IL-6 during ETEC F4 infection.Furthermore,the gene expression of Toll-like receptor 4(TLR4)-mediated upstream related genes of NF-κB signaling pathway has been significantly inhibited.These changes were accompanied by significantly decreased phosphorylation of p65 NF-κB during ETEC F4 infection with B.licheniformis PF9 treatment.The immunofluorescence and western blotting analysis revealed that B.licheniformis PF9 increased the expression levels of zona occludens 1(ZO-1)and occludin(OCLN)in ETEC F4-infected IPEC-J2 cells.Meanwhile,the B.licheniformis PF9 could alleviate the injury of epithelial barrier function assessed by the trans-epithelial electrical resistance(TEER)and cell permeability assay.Interestingly,B.licheniformis PF9 protect IPEC-J2 cells against ETEC F4 infection by decreasing the gene expressions of virulence-related factors(including luxS,estA,estB,and elt)in ETEC F4.Conclusions:Collectively,our results suggest that B.licheniformis PF9 might reduce inflammation-related cytokines through blocking the NF-κB signaling pathways.Besides,B.licheniformis PF9 displayed a significant role in the enhancement of IPEC-J2 cell integrity.展开更多
The solubilization of elastin by Bacillus licheniformis elastase cannot be analyzed by conventional kinetic methods because the biologically relevant substrate is insoluble and the concentration of enzyme-substrate co...The solubilization of elastin by Bacillus licheniformis elastase cannot be analyzed by conventional kinetic methods because the biologically relevant substrate is insoluble and the concentration of enzyme-substrate complex has no physical meaning. In this paper we report the optimization of elastolysis conditions and analysis of elastolytic kinetics. Our results indicated that the hydrolyzing temperature and time are very important factors affecting elastolysis rate. The optimized conditions using central composite design were as follows： elastolysis temperature 50 ℃, elastase concentration 1 × 10^4 U/ml, elastin 80 mg, elastolytic time 4 h. Investigation of the effects of substrate content, elastase concentration and pH was also revealed that low or high elastin content inhibits the elastolysis process. Increasingelastase improves elastin degradation, but high elastase may change the kinetics characterization. Alkaline environment can decrease elastin degradation rate and pH may affect elastolysis by changing elastase reaction pH. To further elucidate the elastolysis process, the logistic model was used to elastolysis kinetics study showing clearly that the logistic model can reasonably explain the elastolysis process, especially under lower elastase concentration. However, there is still need for more investigations with the aid of other methods, such as biochemical and molecular methods.展开更多
Sequential methodology based on the application of three types of experimental designs was used to optimize the fermentation conditions for elastase production from mutant strain ZJUEL31410 of Bacillus licheniformis i...Sequential methodology based on the application of three types of experimental designs was used to optimize the fermentation conditions for elastase production from mutant strain ZJUEL31410 of Bacillus licheniformis in shaking flask cul- tures. The optimal cultivation conditions stimulating the maximal elastase production consist of 220 r/min shaking speed, 25 h fermentation time, 5% (v/v) inoculums volume, 25 ml medium volume in 250 ml Erlenmeyer flask and 18 h seed age. Under the optimized conditions, the predicted maximal elastase activity was 495 U/ml. The application of response surface methodology resulted in a significant enhancement in elastase production. The effects of other factors such as elastin and the growth factor (corn steep flour) on elastase production and cell growth were also investigated in the current study. The elastin had no significant effect on enzyme-improved production. It is still not clear whether the elastin plays a role as a nitrogen source or not. Corn steep flour was verified to be the best and required factor for elastase production and cell growth by Bacillus licheniformis ZJUEL31410.展开更多
Bacillus licheniformis has the biological characteristics of strong resistance to stress, high temperature, high pressure, pH and bile salt, which also has unique advantage in application safety, antibacterial activit...Bacillus licheniformis has the biological characteristics of strong resistance to stress, high temperature, high pressure, pH and bile salt, which also has unique advantage in application safety, antibacterial activity and stability. The recent research results on mechanism of B. licheniformis and its application effect in poultry production are elaborated in the paper.展开更多
文摘[Objective] This study aimed to establish molecular identification methods for Bacillus licheniformis. [Method] Based on clone sequencing and difference analysis for 16S and ITS sequences of B. licheniformis TS-01, specific primers were designed using region sequences as the targets used for amplifying all test strains. [Result] The specific primers of B. licheniformis were designed from the ITS and 16S rDNA regions. The optimal annealing temperature of the specific primers for PCR was 67.2 ℃ with 24 cycles. A 905 bp marker fragment was amplified for B. licheniformis TS-01, while all other test strains showed negative results. This indicated that a specific 16S-ITS marker was obtained, which accurately identified the strain at the species level. [Conclusion] This molecular identification method for B. licheniformis TS-01 has laid the foundation for molecular diagnosis of B. licheniformis.
基金Grants from COMRA(Grant No.DY125-15-T-01 and SOA(Grant No.2010319123366025-4)National High Technology Development Project(863 Project,Grant No.2011AA10A202-2)National Key Technologies R&D Program(Grant No.2011BAE06B04)
文摘Chromatographic separation of the marine-derived bacterium Bacillus licheniformis resulted in the isolation of two new cyclic lipopeptides named ai-Cl6 surfactin （1） and ai-Cl4 surfactin （2）, together with iso-Cm5 surfactin and iso-Cl6 surfactin. The structures of the new cyclic lipopeptides were determined through extensive spectroscopic analysis. The sequences of the amino acids in cyclic nucleus were established by the ESI-MS/MS fragmentation, which provided an efficient method to detect lipopeptides from bacterium extracts without senaration
基金supported by the Agriculture and Agri-Food Canada,AAFC’s IOP project,Manitoba Pork and Swine Innovation PorcCanada Foundation for Innovation(CFI)supported by the Chinese Scholarship Council(CSC).
文摘Background:Enterotoxigenic Escherichia coli(ETEC)F4 commonly colonizes the small intestine and releases enterotoxins that impair the intestinal barrier function and trigger inflammatory responses.Although Bacillus licheniformis(B.licheniformis)has been reported to enhance intestinal health,it remains to be seen whether there is a functional role of B.licheniformis in intestinal inflammatory response in intestinal porcine epithelial cell line(IPEC-J2)when stimulated with ETEC F4.Methods:In the present study,the effects of B.licheniformis PF9 on the release of pro-inflammation cytokines,cell integrity and nuclear factor-κB(NF-κB)activation were evaluated in ETEC F4-induced IPEC-J2 cells.Results:B.licheniformis PF9 treatment was capable of remarkably attenuating the expression levels of inflammation cytokines tumor necrosis factor-α(TNF-α),interleukin(IL)-8,and IL-6 during ETEC F4 infection.Furthermore,the gene expression of Toll-like receptor 4(TLR4)-mediated upstream related genes of NF-κB signaling pathway has been significantly inhibited.These changes were accompanied by significantly decreased phosphorylation of p65 NF-κB during ETEC F4 infection with B.licheniformis PF9 treatment.The immunofluorescence and western blotting analysis revealed that B.licheniformis PF9 increased the expression levels of zona occludens 1(ZO-1)and occludin(OCLN)in ETEC F4-infected IPEC-J2 cells.Meanwhile,the B.licheniformis PF9 could alleviate the injury of epithelial barrier function assessed by the trans-epithelial electrical resistance(TEER)and cell permeability assay.Interestingly,B.licheniformis PF9 protect IPEC-J2 cells against ETEC F4 infection by decreasing the gene expressions of virulence-related factors(including luxS,estA,estB,and elt)in ETEC F4.Conclusions:Collectively,our results suggest that B.licheniformis PF9 might reduce inflammation-related cytokines through blocking the NF-κB signaling pathways.Besides,B.licheniformis PF9 displayed a significant role in the enhancement of IPEC-J2 cell integrity.
基金Project (No. Y304203) supported by the Natural Science Foundationof Zhejiang Province, China
文摘The solubilization of elastin by Bacillus licheniformis elastase cannot be analyzed by conventional kinetic methods because the biologically relevant substrate is insoluble and the concentration of enzyme-substrate complex has no physical meaning. In this paper we report the optimization of elastolysis conditions and analysis of elastolytic kinetics. Our results indicated that the hydrolyzing temperature and time are very important factors affecting elastolysis rate. The optimized conditions using central composite design were as follows： elastolysis temperature 50 ℃, elastase concentration 1 × 10^4 U/ml, elastin 80 mg, elastolytic time 4 h. Investigation of the effects of substrate content, elastase concentration and pH was also revealed that low or high elastin content inhibits the elastolysis process. Increasingelastase improves elastin degradation, but high elastase may change the kinetics characterization. Alkaline environment can decrease elastin degradation rate and pH may affect elastolysis by changing elastase reaction pH. To further elucidate the elastolysis process, the logistic model was used to elastolysis kinetics study showing clearly that the logistic model can reasonably explain the elastolysis process, especially under lower elastase concentration. However, there is still need for more investigations with the aid of other methods, such as biochemical and molecular methods.
文摘Sequential methodology based on the application of three types of experimental designs was used to optimize the fermentation conditions for elastase production from mutant strain ZJUEL31410 of Bacillus licheniformis in shaking flask cul- tures. The optimal cultivation conditions stimulating the maximal elastase production consist of 220 r/min shaking speed, 25 h fermentation time, 5% (v/v) inoculums volume, 25 ml medium volume in 250 ml Erlenmeyer flask and 18 h seed age. Under the optimized conditions, the predicted maximal elastase activity was 495 U/ml. The application of response surface methodology resulted in a significant enhancement in elastase production. The effects of other factors such as elastin and the growth factor (corn steep flour) on elastase production and cell growth were also investigated in the current study. The elastin had no significant effect on enzyme-improved production. It is still not clear whether the elastin plays a role as a nitrogen source or not. Corn steep flour was verified to be the best and required factor for elastase production and cell growth by Bacillus licheniformis ZJUEL31410.
基金Supported by Three New Agriculture Project of Jiangsu Province(SXGC[2012]2012)
文摘Bacillus licheniformis has the biological characteristics of strong resistance to stress, high temperature, high pressure, pH and bile salt, which also has unique advantage in application safety, antibacterial activity and stability. The recent research results on mechanism of B. licheniformis and its application effect in poultry production are elaborated in the paper.
