Isolation and Identification of Antibacterial Lpopeptides from <i>Bacillus subtilis</i> KC-WQ Fermentation Broth and Optimization of Fermentation Conditions

QIAN Rong; XU Xiaoqi; XU Zongqi; XU Hong; LI Sha; XU Zheng; LUO Zhengshan

doi:10.13386/j.issn1002-0306.2021100269

Volume 43 Issue 15

Jul. 2022

Turn off MathJax

Article Contents

Abstract

References

Science and Technology of Food Industry > 2022 > 43(15): 123-131. > DOI: 10.13386/j.issn1002-0306.2021100269

QIAN Rong, XU Xiaoqi, XU Zongqi, et al. Isolation and Identification of Antibacterial Lpopeptides from Bacillus subtilis KC-WQ Fermentation Broth and Optimization of Fermentation Conditions[J]. Science and Technology of Food Industry, 2022, 43(15): 123−131. (in Chinese with English abstract). doi: 10.13386/j.issn1002-0306.2021100269.

Citation:

PDF (11077 KB)

Isolation and Identification of Antibacterial Lpopeptides from Bacillus subtilis KC-WQ Fermentation Broth and Optimization of Fermentation Conditions

College of Food and Light Industry, Nanjing Tech University, Nanjing 211816, China

More Information

Received Date: October 25, 2021
Available Online: May 27, 2022

Graphical Abstract

Abstract

Abstract

This paper studied the antibacterial effect and functional components of the extracellular secretions of a strain of Bacillus subtilis KC-WQ. The experiment found that KC-WQ had obvious antibacterial effect on Escherichia coli, Salmonella enterica subsp. enterica and Aeromonas hydrophila, among which it had the most significant antibacterial effect on Salmonella. On this basis, the acid precipitation method was used to separate the antibacterial components, and the structure was found through thin layer chromatography (TLC), high performance liquid chromatography (HPLC) and matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS). The antibacterial substances secreted by this strain were lipopeptides with a molecular weight distribution between 1030.5 and 1491.4 Da, mainly composed of Iturin, Surfactin and Fengycin, and the minimum inhibitory concentration was 0.8 mg/mL. The lipopeptide component still maintained a good antibacterial effect after heat treatment (105 ℃, 20 min). The response surface method was used to optimize the culture conditions of KC-WQ, and the best fermentation conditions were obtained: The initial pH was 6.0, the fermentation temperature was 36 ℃, the shaker speed was 208 r/min, and the predicted crude product yield was 1.311 g/L. Later verified by experiments, the actual yield was 1.236 g/L. The above results indicated that Bacillus subtilis KC-WQ had good antibacterial and processing properties, could be used as a good antibacterial ingredient, and had a good application prospect in the development of antibacterial and preservation materials in food industry.
- Bacillus subtilis,
- lipopeptide,
- antibacterial activity,
- fermentation condition optimization

FullText(HTML)

References (29)

References

[1]	孙颖颖, 董鹏程, 朱立贤, 等. 食源性致病菌快速检测研究进展[J]. 食品与发酵工业,2020,46(17):264−270. [SUN Y Y, DONG P C, ZHU L X, et al. Research progress in rapid detection of foodborne pathogens[J]. Food and Fermentation Industries,2020,46(17):264−270. SUN Y Y, DONG P C, ZHU L X, et al. Research progress in rapid detection of foodborne pathogens[J]. Food and Fermentation Industries, 2020, 46(17): 264-270.
[2]	金伟伟, 贾振华, 宋水山. 芽胞杆菌产生的脂肽类抗生素的结构和应用[J]. 微生物学杂志,2017,37(3):122−127. [JIN W W, JIA Z H, SONG S S. Structure and the application of lipopeptides antibiotics from Bacillus[J]. Journal of Microbiology,2017,37(3):122−127. doi: 10.3969/j.issn.1005-7021.2017.03.020 JIN W W, JIA Z H, SONG S S. Structure and the application of lipopeptides antibiotics from Bacillus[J]. Journal of Microbiology, 2017 , 37(3): 122-127. doi: 10.3969/j.issn.1005-7021.2017.03.020
[3]	RATHINAKUMAR R, WALKENHORST W F, WIMLEY W C, et al. Broad-spectrum antimicrobial eptides by rational combinatorial design and high-throughput screening the importance of interfacial activity[J]. Journal of the American Chemical Society,2009,131(22):7609−7617. doi: 10.1021/ja8093247
[4]	高晓平, 胡惠, 黄现青. Surfactin抑制乳中大肠杆菌O157活性研究[J]. 食品科学,2009,30(11):91−94. [GAO X P, HU H, HUANG X Q. Sterilization effect of Surfactin on Escherichia coli O157 in fresh milk[J]. Food Science,2009,30(11):91−94. GAO X P, HU H, HUANG X Q. Sterilization effect of Surfactin on Escherichia coli O157 in fresh milk[J]. Food Science, 2009, 30(11):91-94.
[5]	MANDAL S M, BARBOSA A, FRANCO O L. Lipopeptides in microbial infection control: Scope and reality for industry[J]. Biotechnology Advances,2013,31(2):338−345. doi: 10.1016/j.biotechadv.2013.01.004
[6]	HUANG E, YOUSEF A E. The lipopeptide antibiotic paenibacterin binds to the bacterial outer membrane and exerts bactericidal activity through cytoplasmic membrane damage[J]. Appl Environ Microbiol,2014,80(9):2700−2704. doi: 10.1128/AEM.03775-13
[7]	SUGAWARA K, NUMATA K I, KONISHI M, et al. Empedopeptin (BMY-28117), a new depsipeptide antibiotic. II. Structure determination[J]. Journal of Antibiotics,1984,37(9):958−964. doi: 10.7164/antibiotics.37.958
[8]	WASHIO K, LIM S P, ROONGSAWANG N, et al. Identification and characterization of the genes responsible for the production of the cyclic lipopeptide arthrofactin by Pseudomonas sp. MIS38[J]. Journal of the Agricultural Chemical Society of Japan,2010,74(5):992−999.
[9]	SM GUÉRET, MEIER P, ROTH H J. Cyclic carbo-isosteric depsipeptides and peptides as a novel class of peptidomimetics[J]. Organic Letters,2014,16(5):1502−1505. doi: 10.1021/ol5003797
[10]	KIHO T, NAKAYAMA M, YASUDA K, et al. Structure-activity relationships of globomycin analogues as antibiotics[J]. Bioorganic & Medicinal Chemistry,2004,12(2):337−361.
[11]	VILELA W F D, FONSECA S G, FANTINATTI-GARBOGGINI F, et al. Production and properties of a surface-active lipopeptide produced by a new marine Brevibacterium luteolum strain[J]. Applied Biochemistry & Biotechnology,2014,174(6):2245−2256.
[12]	SHARAFI H, ABDOLI M, HAJFARAJOLLAH H, et al. First report of a lipopeptide biosurfactant from thermophilic bacterium Aneurinibacillus thermoaerophilus MK01 newly isolated from municipal landfill site[J]. Applied Biochemistry & Biotechnology,2014,173(5):1236−1249.
[13]	连玲丽, 谢荔岩, 郑璐平, 等. 拮抗菌SB1的鉴定及其抗菌物质的分析[J]. 微生物学通报,2010,37(7):986−991. [LIAN L L, XIE L Y, ZHENG L P, et al. Identification of antagonistic Bacterium SB1 and analysis on its antibacterial substance[J]. Microbiology,2010,37(7):986−991. LIAN L L, XIE L Y, ZHENG L P, et al. Identification of antagonistic Bacterium SB1 and analysis on its antibacterial substance[J]. Microbiology, 2010, 37(7): 986-991.
[14]	CHEN Y, X GAO, WEI Y, et al. Isolation, purification and anti-hypertensive effect of a novel angiotensin i-converting enzyme (ACE) inhibitory peptide from ruditapes philippinarum fermented with Bacillus natto[J]. Food & Function,2018,9(10):1039.
[15]	丁飞鸿, 耿云龙, 樊士德, 等. 抑制食源性致病菌脂肽的分离鉴定及生物学特性分析[J]. 食品与发酵工业,2022,48(6):195−203. [DING F H, GENG Y L, FAN S D, et al. Isolation, identifica- tion and biological characteristics analysis of lipopeptides that inhibit food-borne pathogens[J]. Food and Fermentation Industries,2022,48(6):195−203. doi: 10.13995/j.cnki.11-1802/ts.028471 DING F H, GENG Y L, FAN S D, et al. Isolation, identifica- tion and biological characteristics analysis of lipopeptides that inhib- it food-borne pathogens[J]. Food and Fermentation Industries, 2022, 48(6): 195-203. DOI: 10.13995/j.cnki.11-1802/ts.028471.
[16]	VOLLENBROICH D, PAULI G, OZEL M, et al. Antimycoplasma properties and application in cell culture of surfactin, a lipopeptide antibiotic from Bacillus subtilis[J]. Appl Environ Microbiol,1997,63(1):44−49. doi: 10.1128/aem.63.1.44-49.1997
[17]	高学文, 姚仕义, HUONG Pham, 等. 基因工程菌枯草芽孢杆菌GEB3产生的脂肽类抗生素及其生物活性研究[J]. 中国农业科学,2003,36(12):1496−1501. [GAO X W, YAO S Y, HUONG Pham, et al. Lipopeptide antibiotics produced by the engineered strain Bacillus subtilis GEB3 and detection of its bioactivity[J]. Scientla Agricultura Scinica,2003,36(12):1496−1501. doi: 10.3321/j.issn:0578-1752.2003.12.012 GAO X W, YAO S Y, Huong Pham, et al. Lipopeptide antibiotics produced by the engineered strain Bacillus subtilis GEB3 and detection of its bioactivity[J]. Scientla Agricultura Scinica, 2003, 36(12): 1496-1501. doi: 10.3321/j.issn:0578-1752.2003.12.012
[18]	TSUKAGOSHI N, TAMURA G, ARIMA K. A novel protoplast-bursting factor (surfactin) obtained from Bacillus subtilis IAM 1213. I. The effects of surfactin on Bacillus megaterium KM[J]. Biochimica et Biophysica Acta,1970,196(2):211−214. doi: 10.1016/0005-2736(70)90008-8
[19]	王帅, 高圣风, 高学文, 等. 枯草芽孢杆菌脂肽类抗生素发酵和提取条件[J]. 中国生物防治,2007,23(4):342−347. [WANG S, GAO S F, GAO X W, et al. Fermentation and extraction conditions of Bacillus subtilis lipopeptide antibiotics[J]. Chinese Journal of Biological Control,2007,23(4):342−347. WANG S, GAO S F, GAO X W, et al. Fermentation and extraction conditions of Bacillus subtilis lipopeptide antibiotics[J]. Chinese Journal of Biological Control, 2007, 23(4): 342-347.
[20]	喻钢, 田万红, 董思国, 等. 枯草芽孢杆菌抗菌脂肽的分离纯化及质谱检测[J]. 药物分析杂志,2014(10):56−61. [YU G, TIAN W H, DONG S G, et al. Study on purification and mass spectrometric detection of antimicrobial peptide from Bacillus subtilis[J]. Chinese Journal of Pharmaceutical Analysis,2014(10):56−61. YU G, TIAN W H, DONG S G, et al. Study on purification and mass spectrometric detection of antimicrobial peptide from Bacillus subtilis[J]. Chinese Journal of Pharmaceutical Analysis, 2014(10): 56-61
[21]	白杰, 贠建民, 祝发明, 等. 枯草芽孢杆菌菌株B3抗菌脂肽的分离纯化与鉴定[J]. 食品与发酵工业,2018,44(8):82−89. [BAI J, YUN J M, ZHU F M, et al. Isolation, purification and identification of antibacterial peptide from Bacillus subtilis strain B3[J]. Food and Fermentation Industries,2018,44(8):82−89. BAI J, YUN J M, ZHU F M, et al. Isolation, purification and identification of antibacterial peptide from Bacillus subtilis strain B3[J]. Food and Fermentation Industries, 2018, 44(8): 82-89.
[22]	曾国洪, 丛丽娜, 毕楠. 枯草芽孢杆菌诱变株产抗菌脂肽的特性[J]. 大连工业大学学报,2019,38(4):235−238. [ZENG G H, CONG L N, BI N. Characteristics of antimicrobial lipopeptides produced by Bacillus subtilis mutant strains[J]. Journal of Dalian Polytechnic University,2019,38(4):235−238. ZENG G H, CONG L N, BI N. Characteristics of antimicrobial lipopeptides produced by Bacillus subtilis mutant strains[J]. Journal of Dalian Polytechnic University, 2019, 38(4): 235-238.
[23]	FU T, ISLAM M S, ALI M, et al. Two antimicrobial genes from Aegilops tauschii cosson identified by the Bacillus subtilis expression system[J]. Scientific Reports,2020,10(1):13346. doi: 10.1038/s41598-020-70314-5
[24]	黄君. 海洋枯草芽孢杆菌HS-A38产抗菌脂肽类物质发酵条件的优化[D]. 大连: 大连工业大学, 2018. HUANG J. Optimization of the fermentation conditions of Bacillus subtilis HS-A38[D]. Dalian: Dalian Polytechnic University, 2018.
[25]	金清, 肖明. 新型抗菌脂肽——表面活性素、伊枯草菌素和丰原素[J]. 微生物与感染,2018,13(1):56−64. [JIN Q, XIAO M. Novel antimicrobial peptides: Surfactin, iturin and fengycin[J]. Journal of Microbes and Infections,2018,13(1):56−64. doi: 10.3969/j.issn.1673-6184.2018.01.010 JIN Q, XIAO M. Novel antimicrobial peptides: surfactin, iturin and fengycin[J]. Journal of Microbes and Infections, 2018, 13(1): 56-64. doi: 10.3969/j.issn.1673-6184.2018.01.010
[26]	CHUA B Y, ZENG W, LAU Y F, et al. Comparison of lipopeptide-based immunocontraceptive vaccines containing different lipid groups[J]. Vaccine,2007,25(1):92−101. doi: 10.1016/j.vaccine.2006.07.012
[27]	陈亮, 秦素雅, 衡军影, 等. 微生物抗菌脂肽在食品工业中的应用研究进展[J]. 河南工业大学学报:自然科学版,2018,39(5):119−126. [CHEN L, QIN S Y, HENG J Y, et al. Research and application advances of antimicrobial peptides in food industry[J]. Journal of Henan University of Technology (Natural Science Edition),2018,39(5):119−126. CHEN L, QIN S Y, HENG J Y, et al. Research and application advances of antimicrobial peptides in food industry[J]. Journal of Henan University of Technology (Natural Science Edition), 2018, 39(5): 119-126.
[28]	NITSCHKE M, PASTORE G M. Production and properties of a surfactant obtained from Bacillus subtilis grown on cassava waste[J]. Bioresource Technology,2006,97(2):336−341. doi: 10.1016/j.biortech.2005.02.044
[29]	何岚, 王柳懿, 朱琪, 等. 两种绘制枯草芽孢杆菌和大肠杆菌生长曲线方法的比较[J]. 天津农业科学,2017,23(5):14−18. [HE L, WANG L Y, ZHU Q, et al. Comparison of two methods for drawing the growth curve of Bacillus subtilis and Escherichia coli[J]. Tianjin Agricultural Sciences,2017,23(5):14−18. doi: 10.3969/j.issn.1006-6500.2017.05.004 HE L, WANG L Y, ZHU Q, QIAO J Y, WANG W J. Comparison of two methods for drawing the growth curve of Bacillus subtilis and Escherichia coli[J]. Tianjin Agricultural Sciences, 2017, 23(5): 14-18. doi: 10.3969/j.issn.1006-6500.2017.05.004

[1]	JIN Zishuang, CHEN Jiao, MA Zhiqian, ZUO Jia, WU Yiming, MEI Lin. Effect of Ultra High Pressure Combined with Partial Freezing on the Storage Quality of Chicken Soup[J]. Science and Technology of Food Industry, 2024, 45(15): 332-341. DOI: 10.13386/j.issn1002-0306.2023090187
[2]	MENG Di, JIAO He, ZHAO Anqi, HAN Ying, HE Xue, LI Pengxia, HU Huali. Effect of Vacuum Pre-cooling on the Circulation and Shelf Quality of Postharvest Baby Cabbage[J]. Science and Technology of Food Industry, 2024, 45(9): 296-308. DOI: 10.13386/j.issn1002-0306.2023060008
[3]	ZHAO Zhibing, HUANG Tingting, LÜ Jiahan, DU Xiaoji, ZHANG Changfeng, ZHANG Yu, CAO Sen. Effect of Melatonin Coupling with Eugenol Treatment on Storage Quality of Red Amygdalus persica[J]. Science and Technology of Food Industry, 2022, 43(23): 341-346. DOI: 10.13386/j.issn1002-0306.2022070080
[4]	FU Baoshang, DING Ruosong, SHANG Shan, DONG Xiuping, QI Libo, YU Bo. Effects of Different Preservatives on the Storage Quality of Roasted Flake[J]. Science and Technology of Food Industry, 2021, 42(20): 301-308. DOI: 10.13386/j.issn1002-0306.2021010090
[5]	GUO Shuting, XUE Jianyi, LI Feng, ZHAO Botao, WANG Yongli. Effects of Vacuum Precooling Pressure on Postharvest Preservative Quality of Spinach[J]. Science and Technology of Food Industry, 2021, 42(5): 265-269. DOI: 10.13386/j.issn1002-0306.2020050230
[6]	LI Wenjun, LIU Guangqin, WANG Chengzhang, YE Jianzhong, WANG Zijin. Effects of Compound Preservative on the Postharvest Quality of Carya illinoensis during Storage[J]. Science and Technology of Food Industry, 2021, 42(3): 258-264,271. DOI: 10.13386/j.issn1002-0306.2020040215
[7]	DUAN Zhou-wei, XIE Hui, WANG Shi-ping, HE Ai, DOU Zhi-hao. Effect of vacuum pre-cooling treatment on storage quality of Australia Mango[J]. Science and Technology of Food Industry, 2017, (03): 326-330. DOI: 10.13386/j.issn1002-0306.2017.03.055
[8]	LIAO Cai-hu, SHAN Bin, ZHONG Rui-min, HUANG Guo-qing, LAI Wang-hang. Effect of different final temperature on vacuum cooled fresh beef's quality[J]. Science and Technology of Food Industry, 2013, (24): 334-338. DOI: 10.13386/j.issn1002-0306.2013.24.084
[9]	Effect of oligochitosan on storage qualities of apricot fruit[J]. Science and Technology of Food Industry, 2012, (17): 353-356. DOI: 10.13386/j.issn1002-0306.2012.17.006
[10]	Effect of salicylic acid treatment on storage quality of apricot fruits[J]. Science and Technology of Food Industry, 2012, (15): 335-337. DOI: 10.13386/j.issn1002-0306.2012.15.094

Supplements (0)

Cited By

Cited by

Periodical cited type(4)

1.	刘子夜，武琳霞，刘美玉，王蒙. 山东地区不同品种樱桃营养品质评价. 食品安全质量检测学报. 2025(02): 35-45 .
2.	宋莎，刘方利，解璞，王恩权，张绿萍，金吉林. 玛瑙红樱桃胚败育果实和正常发育果实外观形态比较. 中国果树. 2025(03): 42-46 .
3.	陈丽娟，王东，李洪雯，刘佳，张国薇. 中国樱桃不同品种（优系）果实品质分析. 热带作物学报. 2024(03): 514-523 .
4.	赵新玉，杜文瑜，谭梦男，郑晓冬，刘雪梅，闫新焕. 基于气相离子迁移谱技术分析延安市不同县区的富士苹果挥发性成分的差异. 食品安全质量检测学报. 2024(12): 240-250 .