Bacillus subtilis S21产抗菌脂肽分离鉴定及半固态发酵工艺初探
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摘要
采用酸沉、甲醇萃取和LH-20层析,从Bacillus subtilis S21发酵菌液纯化出两种抗菌脂肽,通过质谱鉴定两种抗菌脂肽分别为Surfactin和Fengycin,二级质谱表明Surfactin由β-OH脂肪酸链(C_(12-15))和Glu-Leu-Leu-Val-Asp-Leu-Leu(Ile)组成,Fengycin由β-OH脂肪酸链(C_(14-16))和Glu-Orn-Tyr-Thr-Glu-Val-Pro-Gln-Tyr-Ile组成。在前期探索试验基础上,以黄豆为主要培养基质,通过正交实验,确定了B. subtilis S21产Surfactin半固态发酵条件:无机盐体系添加20%,葡萄糖添加5%,发酵时间48 h,发酵温度37℃,验证实验表明此条件下Surfactin产量比landy培养基产量增加了180余倍。
Through acid precipitation, alcohol extraction and LH-20 column chromatography, twokinds of antibacterial lipopeptides were purified from fermented bacteria liquid of Bacillus subtilis S21, and mass spectrum revealed the lipopeptides were Surfactin and Fengycin. MS-MS revealed thesequence of Surfactin was β-OH-C_(12-15)-Glu-Leu-Leu-Val-Asp-Leu-Leu (Ile) and Fengycin was β-OH-C_(14-16)-Glu-Orn-Tyr-Thr-Glu-Val-Pro-Gln-Tyr-Ile. On the basis of preliminary exploration test,with soybean as the main culture medium, through orthogonal experiment, the semisolid fermentationconditions of Surfactin producing by B. subtilis S21 were determined, the inorganic ion system wasadded 20%, the glucose was added 5%, the fermentation time was 48 h and the fermentation tempera-ture was 37 ℃, the test showed that the yield of Surfactin increased by 180 times more than that ofthe landy medium.
Through acid precipitation, alcohol extraction and LH-20 column chromatography, twokinds of antibacterial lipopeptides were purified from fermented bacteria liquid of Bacillus subtilis S21, and mass spectrum revealed the lipopeptides were Surfactin and Fengycin. MS-MS revealed thesequence of Surfactin was β-OH-C_(12-15)-Glu-Leu-Leu-Val-Asp-Leu-Leu (Ile) and Fengycin was β-OH-C_(14-16)-Glu-Orn-Tyr-Thr-Glu-Val-Pro-Gln-Tyr-Ile. On the basis of preliminary exploration test,with soybean as the main culture medium, through orthogonal experiment, the semisolid fermentationconditions of Surfactin producing by B. subtilis S21 were determined, the inorganic ion system wasadded 20%, the glucose was added 5%, the fermentation time was 48 h and the fermentation tempera-ture was 37 ℃, the test showed that the yield of Surfactin increased by 180 times more than that ofthe landy medium.
引文
[1]向亚萍,周华飞,刘永锋,等.解淀粉芽孢杆菌B1619脂肽类抗生素的分离鉴定及其对番茄枯萎病菌的抑制作用[J].中国农业科学,2016,49(15):2935-2944.
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[16]龚谷迪,周广田,郭阳,等.脂肽surfactin、iturin、fengycin性质鉴定的研究与展望[J].中国食品添加剂,2013(3):211-215.
[17]金清,肖明.新型抗菌肽--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.
[18]施碧红,商伟伟,郑永标,等.抗菌脂肽BSLP21的分离纯化及其结构与活性分析[J].药物生物技术,2015,22(6):484-488.
[19]Schneider J,Taraz K,Budzikiewicz H,et al.The structure of two fengycins from Bacillus subtilis S499[J].Zeitschrift Für Naturforschung C Journal of Biosciences,1999,54(11):859-865.
[20]黄翔峰,彭开铭,刘佳,等.生物表面活性素分离纯化技术进展[J].化工进展,2009,28(10):1820-1827.
[2]翟少伟,李剑,史庆超.抗菌脂肽Surfactin的抗菌活性及应用[J]动物营养学报,2015,27(5):1333-1340.
[3]黄现青,崔保安,魏战勇,等.生物表面活性素体外抗病毒活性[J].中国兽医学报,2009,29(2):143-145.
[4]金伟伟,贾振华,宋水山.芽孢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.
[5]陈旭波.饲料中添加抗菌脂肽Surfactin对断奶仔猪生长性能及血液生化指标的影响[D].厦门:集美大学,2016.
[6]范少本.抗菌脂肽Surfactin对断奶仔猪肠道健康及血清免疫指标的影响[D].厦门:集美大学,2016.
[7]Fan H,Ru J,Zhang Y,et al.Fengycin produced by Bacillus subtilis 9407 plays a major role in the biocontrol of apple ring rot disease[J].Microbiological Research,2017(199):89-97.
[8]徐红梅.Bacillus amyloliquefaciens M1菌株生产的抗菌脂肽对耐药性海洋病原弧菌抑菌活性的研究[D].青岛:中国海洋大学,2014.
[9]都海明,戚广州,王建军,等.抗菌脂肽对肉鸡生产性能和免疫机能的影响[J].江苏农业学报,2010,26(5):1009-1014.
[10]翟少伟,史庆超,陆鹏,等.不同抗菌脂肽添加物对吉富罗非鱼增重率,血清生化指标和非特异性免疫力的影响[J].广东农业科学,2015,42(21):129-133.
[11]别小妹,陆兆新,吕凤霞,等.Bacillus subtilis fmbR抗菌物质的分离和鉴定[J].中国农业科学,2006,39(11):2327-2334.
[12]庄国宏.产抗菌脂肽GD菌株筛选及脂肽分离、鉴定与应用研究[D].扬州:扬州大学,2014.
[13]钱常娣.枯草芽孢杆菌BAB-1脂肽类物质的分离鉴定及性质分析[D].保定:河北农业大学,2010.
[14]龚庆伟.芽孢杆菌抗菌脂肽的分离纯化及Bacillomycin D抑制黄曲霉作用的研究[D].南京:南京农业大学,2012.
[15]李宝庆,鹿秀云,郭庆港,等.枯草芽孢杆菌BAB-1产脂肽类及挥发性物质的分离和鉴定[J].中国农业科学,2010,43(17):3547-3554.
[16]龚谷迪,周广田,郭阳,等.脂肽surfactin、iturin、fengycin性质鉴定的研究与展望[J].中国食品添加剂,2013(3):211-215.
[17]金清,肖明.新型抗菌肽--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.
[18]施碧红,商伟伟,郑永标,等.抗菌脂肽BSLP21的分离纯化及其结构与活性分析[J].药物生物技术,2015,22(6):484-488.
[19]Schneider J,Taraz K,Budzikiewicz H,et al.The structure of two fengycins from Bacillus subtilis S499[J].Zeitschrift Für Naturforschung C Journal of Biosciences,1999,54(11):859-865.
[20]黄翔峰,彭开铭,刘佳,等.生物表面活性素分离纯化技术进展[J].化工进展,2009,28(10):1820-1827.