枯草芽孢杆菌KC-WQ发酵液中抗菌脂肽的分离鉴定及发酵条件优化

钱荣; 续晓琪; 许宗奇; 徐虹; 李莎; 徐铮; 罗正山

doi:10.13386/j.issn1002-0306.2021100269

枯草芽孢杆菌KC-WQ发酵液中抗菌脂肽的分离鉴定及发酵条件优化

doi: 10.13386/j.issn1002-0306.2021100269

南京工业大学食品与轻工学院，江苏南京 211816

基金项目: 国家自然科学基金（31801496）；江苏省自然科学基金面上项目（BK20201358）；中国博士后基金面上项目（2021M690191）山东省博士后创新项目（202102056）。

详细信息

作者简介:
钱荣（1997−），男，硕士研究生，研究方向：食品微生物，E-mail：297888741@qq.com

通讯作者:
续晓琪（1990−），女，博士，副教授，研究方向：食品微生物与营养支持，E-mail：xiaoqi_xu@njtech.edu.cn

中图分类号: TS201.3
计量
- 文章访问数: 27
- HTML全文浏览量: 18
- PDF下载量: 5
- 被引次数: 0
出版历程
- 收稿日期: 2021-10-26
- 网络出版日期: 2022-06-19
- 刊出日期: 2022-08-03

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

摘要

摘要: 本文研究了一株枯草芽孢杆菌KC-WQ的胞外分泌物的抑菌效果及功效成分。实验发现枯草芽孢杆菌KC-WQ对大肠杆菌（Escherichia coli）、沙门氏菌（Salmonella enterica subsp. enterica）和嗜水气单胞菌（Aeromonas hydrophila）均有明显的抑菌效果，其中对沙门氏菌的抑制效果最为显著。在此基础上，采用酸沉淀法分离抗菌成分，通过薄层色谱法（TLC）、高效液相色谱法（HPLC）、基质辅助激光解吸电离飞行时间质谱（MALDI-TOF-MS）分析结构发现，该菌株分泌抗菌物质为脂肽类，分子量分布在1030.5~1491.4 Da之间，主要由伊枯草菌素（Iturin）、表面活性素（Surfactin）和芬荠素（Fengycin）组成，最小抑菌浓度为0.8 mg/mL。该脂肽成分经热处理（105 ℃，20 min）后，仍保持良好的抗菌效果。采用响应面法对KC-WQ的发酵条件进行优化，得到最佳发酵条件为：初始pH为6.0，发酵温度为36 ℃，摇床转速为208 r/min，预测粗产物产量为1.311 g/L，后经实验验证，实际产量可达1.236 g/L。上述结果表明，枯草芽孢杆菌KC-WQ具有良好的抗菌性能和加工性能，可作为抑菌成分，在食品保藏和抗菌包装材料开发中具有应用前景。
- 枯草芽孢杆菌 /
- 脂肽 /
- 抑菌活性 /
- 发酵条件优化
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

HTML全文

图 1 枯草芽孢杆菌KC-WQ胞外发酵液抑菌图

Figure 1. Antibacterial diagram of extracellular fermentation broth of Bacillus subtilis KC-WQ

注：a为枯草芽孢杆菌胞外发酵液抑制沙门氏菌，b为枯草芽孢杆菌胞外发酵液抑制大肠杆菌，c为枯草芽孢杆菌胞外发酵液抑制嗜水气单胞菌；（Ⅰ）为对照组，50 μL LB液体培养基，（Ⅱ）组添加50 μL发酵液，（Ⅲ）组添加100 μL发酵液，（Ⅳ）组添加150 μL发酵液。

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图 2 脂肽粗提物TLC结果

Figure 2. TLC results of crude lipopeptide extract

注：A、B为平行组。

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图 3 脂肽粗提物与标准品HPLC保留时间对比

Figure 3. Comparison of HPLC retention time between crude lipopeptide extract and standard product

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图 4 脂肽粗提物MALDI-TOF结果

Figure 4. MALDI-TOF analysis results of crude lipopeptide extract

注：a：Iturin族和Surfactin族；b：Fengycin族。

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图 5 抗菌脂肽耐热效果评价

Figure 5. Evaluation of the heat resistance of lipopeptides

注：对照组样品为未经热处理的粗脂肽。

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图 6 碳源（a）、氮源（b）优化对脂肽粗产量及OD_{600 nm}的影响

Figure 6. The influence of carbon source （a） and nitrogen source （b） optimization on the crude lipopeptide yield and OD_{600 nm}

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图 7 摇床发酵条件优化结果

Figure 7. Optimized results of shaker fermentation conditions

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图 8 脂肽粗产量的响应面曲线图及等高线图

Figure 8. Response surface curve and contour plot of crude lipopeptide yield

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表 1 各变量及其高低水平设计

Table 1. Variables and their high and low level design

编号	因素	−1	0	+1
X₁	初始pH	6	7	8
X₂	发酵温度（℃）	33	35	37
X₃	摇床转速（r/min）	150	200	250

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表 2 枯草芽孢杆菌KC-WQ抑菌圈直径

Table 2. Inhibition zone diameter of Bacillus subtilis KC-WQ

	抑菌圈直径（mm）
	Ⅰ	Ⅱ	Ⅲ	Ⅳ
沙门氏菌	0.00	11.00±1.00^a	14.83±2.47^b	16.50±2.18^c
大肠杆菌	0.00	11.33±1.53^a	12.00±2.65^b	12.83±2.37^c
嗜水气单胞菌	0.00	10.00±2.00^a	14.40±1.04^b	15.77±1.57^c
注：具有显著性差异的组用不同的字母标记。

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表 3 脂肽粗提物样品HPLC保留时间及面积

Table 3. HPLC retention time and area of crude lipopeptide extract sample

	保留时间（峰尖，min）	面积	峰面积占比（%）
1	9.977	836.250	4.275
2	10.402	299.384	1.530
3	14.149	2971.641	15.191
4	15.301	1701.197	8.697
5	15.859	841.169	4.300
6	17.189	241.511	1.235
7	19.508	1170.928	5.986
8	20.897	873.000	4.463

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表 4 MS结果分析

Table 4. MS result analysis

Family	m/z	（M+H⁺）	（M+Na⁺）	（M+K⁺）
Iturin	C13~C16	1030.477	1052.447	1068.357
		1044.482	1066.356	1082.319
		1058.486	1080.364	1096.326
		1072.494	1094.365	1110.332
	C13~C15 Bacillomycin D	1031.478	1053.312	1069.408
		1045.483	1067.353	1083.316
		1059.486	1081.322	1097.322
Surfactin	C12~C15 Surfactin	−	1016.472	1032.437
		−	1030.477	1046.441
		−	1044.482	1060.443
		−	1058.486	1074.449
Fengycin	C15~C18 Fengycin A	−	1471.366	1487.338
		−	1485.386	1501.355
		−	1499.394	1515.385
		−	1513.406	1529.373

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表 5 不同浓度的KC-WQ抗菌脂肽对沙门氏菌的抑菌效果

Table 5. Inhibition of different concentrations of KC-WQ crude lipopeptide against Salmonella

抗菌脂肽质量浓度（mg·mL⁻¹）	抑菌圈直径（mm）
0.4	−
0.8	10.0±0.1
1.6	11.0±0.1
3.1	11.5±0.3
6.2	12.6±0.3
12.2	14.7±0.3
25	16.7±0.2

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表 6 响应值试验及结果

Table 6. Response value test and results

实验号	因素			脂肽粗产量（g/L）
实验号	X₁	X₂	X₃	脂肽粗产量（g/L）
1	7	35	200	1.256±0.06
2	7	37	250	1.224±0.06
3	6	35	250	1.021±0.05
4	6	37	200	0.976±0.04
5	7	35	200	1.324±0.06
6	8	37	200	0.889±0.04
7	7	37	150	1.132±0.05
8	8	35	150	1.024±0.05
9	7	35	200	1.256±0.06
10	8	33	200	0.932±0.04
11	7	35	200	1.256±0.06
12	7	35	200	1.256±0.06
13	7	33	150	1.004±0.05
14	6	35	150	0.952±0.04
15	7	33	250	1.136±0.05
16	8	35	250	1.044±0.05
17	6	33	200	0.951±0.04

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表 7 二次多项回归模型方差结果

Table 7. Quadratic polynomial regression model variance results

方差来源	平方和	自由度	均方	F值	P	显著性
模型	0.31	9	0.034	15.15	0.0008	***
X₁	0.071	1	0.071	31.68	0.0008	***
X₂	0.053	1	0.053	23.84	0.0018	**
X₃	3.433E-003	1	3.433E-003	1.53	0.2560	不显著
X₁X₂	2.712E-003	1	2.712E-003	1.21	0.3080	不显著
X₁X₃	1.211E-003	1	1.211E-003	0.54	0.4865	不显著
X₂X₃	4.000E-004	1	4.000E-004	0.18	0.6855	不显著
X₁²	0.20	1	0.20	87.94	<0.0001	***
X₂²	0.050	1	0.050	22.47	0.0021	**
X₃²	5.510E-003	1	5.510E-003	2.46	0.1611	不显著
残差	0.016	7	2.244E-003
失拟项	0.012	3	4.002E-003	4.33	0.0954	不显著
纯误差	3.699E-003	4	9.248E-003
总和	0.32	16
注：R²=0.9512，R²_Adj=0.8884，P<0.05，P<0.01，** P<0.001。

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