Antibacterial activities and optimization of fermentation conditions of lipopeptides produced by Aureobasidium pullulans PA-2

doi:10.3969/j.issn.1004-1524.2021.03.13

Acta Agriculturae Zhejiangensis ›› 2021, Vol. 33 ›› Issue (3): 479-489.DOI: 10.3969/j.issn.1004-1524.2021.03.13

• Plant Protection • Previous Articles Next Articles

Antibacterial activities and optimization of fermentation conditions of lipopeptides produced by Aureobasidium pullulans PA-2

YUE Denggao, CHENG Liang^*(), GUO Qingyun

Academy of Agriculture and Forestry Science of Qinghai University, Key Laboratory of Agricultural Integrated Pest Management of Qinghai Province, Scientific Observing and Experimental Station of Crop Pest of Ministry of Agriculture and Rural Affairs, Xining 810016, China

Received:2020-07-26 Online:2021-04-02 Published:2021-03-25
Contact: CHENG Liang

Abstract

Abstract:

The antibacterial activities of lipopeptides from Aureobasidium pullulans PA-2 was explored, and the fermentation conditions were optimized with the output of lipopeptides as the response value, laying a theoretical foundation for the production and application of lipopeptides. The lipopeptides was crudely extracted by acid precipitation method. The crude extract of lipopeptide was qualitatively detected by in-situ acid hydrolysis and ninhydrin colorimetry. Antibacterial activities were measured by the method of agar drilling diffusion. The fermentation conditions were optimized by central composite design (CCD). Finally, the crude extract was identified as cyclic lipopeptide. The lipopeptides had obvious effects on Staphylococcus aureus, Saccharomyces cerevisiae, Escherichia coli, Mycosphaerella cerasella, Pyrenophorateres drechsler and antibacterial circle were 3.65, 1.95, 2.15, 1.35 and 2.18 cm respectively. The optimal fermentation conditions were: inoculation amount 6.8%, rotation speed 216 r·min ^-1, culture temperature 26 ℃, liquid volume 125 mL and initial pH 7.Under this condition, predicting yield of antibacterial lipopeptides by the model was 0.94 g·L ^-1, actually 0.92 g·L ^-1, which was 51% more than that of before(0.61 g·L ^-1). The optimized fermentation conditions increase the yield of the lipopeptide and reduce the fermentation cost, which may be applied to the control of strains with inhibitory effect above

Key words: Aureobasidium pullulans PA-2, lipopeptides, antibacterial activity, optimization of fermentation

CLC Number:

S476.8

YUE Denggao, CHENG Liang, GUO Qingyun. Antibacterial activities and optimization of fermentation conditions of lipopeptides produced by Aureobasidium pullulans PA-2[J]. Acta Agriculturae Zhejiangensis, 2021, 33(3): 479-489.

Figures/Tables 8

References 29

[1]	廖全山 . 我国抗生素滥用现状、原因及对策综述[J]. 世界最新医学信息文摘, 2016,16(57):41-42.
	LIAO Q S . Summary of the current situation, causes and countermeasures of antibiotic abuse in my country[J]. World Latest Medicine Information, 2016,16(57):41-42. (in Chinese)
[2]	唐胜球, 董小英, 邹晓庭 . 微生物脂肽的研究概况[J]. 黑龙江畜牧兽医, 2007(5):28-29.
	TANG S Q, DONG X Y, ZOU X T . Research overview of microbial lipopeptides[J]. Heilongjiang Animal Science and Veterinary Medicine, 2007(5):28-29. (in Chinese)
[3]	KOUMOUTSI A, CHEN X H, HENNE A , et al. Structural and functional characterization of gene clusters directing nonribosomal synjournal of bioactive cyclic lipopeptides in Bacillus amyloliquefaciens strain FZB42[J]. Journal of Bacteriology, 2004,186(4):1084-1096. DOI URL PMID
[4]	庄国宏, 宋涛, 陆金荣 , 等. 产抗大肠杆菌脂肽化合物枯草芽孢杆菌筛选及脂肽化合物的分离纯化[J]. 中国家禽, 2019,41(13):20-25.
	ZHUANG G H, SONG T, LU J R , et al. Screening of Bacillus subtilis with lipopeptide compounds against E. coli and purification and separation of compounds[J]. China Poultry, 2019,41(13):20-25.(in Chinese with English abstract)
[5]	陈倩倩, 刘波, 王阶平 , 等. 芽胞杆菌FJAT-28592抗真菌脂肽的研究[J]. 农业生物技术学报, 2016,24(2):261-269.
	CHEN Q Q, LIU B, WANG J P , et al. Anti-fungal Lipopetides Produced by Bacillus siamensis FJAT-28592[J]. Journal of Agricultural Biotechnology, 2016,24(2):261-269.(in Chinese with English abstract)
[6]	SINGH P, CAMEOTRA S S . Potential applications of microbial surfactants in biomedical sciences[J]. Trends in Biotechnology, 2004,22(3):142-146. DOI URL
[7]	孙艳新 . 脂肽化合物的分离及其抑制胡麻枯萎病病原菌的研究[D]. 呼和浩特: 内蒙古大学, 2019.
	SUN Y X . Isolation of lipopeptide compounds and their inhibition of pathogens of flax blight[D]. Hohhot: Inner Mongolia University, 2019.(in Chinese with English abstract)
[8]	文炜涛, 王新伟, 蔡婷 , 等. 脂肽生物表面活性剂作用下多菌种石油生物降解影响因素优化[J]. 环境工程学报, 2018,12(12):3520-3530.
	WEN W T, WANG X W, CAI T , et al. Factors optimization for multi-strain biodegradation of petroleum under the effect of surfactin[J]. Chinese Journal of Environmental Engineering, 2018,12(12):3520-3530.(in Chinese with English abstract)
[9]	沈素 . 环脂肽类抗生素临床应用及评价[J]. 中国医院用药评价与分析, 2011,11(8):677-679.
	SHEN S . Evaluation on clinical application of cyclic lipopeptide antibiotics[J]. Evaluation and Analysis of Drug-Use in Hospitals of China, 2011,11(8):677-679.(in Chinese with English abstract)
[10]	高兆建, 王秋芬, 胡鑫强 , 等. 凝结芽孢杆菌XZQ-16抗菌脂肽分离鉴定及抗菌特性[J]. 食品工业科技, 2021(3):36-42.
	GAO Z J, WANG Q F, HU X Q , et al. Isolation, identification and antibacterial properties of antibacterial lipopeptides from Bacillus coagulans XZQ-16[J]. Food Industry Technology, 2021(3):36-42. (in Chinese with English abstract)
[11]	吴艳清, 王游游, 王畅 , 等. 枯草芽孢杆菌WL2脂肽粗提物对致病疫霉的抑制作用及其分离鉴定[J]. 河北大学学报(自然科学版), 2018,38(6):632-639.
	WU Y Q, WANG Y Y, WANG C , et al. Inhibitory effect of lipopeptide crude extract produced by Bacillus subtilis WL2 on Phytophthora infestans and its isolation and identification[J]. Journal of Hebei University (Natural Science Edition), 2018,38(6):632-639.(in Chinese with English abstract)
[12]	赵红霞, 苟萍, 刘小平 , 等. 短梗霉素A对灰葡萄孢菌生长的影响[J]. 植物保护, 2015,41(6):44-48, 59.
	ZHAO H X, GOU P, LIU X P , et al. Effects of aureobasidin A on Botrytis cinerea's growth[J]. Plant Protection, 2015,41(6):44-48, 59.(in Chinese with English abstract)
[13]	刘小平 . 短梗霉素A对水果产后病原真菌的抑制作用[D]. 乌鲁木齐: 新疆大学, 2008.
	LIU X P . Inhibition effect of postharvest pathogens fungi of fruit by aureobasidin A[D]. Urumqi: Xinjiang University, 2008.(in Chinese with English abstract)
[14]	姬婧媛, 杨洁, 高小宁 , 等. 植物内生枯草芽孢杆菌E1R-j脂肽类化合物的分离鉴定及抑菌作用[J]. 农药学学报, 2015,17(2):172-178.
	JI J Y, YANG J, GAO X N , et al. Isolation and identification of lipopeptides produced by endophytic bacteria Bacillus subtilis ElR-j and its anti-fungal mechanism studies[J]. Chinese Journal of Pesticide Science, 2015,17(2):172-178.(in Chinese with English abstract)
[15]	桑建伟, 杨扬, 陈奕鹏 , 等. 内生解淀粉芽孢杆菌BEB17脂肽类和聚酮类化合物的抑菌活性分析[J]. 植物病理学报, 2018,48(3):402-412.
	SANG J W, YANG Y, CHEN Y P , et al. Antibacterial activity analysis of lipopeptide and polyketide compounds produced by endophytic bacteria Bacillus amyloliquefaciens BEB17[J]. Acta Phytopathologica Sinica, 2018,48(3):402-412.(in Chinese with English abstract)
[16]	别小妹, 吕凤霞, 陆兆新 , 等. Bacillus subtilis fmbJ脂肽类抗菌物质的分离和鉴定[J]. 生物工程学报, 2006,22(4):644-649. PMID
	BIE X M, LYU F X, LU Z X , et al. Isolation and identification of lipopeptides produced by Bacillus subtilis fmb[J][J]. Chinese Journal of Biotechnology, 2006,22(4):644-649.(in Chinese with English abstract) URL PMID
[17]	NANJUNDAN J, RAMASAMY R, UTHANDI S , et al. Antimicrobial activity and spectroscopic characterization of surfactin class of lipopeptides from Bacillus amyloliquefaciens SR1[J]. Microbial Pathogenesis, 2019,128:374-380. DOI URL PMID
[18]	高兆建, 秦宸锴, 黄亮浩 . 腊肉拮抗菌分离及抗真菌脂肽特性分析[J/OL].食品科学:1-17[2020-07-12]. https://kns.cnki.netkcmsdetail/detail.aspx?dbcode=CAPJ&dbname=CAPJLAST&filename=SPKX20200529015&v=17WwU59A5kCGGlzKOxZG2jTBWLL6Z5bEr1fK5k26yVFX%25mmd2F%25mmd2BDMVzNkR9uipE8pxciB.
	GAO Z J, QIN C K, HUANG L H . Antagonistic antibacterial separation of bacon and analysis of antifungal lipopeptide properties[J]. Food Science:1-17[2020-07-12]. https://kns.cnki.netkcmsdetail/detail.aspx?dbcode=CAPJ&dbname=CAPJLAST&filename=SPKX20200529015&v=17WwU59A5kCGGlzKOxZG2jTBWLL6Z5bEr1fK5k26yVFX%25mmd2F%25mmd2BDMVzNkR9uipE8pxciB .(in Chinese with English abstract)
[19]	刘畅 . 出芽短梗霉的发酵条件及其糖酵解研究[D]. 无锡: 江南大学, 2012.
	LIU C . Study on fermentation conditions and glycolytic pathway of Aureobasidium pullulans[D]. Wuxi, China: Jiangnan University, 2012.(in Chinese with English abstract)
[20]	ONGENA M, JACQUES P . Bacillus lipopeptides: versatile weapons for plant disease biocontrol[J]. Trends in Microbiology, 2008,16(3):115-125. DOI URL
[21]	NIHORIMBERE V, CAWOY H, SEYER A , et al. Impact of rhizosphere factors on cyclic lipopeptide signature from the plant beneficial strain Bacillus amyloliquefaciens S499[J]. FEMS Microbiology Ecology, 2012,79(1):176-191. DOI URL
[22]	DELEU M, BOUFFIOUX O, RAZAFINDRALAMBO H , et al. Interaction of surfactin with membranes: a computational approach[J]. Langmuir, 2003,19(8):3377-3385. DOI URL
[23]	HEERKLOTZ H, WIEPRECHT T, SEELIG J . Membrane perturbation by the lipopeptide surfactin and detergents as studied by deuterium NMR[J]. The Journal of Physical Chemistry B, 2004,108(15):4909-4915. DOI URL
[24]	YAO S Y, GAO X W, FUCHSBAUER N , et al. Cloning, sequencing, and characterization of the genetic region relevant to biosynjournal of the lipopeptides iturin A and surfactin in Bacillus subtilis[J]. Current Microbiology, 2003,47(4):0272-0277. DOI URL
[25]	杨莹, 庄新亚, 程亮 , 等. 出芽短梗霉菌PA-2发酵培养基及发酵条件优化[J]. 南方农业学报, 2019,50(9):1998-2008.
	YANG Y, ZHUANG X Y, CHENG L , et al. Fermentation medium of Aureobasidium pullulans PA-2 and optimization of fermentation conditions[J]. Journal of Southern Agriculture, 2019,50(9):1998-2008.(in Chinese with English abstract)
[26]	李睿颖, 乔长晟, 国华 , 等. 出芽短梗霉发酵合成聚苹果酸的研究[J]. 食品与发酵科技, 2011,47(5):68-71.
	LI R Y, QIAO C S, GUO H , et al. Polymalic acid synjournal with Aureobasidium pullulans fermentation experiments[J]. Food and Fermentation Technology, 2011,47(5):68-71.(in Chinese with English abstract)
[27]	何太波, 袁恺, 周卫强 , 等. 出芽短梗霉发酵制备聚苹果酸研究[J]. 生物化工, 2020,6(3):133-139.
	HE T B, YUAN K, ZHOU W Q , et al. Research progress on fermentation of polymalic acid from Aureobasidium pullulans[J]. Biological Chemical Engineering, 2020,6(3):133-139.(in Chinese with English abstract)
[28]	沈琦, 张殿鹏, 郝雅荞 , 等. 出芽短梗霉新菌株RM1603产普鲁兰多糖条件优化及多糖分析[J]. 生物技术通讯, 2019,30(3):385-390.
	SHEN Q, ZHANG D P, HAO Y Q , et al. Optimum conditions and identification of pullulan produced by a new finding strain Aureobasidium pullulans RM1603[J]. Letters in Biotechnology, 2019,30(3):385-390.(in Chinese with English abstract)
[29]	杨洁 . 枯草芽孢杆菌E1R-j产抗菌脂肽的发酵条件优化及分离纯化[D]. 杨凌:西北农林科技大学, 2012.
	YANG J . Optimization of fermentation conditions and separation and purification of antibacterial lipopeptide produced by Bacillus subtilis E1R-j[D]. Yangling: Northwest A&F University, 2012. (in Chinese with English abstract)

试验因素 Factor	试验水平Level
试验因素 Factor	-2.378	-1	0	+1	+2.378
接种量Inoculation amount (A)/%	2.24	5	7	9	11.76
转速Rotation speed(B)/(r·min^-1)	118.66	160	190	220	261.34
温度Temperature(C)/℃	20.24	23	25	27	29.80
装液量Liquid volume (D)/mL	31.10	100	150	200	268.90
初始pH Initial pH(E)	4.62	6	7	8	9.38

试验因素 Factor	试验水平Level
试验因素 Factor	-2.378	-1	0	+1	+2.378
接种量Inoculation amount (A)/%	2.24	5	7	9	11.76
转速Rotation speed(B)/(r·min^-1)	118.66	160	190	220	261.34
温度Temperature(C)/℃	20.24	23	25	27	29.80
装液量Liquid volume (D)/mL	31.10	100	150	200	268.90
初始pH Initial pH(E)	4.62	6	7	8	9.38

靶标菌 Target bacteria	平均抑菌圈直径 Antibacterial circle diameter/cm
金黄色葡萄球菌Staphylococcus aureus	3.65
酿酒酵母菌Saccharomyces cerevisiae	1.95
大肠埃希菌Escherichia coli	2.15
白色念珠菌Candida albicans	0
樱桃球腔菌Mycosphaerella cerasella	1.35
大麦网斑病Pyrenophora teres	2.18

靶标菌 Target bacteria	平均抑菌圈直径 Antibacterial circle diameter/cm
金黄色葡萄球菌Staphylococcus aureus	3.65
酿酒酵母菌Saccharomyces cerevisiae	1.95
大肠埃希菌Escherichia coli	2.15
白色念珠菌Candida albicans	0
樱桃球腔菌Mycosphaerella cerasella	1.35
大麦网斑病Pyrenophora teres	2.18

方差来源 Soruces of variation	平方和 Sum of squares	自由度 Degree of freedom	方差 Variance	F值 F value	P值 P value	显著性 Significance
回归模型Regression model	2.06	20	0.10	89.28	<0.000 1	**
A	1.426×10^-3	1	1.426×10^-3	1.23	0.275 6
B	0.51	1	0.51	439.53	<0.000 1	**
C	0.029	1	0.029	24.76	<0.000 1	**
D	0.028	1	0.028	24.26	<0.000 1	**
E	3.887×10^-3	1	3.887×10^-3	3.37	0.076 9
AB	1.125×10^-4	1	1.125×10^-4	0.097	0.757 2
AC	0.025	1	0.025	21.92	<0.000 1	**
AD	1.800×10^-3	1	1.800×10^-3	1.56	0.221 9
AE	3.613×10^-3	1	3.613×10^-3	3.13	0.087 5
BC	0.016	1	0.016	14.03	0.000 8	**
BD	0.019	1	0.019	16.46	0.000 3	**
BE	2.450×10^-3	1	2.450×10^-3	2.12	0.156 0
CD	0.20	1	0.20	169.10	<0.000 1	**
CE	4.500×10^-4	1	4.500×10^-4	0.39	0.537 4
DE	1.125×10^-4	1	1.125×10^-4	0.097	0.757 2
A2	0.32	1	0.32	275.38	<0.000 1	**
B2	0.15	1	0.15	129.08	<0.000 1	**
C2	0.30	1	0.30	256.42	<0.000 1	**
D2	0.54	1	0.54	468.04	<0.000 1	**
E2	0.51	1	0.51	443.22	<0.000 1	**
残差Residual	0.033	29	1.155×10^-3
失拟项Lack of fit	0.026	22	1.161×10^-3	1.02	0.528 6
纯误差Pure error	7.950×10^-3	7	1.136×10^-3
合计Cor total	2.10	49

Antibacterial activities and optimization of fermentation conditions of lipopeptides produced by Aureobasidium pullulans PA-2

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References 29

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序号 No.	A	B	C	D	E	脂肽产量 Lipopeptide yield/(g·L^-1)	预测值 Predicted value/(g·L^-1)
1	0	-2.38	0	0	0	0.32	0.31
2	0	2.38	0	0	0	0.82	0.83
3	1	-1	1	1	-1	0.26	0.25
4	1	-1	1	1	1	0.25	0.26
5	0	0	0	0	0	0.83	0.86
6	0	0	0	2.38	0	0.25	0.24
7	0	0	0	0	0	0.89	0.86
8	0	0	0	0	0	0.82	0.86
9	-1	-1	1	-1	1	0.45	0.48
10	1	-1	-1	1	-1	0.46	0.45
11	-1	1	1	-1	-1	0.77	0.76
12	-1	-1	-1	1	1	0.45	0.44
13	-1	-1	-1	-1	1	0.28	0.27
14	1	1	1	1	1	0.48	0.45
15	1	1	-1	-1	1	0.52	0.53
16	-1	1	-1	1	-1	0.56	0.51
17	1	1	-1	1	-1	0.59	0.58
18	0	0	0	0	2.38	0.36	0.34
19	-1	1	1	1	-1	0.50	0.52
20	1	1	1	-1	-1	0.78	0.75
21	-2.38	0	0	0	0	0.45	0.42
22	-1	-1	1	1	-1	0.34	0.28
23	-1	1	-1	1	1	0.54	0.55
24	-1	-1	1	1	1	0.33	0.34
25	-1	1	-1	-1	1	0.45	0.47
26	0	0	0	-2.38	0	0.36	0.36
27	-1	-1	1	-1	-1	0.39	0.43
28	1	-1	-1	-1	1	0.33	0.33
29	-1	1	1	-1	1	0.81	0.77
30	-1	1	-1	-1	-1	0.43	0.45
31	-1	1	1	1	1	0.49	0.54
32	0	0	-2.38	0	0	0.38	0.39
33	1	-1	1	-1	-1	0.42	0.43
34	1	1	1	1	-1	0.45	0.48
35	1	-1	-1	-1	-1	0.34	0.31
36	0	0	2.38	0	0	0.52	0.51
37	2.38	0	0	0	0	0.42	0.45
38	0	0	0	0	-2.38	0.28	0.30
39	1	-1	1	-1	1	0.44	0.43
40	0	0	0	0	0	0.84	0.86
41	1	-1	-1	1	1	0.46	0.47
42	-1	-1	-1	-1	-1	0.23	0.21
43	0	0	0	0	0	0.85	0.86
44	-1	-1	-1	1	-1	0.30	0.37
45	1	1	1	-1	1	0.71	0.72
46	1	1	-1	1	1	0.60	0.58
47	0	0	0	0	0	0.86	0.86
48	0	0	0	0	0	0.90	0.86
49	0	0	0	0	0	0.91	0.86
50	1	1	-1	-1	-1	0.55	0.55

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[2]	JIANG Han, LIU Jiao, LI Ping, GU Qing. Heterologous expression and purification of plantaricin JK and analysis of its antibacterial activity [J]. , 2017, 29(2): 332-337.
[3]	HUANG Huayi, WANG Jialin, MA Rong, LIANG Yingmei, TIAN Chengming. Antifungal activities of Bacillus subtilis STO-12 and analysis on its antifungal substances [J]. , 2017, 29(1): 81-88.
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[6]	LIU Hui-hui;LI Shu-ping;ZHAO Qian;ZHAO Shu-jiang;*. Identification of marine fungal strain NJ0104 with the anti-microbial activities of their metabolites to bacterial pathogen of Pseudosciaena [J]. , 2012, 24(5): 0-807.