一株后生元菌株的抑菌特性研究及其细菌素基因簇的挖掘

张晓妍; 沙沈菲; 郭丽丹; 贾爽; 周婉婷; 陈雨滢; 汪立平

doi:10.13386/j.issn1002-0306.2022030280

一株后生元菌株的抑菌特性研究及其细菌素基因簇的挖掘

doi: 10.13386/j.issn1002-0306.2022030280

张晓妍^1,,
沙沈菲¹,
郭丽丹¹,
贾爽¹,
周婉婷¹,
陈雨滢¹,
汪立平^{1, 2, 3,*, ,}

1.
上海海洋大学食品学院，上海 201306
2.
农业部水产品贮藏保鲜质量安全风险评估实验室，上海 201306
3.
上海海洋大学食品热加工工程技术研究中心，上海 201306

基金项目: 2019年教育部高等教育司第二批产学合作协同育人项目资助（201902187007）；大学生创新创业训练计划项目（X202110264048）。

详细信息

作者简介:
张晓妍（1996−），女，硕士研究生，研究方向：益生菌及其代谢产物，E-mail：seranne_run@163.com

通讯作者:
汪立平（1968−），女，博士，副教授，研究方向：益生元、益生菌及其代谢产物、传统发酵食品，E-mail：lpwang@shou.edu.cn

中图分类号: TS201.3
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出版历程
- 收稿日期: 2022-03-23
- 网络出版日期: 2023-02-22

Antimicrobial Properties of A Postbiotic Strain and Gene Cluster Mining of Its Bacteriocin

1.
School of Food Science and Technology, Shanghai Ocean University, Shanghai 201306, China
2.
Laboratory of Quality and Safety Risk Assessment for Aquatic Products on Storage and Preservation, Ministry of Agriculture, Shanghai 201306, China
3.
Engineering Center of Food Thermal-processing Technology, Shanghai Ocean University, Shanghai 201306, China

摘要

摘要: 本文旨在筛选一株可拮抗中国大鲵源嗜水气单胞菌的后生元菌株，并对其进行抑菌特性的研究和细菌素基因簇的挖掘。以中国大鲵病原性嗜水气单胞菌（Aeromonas hydrophila, Ah2）为指示菌，筛选一株产细菌素乳酸菌株并评估其药敏特性；采用有机溶剂萃取法初步纯化菌株产细菌素；通过pH、温度和消化酶耐受性、贮藏稳定性、抑菌谱及最小抑菌（MIC）和杀菌浓度（MBC）共六类指标评价细菌素的抑菌特性；溶血反应与细胞毒性实验测试细菌素对Ah2的抑菌效果，经扫描电子显微镜初步探究细菌素的抑菌机制；经由紫外全波长扫描定性细菌素以及Tricine-SDS-PAGE电泳测定细菌素的分子量范围；最后根据菌株的全基因组序列挖掘其潜在的细菌素基因簇（RiPPs）。结果显示，从青岛市售腐乳中筛出一株产细菌素植物乳植物杆菌M4L1（Lactiplantibacillus plantarum M4L1）。初步纯化M4L1产细菌素并命名为LP01。细菌素LP01具备良好的消化酶耐受性，且在pH2~10、−20~121 ℃和9个月贮藏期内均表现出稳定的抑菌活性，并对单增李斯特菌、弗氏柠檬酸杆菌等14株革兰氏阳性和阴性菌均有不同程度的抑制作用；另外，LP01对Ah2的MIC和MBC分别为12.94和25.88 μg/mL。经MBC浓度的LP01处理后的Ah2，溶血活性和细胞毒性均得到明显缓解；SEM观察其通过破坏Ah2的细胞壁具有抑制或杀伤作用。LP01在波长200~220 nm处的肽类特征吸收峰显著，电泳后条带显示其分子量在3.3~4.0 kDa，LP01为小分子肽类细菌素。此外，基因注释到M4L1有2个细菌素基因簇（RiPPs），对应产物分别为Plantaricin K和Plantaricin E，均属于植物乳杆菌II类细菌素。菌株M4L1不仅含有多种抗菌物质相关基因簇，而且其细菌素LP01具备了优良的抑菌性能，能有效抑制多种水产病原菌、食物腐败菌及食源性致病菌等。产细菌素植物乳植物杆菌M4L1作为一株潜在的后生元菌株具有较好的应用前景。
- 嗜水气单胞菌 /
- 植物乳植物杆菌 /
- 后生元 /
- 细菌素 /
- 基因挖掘
Abstract: The aim of this study was to screen a postbiotic strain against Aeromonas hydrophila, and analyze its antibacterial characteristics and bacteriocin related gene cluster (RiPPs). The pathogen Aeromonas hydrophila (Ah2) from Andrias davidianus was used as an indicator for the screening of an excellent lactic acid bacterium (LAB), then the antibiotic susceptibility of the LAB to 10 common antibiotics was evaluated. The crude extract of bacteriocin (CEB) produced by the LAB was preliminarily purified by organic solvent extraction. Antibacterial properties of the CEB were then evaluated by 6 metrics below: pH, temperature and digestive enzyme tolerance, storage stability, antimicrobial spectrum, minimum inhibitory concentration (MIC) and maximum inhibitory concentration (MBC). CEB of two concentrations above was used for hemolysis and cytotoxicity experiment to further verify the bacteriocin’s effect on Ah2, and for scanning electron microscope (SEM) observation to preliminary explore the antimicrobial mechanism of the bacteriocin. Then through UV full-wavelength scanning and Tricine-SDS-PAGE, the qualitation and molecular weight of the bacteriocin was seprately identified and estimated. Finally, the whole genome sequence of the LAB was determined and then its potential bacteriocin gene cluster (RiPPs) mining was predicted in the core peptide database BAGEL4. Results showed that the excellent bacteriocin-producing strain M4L1 was isolated and screened from the Sufu sold in Qingdao, and was identified as Lactiplantibacillus plantarum (L. plantarum). The bacteriocin of L. plantarum M4L1 was half-purified and named as LP01. Bacteriocin LP01 exhibited good digestive enzyme tolerance and relatively stable antibacterial activity at pH2~12, −20~121 ℃ and 9 months storage, it also presented a wide antimicrobial spectrum on 14 Gram positive and negative strains including 7 aquatic pathogens, such as Listeria monocytogenes, Citrobacter freundii and etc. Moreover, the MIC and MBC of LP01 against Ah2 was 12.94 and 25.88 μg/mL, respectively. After treatment with LP01, the hemolytic activity and cytotoxicity caused by Ah2 were significantly relieved as confirmed, which presented bacteriocin LP01’s excellent antibacterial effect. Especially, the SEM results showed that LP01 could inhibit or kill Ah2 by damaging cell wall. LP01 showed significantly characteristic peak at 200~220 nm corresponding to peptides, and electrophoresis band indicated the molecular weight range of LP01 was 3.3~4.0 kDa. LP01 was identified as small-peptide bacteriocin. There are 2 bacteriocin gene clusters (RiPPs) corresponding to the class II bacteriocins, L. plantarum Plantaricin K and Plantaricin E, were found in the genome of strain M4L1. In all, the bacteriocin-producing L. plantarum M4L1 not only owned bacteriocin gene clusters, but was also equipped with excellent antimicrobial characteristics and could inhibit a variety of aquatic pathogens, which has the potential to be developed as a postbiotic candidate strain in aquaculture.
- Aeromonas hydrophila /
- Lactiplantibacillus plantarum /
- postbiotics /
- bacteriocin /
- gene mining

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图 1 四株乳酸菌对Ah2的抑菌效果对比

注：QD5、SH7、M4D2和M4L1为对Ah2有较好抑菌效果的4株乳酸菌。

Figure 1. Comparison of inhibition effect on four strains of lactic acid bacteria against Ah2

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图 2 菌株M4L1的菌落形态和显微形态图

Figure 2. Colony and microscopic morphology of M4L1

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图 3 植物乳植物杆菌M4L1的生长动态及抑菌活性规律

Figure 3. Growth dynamics and antimicrobial regularity of L. plantarum M4L1

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图 4 细菌素LP01的耐受性

注：A：pH耐受性；B：温度耐受性；C：贮藏稳定性；D：酶敏感性；不同小写字母说明具有显著差异（P<0.05）。

Figure 4. Tolerance of bacteriocin LP01

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图 5 细菌素LP01对Ah2引起的溶血活性和细胞毒性的影响

注：A：溶血反应检测；B：细胞毒性试验；MIC和MBC：LP01处理组；NC：阴性无Ah2对照组；PC：阳性有Ah2对照组。

Figure 5. Effect of bacteriocin LP01 on hemolysis and cytotoxicity caused by Ah2

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图 6 指示菌Ah2的SEM图

注：A：未经处理的Ah2；B：经MIC处理1 h；C：经MBC处理1 h；1：单个细胞；2：群体细胞。

Figure 6. SEM images of indicator Ah2

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图 7 细菌素LP01的紫外全波长扫描图

注：肽类波长：200~220 nm；蛋白类波长：280 nm。

Figure 7. UV full-wavelength scanning of bacteriocin LP01

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图 8 细菌素LP01的Tricine-SDS-PAGE电泳图

注：Marker：超低分子量蛋白质标准；1：经过染色的样品谱带；2：凝胶原位检测样品条带。

Figure 8. Tricine-SDS-PAGE of bacteriocin LP01

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图 9 植物乳植物杆菌M4L1的细菌素基因簇图谱

Figure 9. Genetic organization map of bacteriocin gene clusters of L. plantarum M4L1

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表 1 四株乳酸菌的抑菌活性

Table 1. Antimicrobial activity of four strains of lactic acid bacteria

菌株编号	抑菌圈直径（mm）
QD5	19.21±0.13^c
SH7	16.85±0.14^d
M4D2	23.45±0.32^b
M4L1	25.13±0.27^a
注：不同小写字母说明具有显著差异（P<0.05）。

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表 2 植物乳植物杆菌M4L1的药敏特性

Table 2. Antibiotic susceptbility of L. plantarum M4L1

抗生素	抑菌圈直径判断标准（mm）			药物含量（μg/片）	抑菌圈直径（mm）	敏感性
抗生素	耐药（R）	中度敏感（I）	高度敏感（S）	药物含量（μg/片）	抑菌圈直径（mm）	敏感性
复方新诺明Cotrimoxazole	≤10	11~15	≥16	23.75	18.61±0.25	S
头孢曲松 Ceftriaxone	≤13	14~20	≥21	30	31.42±0.16	S
环丙沙星 Ciprofloxacin	≤15	16~20	≥21	5	0	R
氨苄西林 Ampicillin	≤15	16~22	≥23	10	23.96±0.32	S
庆大霉素 Gentamicin	≤12	13~14	≥16	10	0	R
林可霉素 Lincomycin	≤14	15~20	≥21	2	13.63±0.22	R
氯霉素 Chloroamphenicol	≤12	13~17	≥18	30	21.34±0.11	S
四环素 Tetracycline	≤14	15~18	≥19	30	20.25±0.19	S
红霉素 Erythromycin	≤13	14~22	≥23	15	20.92±0.38	I
青霉素 Penicillin	≤19	20~22	≥23	100	16.98±0.30	R

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表 3 细菌素LP01的抑菌谱

Table 3. Antibacterial spectrum of bacteriocin LP01

类别	菌株	抑菌圈直径（mm）	类别	菌株	抑菌圈直径（mm）
G⁺	蜡样芽孢杆菌 Bacillus cereus	21.50±0.48	G^-	副溶血性弧菌 Vibrio parahaemolyticus^®	30.67±0.29
	腐生葡萄球菌 Staphylococcus saprophysis	16.13±0.42		腐败希瓦氏菌 Shewanella putrefaciens^®	20.37±0.32
	金黄色葡萄球菌 Staphylococcus aureus^®	21.05±0.32		嗜水气单胞菌（鲫源） Aeromonas hydrophila	32.83±0.29
	单增李斯特氏菌 Listeria monocytogenes^®	36.67±0.29		弗氏柠檬酸杆菌 Citrobacter freundii	26.13±0.40
	变形链球菌 Streptococcus mutans	28.90±0.36		医院不动杆菌 Acinetobacter nosocomialis	28.90±0.36
	大肠杆菌 Escherichia coli^®	26.43±0.40		鲍曼不动杆菌 Acinetobacter baumannii	32.53±0.22
真菌	酿酒酵母 Saccharomyceb cerevisiae	0		沙门氏菌 salmonella enterica^®	31.77±0.25
真菌	德巴利汉逊酵母 Debaryomyceb hansenii	9.16±0.35		摩根氏菌 Morganella morganii	26.3±0.35
注：G⁺和G^-表示革兰氏阳性和阴性菌；符号‘^®’表示ATCC菌株，未标记的菌为实验室分离；抑菌圈直径数值包含孔的直径（6.02±0.18）mm。

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参考文献(31)

[1]	ASSEFA A, ABUNNA F. Maintenance of fish health in aquaculture: Review of epidemiological approaches for prevention and control of infectious disease of fish[J]. Veterinary Medicine International,2018:1−10.
[2]	张静, 陈红莲, 鲍俊杰, 等. 水产养殖中嗜水气单胞菌拮抗菌的研究进展[J]. 江苏农业科学,2020,48(17):21−33. [ZHANG J, CHEN H L, BAO J J, et al. Research progress on antagonistic microbes of Aeromonas hydrophila in aquaculture[J]. Jiangsu Agricultural Sciences,2020,48(17):21−33.
[3]	杜明洋, 叶仕根, 刘娟, 等. 水产动物病原拮抗微生物及其应用研究进展[J]. 大连海洋大学学报,2017,32(6):753−758. [DU M Y, YE S G, LIU J, et al. Research advances on antagonistic microbes and application in aquatic animal pathogens: A review[J]. Journal of Dalian Ocean University,2017,32(6):753−758.
[4]	HAI N V. The use of probiotics in aquaculture[J]. Journal of Applied Microbiology,2015,119(4):917−935. doi: 10.1111/jam.12886
[5]	WILKINS T, SEQUOIA J. Probiotics for gastrointestinal conditions: A summary of the evidence[J]. American Family Physician,2017,96(3):170−178.
[6]	KLEMASHEVICH C, WU C, HOWSMON D, et al. Rational identification of diet-derived postbiotics for improving intestinal microbiota function[J]. Current Opinion in Biotechnology,2014,26:85−90. doi: 10.1016/j.copbio.2013.10.006
[7]	CARRIE A M W, SHARON Y G, JAN K, et al. Postbiotics and their potential applications in early life nutrition and beyond[J]. International Journal of Molecular Sciences,2019,20(19):4673. doi: 10.3390/ijms20194673
[8]	CHRISTOPHE L L, BENOIT F, RIADH H, et al. On Lactococcus lactis UL719 competitivity and nisin (Nisaplin^®) capacity to inhibit Clostridium difficile in a model of human colon[J]. Frontiers in Microbiology,2015,6:1020.
[9]	FUJIKI T, HIROSE Y, YAMAMOTO Y, et al. Enhanced immunomodulatory activity and stability in simulated digestive juices of Lactobacillus plantarum L-137 by heat treatment[J]. Bioscience, Biotechnology, and Biochemistry,2012,76(5):918−922. doi: 10.1271/bbb.110919
[10]	ARIMORI Y, NAKAMURA R, HIROSE Y, et al. Daily intake of heat-killed Lactobacillus plantarum L-137 enhances type I interferon production in healthy humans and pigs[J]. Immunopharmacology and Immunotoxicology,2012,34(6):937−943. doi: 10.3109/08923973.2012.672425
[11]	HIROSE Y, YAMAMOTO Y, YOSHIKAI Y, et al. Oral intake of heat-killed Lactobacillus plantarum L-137 decreases the incidence of upper respiratory tract infection in healthy subjects with high levels of psychological stress[J]. Journal of Nutritional Science,2013,2:e39. doi: 10.1017/jns.2013.35
[12]	全国生化检测标准化技术委员会(SAC/TC 387). GB/T 39101-2020 多肽抗菌性测定抑菌圈法[S]. 国家市场监督管理总局, 国家标准化管理委员会, 2020 National Biochemical Testing Standardization Technical Committee (SAC/TC 387). GB/T 39101-2020 Determination of antimicrobial activity for polypeptides-Inhibition zone method[S]. The State Administration of Market Supervision & Administration, National Standardization Management Committee, 2020.
[13]	谭才邓, 朱美娟, 杜淑霞, 等. 抑菌试验中抑菌圈法的比较研究[J]. 食品工业,2016,37(11):122−125. [TAN C D, ZHU M J, DU S X, et al. Study on the inhibition zone method in antimicrobial test[J]. Food Industry,2016,37(11):122−125.
[14]	WS/T 6392018 抗菌药物敏感性试验的技术要求[S]. 中华人民共和国国家卫生健康委员会, 2019 WS/T 639-2018 Technical requirements for antimicrobial susceptibility test[S]. National Health Commission of the People 's Republic of China, 2019.
[15]	李治学, 汪以真, 韩菲菲, 等. 微量测定阳离子抗菌肽最小抑菌和最小杀菌浓度的方法[P]. 浙江省: CN103173517B, 2014-07-30 LI Z X, WANG Y Z, HAN F F, et al. Method for measuring minimum inhibitory and minimum bactericidal concentration of cationic antimicrobial peptides in trace amounts[P]. Zhejiang Province: CN103173517B, 2014-07-30.
[16]	CAO L, PAN L, GONG L, et al. Interaction of a novel Bacillus velezensis (BvL03) against Aeromonas hydrophila in vitro and in vivo in grass carp[J]. Applied Microbiology and Biotechnology,2019,103(21-22):8987−8999. doi: 10.1007/s00253-019-10096-7
[17]	辛维岗, 江宇航, 陈诗雨, 等. 滇池金线鲃肠道产细菌素细菌的筛选鉴定及细菌素LSP01的抑菌作用[J]. 微生物学通报,2022,49(1):242−255. [XIN W G, JIANG Y H, CHEN S Y, et al. Screening and identification of bacteriocin-producing bacteria in the intestines of Sinocyclocheilus grahami in Dianchi and the antibacterial effect of bacteriocin[J]. Microbiology China,2022,49(1):242−255. doi: 10.13344/j.microbiol.china.210290
[18]	SCHÄGGER H, VON JAGOW G. Tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis for the separation of proteins in the range from 1 to 100 kDa[J]. Analytical Biochemistry,1987,166(2):368−379. doi: 10.1016/0003-2697(87)90587-2
[19]	邓小娟, 曹阳, 钟仰进, 等. SDS-PAGE凝胶原位检测抗菌蛋白的活性——生物自显影技术[J]. 西南农业大学学报,2005,27(1):136−137. [DENG X J, CAO Y, ZHONG Y J, et al. Detection of antimicrobial protein activity in situ on SDS-PAGE gel—bioautography technique[J]. South China Agricultural Univ,2005,27(1):136−137. doi: 10.3969/j.issn.1673-9868.2005.01.033
[20]	BIBB M J. Regulation of secondary metabolism in streptomycetes[J]. Current Opinion in Microbiology,2005,8(2):208−215. doi: 10.1016/j.mib.2005.02.016
[21]	PARENTE E, CIOCIA F, RICCIARDI A, et al. Diversity of stress tolerance in Lactobacillus plantarum, Lactobacillus pentosus and Lactobacillus paraplantarum: A multivariate screening study[J]. International Journal of Food Microbiology,2010,144(2):270−279. doi: 10.1016/j.ijfoodmicro.2010.10.005
[22]	李平兰, 潘伟好, 吕艳妮, 等. 微生态制剂中常用乳酸菌对抗生素的药敏性研究[J]. 中国农业大学学报,2004(1):16−20. [LI P L, PAN W H, LÜ Y N, et al. Antibiotic susceptibility of lactic acid bacteria commonly used in microecologics[J]. Journal of China Agricultural University,2004(1):16−20. doi: 10.3321/j.issn:1007-4333.2004.01.004
[23]	李禤, 贾丹, 刘军龙, 等. 新分离植物乳植物杆菌的药敏性和抑菌性试验[J]. 中国兽医科学,2019,49(7):879−886. [LI X, JIA D, LIU J L, et al. Antibiotic susceptibility and antimicrobial test of newly isolated Lactobacillus plantarum in vitro[J]. China Veterinary Science,2019,49(7):879−886.
[24]	RAYMAN M K, ARIS B, HURST A. Nisin: A possible alternative or adjunct to nitrite in the preservation of meats[J]. Applied and Environmental Microbiology,1981,41(2):375−380. doi: 10.1128/aem.41.2.375-380.1981
[25]	陈佰义, 何礼贤, 胡必杰, 等. 中国鲍曼不动杆菌感染诊治与防控专家共识[J]. 中国医药科学,2012,2(8):3−8. [CHEN B Y, HE L X, HU B J, et al. The interpretation of Chinese expert consensus for the diagnosis, treatment, prevention and control of Acinetobacter baumannii infection[J]. Chinese Journal of Medical Sciences,2012,2(8):3−8.
[26]	MESSI P, BONDI M, SABIA C, et al. Detection and preliminary characterization of a bacteriocin (plantaricin 35d) produced by a strain[J]. International Journal of Food Microbiology,2001,64(1−2):193−198. doi: 10.1016/S0168-1605(00)00419-0
[27]	WANG Y, QIN Y, ZHANG Y, et al. Antibacterial mechanism of plantaricin LPL-1, a novel class IIa bacteriocin against Listeria monocytogenes[J]. Food Control,2019,97:87−93. doi: 10.1016/j.foodcont.2018.10.025
[28]	SOUSA E L D E, ASSANE I M, SANTOS-FILHO N A, et al. Haematological, biochemical and immunological biomarkers, antibacterial activity, and survival in Nile tilapia Oreochromis niloticus after treatment using antimicrobial peptide LL-37 against Streptococcus agalactiae[J]. Aquaculture,2021,533:736181. doi: 10.1016/j.aquaculture.2020.736181
[29]	YI L, LI X, LUO L, et al. A novel bacteriocin BMP11 and its antibacterial mechanism on cell envelope of Listeria monocytogenes and Cronobacter sakazakii[J]. Food Control,2018,91:160−169. doi: 10.1016/j.foodcont.2018.03.038
[30]	DIEP D B, MYHRE R, JOHNSBORG O, et al. Inducible bacteriocin production in Lactobacillus is regulated by differential expression of the pln operons and by two antagonizing response regulators, the activity of which is enhanced upon phosphorylation: Inducible bacteriocidin production in Lactobacillus[J]. Molecular Microbiology,2003,47(2):483−494. doi: 10.1046/j.1365-2958.2003.03310.x
[31]	TIETZ J I, SCHWALEN C J, PATEL P S, et al. A new genome-mining tool redefines the lasso peptide biosynthetic landscape[J]. Nature Chemical Biology,2017,13(5):470−478.