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草鱼源乳酸菌的分离鉴定及其生物学特性研究

王楠 尹纪元 王英英 李莹莹 吴斯宇 石存斌 李家豪 曹际振 王庆

王楠, 尹纪元, 王英英, 李莹莹, 吴斯宇, 石存斌, 李家豪, 曹际振, 王庆. 草鱼源乳酸菌的分离鉴定及其生物学特性研究[J]. 南方水产科学. doi: 10.12131/20210039
引用本文: 王楠, 尹纪元, 王英英, 李莹莹, 吴斯宇, 石存斌, 李家豪, 曹际振, 王庆. 草鱼源乳酸菌的分离鉴定及其生物学特性研究[J]. 南方水产科学. doi: 10.12131/20210039
Nan WANG, Jiyuan YIN, Yingying WANG, Yingying LI, Siyu WU, Cunbin SHI, Jiahao LI, Jizhen CAO, Qing WANG. Isolation, identification and biological characteristics of lactobacillus from grass carp[J]. South China Fisheries Science. doi: 10.12131/20210039
Citation: Nan WANG, Jiyuan YIN, Yingying WANG, Yingying LI, Siyu WU, Cunbin SHI, Jiahao LI, Jizhen CAO, Qing WANG. Isolation, identification and biological characteristics of lactobacillus from grass carp[J]. South China Fisheries Science. doi: 10.12131/20210039

草鱼源乳酸菌的分离鉴定及其生物学特性研究

doi: 10.12131/20210039
基金项目: 国家重点研发计划项目 (2019YFD0900102);广东省促进经济高质量发展专项资金海洋经济发展项目 (GDOE〔2019〕A29);中国水产科学研究院珠江水产研究所基本科研业务费 (2019XN-001);财政部和农业农村部国家现代农业产业技术体系资助 (CARS-45)
详细信息
    作者简介:

    王楠:王 楠 (1997—),女,硕士研究生,研究方向为水产动物病害与防控。E-mail: 1987897100@qq.com

    通讯作者:

    尹纪元 (1985—),男,博士,助理研究员,从事水产病害与黏膜免疫研究。E-mail: neauyjy@126.com

    王 庆 (1976—),女,博士,研究员,从事水产动物病毒学研究。E-mail: sunny_929@163.com

  • 中图分类号: S 917.1

Isolation, identification and biological characteristics of lactobacillus from grass carp

  • 摘要: 随着水产养殖病害问题日益严重,抗生素和药物使用对环境造成的危害越来越大,开展绿色生态防控技术研究是实现水产健康养殖的重要途径。该研究从健康草鱼 (Ctenopharyngodon idella) 肠道分离获得的8株乳酸菌 (Lactobacillus),经过生化反应和16S RNA鉴定,均为植物乳杆菌 (L. plantarum)。生物学特性评价结果表明植物乳杆菌分离株Y190430的发酵性能最好,且具有良好的抗生素敏感性;与植物乳杆菌标准株ATCC8014和商品乳酸菌相比较,分离株Y190430对酸、碱、盐、温度等环境胁迫具有更好的耐受性,同时具有更快的产酸速率,并通过代谢产物抑制病原菌生长,体外抑菌试验表明分离株Y190430对常见水产病原菌拮抗能力更强。
  • 图  1  分离菌株的菌落形态特征 (a) 及革兰氏染色 (b)

    Figure  1.  Colony morphological characteristics (a) and gram staining (b) of isolated strains

    图  2  分离菌株的16S rRNA序列的PCR扩增结果

    Figure  2.  PCR amplification results of 16S rRNA sequence of isolated strains

    M. DL2 000 Marker; 1. C13; 2. C14; 3. C15; 4. C16; 5. C17; 6. C18; 7. C20; 8. C35

    图  3  基于分离菌株16S rRNA序列的系统发育树

    Figure  3.  Phylogenetic tree based on 16S rRNA sequences of isolated strains

    图  4  分离菌株的发酵生长曲线

    Figure  4.  Fermentation growth curve of isolated strains

    图  5  分离菌株Y190430的环境耐受性分析结果:温度耐受性 (a); 酸碱耐受性 (b); 渗透压耐受性 (c)

    Figure  5.  Environmental tolerance analysis results of Y190430 strain: Temperature tolerance (a); Acid base tolerance (b); Osmotic pressure tolerance (c)

    图  6  分离株Y190430的产酸性能

    a. 与标准株相比较具有显著性差异;b. 与商品株相比较具有显著性差异;ab. 与标准株、商品株相比较均具有显著性差异

    Figure  6.  Ability to produce acid of Y190430 strain

    a. Compared with standard strain, there is a significant difference; b. Compared with commercial lactobacillus, there is a significant difference; ab. Compared with standard strain and commercial lactobacillus, there is a significant difference.

    表  2  分离菌株的生理生化特征

    Table  2.   Physiological and biochemical characteristics of isolated strains

    生化项目
    Biochemical item
    分离菌株
    Isolated strain
    C13C14C15C16C17C18C20C35
    苦杏仁苷Amy gdalin + + + + + + + +
    阿拉伯糖 Pectinose + + + + + + + +
    纤维二糖Cellobiose + + + + + + + +
    七叶苷Aesculin + + + + + + + +
    果糖Fructose + + + + + + + +
    半乳糖Galactose + + + + + + + +
    葡萄糖Glucose + + + + + + + +
    葡萄糖酸盐Gluconic acid
    乳糖Lactose + + + + + + + +
    麦芽糖Maltose + + + + + + + +
    甘露醇Mannitol + + + + + + + +
    甘露糖Mannose + + + + + + + +
    松三糖Melezitose + + + + + + + +
    蜜二糖Melibiose + + + + + + + +
    棉籽糖Raffinose
    鼠李糖Rhamnose
    核糖Ribose + + + + + + + +
    水杨素Salicin + + + + + + + +
    山梨糖Sorbose
    蔗糖Sucrose + + + + + + + +
    蕈糖Trehalose + + + + + + + +
    木糖Xylose
    注:“+”表示阳性; “−”表示阴性 Note: “+” represents positive reaction; “−” represents negative reaction
    下载: 导出CSV

    表  3  分离菌株Y190430对酸碱的耐受性

    Table  3.   Acid base tolerance of Y190430 strain $ \overline { X}\pm { \rm {SD}}$

    培养pH
    Incubation pH
    log CFU·mL−1
    植物乳杆菌Y190430
    L. plantarum Y190430
    植物乳杆菌ATCC8014
    L. plantarum ATCC8014
    商品乳酸菌
    Commercial lactobacillus
    2.02.50±0.08AB1.10±0.031.70±0.05
    3.03.50±0.05AB2.90±0.023.20±0.05
    4.07.30±0.04AB5.90±0.047.00±0.07
    5.08.30±0.02AB8.10±0.078.10±0.07
    5.68.90±0.248.80±0.028.80±0.07
    6.08.80±0.04AB8.50±0.048.60±0.07
    7.08.50±0.02A8.30±0.028.40±0.08
    8.08.20±0.04B8.10±0.078.00±0.07
    9.08.00±0.04AB7.50±0.027.70±0.02
    注:“A”表示植物乳杆菌Y190430与植物乳杆菌ATCC8014相比较具有显著性差异;“B”表示Y190340与商品株相比较具有显著性差异; Note: “A” represents a significant difference between L. plantarum Y190430 and L. plantarum ATCC8014; “B” represents a significant difference between L. plantarum Y190430 and commercial lactobacillus.
    下载: 导出CSV

    表  4  分离菌株Y190430对渗透压的耐受性

    Table  4.   Osmotic pressure tolerance of Y190430 strain $ \overline { X}\pm { \rm {SD}}$

    NaCl质量分数
    NaCl mass fraction/%
    Log CFU·mL−1/()
    植物乳杆菌Y190430
    L. plantarum Y190430
    植物乳杆菌ATCC8014
    L. plantarum ATCC8014
    商品乳酸菌
    Commercial lactobacillus
    0.98.90±0.08B8.80±0.088.50±0.16
    2.08.70±0.16B8.50±0.167.90±0.16
    4.07.10±0.166.90±0.086.90±0.16
    6.06.50±0.086.70±0.166.40±0.16
    8.05.10±0.245.00±0.164.70±0.20
    注:“B”表示植物乳杆菌Y190430与商品株相比具有显著性差异 (P<0.05) Note: Note: “B” represents a significant difference between L. plantarum Y190430 and commercial lactobacillus (P<0.05).
    下载: 导出CSV

    表  5  分离株Y190430的药敏测试结果

    Table  5.   The results of antibiotic sensitivity of Y190430 strain

    抗生素
    Antibiotics
    标准抑菌直径
    Standard inhibition zone diameter/mm
    植物乳杆菌Y190430
    L. plantarum Y190430
    耐药 (R)
    Resistance
    中等敏感 (I)
    Intermediate
    敏感 (S)
    Susceptible
    抑菌圈直径
    Inhibition zone diameter/mm
    敏感性
    Susceptibility
    青霉素 Penicillin ≤19 20~27 ≥28 25.85±0.11 I
    氨苄西林 Ampicillin ≤28 ≥29 34.03±0.09 S
    多西环 Doxycycline ≤12 13~15 ≥16 21.26±0.07 S
    四环素 Tetracycline ≤14 15~18 ≥19 18.37±0.11 I
    氯霉素 Chloramphenicol ≤12 13~17 ≥18 27.62±0.10 S
    氟苯尼考 Florfenicol ≤12 13~17 ≥18 30.95±0.08 S
    红霉素 Erythromycin ≤13 14~22 ≥23 25.71±0.05 S
    林可霉素 Lincomycin ≤14 15~20 ≥21 28.25±0.07 S
    下载: 导出CSV

    表  1  分离菌株的菌落形态特征

    Table  1.   Colony morphological characteristics of isolated strains

    编号
    No.
    是否光滑
    Smooth or
    rough
    边缘形状
    Edge
    shape
    菌落颜色
    Strain
    colour
    透明度
    Transparency
    菌落形状
    Strain
    shape
    C13整齐白色圆形
    C14整齐白色圆形
    C15整齐白色圆形
    C16整齐白色圆形
    C17整齐白色圆形
    C18整齐白色圆形
    C20整齐白色圆形
    C35整齐白色圆形
    下载: 导出CSV

    表  6  分离菌株Y190340对水产养殖常见致病菌的抑制作用

    Table  6.   Inhibitory effect of Y190340 strain on common pathogenic bacteria in aquaculture

    致病菌
    Pathogen
    平均抑菌圈直径 Average inhibition zone diameter/mm
    植物乳杆菌Y190430
    L. plantarum Y190430
    植物乳杆菌ATCC8014
    L. plantarum ATCC8014
    商品乳酸菌
    Commercial lactobacillus
    舒伯特气单胞菌 A. schubertii 8.11±0.02 8.53±0.02 8.32±0.06
    嗜水气单胞菌 A. hydrophila 8.89±0.07 7.69±0.08 9.76±0.05
    迟缓爱德华氏菌 E. tarda 9.03±007 8.51±0.07 7.96±0.04
    铜绿假单胞菌 P. aeruginosa 12.77±0.05 10.32±0.06 11.43±0.04
    维氏气单胞菌 A. veronii 9.89±0.02 7.53±0.07 9.76±0.07
    植物乳杆菌ATCC8014 L. plantarum ATCC8014
    乳酸乳球菌NZ9000 L. lactis NZ9000
    注:“—”为无抑菌作用 Note: Note: “—” represents no bacteriostatic effect.
    下载: 导出CSV
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出版历程
  • 收稿日期:  2021-01-19
  • 修回日期:  2021-04-28
  • 网络出版日期:  2021-06-05

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