Isolation, identification and biological characteristics of lactobacillus from grass carp
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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8株乳酸菌 (Lactobacillus)，经生化反应和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，并可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对常见水产病原菌的拮抗能力更强。Abstract: As the problem of aquaculture diseases is growing, the harm of using antibiotics and drugs is more and more serious and the research on ecological control technology is an important way to achieve healthy aquaculture. In this study, eight lactobacillus strains were isolated from the intestinal tract of the healthy grass carps (Ctenopharyngodon idella). The identification result indicates that all the eight strains were Lactobacillus plantarum. The results of biological characteristics evaluation show that L. plantarum strain Y190430 had a high growth rate, and it was sensitivity to common antibiotics. In addition, compared with the standard strain and commercial lactobacillus, L. plantarum Y190430 represented better tolerance to environmental stress such as acid, alkali, salt and temperature. Besides, it had a higher acid production rate and inhibitted the growth of pathogenic bacterias better. The results provide references for the research on ecological control of aquatic diseases.
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Key words:
- Lactobacillus /
- Fermenting /
- Antibiotic sensitivity /
- Ecological prevention and control /
- Probiotics
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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 5 分离菌株Y190430的环境耐受性分析结果
a. 温度耐受性; b. 酸碱耐受性; c. 渗透压耐受性。
Figure 5. Environmental tolerance analysis results of Y190430 strain
a. Temperature tolerance; b. Acid base tolerance; c. Osmotic pressure tolerance.
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 strainC13 C14 C15 C16 C17 C18 C20 C35 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：“+”阳性; “−”阴性。 Note: "+" Positive reaction; "−" Negative reaction. 
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表 3 分离菌株Y190430对酸碱的耐受性
Table 3. Acid base tolerance of Y190430 strain
$ \overline { \boldsymbol{X}}\pm { \bf {SD}}$ 培养pH
Incubation pH菌落总数 CFU logCFU·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 lactobacillus2.0 2.50±0.08AB 1.10±0.03 1.70±0.05 3.0 3.50±0.05AB 2.90±0.02 3.20±0.05 4.0 7.30±0.04AB 5.90±0.04 7.00±0.07 5.0 8.30±0.02AB 8.10±0.07 8.10±0.07 5.6 8.90±0.24 8.80±0.02 8.80±0.07 6.0 8.80±0.04AB 8.50±0.04 8.60±0.07 7.0 8.50±0.02A 8.30±0.02 8.40±0.08 8.0 8.20±0.04B 8.10±0.07 8.00±0.07 9.0 8.00±0.04AB 7.50±0.02 7.70±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. Significant difference between L. plantarum Y190430 and L. plantarum ATCC8014; B. Significant difference between L. plantarum Y190430 and commercial lactobacillus.
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表 4 分离菌株Y190430对渗透压的耐受性
Table 4. Osmotic pressure tolerance of Y190430 strain
$ \overline { \boldsymbol{X}}\pm { \bf {SD}}$ 氯化钠
质量分数
NaCl mass fraction/%菌落总数 CFU
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 lactobacillus0.9 8.90±0.08B 8.80±0.08 8.50±0.16 2.0 8.70±0.16B 8.50±0.16 7.90±0.16 4.0 7.10±0.16 6.90±0.08 6.90±0.16 6.0 6.50±0.08 6.70±0.16 6.40±0.16 8.0 5.10±0.24 5.00±0.16 4.70±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: B. Significant difference between L. plantarum Y190430 and commercial lactobacillus (P<0.05).
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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
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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
shapeC13 是 整齐 白色 无 圆形 C14 是 整齐 白色 无 圆形 C15 是 整齐 白色 无 圆形 C16 是 整齐 白色 无 圆形 C17 是 整齐 白色 无 圆形 C18 是 整齐 白色 无 圆形 C20 是 整齐 白色 无 圆形 C35 是 整齐 白色 无 圆形
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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 A 7.69±0.08 9.76±0.05 迟缓爱德华氏菌 E. tarda 9.03±007 AB 8.51±0.07 7.96±0.04 铜绿假单胞菌 P. aeruginosa 12.77±0.05 B 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；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。 Note: “—” represent no bacteriostatic effect; A. Compared with standard strain, there was significant difference; B. Compared with commercial lactobacillus, there was significant difference; AB. Compared with standard strain and commercial lactobacillus, there was significant difference.
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