一株家蚕肠道分离乳酸菌的鉴定及生物学特性

Identification and biological characterization of a lactic acid bacterial strain isolated from the gut of Bombyx mori

摘要:

【背景】乳酸菌(lactic acid bacteria)作为益生菌可用作动物微生态制剂，家蚕(Bombyx mori)作为鳞翅目的模式昆虫，其肠道内的乳酸菌还有待研究。【目的】从家蚕肠道菌中筛选出性能良好的乳酸菌作为微生态制剂潜力菌株。【方法】通过形态学特征和生化试验筛选出家蚕肠道分离菌中的乳酸菌，结合16S rRNA基因序列分析鉴定到种；通过溶血试验评估其安全性；通过生长曲线及产酸曲线测定其生长特征和产酸情况；通过温度及pH敏感性试验测定其抗逆性；通过测定自聚合能力、疏水能力、病原菌共凝聚能力评估其黏附性；通过药敏试验测定其耐药性，并检测其耐药基因。【结果】从家蚕肠道分离出的56个菌株中筛选出一株乳酸菌A2，经鉴定为粪肠球菌(Enterococcus faecalis)。该菌株不溶血，接种后3−9 h生长迅速且产酸最快；在80 ℃高温条件下仍能存活；pH<4.0时存活率显著下降，但当pH 10.0时存活率>78%，具有极强的耐碱性；黏附性较强，自聚合能力为18%，疏水能力为70%，与大肠杆菌(Escherichia coli)的共凝聚性为32%、与沙门氏菌(Salmonella)的共凝聚性为34%、与金黄色葡萄球菌(Staphylococcus aureus)的共凝聚性为40%，对氨苄西林、红霉素和万古霉素有耐药性，对庆大霉素中度敏感，对磺胺异恶唑、氯霉素、四环素和氧氟沙星敏感。此外，从该菌株中检测到4类抗生素的5个耐药基因，它们分别是：β-内酰胺类bla_SHV、bla_TEM、氯霉素类cat、氨基糖苷类aph(3')-IIa和四环素类tetE。【结论】该乳酸菌安全、耐高温、耐碱、黏附性较强，可作为后续开发微生物发酵饲料及家蚕微生态制剂的菌种，为肠道益生菌的开发利用提供科学依据和优质资源。

关键词:家蚕;乳酸菌;生物学特性;耐药基因

Abstract:

[Background] Lactic acid bacteria as probiotics can be developed as microecological preparations for animals. The lactic acid bacteria in the gut of Bombyx mori, a model insect of Lepidoptera, remain to be studied. [Objective] To isolate the lactic acid bacteria with good biological properties from the gut of B. mori for developing microecological preparations. [Methods] The lactic acid bacteria isolated from the gut of B. mori were screened based on morphological, physiological, and biochemical characteristics and identified by 16S rRNA gene sequencing. The safety of the strain screened out was evaluated by the hemolysis test, and the growth characteristics and acid production of the strain were determined by growth curve and acid production curve. The stress tolerance of the strain was determined by temperature and pH sensitivity tests. The adhesion of the strain was evaluated based on self-aggregation, hydrophobicity, and coagglutination with pathogens. Furthermore, the drug resistance of the strain was examined by the drug sensitivity test, and the drug resistance genes carried by the strain were detected. [Results] Among the 56 strains isolated, one strain A2 was screened out and identified as Enterococcus farcalis. The strain did not present hemolysis, experienced rapid growth and acid production 3–9 h after inoculation. It could survive at a high temperature of 80 ℃. The survival rate of the strain decreased in the case of below pH 4.0 and was over 78% at pH 10.0, which indicated that the strain had strong alkaline tolerance. The strain demonstrated strong adhesion, with the self-aggregation of 18%, the hydrophobicity of 70%, and the coagglutination of 32%, 34%, and 40% with Escherichia coli, Salmonella, and Staphylococcus aureus, respectively. It was resistant to ampicillin, erythromycin, and vancomycin, moderately sensitive to gentamicin, and sensitive to sulfafurazole, chloramphenicol, tetracycline, and ofloxacin. The strain carried five drug resistance genes conferring resistance to four categories of antibiotics: β-lactam (bla_SHV and bla_TEM), chloramphenicol (cat), aminoglycosides (aph(3')-IIa), and tetracycline (tetE). [Conclusion] The lactic acid bacterial strain isolated from the gut of B. mori was safe, thermostable, and alkali-tolerant and it had strong adhesion. Therefore, it served as a candidate strain for the subsequent development of microbial fermented feed and microecological preparations for silkworm. This study provided a scientific basis and high-quality resources for the development and utilization of gut probiotics.

Key words:Bombyx mori;lactic acid bacteria;biological characteristics;drug resistance gene