圈养大熊猫肠道乳酸菌分离及益生特性

Isolation and probiotic characteristics of lactic acid bacteria from intestinal tract of giant panda in captivity

摘要:

【背景】大熊猫数量稀少、繁殖困难，在其生长过程中极易感染消化系统疾病甚至死亡。【目的】分析圈养大熊猫肠道可培养乳酸菌的群落结构与功能，筛选具有益生特性的乳酸菌菌株，为大熊猫消化系统疾病的预防和肠道微生物研究提供理论参考及菌种资源。【方法】采用3种培养基分离大熊猫粪便中的乳酸菌，通过革兰氏染色镜检和过氧化氢试验对分离的菌株进行初步鉴定，基于BOXA1R-PCR图谱遗传多样性选取代表菌株进行16S rRNA基因测序分析并进行主成分分析(principal component analysis, PCA)，同时分析乳酸菌菌株安全性和益生特性。【结果】通过初步鉴定共分离获得58株乳酸菌，根据BOXA1R-PCR结果挑选20株菌进行测序，结果显示20株菌分属于明串珠菌属(Leuconostoc)、肠球菌属(Enterococcus)、魏斯氏菌属(Weissella)和链球菌属(Streptococcus)这4个属，主成分分析结果表明不同年龄段的大熊猫肠道乳酸菌群落结构存在差异。20株乳酸菌均不溶血，17株乳酸菌对11种抗生素均敏感；11株菌耐pH值2.0酸性条件，14株菌对0.3%的胆盐具有良好的耐受性。5株菌SW-51、SW-48、MQ-41、SW-58和MX-23对3种指示菌均具有抑菌效果，50%以上的供试菌株具有自凝聚和共凝聚能力，3株菌的产酸和生长速率较一致，其中乳酸明串珠菌(Leuconostoc lactis) MX-23生长最快且产酸能力最强。【结论】大熊猫肠道内蕴藏着丰富的乳酸菌资源，其结构与组成受其年龄影响，具有较好的益生特性和应用前景。

Abstract:

[Background] The giant pandas are rare, difficult to be bred, and susceptible to digestive system diseases during the growth process. [Objective] To reveal the community structure features and the roles of lactic acid bacteria (LAB) in the intestinal tract of the giant panda and screen out the LAB strains with probiotic characteristics for the prevention of digestive system diseases and the research on intestinal flora in the giant panda. [Methods] Three culture media were employed to isolate the LAB from the intestines of giant pandas in captivity. The isolates were preliminarily identified by Gram staining microscopy and hydrogen peroxide test. The representative strains were selected for 16S rRNA gene sequencing and Principal component analysis (PCA) based on the genetic diversity of BOXA1R-PCR spectrum, and the safety and probiotics of LAB were further analyzed. [Results] A total of 58 LAB strains were isolated, from which 20 representative strains were selected for 16S rRNA gene sequencing according to BOXA1R-PCR results. The 20 strains belonged to Streptococcus, Leuconostoc, Weissella, and Enterococcus, respectively. The principal component analysis showed that the community structure of LAB varied in the giant pandas at different ages. All the 20 strains showed negative results in hemolysis, and 17, 11, and 14 strains were susceptible to 11 antibiotics, tolerant to acidic conditions of pH 2.0, and tolerant to 0.3% bile salts, respectively. Strains SW-51, SW-48, MQ-41, SW-58, and MX-23 demonstrated strong inhibitory activities to 3 pathogenic bacterial species. Over 50% of the strains showed high auto-aggregation and co-aggregation capacities. Three strains showed consistent acid production and growth rate, among which Leuconostoc lactis MX-23 showed the best performance in growth and acid production. [Conclusion] The intestines of giant pandas harbor abundant LAB with valuable probiotic characteristics and application prospects, and the structure and composition of the LAB were influenced by age.