一株瘤胃源产丁酸肠道罗斯拜瑞氏菌的筛选鉴定及培养条件优化

Screening, identification, and culture condition optimization of a rumen-derived butyrate-producing Roseburia intestinalis strain

摘要:

【背景】产丁酸菌是宿主胃肠道中一类重要的功能微生物群，其代谢产物能够促进反刍动物胃肠道的健康发育。【目的】分离筛选瘤胃源产丁酸菌株，明确其益生特性并优化培养条件，为后续开发利用提供菌种资源和理论基础。【方法】自中国农业科学院昌平试验基地采集成年羊新鲜瘤胃内容物，先用强化梭菌培养基(reinforced Clostridium medium,RCM)初筛，再以丁酸产量为标准复筛得到产丁酸效果最好的菌株，分析其生长特性、益生特性，利用单因素和正交试验对菌株培养条件进行优化。【结果】筛选到一株高效产丁酸的菌株，经鉴定为肠道罗斯拜瑞氏菌(Roseburia intestinalis)，命名为R. intestinalis F12-9。R. intestinalis F12-9种子液以3%的接种量接入RCM培养基，培养温度37℃、初始pH 6.8，0−10 h为对数生长期。R. intestinalis F12-9在6.0% NaCl中的存活率高达(63.88±3.16)%，菌株在3.0 g/L胆盐中的存活率为(200.82±11.19)%，在模拟胃液(pH 2.5、pH 3.0)以及胰液中的存活率分别为(110.11±9.44)%、(100.18±8.44)%和(175.05±5.20)%。经正交试验优化后，菌株最佳培养条件为：接种量5%、pH 7.0、培养时间12 h，OD₆₀₀为1.913，较未优化前提升了1.34倍。【结论】瘤胃源R. intestinalis F12-9为一株新的产丁酸菌株，其产丁酸能力强，无溶血，抗生素耐药性风险低，耐受性强，具有良好的开发前景。

Abstract:

[Background] Butyrate-producing bacteria are an important of functional microorganisms in the host gastrointestinal tract, and their metabolites can promote the healthy development of the gastrointestinal tract of ruminants.[Objective] This study aimed to isolate and screen rumen-derived butyrate-producing strains, clarify their probiotic characteristics and optimize culture conditions, thus providing strain resources and a theoretical basis for subsequent development and application. [Methods] Fresh rumen contents were collected from adult sheep at the Changping Base of Chinese Academy of Agricultural Sciences. The strains with high butyrate production were obtained by primary screening with reinforced Clostridium medium (RCM) and secondary screening with butyrate production as the standard. Growth and probiotic characteristics of the strains were analyzed, and the optimal culture conditions were determined by single factor and orthogonal tests. [Results] A strain with high butyrate production was screened and identified as Roseburia intestinalis, named R. intestinalis F12-9. The seed liquid of R. intestinalis F12-9 was inoculated into the RCM at an inoculation amount of 3% and cultured at 37 ℃ and initial pH 6.8, under which the strain exhibited a logarithmic growth phase within 0–10 h. The survival rate of R. intestinalis F12-9 exposed to 6.0% NaCl and 3.0 g/L bile salt were (63.88±3.16)% and (200.82±11.19)%, respectively. The survival rates in simulated gastric juice (pH 2.5, pH 3.0) and pancreatic juice were (110.11±9.44)%, (100.18±8.44)%, and (175.05±5.20)%, respectively. The optimized culture conditions of the strain were as follows: inoculation amount of 5%, pH 7.0, and culture time of 12 h, resulted in an OD₆₀₀ value of 1.913, which was 1.34 times higher than that before optimization. [Conclusion] Rumen-derived R. intestinalis F12-9 is a new butyrate-producing strain, with a strong ability to produce butyrate, no hemolysis, a low risk of antibiotic resistance, and strong tolerance, demonstrating its broad development prospects.