太子参内生细菌RPB-32的分类鉴定及其代谢物对小鼠肠道微生物群落的影响

【背景】肠道微生物在宿主肠道微环境稳态中起着至关重要的作用。众多因子,如抗生素、饮食和年龄等会干扰这种微平衡,引起菌群平衡发生改变,进而影响到机体健康状况。太子参具有心肌保护、增加免疫、抗氧化、抗糖尿病、抗应激、抗疲劳等药理活性,而太子参内生菌代谢物对肠道微生物的调控作用以及可能对机体健康的影响目前还未见报道。【目的】研究太子参内生菌RPB-32分类地位及其代谢提取物对小鼠肠道微生物的调节作用,以探讨其对机体健康的影响。【方法】对RPB-32进行传统方法和分子鉴定。将120只KM小鼠随机分为溶剂对照组(3%乙醇)及石油醚提取物、正丁醇提取物和乙酸乙酯提取物低、中、高剂量组(n=12)。小鼠灌胃处理14 d后采集粪便,通过选择性培养基培养及宏基因组测序溶剂对照(S.F.1为3%乙醇溶液)、乙酸乙酯提取物高剂量组(S.F.2)]检测灌胃前后小鼠肠道微生物的变化。【结果】常规传统方法与分子鉴定结果表明RPB-32为芽孢杆菌(Bacillus sp.)。与空白对照组相比,给予内生菌代谢提取物的乙酸乙酯中剂量组、乙酸乙酯高剂量组、正丁醇低剂量组、正丁醇中剂量组及正丁醇高剂量组乳酸菌数量明显增加,正丁醇中剂量组的肠球菌和正丁醇高剂量组的肠杆菌数量明显减少。S.F.2与S.F.1基于门、属和种水平上的微生物群落结构相比表明:S.F.2的拟杆菌门(Bacteroidetes)数量明显增加,而厚壁菌门(Firmicutes)数量明显减少;S.F.2的Bacteroides、Prevotella、Parabacteroides、Akkermansia、Helicobacter、Butyricimonas、Alistipes、Ruminococcus、Muribaculum、Barnesiella、Odoribacter、Azospirillum、Mucispirillum和Lactobacillus数量明显增多,Rikenella、Angelakisella、Clostridium、Oscillibacter、Pseudoflavonifractor、Flavonifractor、Mailhella、Desulfovibrio、Faecalibacterium、Eubacterium、Lachnoclostridium、Hungatella、Butyrivibrio、Blautia、Dorea、Ruminiclostridium、Roseburia、Anaerotruncus和Butyricicoccus等数量减少;S.F.2的Bacteroides sartorii、Bacteroides caecimuris、Bacteroides uniformis、Parabacteroides chinchillae、Parabacteroides glodsteinii、Helicobacter bilis、Bacteroides fragilis、Bacteroides vulgatus、Bacteroides thetaiotaomicron和Butyricimonas virosa等数量明显增多,Faecalibacterium prausnitzii、Rikenella microfusus、Flavonifractor plautii、Mailhella massiliensis和Clostridium clostridioforme等数量减少。【结论】初步判断太子参内生芽孢杆菌RPB-32代谢物(BM)对肠道微生物组成有明显影响,可能对机体糖脂降解、抗感染和抗炎等方面有改善作用。

Identification of endogenous bacteria RPB-32 of Radix pseudostellariae and its metabolites effect on intestinal microorganism community of mice

Background]Intestinal microorganisms play a vital role in homeostasis of the host’s intestinal microenvironment.Many factors,such as antibiotics,diet and age,can interfere with this microbalance,causing changes in the microflora balance,which in turn affects the health of the body.There exist many pharmacological activities such as myocardial protection,immunity improvement,anti-oxidation,anti-diabetes,anti-stress,and anti-fatigue on Radix pseudostellariae.However,the regulation effect of endophytic metabolites of Radix pseudostellariae on intestinal microbes and their possible impacts on body health have not been studied so far.Objective]The taxonomic status of endophytic bacterium RPB-32 from Radix pseudostellariae and the regulation of its metabolic extracts on the intestinal microbes of mice were studied to explore its impact on body health.Methods]Strain RPB-32 was identified based on the traditional and molecular methods.120 KM mice were randomly divided into four groups,viz.solvent control group(3%ethanol),the low,medium and high-dose groups of petroleum ether extract,ethyl acetate extract and n-butanol extract(n=12).After 14 days of intragastric treatments,the mice feces were collected aseptically,and the changes of intestinal microorganisms before and after intragastric gavage were detected by selective medium culture and metagenomic sequencing(solvent control(S.F.1),high-dose ethyl acetate extract group(S.F.2)).Results]Strain RPB-32 was identified as Bacillus sp.according to the conventional and molecular methods.Compared with the blank control group,the amounts of lactic acid bacteria significantly increased in the tested groups of medium-dose ethyl acetate extracts,high-dose ethyl acetate extracts,low-dose n-butanol extracts,medium-dose n-butanol extracts and high-dose n-butanol extracts,while the amounts of Enterococcus and Enterobacter in the groups of middle-dose n-butanol extracts and high-dose n-butanol extracts were significantly reduced,respectively.The comparison of the microbial community structure showed that the amount of Firmicutes obviously reduced,but that of Bacteroidetes significantly increased in S.F.2 collated with S.F.1 at the phylum level;the amounts of Bacteroides,Prevotella,Parabacteroides,Akkermansia,Helicobacter,Butyricimonas,Alistipes,Ruminococcus,Muribaculum,Barnesiella,Odoribacter,Azospirillum,Mucispirillum,Lactobacillus obviously increased,while those of Rikenella,Angelakisella,Clostridium,Oscillibacter,Pseudoflavonifractor,Flavonifractor,Mailhella,Desulfovibrio,Faecalibacterium,Eubacterium,Lachnoclostridium,Hungatella,Butyrivibrio,Blautia,Dorea,Ruminiclostridium,Roseburia,Anaerotruncus,Butyricicoccus significantly decreased in S.F.2 contrasted with S.F.1 at the genus level;the amounts of Bacteroides sartorii,Bacteroides caecimuris,Bacteroides uniformis,Parabacteroides chinchillae,Parabacteroides glodsteinii,Helicobacter bilis,Bacteroides fragilis,Bacteroides vulgatus,Bacteroides thetaiotaomicron,Butyricimonas virosa strikingly increased,while those of Faecalibacterium prausnitzii,Rikenella microfusus,Flavonifractor plautii,Mailhella massiliensis,Clostridium clostridioforme significantly decreased in S.F.2 in comparison with S.F.1 at the species level.Conclusion]It was preliminarily determined that the metabolites of endophytic Bacillus sp.RPB-32(BM)from Radix pseudostellariae had the significant effects on the composition of intestinal microorganisms in mice,and might improve the degradation of glucose and lipid,anti-infective and anti-inflammatory activities in body.