内生菌与人类肠道微生物群使用体外浴发酵系统的相互作用分析
Summary
本文描述了一种使用体外批次发酵系统研究内生菌与人类肠道微生物群之间的相互作用的协议。
Abstract
人类肠道微生物已成为促进人类健康、预防疾病的重要研究对象。因此，研究内生菌（如药物和益生菌）与肠道微生物群之间的相互作用已成为一个重要的研究课题。然而，由于生物伦理学和经济限制，在体内与人类志愿者进行的实验对于此类研究来说并不理想。因此，动物模型被用来评估体内的这些相互作用。然而，动物模型研究仍然受到生物伦理学考虑的限制，此外，动物和人类的微生物群的组成和多样性也各不相同。另一种研究策略是使用批量发酵实验，允许评估体外生物和肠道微生物群之间的相互作用。为了评估这一策略，双歧杆菌（比夫）外聚糖（EPS）被用作具有代表性的异种生物。然后，利用薄层色谱（TLC）、16S rRNA基因高通量测序的细菌群落成分分析、气相色谱等多种方法，对Bif EPS与人类肠道微生物群的相互作用进行了研究。短链脂肪酸（SCFA）。这里介绍的是一个协议，研究内生菌和人类肠道微生物群之间的相互作用，使用体外批次发酵系统。重要的是，该协议也可以修改，以研究其他内生菌和肠道微生物群之间的一般相互作用。
Introduction
胃微生物群在人类肠道的功能和宿主健康中起着重要的作用。因此，肠道微生物群最近成为疾病预防和治疗的重要目标。此外，肠道细菌与宿主肠道细胞相互作用，调节基本宿主过程，包括代谢活动、营养利用、免疫系统调节，甚至大脑功能和决策2，3.内生菌具有影响肠道微生物群的细菌组成和多样性的巨大潜力。因此，内生菌和人类肠道微生物群之间的相互作用吸引了越来越多的研究关注4，5，6，7，8，9。
由于生物伦理学和经济限制，很难评估内生菌与体内人类肠道微生物群之间的相互作用。例如，未经美国食品和药物管理局许可，无法进行调查内生菌与人类肠道微生物群之间相互作用的实验，招募志愿者的成本很高。因此，动物模型经常用于此类调查。然而，由于动物与人类相关的社区成分不同，且多样性，动物模型的使用受到限制。探索内生菌与人类肠道微生物群相互作用的体外替代方法是利用批量培养实验。
外聚糖（EPS）是益生菌，对维持人类健康有重大贡献10。由不同的单糖组合物和结构组成的不同 EPS 可以表现出不同的功能。以前的分析已经确定了Bif EPS的组成，这是当前研究11中具有代表性的异种生物。然而，在EPS组成和多样性方面，没有考虑到宿主相关的代谢效应。
此处描述的协议使用来自 12 名志愿者的粪便微生物群来发酵 Bif EPS。然后，结合使用薄层色谱法 （TLC）、16S rRNA 基因高通量测序和气相色谱 （GC） 来研究 EPS 与人类肠道微生物群之间的相互作用。与体内实验相比，该协议的明显优点是成本低，避免了宿主新陈代谢的干扰作用。此外，所述协议可用于其他研究，研究内生菌和人类肠道微生物群之间的相互作用。
Protocol
本议定书遵循湖南科学与工程大学（中国湖南）和浙江公学（浙江，中国）伦理委员会的指导方针。 1. 细菌的制备 制备双歧杆菌中溴 在950 mL蒸馏水中结合以下成分：肉提取物，5 g/L;酵母提取物，5克/升;病例蛋白，10克/升;大豆，5克/升;葡萄糖，10克/升;K2HPO4， 2.04 g/L;MgSO4±7H2O，0.22 g/L;MnSO4.H20， 0.05 g/L;纳Cl，5克/升;?…
Representative Results
在厌氧孵育72小时后，在PYG板上的B.长膜培养中观察到粘膜EPS的产生（图1A）。培养物的离心，随后是乙醇沉淀和干燥，导致纤维素样EPS的收集（图1B）。干燥EPS和可溶性淀粉然后用作发酵培养的碳源。TLC因其成本低、结果周转快而用于寡糖分离和纯度分析。虽然人类粪便微生物群淀粉的降解速度比Bif EPS快（图2）…
Discussion
在过去十年中，在了解人类肠道微生物群的组成和活动方面取得了重大进展。由于这些研究，全息生物概念已经出现，它代表宿主和相关微生物群落之间的相互作用，如在人类和他们的肠道微生物群之间19，20。此外，人类甚至现在被视为超级生物21，其中肠道微生物群已被确认为人类功能器官之一22，23。<sup …
Declarações
The authors have nothing to disclose.
Acknowledgements
这项研究由中国自然科学基金（第31741109号）、湖南省自然科学基金（第2018JJ3200号）和湖南科技大学应用特色学科建设项目资助。我们感谢LetPub（www.letpub.com）在编写本手稿期间提供的语言帮助。
Materials
| 0.22 µm membrane filters | Millipore | SLGP033RB | Use to filter samples |
| 0.4-mm Sieve | Thermo Fischer | 308080-99-1 | Use to prepare human fecal samples |
| 5-bromo-4-chloro-3-indolyl β-D-galactopyranoside (X-Gal) | Solarbio | X1010 | Use to prepare color plate |
| Acetic | Sigma-Aldrich | 71251 | Standard sample for SCFA |
| Agar | Solarbio | YZ-1012214 | The component of medium |
| Anaerobic chamber | Electrotek | AW 400SG | Bacteria culture and fermentation |
| Autoclave | SANYO | MLS-3750 | Use to autoclave |
| Bacto soytone | Sigma-Aldrich | 70178 | The component of medium |
| Baking oven | Shanghai Yiheng Scientific Instruments Co., Ltd | DHG-9240A | Use to heat and bake |
| Beef Extract | Solarbio | G8270 | The component of medium |
| Bifidobacterium longum Reuter | ATCC | ATCC® 51870™ | Bacteria |
| Bile Salts | Solarbio | YZ-1071304 | The component of medium |
| Butyric | Sigma-Aldrich | 19215 | Standard sample for SCFA |
| CaCl2 | Solarbio | C7250 | Salt solution of medium |
| Capillary column | SHIMADZU-GL | InertCap FFAP (0.25 mm × 30 m × 0.25 μm) | Used to SCFA detection |
| Casein Peptone | Sigma-Aldrich | 39396 | The component of medium |
| Centrifuge | Thermo Scientific | Sorvall ST 8 | Use for centrifugation |
| CoSO4.7H2O | Solarbio | C7490 | The component of medium |
| CuSO4.5H2O | Solarbio | 203165 | The component of medium |
| Cysteine-HCl | Solarbio | L1550 | The component of medium |
| Ethanol | Sigma-Aldrich | E7023 | Use to prepare vitamin K1 |
| FeSO4.7H2O | Solarbio | YZ-111614 | The component of medium |
| Formic Acid | Sigma-Aldrich | 399388 | Used to TLC |
| Gas chromatography | Shimadzu Corporation | GC-2010 Plus | Used to SCFA detection |
| Glass beaker | Fisher Scientific | FB10050 | Used for slurry preparation |
| Glucose | Solarbio | G8760 | The component of medium |
| Haemin | Solarbio | H8130 | The component of medium |
| HCl | Sigma-Aldrich | 30721 | Basic solution used to adjust the pH of the buffers |
| Isobutyric | Sigma-Aldrich | 46935-U | Standard sample for SCFA |
| Isovaleric Acids | Sigma-Aldrich | 129542 | Standard sample for SCFA |
| K2HPO4 | Solarbio | D9880 | Salt solution of medium |
| KCl | Solarbio | P9921 | The component of medium |
| KH2PO4 | Solarbio | P7392 | Salt solution of medium |
| LiCl.3H2O | Solarbio | C8380 | Use to prepare color plate |
| Meat Extract | Sigma-Aldrich-Aldrich | 70164 | The component of medium |
| Metaphosphoric Acid | Sigma-Aldrich | B7350 | Standard sample for SCFA |
| MgCl2.6H2O | Solarbio | M8160 | The component of medium |
| MgSO4.7H2O | Solarbio | M8300 | Salt solution of medium |
| MISEQ | Illumina | MiSeq 300PE system | DNA sequencing |
| MnSO4.H20 | Sigma-Aldrich | M8179 | Salt solution of medium |
| Mupirocin | Solarbio | YZ-1448901 | Antibiotic |
| NaCl | Solarbio | YZ-100376 | Salt solution of medium |
| NaHCO3 | Sigma-Aldrich | 792519 | Salt solution of medium |
| NanoDrop ND-2000 | NanoDrop Technologies | ND-2000 | Determine DNA concentrations |
| NaOH | Sigma-Aldrich | 30620 | Basic solution used to adjust the pH of the buffers |
| n-butanol | ChemSpider | 71-36-3 | Used to TLC |
| NiCl2 | Solarbio | 746460 | The component of medium |
| Orcinol | Sigma-Aldrich | 447420 | Used to prepare orcinol reagents |
| Propionic | Sigma-Aldrich | 94425 | Standard sample for SCFA |
| QIAamp DNA Stool Mini Kit | QIAGEN | 51504 | Extract bacterial genomic DNA |
| Ready-to-use PBS powder | Sangon Biotech (Shanghai) Co., Ltd. | A610100-0001 | Used to prepare the lipid suspension |
| Resazurin | Solarbio | R8150 | Anaerobic Equipment |
| Speed Vacuum Concentrator | LABCONCO | CentriVap | Use to prepare EPSs |
| Starch | Solarbio | YZ-140602 | Use to the carbon source |
| Sulfuric Acid | Sigma-Aldrich | 150692 | Used to prepare orcinol reagents |
| T100 PCR | BIO-RAD | 1861096 | PCR amplification |
| TLC aluminium sheets | MerckMillipore | 116835 | Used to TLC |
| Trypticase Peptone | Sigma-Aldrich | Z699209 | The component of medium |
| Tryptone | Sigma-Aldrich | T7293 | The component of medium |
| Tween 80 | Solarbio | T8360 | Salt solution of medium |
| Valeric | Sigma-Aldrich | 75054 | Standard sample for SCFA |
| Vitamin K1 | Sigma-Aldrich | V3501 | The component of medium |
| Vortex oscillator | Scientific Industries | Vortex.Genie2 | Use to vortexing |
| Yeast Extract | Sigma-Aldrich | Y1625 | The component of medium |
| ZnSO4.7H2O | Sigma-Aldrich | Z0251 | The component of medium |
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