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Described here is a protocol to investigate the interactions between endobiotics and human gut microbiota using in vitro batch fermentation systems.
Human intestinal microorganisms have recently become an important target of research in promoting human health and preventing diseases. Consequently, investigations of interactions between endobiotics (e.g., drugs and prebiotics) and gut microbiota have become an important research topic. However, in vivo experiments with human volunteers are not ideal for such studies due to bioethics and economic constraints. As a result, animal models have been used to evaluate these interactions in vivo. Nevertheless, animal model studies are still limited by bioethics considerations, in addition to differing compositions and diversities of microbiota in animals vs. humans. An alternative research strategy is the use of batch fermentation experiments that allow evaluation of the interactions between endobiotics and gut microbiota in vitro. To evaluate this strategy, bifidobacterial (Bif) exopolysaccharides (EPS) were used as a representative xenobiotic. Then, the interactions between Bif EPS and human gut microbiota were investigated using several methods such as thin-layer chromatography (TLC), bacterial community compositional analysis with 16S rRNA gene high-throughput sequencing, and gas chromatography of short-chain fatty acids (SCFAs). Presented here is a protocol to investigate the interactions between endobiotics and human gut microbiota using in vitro batch fermentation systems. Importantly, this protocol can also be modified to investigate general interactions between other endobiotics and gut microbiota.
Gut microbiota play an important role in the functioning of human intestines and in host health. Consequently, gut microbiota have recently become an important target for disease prevention and therapy1. Moreover, gut bacteria interact with host intestinal cells and regulate fundamental host processes, including metabolic activities, nutrient availabilities, immune system modulation, and even brain function and decision-making2,3. Endobiotics have considerable potential to influence the bacterial composition and diversity of gut microbiota. Thus, interactions between endobiotics and hum....
This protocol follows the guidelines of the ethics committee of Hunan University of Science and Engineering (Hunan, China), and the Zhejiang Gongshang University (Zhejiang, China).
1. Preparation of bacteria
The production of mucoid EPS could be observed in B. longum cultures on PYG plates after anaerobic incubation for 72 h (Figure 1A). Centrifugation of culture scrapes, followed by ethanol precipitation and drying, resulted in the collection of cellulose-like EPS (Figure 1B). Dried EPS and soluble starch were then used as carbon sources for fermentation cultures. TLC was used for oligosaccharide separation and purity analysis due to its low cost and rapid.......
Significant progress has been made towards understanding human gut microbiota composition and activities over the last decade. As a consequence of these studies, the holobiont concept has emerged, which represents the interactions between hosts and associated microbial communities, such as in between humans and their gut microbiota19,20. Furthermore, humans are even now regarded as superorganisms21, wherein the gut microbiota have been rec.......
This study was funded by the National Nature Science Foundation of China (No. 31741109), the Hunan Natural Science Foundation (No. 2018JJ3200), and the construct program of applied characteristic discipline in Hunan University of Science and Engineering. We thank LetPub (www.letpub.com) for its linguistic assistance during the preparation of this manuscript.
....Name | Company | Catalog Number | Comments |
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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