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.

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
<ol>
<li>Preparation of bifidobacterium medium broth
<ol>
<li>Combine the following components in 950 mL of distilled water: meat extract, 5 g/L; yeast extract, 5 g/L; casein peptone, 10 g/L; soytone, 5 g/L; glucose, 10 g/L; K2HPO4, 2.04 g/L; MgSO4&#183;7H2O, 0.22 g/L; MnSO4.H20, 0.05 g/L; NaCl, 5 g/L; Tween 80, 1 mL; salt solution, 40 mL (CaCl2.2H2O, 0.25 g/L; KH2PO4, 1 g/L; NaHCO3, 10 g/L; NaCl, 2 g/L); and resazurin, 0.4 mL (2.5 mg/L). Adjust the pH to 6.8 with 2 M NaOH.</li>
<li>Autoclave at 121 &#176;C for 15 min and allow the broth to cool to room temperature (RT) under anaerobic conditions (10% H2, 10% CO2, 80% N2). Add filter-sterilized cysteine-HCl (0.5 g/L) and mupirocin (5 mg/L) to the medium.</li>
</ol>
</li>
<li>Add an aliquot (50 &#956;L) of frozen Bifidobacterium longum to a culture tube with 5 mL of bifidobacterium medium broth under anaerobic conditions, then culture in an anaerobic incubator for 24 h at 37 &#176;C.</li>
</ol>
2. Preparation of bifidobacterial EPSs
<ol>
<li>Preparation of PYG agar medium
<ol>
<li>Combine the following: peptone, 20 g/L; yeast extract, 10 g/L; glucose, 5 g/L; NaCl, 0.08 g/L; CaCl2, 0.008 g/L; MgSO4&#183;7H2O, 0.008 g/L; K2HPO4, 0.04 g/L; KH2PO4, 0.04 g/L; NaHCO3, 0.4 g/L; agar, 12 g/L. Adjust the pH to 7.2 using 10 M NaOH.</li>
<li>Autoclave the media at 121 &#176;C for 15 min and cool to ~50 &#176;C. Then, per 1 L of medium, add 0.5 mL of filter-sterilized vitamin K1 solution (1 g of vitamin K1 dissolved in 99 mL of 99% ethanol), 5 mL of haemin solution (0.5 g of haemin dissolved in 1 mL of 1 mol/L NaOH, then brought up to 100 mL with distilled water), and 0.5 g of cysteine-HCl.</li>
<li>Before pouring the PYG plates, add filter-sterilized 5-bromo-4-chloro-3-indolyl &#946;-D-galactopyranoside (X-Gal, 0.06 g/L), LiCl&#183;3H2O (5.7 g/L) and mupirocin (5 mg/L) to the medium. NOTE: X-Gal and LiCl.3H2O allow the identification of B. longum colonies on plates via coloration changes.</li>
</ol>
</li>
<li>Inoculate 20 &#956;L of B. longum strains (step 1.2) to PYG plates and place in an anaerobic incubator at 37 &#176;C for 72 h.</li>
<li>Collect mucoid bacterial colonies from the PYG plates using a weighing scoop, then completely resuspend in 10 mL of phosphate-buffered saline (PBS) using a vortex oscillator. NOTE: The bacterial and EPS mixtures should be resuspended completely by vortexing or pipetting up and down repeatedly until the fibers are completely dissolved in PBS.</li>
<li>Centrifuge the suspension at 6,000 x g for 5 min.</li>
<li>Carefully transfer the supernatants to a new centrifuge tube and mix completely with three volumes of cold 99% ethanol by repeated inversion and blending.</li>
<li>Centrifuge the mixture at 6,000 x g for 5 min and completely remove the supernatants.</li>
<li>Remove the precipitates from the centrifuge tubes by scraping and drying the EPS extracts overnight using a speed vacuum.</li>
</ol>
3. Preparation of fermentation medium
<ol>
<li>Preparation of basic culture medium VI
<ol>
<li>Combine the following: peptone, 3 g/L; tryptone, 3 g/L; yeast extract, 4.5 g/L; mucin, 0.5 g/L; bile salts No. 3, 0.4 g/L; NaCl, 4.5 g/L; KCl, 2.5 g/L; MgCl2&#183;6H2O, 4.5 g/L; 1 mL Tween 80; CaCl2.6H2O, 0.2 g/L; KH2PO4, 0.4 g/L; MgSO4&#183;7H2O, 3.0 g/L; MnCl2&#183;4H2O, 0.32 g/L; FeSO4&#183;7H2O, 0.1 g/L; CoSO4&#183;7H2O, 0.18 g/L; CaCl2&#183;2H2O, 0.1 g/L; ZnSO4&#183;7H2O, 0.18 g/L; CuSO4&#183;5H2O, 0.01 g/L; and NiCl2&#183;6H2O, 0.092 g/L. Adjust the pH to 6.5 with 1 M HCl.</li>
<li>Prepare haemin and cysteine as done in section 2.1 and add after autoclaving and cooling.</li>
</ol>
</li>
<li>Prepare culture media that contains different carbon sources with a VI base media. Prior to autoclaving, add 8 g/L of Bif EPS fibers to medium VI, comprising group VI_Bif. In addition, add 8 g/L starch to medium VI to represent group VI_Starch. Finally, medium VI without addition of a carbon source is used as the control (group VI). NOTE: Bif EPS and starch are first dissolved in hot water using a magnetic agitator, then mixed with prepared VI medium.</li>
<li>Autoclave all media at 121 &#176;C for 15 min and allow to cool to RT.</li>
<li>Transfer a subsample (5 mL) of each medium to culture tubes in an anaerobic incubator, and store the remaining media at 4 &#176;C.</li>
</ol>
4. Human fecal sample preparation
<ol>
<li>Collect fresh fecal samples immediately following fresh defecation from healthy adult human volunteers using feces containers, and subsequently use for slurry preparation. NOTE: Prior to sample collection, all the volunteers should be screened to ensure no receiving of antibiotics, probiotics, or prebiotic treatments for at least 3 months prior to donating samples. In addition, all donors must provide informed, written consent.</li>
<li>Transfer a fresh fecal sample (1 g) to 10 mL of 0.1 M anaerobic PBS (pH 7.0) into glass beakers, then use glass rods to prepare a 10% (w/v) slurry.</li>
<li>Use a 0.4 mM sieve to filter the fecal slurry. Then, use a subsample of the filtered slurry to inoculate batch culture fermentation experiments, and store the remainder at -80 &#176;C for further analyses. NOTE: Steps 4.2&#8211;4.3 are conducted in an anaerobic chamber.</li>
</ol>
5. In vitro batch fermentation
<ol>
<li>Add filtered fecal slurry (500 &#956;L) to the fermentation medium prepared in step 3.2 within an anaerobic chamber, then incubate at 37 &#176;C.</li>
<li>Collect 2 mL of fermented samples at 24 h and 48 h in the anaerobic chamber and then centrifuge outside of the chamber at 6,000 x g for 3 min.</li>
<li>Carefully transfer the supernatants to a new centrifuge tube that will be used for polysaccharide degradation analysis and short-chain fatty acids (SCFAs) measurements.</li>
<li>Store the centrifugation pellets at -80 &#176;C and subsequently use for bacterial genomic DNA extraction.</li>
</ol>
6. EPS degradation by human fecal microbiota
<ol>
<li>Load 0.2 &#956;L of fermented supernatants onto pre-coated silica gel-60 TLC aluminum plates, then dry using a hair drier.</li>
<li>Develop the plates in 20 mL of a formic acid/n-butanol/water (6:4:1, v:v:v) solution and dry using a hair drier.</li>
<li>Soak the plates in the orcinol reagent to dye, then dry using a hair drier.
<ol>
<li>Prepare orcinol reagents by dissolving 900 mg of Lichenol in 25 mL of distilled water then adding 375 mL of ethanol. Subsequently, concentrated sulfuric acid should be slowly added and the solution thoroughly mixed.</li>
</ol>
</li>
<li>Heat plates at 120 &#176;C for 3 min in a baking oven and evaluate degradation of EPS by measuring TLC bands.</li>
</ol>
7. Effects of EPS on human intestinal microbiota
<ol>
<li>Freeze-thaw the original fecal samples prepared in step 4.3 and fermented samples prepared in step 5.4.</li>
<li>Extract bacterial genomic DNA (gDNA) from all the samples using a stool bacterial genomic DNA extraction kit following the manufacturer&#8217;s instructions.</li>
<li>Determine DNA concentrations, integrities, and size distributions using a micro-spectrophotometer and agar gel electrophoresis.</li>
<li>Conduct PCR of bacterial 16S rRNA genes from the extracted gDNA using the following previously described forward and reverse primers12: -forward primer (barcoded primer 338F): ACTCCTACGGGAGGCAGCA -reverse primer (806R): GGACTACHVGGGTWTCTAAT Use the following thermal cycler conditions:
<ol>
<li>94 &#176;C for 5 min.</li>
<li>94 &#176;C for 30 s.</li>
<li>55 &#176;C for 30 s.</li>
<li>72 &#176;C for 1 min.</li>
<li>Repeat 2&#8211;4 for 35 cycles</li>
<li>72 &#176;C for 5 min.</li>
<li>4 &#176;C hold until removal from thermal cycler.</li>
</ol>
</li>
<li>Conduct high-throughput sequencing of PCR products at a DNA sequencing company using ultra-high throughput microbial community analysis.</li>
<li>Obtain clean, high quality sequences using the Quantitative Insights into Microbial Ecology (QIIME) sequence analysis pipeline13.</li>
<li>Define operational taxonomic units (OTUs) for 16S rRNA gene sequences with greater than 97% nucleotide similarity using bioinformatics tools such as the Mothur software suite14.</li>
<li>Choose a representative sequence from each OTU and use the RDP classifier along with the SILVA taxonomic database to classify representative sequences15.</li>
<li>Calculate Good&#8217;s coverage, alpha diversity metrics (including Simpson and Shannon index), and richness (observed number of OTUs) using bioinformatics tools16.</li>
</ol>
8. Effects of EPS on SCFA production by human intestinal microbiota
<ol>
<li>Add fermented supernatants (1 mL) prepared in step 5.3 to 2 mL centrifuge tubes.</li>
<li>Add 0.2 mL of 25% (w/v) metaphosphoric acid to each of the samples and thoroughly mix the solutions by vortexing.</li>
<li>Centrifuge the mixtures at 13,000 x g for 20 min and transfer the supernatants to fresh tubes.</li>
<li>Concomitantly, prepare solutions of 120 mM acetic, propionic, butyric, isobutyric, valeric and isovaleric acids. Then, add 1 mL of each prepared acid to 1.2 mL of 25% (w/v) metaphosphoric acid and use as the standard cocktails.</li>
<li>Filter the samples using a 0.22 &#956;M membrane.</li>
<li>Detect SCFA concentrations using high performance gas chromatography according to previously described protocols11,17. NOTE: An InertCap FFAP column (0.25 mM x 30 m x 0.25 &#956;M) is used for gas chromatography (GC). SCFA concentrations are then quantified based on peak areas using the single-point internal standard method in the GC Solution software package.</li>
</ol>

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The authors declare that they have no conflicts of interest. The figures were cited in Yin et al.11.

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...

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 human gut microbiota have attracted increasing research attention4,5,6,7,8,9.
It is difficult to evaluate interactions between endobiotics and human gut microbiota in vivo due to bioethics and economic constraints. For example, experiments investigating the interactions between endobiotics and human gut microbiota cannot be performed without permission of the Food and Drug Administration, and recruitment of volunteers is expensive. Consequently, animal models are often used for such investigations. However, the use of animal models is limited due to different microbiota compositions and diversity in animal- vs. human-associated communities. An alternative in vitro method to explore the interactions between endobiotics and human gut microbiota is through the use of batch culture experiments.
Exopolysaccharides (EPSs) are prebiotics that significantly contribute to the maintenance of human health10. Distinct EPSs that consist of different monosaccharide compositions and structures can exhibit distinct functions. Previous analyses have determined the composition of Bif EPSs, which are the representative xenobiotic targeted in the current study11. However, host-associated metabolic effects have not been considered with regard to EPS composition and diversity.
The protocol described here uses the fecal microbiota from 12 volunteers to ferment Bif EPSs. Thin-layer chromatography (TLC), 16S rRNA gene high-throughput sequencing, and gas chromatography (GC) are then used in combination to investigate the interactions between EPSs and human gut microbiota. Distinct advantages of this protocol compared to in vivo experiments are its low cost and avoidance of interfering effects from the host&#x2019;s metabolism. Furthermore, the described protocol can be used in other studies that investigate interactions between endobiotics and human gut microbiota.

<table><tbody><tr><td>0.22 &amp;micro;m membrane filters</td><td>Millipore</td><td>SLGP033RB</td><td>Use to filter samples</td></tr><tr><td>0.4-mm Sieve</td><td>Thermo Fischer</td><td>308080-99-1</td><td>Use to prepare human fecal samples</td></tr><tr><td>5-bromo-4-chloro-3-indolyl &amp;beta;-D-galactopyranoside (X-Gal)</td><td>Solarbio</td><td>X1010</td><td>Use to prepare color plate</td></tr><tr><td>Acetic</td><td>Sigma-Aldrich</td><td>71251</td><td>Standard sample for SCFA</td></tr><tr><td>Agar</td><td>Solarbio</td><td>YZ-1012214</td><td>The component of medium</td></tr><tr><td>Anaerobic chamber</td><td>Electrotek&amp;nbsp;</td><td>AW 400SG</td><td>Bacteria culture and fermentation</td></tr><tr><td>Autoclave</td><td>SANYO</td><td>MLS-3750</td><td>Use to autoclave</td></tr><tr><td>Bacto soytone</td><td>Sigma-Aldrich</td><td>70178</td><td>The component of medium</td></tr><tr><td>Baking oven</td><td>Shanghai Yiheng Scientific Instruments Co., Ltd</td><td>DHG-9240A</td><td>Use to heat and bake</td></tr><tr><td>Beef Extract</td><td>Solarbio</td><td>G8270</td><td>The component of medium</td></tr><tr><td>&lt;em&gt;Bifidobacterium longum Reuter&lt;/em&gt;</td><td>ATCC</td><td>ATCC&amp;reg; 51870&amp;trade;</td><td>Bacteria</td></tr><tr><td>Bile Salts</td><td>Solarbio</td><td>YZ-1071304</td><td>The component of medium</td></tr><tr><td>Butyric</td><td>Sigma-Aldrich</td><td>19215</td><td>Standard sample for SCFA</td></tr><tr><td>CaCl&lt;sub&gt;2&lt;/sub&gt;</td><td>Solarbio</td><td>C7250</td><td>Salt solution of medium</td></tr><tr><td>Capillary column</td><td>SHIMADZU-GL</td><td>InertCap FFAP (0.25 mm &amp;times; 30 m &amp;times; 0.25 &amp;mu;m)</td><td>Used to SCFA detection</td></tr><tr><td>Casein Peptone</td><td>Sigma-Aldrich</td><td>39396</td><td>The component of medium</td></tr><tr><td>Centrifuge</td><td>Thermo Scientific</td><td>Sorvall ST 8</td><td>Use for centrifugation</td></tr><tr><td>CoSO&lt;sub&gt;4&lt;/sub&gt;.7H&lt;sub&gt;2&lt;/sub&gt;O</td><td>Solarbio</td><td>C7490</td><td>The component of medium</td></tr><tr><td>CuSO&lt;sub&gt;4&lt;/sub&gt;.5H&lt;sub&gt;2&lt;/sub&gt;O</td><td>Solarbio</td><td>203165</td><td>The component of medium</td></tr><tr><td>Cysteine-HCl</td><td>Solarbio</td><td>L1550</td><td>The component of medium</td></tr><tr><td>Ethanol</td><td>Sigma-Aldrich</td><td>E7023</td><td>Use to prepare vitamin K&lt;sub&gt;1&lt;/sub&gt;</td></tr><tr><td>FeSO&lt;sub&gt;4&lt;/sub&gt;.7H&lt;sub&gt;2&lt;/sub&gt;O</td><td>Solarbio</td><td>YZ-111614</td><td>The component of medium</td></tr><tr><td>Formic Acid</td><td>Sigma-Aldrich</td><td>399388</td><td>Used to TLC</td></tr><tr><td>Gas chromatography</td><td>Shimadzu Corporation</td><td>GC-2010 Plus</td><td>Used to SCFA detection</td></tr><tr><td>Glass beaker</td><td>Fisher Scientific</td><td>FB10050</td><td>Used for slurry preparation</td></tr><tr><td>Glucose</td><td>Solarbio</td><td>G8760</td><td>The component of medium</td></tr><tr><td>Haemin</td><td>Solarbio</td><td>H8130</td><td>The component of medium</td></tr><tr><td>HCl</td><td>Sigma-Aldrich</td><td>30721</td><td>Basic solution used to adjust the pH of the buffers</td></tr><tr><td>Isobutyric</td><td>Sigma-Aldrich</td><td>46935-U</td><td>Standard sample for SCFA</td></tr><tr><td>Isovaleric Acids</td><td>Sigma-Aldrich</td><td>129542</td><td>Standard sample for SCFA</td></tr><tr><td>K&lt;sub&gt;2&lt;/sub&gt;HPO&lt;sub&gt;4&lt;/sub&gt;</td><td>Solarbio</td><td>D9880</td><td>Salt solution of medium</td></tr><tr><td>KCl</td><td>Solarbio</td><td>P9921</td><td>The component of medium</td></tr><tr><td>KH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;</td><td>Solarbio</td><td>P7392</td><td>Salt solution of medium</td></tr><tr><td>LiCl.3H&lt;sub&gt;2&lt;/sub&gt;O</td><td>Solarbio</td><td>C8380</td><td>Use to prepare color plate</td></tr><tr><td>Meat Extract</td><td>Sigma-Aldrich-Aldrich</td><td>70164</td><td>The component of medium</td></tr><tr><td>Metaphosphoric Acid</td><td>Sigma-Aldrich</td><td>B7350</td><td>Standard sample for SCFA</td></tr><tr><td>MgCl&lt;sub&gt;2&lt;/sub&gt;.6H&lt;sub&gt;2&lt;/sub&gt;O</td><td>Solarbio</td><td>M8160</td><td>The component of medium</td></tr><tr><td>MgSO&lt;sub&gt;4&lt;/sub&gt;.7H&lt;sub&gt;2&lt;/sub&gt;O</td><td>Solarbio</td><td>M8300</td><td>Salt solution of medium</td></tr><tr><td>MISEQ</td><td>Illumina</td><td>MiSeq 300PE system</td><td>DNA sequencing</td></tr><tr><td>MnSO&lt;sub&gt;4&lt;/sub&gt;.H&lt;sub&gt;2&lt;/sub&gt;0</td><td>Sigma-Aldrich</td><td>M8179</td><td>Salt solution of medium</td></tr><tr><td>Mupirocin</td><td>Solarbio</td><td>YZ-1448901</td><td>Antibiotic</td></tr><tr><td>NaCl</td><td>Solarbio</td><td>YZ-100376</td><td>Salt solution of medium</td></tr><tr><td>NaHCO&lt;sub&gt;3&lt;/sub&gt;</td><td>Sigma-Aldrich</td><td>792519</td><td>Salt solution of medium</td></tr><tr><td>NanoDrop ND-2000</td><td>NanoDrop Technologies</td><td>ND-2000</td><td>Determine DNA concentrations</td></tr><tr><td>NaOH</td><td>Sigma-Aldrich</td><td>30620</td><td>Basic solution used to adjust the pH of the buffers</td></tr><tr><td>n-butanol</td><td>ChemSpider</td><td>71-36-3</td><td>Used to TLC</td></tr><tr><td>NiCl&lt;sub&gt;2&lt;/sub&gt;</td><td>Solarbio</td><td>746460</td><td>The component of medium</td></tr><tr><td>Orcinol</td><td>Sigma-Aldrich</td><td>447420</td><td>Used to prepare orcinol reagents</td></tr><tr><td>Propionic</td><td>Sigma-Aldrich</td><td>94425</td><td>Standard sample for SCFA</td></tr><tr><td>QIAamp DNA Stool Mini Kit</td><td>QIAGEN</td><td>51504</td><td>Extract bacterial genomic DNA</td></tr><tr><td>Ready-to-use PBS powder</td><td>Sangon Biotech (Shanghai) Co., Ltd.</td><td>A610100-0001</td><td>Used to prepare the lipid suspension</td></tr><tr><td>Resazurin</td><td>Solarbio</td><td>R8150</td><td>Anaerobic Equipment</td></tr><tr><td>Speed Vacuum Concentrator</td><td>LABCONCO</td><td>CentriVap</td><td>Use to prepare EPSs</td></tr><tr><td>Starch</td><td>Solarbio</td><td>YZ-140602</td><td>Use to the carbon source</td></tr><tr><td>Sulfuric Acid</td><td>Sigma-Aldrich</td><td>150692</td><td>Used to prepare orcinol reagents</td></tr><tr><td>T100 PCR</td><td>BIO-RAD</td><td>1861096</td><td>PCR amplification</td></tr><tr><td>TLC aluminium sheets</td><td>MerckMillipore</td><td>116835</td><td>Used to TLC</td></tr><tr><td>Trypticase Peptone</td><td>Sigma-Aldrich</td><td>Z699209</td><td>The component of medium</td></tr><tr><td>Tryptone</td><td>Sigma-Aldrich</td><td>T7293</td><td>The component of medium</td></tr><tr><td>Tween 80</td><td>Solarbio</td><td>T8360</td><td>Salt solution of medium</td></tr><tr><td>Valeric</td><td>Sigma-Aldrich</td><td>75054</td><td>Standard sample for SCFA</td></tr><tr><td>Vitamin K&lt;sub&gt;1&lt;/sub&gt;</td><td>Sigma-Aldrich</td><td>V3501</td><td>The component of medium</td></tr><tr><td>Vortex oscillator</td><td>Scientific Industries</td><td>Vortex.Genie2</td><td>Use to vortexing</td></tr><tr><td>Yeast Extract</td><td>Sigma-Aldrich</td><td>Y1625</td><td>The component of medium</td></tr><tr><td>ZnSO&lt;sub&gt;4&lt;/sub&gt;.7H&lt;sub&gt;2&lt;/sub&gt;O</td><td>Sigma-Aldrich</td><td>Z0251</td><td>The component of medium</td></tr></tbody></table>

analysis of interactions between endobiotics and human gut microbiota using in vitro bath 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.

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...

Watch this Scientific Journal Video about Analysis of Interactions between Endobiotics and Human Gut Microbiota Using In Vitro Bath Fermentation Systems at JoVE.com

Analysis of Interactions between Endobiotics and Human Gut Microbiota Using In Vitro Bath Fermentation Systems

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, ...

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Research

JoVE Journal

Immunology and Infection

6.9K Views.  Hunan University of Science and Engineering. Described here is a protocol to investigate the interactions between endobiotics and human gut microbiota using in vitro batch fermentation systems.

Video: Analysis of Interactions between Endobiotics and Human Gut Microbiota Using In Vitro Bath Fermentation Systems

Human T Lymphocyte Isolation, Culture and Analysis of Migration In Vitro

The migration of T lymphocytes involves the adhesive interaction of cell surface integrins with ligands expressed on other cells or with extracellular matrix proteins. The precise spatiotemporal activation of integrins from a low affinity state to a high affinity state at the cell leading edge is important for T lymphocyte migration 1. Likewise, retraction of the cell trailing edge, or uropod, is a necessary step in maintaining persistent integrin-dependent T lymphocyte motility 2. Many therapeutic approaches to autoimmune or inflammatory diseases target integrins as a means to inhibit the excessive recruitment and migration of leukocytes 3. To study the molecular events that regulate human T lymphocyte migration, we have utilized an in vitro system to analyze cell migration on a two-dimensional substrate that mimics the environment that a T lymphocyte encounters during recruitment from the vasculature. T lymphocytes are first isolated from human donors and are then stimulated and cultured for seven to ten days. During the assay, T lymphocytes are allowed to adhere and migrate on a substrate coated with intercellular adhesion molecule-1 (ICAM-1), a ligand for integrin LFA-1, and stromal cell-derived factor-1 (SDF-1). Our data show that T lymphocytes exhibit a migratory velocity of ~15 &mu;m/min. T lymphocyte migration can be inhibited by integrin blockade 1 or by inhibitors of the cellular actomyosin machinery that regulates cell migration 2.

Human T Lymphocyte Isolation, Culture and Analysis of Migration In Vitro

T lymphocyte migration occurs during homing to lymphoid organs, exit from the vasculature, and entering into peripheral tissues. Here, we describe a protocol that can be used to analyze T lymphocyte migration in vitro.

The migration of T lymphocytes involves the adhesive interaction of cell surface integrins with ligands expressed on other cells or with extracellular ...

Invasion of Human Cells by a Bacterial Pathogen

Here we will describe how we study the invasion of human endothelial cells by bacterial pathogen Staphylococcus aureus . The general protocol can be applied to the study of cell invasion by virtually any culturable bacterium. The stages at which specific aspects of invasion can be studied, such as the role of actin rearrangement or caveolae, will be highlighted. Host cells are grown in flasks and when ready for use are seeded into 24-well plates containing Thermanox coverslips. Using coverslips allows subsequent removal of the cells from the wells to reduce interference from serum proteins deposited onto the sides of the wells (to which S. aureus would attach). Bacteria are grown to the required density and washed to remove any secreted proteins (e.g. toxins). Coverslips with confluent layers of endothelial cells are transferred to new 24-well plates containing fresh culture medium before the addition of bacteria. Bacteria and cells are then incubated together for the required amount of time in 5% CO2 at 37&deg;C. For S. aureus this is typically between 15-90 minutes. Thermanox coverslips are removed from each well and dip-washed in PBS to remove unattached bacteria. If total associated bacteria (adherent and internalised) are to be quantified, coverslips are then placed in a fresh well containing 0.5% Triton X-100 in PBS. Gentle pipetting leads to complete cell lysis and bacteria are enumerated by serial dilution and plating onto agar. If the number of bacteria that have invaded the cells is needed, coverslips are added to wells containing 500 &mu;l tissue culture medium supplemented with gentamicin and incubation continued for 1 h, which will kill all external bacteria. Coverslips can then be washed, cells lysed and bacteria enumerated by plating onto agar as described above. If the experiment requires direct visualisation, coverslips can be fixed and stained for light, fluorescence or confocal microscopy or prepared for electron microscopy.

A general protocol for the study of invasion of host cells by a bacterial pathogen, focusing on Staphylococcus aureus and human endothelial cells.

Here we will describe how we study the invasion of human endothelial cells by bacterial pathogen Staphylococcus aureus . The general protocol can be ...

Assessing the Viability of a Synthetic Bacterial Consortium on the In Vitro Gut Host-microbe Interface

The interplay between host and microbiota has been long recognized and extensively described. The mouth is similar to other sections of the gastrointestinal tract, as resident microbiota occurs and prevents colonisation by exogenous bacteria. Indeed, more than 600 species of bacteria are found in the oral cavity, and a single individual may carry around 100 different at any time. Oral bacteria possess the ability to adhere to the various niches in the oral ecosystem, thus becoming integrated within the resident microbial communities, and favouring growth and survival. However, the flow of bacteria into the gut during swallowing has been proposed to disturb the balance of the gut microbiota. In fact, oral administration of P. gingivalis shifted bacterial composition in the ileal microflora. We used a synthetic community as a simplified representation of the natural oral ecosystem, to elucidate the survival and viability of oral bacteria subjected to simulated gastrointestinal transit conditions. Fourteen species were selected, subjected to in vitro salivary, gastric, and intestinal digestion processes, and presented to a multicompartment cell model containing Caco-2 and HT29-MTX cells to simulate the gut mucosal epithelium. This model served to unravel the impact of swallowed bacteria on cells involved in the enterohepatic circulation. Using synthetic communities allows for controllability and reproducibility. Thus, this methodology can be adapted to assess pathogen viability and subsequent inflammation-associated changes, colonization capacity of probiotic mixtures, and ultimately, potential bacterial impact on the presystemic circulation.

Assessing the Viability of a Synthetic Bacterial Consortium on the In Vitro Gut Host-microbe Interface

Gut host-microbe interactions were assessed using a novel approach combining a synthetic oral community, in vitro gastrointestinal digestion, and a model of the small intestine epithelium. We present a method that can be adapted to evaluate cell invasion of pathogens and multi-species biofilms, or even to test probiotic formulations&#39; survivability.

The interplay between host and microbiota has been long recognized and extensively described. The mouth is similar to other sections of the ...

Biochemistry

A Novel In vitro Model for Studying the Interactions Between Human Whole Blood and Endothelium

The majority of all known diseases are accompanied by disorders of the cardiovascular system. Studies into the complexity of the interacting pathways activated during cardiovascular pathologies are, however, limited by the lack of robust and physiologically relevant methods. In order to model pathological vascular events we have developed an in vitro assay for studying the interaction between endothelium and whole blood. The assay consists of primary human endothelial cells, which are placed in contact with human whole blood. The method utilizes native blood with no or very little anticoagulant, enabling study of delicate interactions between molecular and cellular components present in a blood vessel.
We investigated functionality of the assay by comparing activation of coagulation by different blood volumes incubated with or without human umbilical vein endothelial cells (HUVEC). Whereas a larger blood volume contributed to an increase in the formation of thrombin antithrombin (TAT) complexes, presence of HUVEC resulted in reduced activation of coagulation. Furthermore, we applied image analysis of leukocyte attachment to HUVEC stimulated with tumor necrosis factor (TNF&#945;) and found the presence of CD16+ cells to be significantly higher on TNF&#945; stimulated cells as compared to unstimulated cells after blood contact. In conclusion, the assay may be applied to study vascular pathologies, where interactions between the endothelium and the blood compartment are perturbed.

A Novel In vitro Model for Studying the Interactions Between Human Whole Blood and Endothelium

The accessibility of reliable models to investigate vascular blood interactions in humans is lacking. We present an in vitro model of cultured primary human endothelial cells combined with human whole blood to investigate cellular interactions both in the blood (ELISA) and the vascular compartment (microscopy).

The majority of all known diseases are accompanied by disorders of the cardiovascular system. Studies into the complexity of the interacting pathways ...

Co-culture of Living Microbiome with Microengineered Human Intestinal Villi in a Gut-on-a-Chip Microfluidic Device

Here, we describe a protocol to perform long-term co-culture of multi-species human gut microbiome with microengineered intestinal villi in a human gut-on-a-chip microphysiological device. We recapitulate the intestinal lumen-capillary tissue interface in a microfluidic device, where physiological mechanical deformations and fluid shear flow are constantly applied to mimic peristalsis. In the lumen microchannel, human intestinal epithelial Caco-2 cells are cultured to form a &#39;germ-free&#39; villus epithelium and regenerate small intestinal villi. Pre-cultured microbial cells are inoculated into the lumen side to establish a host-microbe ecosystem. After microbial cells adhere to the apical surface of the villi, fluid flow and mechanical deformations are resumed to produce a steady-state microenvironment in which fresh culture medium is constantly supplied and unbound bacteria (as well as bacterial wastes) are continuously removed. After extended co-culture from days to weeks, multiple microcolonies are found to be randomly located between the villi, and both microbial and epithelial cells remain viable and functional for at least one week in culture. Our co-culture protocol can be adapted to provide a versatile platform for other host-microbiome ecosystems that can be found in various human organs, which may facilitate in vitro study of the role of human microbiome in orchestrating health and disease.

We describe an in vitro protocol to co-culture gut microbiome and intestinal villi for an extended period using a human gut-on-a-chip microphysiological system.

Here, we describe a protocol to perform long-term co-culture of multi-species human gut microbiome with microengineered intestinal villi in a human ...

Bioengineering

Applying Advanced In Vitro Culturing Technology to Study the Human Gut Microbiota

The human gut microbiota plays a vital role in both human health and disease. Studying the gut microbiota using an in vivo model, is difficult due to its complex nature, and its diverse association with mammalian components. The goal of this protocol is to culture the gut microbiota in vitro, which allows for the study of the gut microbiota dynamics, without having to consider the contribution of the mammalian milieu. Using in vitro culturing technology, the physiological conditions of the gastro intestinal tract are simulated, including parameters such as pH, temperature, anaerobiosis, and transit time. The intestinal surface of the colon is simulated by adding mucin-coated carriers, creating a mucosal phase, and adding further dimension. The gut microbiota is introduced by inoculating with the human fecal material. Upon inoculation with this complex mixture of bacteria, specific microbes are enriched in the different longitudinal (ascending, transverse and descending colons) and transversal (luminal and mucosal) environments of the in vitro model. It is crucial to allow the system to reach a steady state, in which the community and the metabolites produced remain stable. The experimental results in this manuscript demonstrate how the inoculated gut microbiota community develops into a stable community over time. Once steady state is achieved, the system can be used to analyze bacterial interactions and community functions or to test the effects of any additives on the gut microbiota, such as food, food components, or pharmaceuticals.

Here, we present a protocol for culturing the gut microbiota of the colon in vitro, using a series of bioreactors that simulate the physiological conditions of the gastro intestinal tract.

The human gut microbiota plays a vital role in both human health and disease. Studying the gut microbiota using an in vivo model, is difficult due to its ...

An In Vitro Batch-culture Model to Estimate the Effects of Interventional Regimens on Human Fecal Microbiota

The emerging role of the gut microbiome in several human diseases demands a breakthrough of new tools, techniques and technologies. Such improvements are needed to decipher the utilization of microbiome modulators for human health benefits. However, the large-scale screening and optimization of modulators to validate microbiome modulation and predict related health benefits may be practically difficult due to the need for large number of animals and/or human subjects. To this end, in vitro or ex vivo models can facilitate preliminary screening of microbiome modulators. Herein, it is optimized and demonstrated an ex vivo fecal microbiota culture system that can be used for examining the effects of various interventions of gut microbiome modulators including probiotics, prebiotics and other food ingredients, aside from nutraceuticals and drugs, on the diversity and composition of the human gut microbiota. Inulin, one of the most widely studied prebiotic compounds and microbiome modulators, is used as an example here to examine its effect on the healthy fecal microbiota composition and its metabolic activities, such as fecal pH and the fecal levels of organic acids including lactate and short-chain fatty acids (SCFAs). The protocol may be useful for studies aimed at estimating the effects of different interventions of modulators on fecal microbiota profiles and at predicting their health impacts.

This protocol describes an in vitro batch-culture fermentation system of human fecal microbiota, using inulin (a well-known prebiotic and one of the most widely studied microbiota modulators) to demonstrate the use of this system in estimating effects of specific interventions on fecal microbiota composition and metabolic activities.

The emerging role of the gut microbiome in several human diseases demands a breakthrough of new tools, techniques and technologies. Such improvements are ...

An Intestinal Gut Organ Culture System for Analyzing Host-Microbiota Interactions

The structure of the gut tissue facilitates close and mutualistic interactions between the host and the gut microbiota. These cross-talks are crucial for maintaining local and systemic homeostasis; changes to gut microbiota composition (dysbiosis) associate with a wide array of human diseases. Methods for dissecting host-microbiota interactions encompass an inherent tradeoff among preservation of physiological tissue structure (when using in vivo animal models) and the level of control over the experiment factors (as in simple in vitro cell culture systems). To address this tradeoff, Yissachar et al. recently developed an intestinal organ culture system. The system preserves a naive colon tissue construction and cellular mechanisms and it also permits tight experimental control, facilitating experimentations that cannot be readily performed in vivo. It is optimal for dissecting short-term responses of various gut components (such as epithelial, immunological and neuronal elements) to luminal perturbations (including anaerobic or aerobic microbes, whole microbiota samples from mice or humans, drugs and metabolites). Here, we present a detailed description of an optimized protocol for organ culture of multiple gut fragments using a custom-made gut culture device. Host responses to luminal perturbations can be visualized by immunofluorescence staining of tissue sections or whole-mount tissue fragments, fluorescence in-situ hybridization (FISH), or time-lapse imaging. This system supports a wide array of readouts, including next-generation sequencing, flow cytometry, and various cellular and biochemical assays. Overall, this three-dimensional organ culture system supports the culture of large, intact intestinal tissues and has broad applications for high-resolution analysis and visualization of host-microbiota interactions in the local gut environment.

This article presents a unique method for analyzing host-microbiome interactions using a novel gut organ culture system for ex vivo experiments.

The structure of the gut tissue facilitates close and mutualistic interactions between the host and the gut microbiota. These cross-talks are crucial for ...

Bioreactor Assembly for Continuous Culture of Complex Fecal Communities

The microbiota, especially bacteria, respond to various environmental exposures such as micro and macronutrients, pharmacological compounds, and inflammatory mediators, which dynamically alter community composition and microbial metabolic output. Understanding how physiological culture conditions affect microbial communities and diversity and their metabolic capacity will contribute important knowledge of their impact on health and diseases. Here, we present a protocol adapted from the template published by Auchtung et al. to create mini-bioreactor arrays that can cultivate complex fecal communities, define bacterial consortiums or single strains under precise conditions, including nutrient availability, temperature, flow rate, pH, and oxygen content. We describe the process for building the mini-bioreactor system, including adaptations to improve limitations in the published protocol. We also discuss the setup of the mini-bioreactor system under anaerobic conditions with the use of MEGA media (adapted from Han et al.), which is a rich media supporting the growth of diverse bacteria. We describe the inoculation of gut humanized mouse fecal samples into the mini-bioreactor system, followed by the establishment of complex community cultures within the mini-bioreactor system, which can be grown under continuous flow with aseptic sampling to monitor community composition. This system is adaptable to dietary changes and other cultural modifications. The techniques described here allow for the characterization of diverse, fastidious fecal communities under dynamic conditions or in response to perturbation in isolation from host-derived factors.

This protocol details the assembly of mini-bioreactor arrays to be utilized for continuous flow culture of complex fecal communities under anaerobic conditions. We also discuss the methods for assembly, inoculation, and sampling of the reactors for further analysis.

The microbiota, especially bacteria, respond to various environmental exposures such as micro and macronutrients, pharmacological compounds, and ...

An Ex Vivo Gut Organ Culture System to Study Host-Microbiota Interactions

To begin, insert the blunt-end needles to the appropriate position within the device mold and cast approximately 20g of polydimethylsiloxane or PDMS mix for one set of the device and lid. Place the molds in a vacuum chamber for 30 minutes to remove air bubbles from the PDMS mix, then, incubate the molds at 55 degrees Celsius overnight to complete PDMS polymerization.
When the PDMS is set, pull out the needles from the mold and carefully release the culture device and lid from the plastic molds. Remove PDMS residues from the well outline using a surgical blade.
Attach PDMS device in the device cover onto a cover glass of 75 by 50-millimeter microslides using non-toxic silicone adhesive, and leave the parts to set overnight. Apply the glue to the smooth side of the device. Insert 12 22-gauge needles for the lumen and 12 18-gauge needles for the well. Fix all the needles in place using silicone and let it set overnight.
Purge the input syringes, and make sure that the well medium flows out from all tubes into a waste glass. Then, purge the input syringes, and make sure that the stimulations flow out of all the tubes into a waste glass, taking care to not contaminate the different stimulations.
After sacrificing the mouse, use sharp scissors and forceps to dissect it, and take out the digestive tract from the stomach to the anus by cutting all the fat and connective tissues. Cut the colon and place it on a new plate. Minimize contact while holding the tissue gently and only at the edges. Perform the colon flush under the dissection microscope.
Gently flush the colon content with sterile IMDM with the prepared 10-milliliter syringe as described in the text. After removing the feces from the intestinal tissue, place the colon in a new 6-well plate filled with 0.5 milliliters of sterile IMDM. Next, take the colon tissue and carefully connect it to the 22-gauge needle, making a tight tie with the two threads.
Maintain the correct orientation of the colon to the lumen flow such that the proximal and distal is equal to input and output respectively. Repeat colon flushing and needle tying for all the tissues. Connect the input and output tubes to the device, then begin the experiment by starting the pumps at the desired rates.

Analysis of Interactions between Endobiotics and Human Gut Microbiota Using In Vitro Bath Fermentation Systems

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Human T Lymphocyte Isolation, Culture and Analysis of Migration In Vitro

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Co-culture of Living Microbiome with Microengineered Human Intestinal Villi in a Gut-on-a-Chip Microfluidic Device

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System hodowli narządów jelitowych ex vivo do badania interakcji między gospodarzem a mikrobiotą