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 &#181;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 &#946;-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&#160;</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>Bifidobacterium longum Reuter</td>
			<td>ATCC</td>
			<td>ATCC&#174; 51870&#8482;</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>CaCl2</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 &#215; 30 m &#215; 0.25 &#956;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>CoSO4.7H2O</td>
			<td>Solarbio</td>
			<td>C7490</td>
			<td>The component of medium</td>
		</tr>
		<tr>
			<td>CuSO4.5H2O</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 K1</td>
		</tr>
		<tr>
			<td>FeSO4.7H2O</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>K2HPO4</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>KH2PO4</td>
			<td>Solarbio</td>
			<td>P7392</td>
			<td>Salt solution of medium</td>
		</tr>
		<tr>
			<td>LiCl.3H2O</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>MgCl2.6H2O</td>
			<td>Solarbio</td>
			<td>M8160</td>
			<td>The component of medium</td>
		</tr>
		<tr>
			<td>MgSO4.7H2O</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>MnSO4.H20</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>NaHCO3</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>NiCl2</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 K1</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>ZnSO4.7H2O</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, ...

analysis-interactions-between-endobiotics-human-gut-microbiota-using

Universidad de Cartagena UDEC

Research

JoVE Journal

Immunology and Infection

6.8K 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 ...

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 ...

A New Method for Qualitative Multi-scale Analysis of Bacterial Biofilms on Filamentous Fungal Colonies Using Confocal and Electron Microscopy

Bacterial biofilms frequently form on fungal surfaces and can be involved in numerous bacterial-fungal interaction processes, such as metabolic cooperation, competition, or predation. The study of biofilms is important in many biological fields, including environmental science, food production, and medicine. However, few studies have focused on such bacterial biofilms, partially due to the difficulty of investigating them. Most of the methods for qualitative and quantitative biofilm analyses described in the literature are only suitable for biofilms forming on abiotic surfaces or on homogeneous and thin biotic surfaces, such as a monolayer of epithelial cells.
While laser scanning confocal microscopy (LSCM) is often used to analyze in situ and in vivo biofilms, this technology becomes very challenging when applied to bacterial biofilms on fungal hyphae, due to the thickness and the three dimensions of the hyphal networks. To overcome this shortcoming, we developed a protocol combining microscopy with a method to limit the accumulation of hyphal layers in fungal colonies. Using this method, we were able to investigate the development of bacterial biofilms on fungal hyphae at multiple scales using both LSCM and scanning electron microscopy (SEM). This report describes the protocol, including microorganism cultures, bacterial biofilm formation conditions, biofilm staining, and LSCM and SEM visualizations.

This protocol describes a new method to grow and qualitatively analyze bacterial biofilms on fungal hyphae by confocal and electron microscopy.

Bacterial biofilms frequently form on fungal surfaces and can be involved in numerous bacterial-fungal interaction processes, such as metabolic ...

Analysis of Lymphocyte Extravasation Using an In Vitro Model of the Human Blood-brain Barrier

Lymphocyte extravasation into the central nervous system (CNS) is critical for immune surveillance. Disease-related alterations of lymphocyte extravasation might result in pathophysiological changes in the CNS. Thus, investigation of lymphocyte migration into the CNS is important to understand inflammatory CNS diseases and to develop new therapy approaches. Here we present an in vitro model of the human blood-brain barrier to study lymphocyte extravasation. Human brain microvascular endothelial cells (HBMEC) are confluently grown on a porous polyethylene terephthalate transwell insert to mimic the endothelium of the blood-brain barrier. Barrier function is validated by zonula occludens immunohistochemistry, transendothelial electrical resistance (TEER) measurements as well as analysis of evans blue permeation. This model allows investigation of the diapedesis of rare lymphocyte subsets such as CD56brightCD16dim/- NK cells. Furthermore, the effects of other cells, cytokines and chemokines, disease-related alterations, and distinct treatment regimens on the migratory capacity of lymphocytes can be studied. Finally, the impact of inflammatory stimuli as well as different treatment regimens on the endothelial barrier can be analyzed.

Analysis of Lymphocyte Extravasation Using an In Vitro Model of the Human Blood-brain Barrier

Here, we describe a human blood-brain barrier model enabling to investigate lymphocyte transmigration into the central nervous system in vitro.

Lymphocyte extravasation into the central nervous system (CNS) is critical for immune surveillance. Disease-related alterations of lymphocyte ...

In Vitro Methods for Comparing Target Binding and CDC Induction Between Therapeutic Antibodies: Applications in Biosimilarity Analysis

Therapeutic monoclonal antibodies (mAbs) are relevant to the treatment of different pathologies, including cancers. The development of biosimilar mAbs by pharmaceutical companies is a market opportunity, but it is also a strategy to increase drug accessibility and reduce therapy-associated costs. The protocols detailed here describe the evaluation of target binding and CDC induction by rituximab in Daudi cells. These two functions require different structural regions of the antibody and are relevant to the clinical effect induced by rituximab. The protocols allow the side-to-side comparison of a reference rituximab and a marketed rituximab biosimilar. The evaluated products showed differences both in target binding and CDC induction, suggesting that there are underlying physicochemical differences and highlighting the need to analyze the impact of those differences in the clinical setting. The methods reported here constitute simple and inexpensive in vitro models for the evaluation of the activity of rituximab biosimilars. Thus, they can be useful during biosimilar development, as well as for quality control in biosimilar production. Furthermore, the presented methods can be extrapolated to other therapeutic mAbs.

In Vitro Methods for Comparing Target Binding and CDC Induction Between Therapeutic Antibodies: Applications in Biosimilarity Analysis

This protocol describes the in vitro comparison of two key functional characteristics of rituximab: target binding and complement-dependent cytotoxicity (CDC) induction. The methods were employed for a side-to-side comparison between reference rituximab and a rituximab biosimilar. These assays can be employed during biosimilar development or as a quality control in their production.

Therapeutic monoclonal antibodies (mAbs) are relevant to the treatment of different pathologies, including cancers. The development of biosimilar mAbs by ...

Human Colonoid Monolayers to Study Interactions Between Pathogens, Commensals, and Host Intestinal Epithelium

Human 3-dimensional (3D) enteroid or colonoid cultures derived from crypt base stem cells are currently the most advanced ex vivo model of the intestinal epithelium. Due to their closed structures and significant supporting extracellular matrix, 3D cultures are not ideal for host-pathogen studies. Enteroids or colonoids can be grown as epithelial monolayers on permeable tissue culture membranes to allow manipulation of both luminal and basolateral cell surfaces and accompanying fluids. This enhanced luminal surface accessibility facilitates modeling bacterial-host epithelial interactions such as the mucus-degrading ability of enterohemorrhagic E. coli (EHEC) on colonic epithelium. A method for 3D culture fragmentation, monolayer seeding, and transepithelial electrical resistance (TER) measurements to monitor the progress towards confluency and differentiation are described. Colonoid monolayer differentiation yields secreted mucus that can be studied by the immunofluorescence or immunoblotting techniques. More generally, enteroid or colonoid monolayers enable a physiologically-relevant platform to evaluate specific cell populations that may be targeted by pathogenic or commensal microbiota.

Here, we present a protocol to culture human enteroid or colonoid monolayers that have intact barrier function to study host epithelial-microbiota interactions at the cellular and biochemical level.

Human 3-dimensional (3D) enteroid or colonoid cultures derived from crypt base stem cells are currently the most advanced ex vivo model of the intestinal ...

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 ...

Analysis of Group IV Viral SSHHPS Using In Vitro and In Silico Methods

Alphaviral enzymes are synthesized in a single polypeptide. The nonstructural polyprotein (nsP) is processed by its nsP2 cysteine protease to produce active enzymes essential for viral replication. Viral proteases are highly specific and recognize conserved cleavage site motif sequences (~6-8 amino acids). In several Group IV viruses, the nsP protease(s) cleavage site motif sequences can be found in specific host proteins involved in generating the innate immune responses and, in some cases, the targeted proteins appear to be linked to the virus-induced phenotype. These viruses utilize short stretches of homologous host-pathogen protein sequences (SSHHPS) for targeted destruction of host proteins. To identify SSHHPS the viral protease cleavage site motif sequences can be inputted into BLAST and the host genome(s) can be searched. Cleavage initially can be tested using the purified nsP viral protease and fluorescence resonance energy transfer (FRET) substrates made in E. coli. The FRET substrates contain cyan and yellow fluorescent protein and the cleavage site sequence (CFP-sequence-YFP). This protease assay can be used continuously in a plate reader or discontinuously in SDS-PAGE gels. Models of the bound peptide substrates can be generated in silico to guide substrate selection and mutagenesis studies. CFP/YFP substrates have also been utilized to identify protease inhibitors. These in vitro and in silico methods can be used in combination with cell-based assays to determine if the targeted host protein affects viral replication.

We present a general protocol for identifying short stretches of homologous host-pathogen protein sequences (SSHHPS) embedded in the viral polyprotein. SSHHPS are recognized by viral proteases and direct the targeted destruction of specific host proteins by several Group IV viruses.

Alphaviral enzymes are synthesized in a single polypeptide. The nonstructural polyprotein (nsP) is processed by its nsP2 cysteine protease to produce ...

Analysis of SAMHD1 Restriction by Flow Cytometry in Human Myeloid U937 Cells

Sterile &#945;-motif/histidine-aspartate domain-containing protein 1 (SAMHD1) inhibits replication of HIV-1 in quiescent myeloid cells. U937 cells are widely used as a convenient cell system for analyzing SAMHD1 activity due to a low level of SAMHD1 RNA expression, leading to undetectable endogenous protein expression. Based on similar assays developed in the Stoye laboratory to characterize other retroviral restriction factors, the Bishop lab developed a two-color restriction assay to analyze SAMHD1 in U937 cells. Murine Leukaemia Virus-like particles expressing SAMHD1, alongside YFP expressed from an IRES, are used to transduce U937 cells. Cells are then treated with phorbol myristate acetate to induce differentiation to a quiescent phenotype. Following differentiation, cells are infected with HIV-1 virus-like particles expressing a fluorescent reporter. After 48 h, cells are harvested and analyzed by flow cytometry. The proportion of HIV-infected cells in the SAMHD1-expressing population is compared to that in internal control cells lacking SAMHD1. This comparison reveals a restriction ratio. SAMHD1 expression leads to a five-fold reduction in HIV infection, corresponding to a restriction ratio of 0.2. Our recent substitution of RFP for the original GFP as the reporter gene for HIV infection has facilitated flow cytometry analysis.
This assay has been successfully used to characterize the effect of amino acid substitutions on SAMHD1 restriction by transducing with viruses encoding altered SAMHD1 proteins, derived from site-directed mutagenesis of the expression vector. For example, the catalytic site substitutions HD206-7AA show a restriction phenotype of 1, indicating a loss of restriction activity. Equally, the susceptibility of different tester viruses can be determined. The assay can be further adapted to incorporate the effect of differentiation status, metabolic status, and SAMHD1 modifiers to better understand the relationship between SAMHD1, cell metabolic state, and viral restriction.

Described here is an established method to determine the extent of HIV-1 restriction by the cellular inhibitory protein SAMHD1. Human myeloid lineage U937 cells are transduced with a SAMHD1 expression vector co-expressing YFP, differentiated and then challenged with HIV-RFP. The level of restriction is determined by flow cytometry analysis.

Sterile &#945;-motif/histidine-aspartate domain-containing protein 1 (SAMHD1) inhibits replication of HIV-1 in quiescent myeloid cells. U937 cells are ...

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Analysis of SAMHD1 Restriction by Flow Cytometry in Human Myeloid U937 Cells