1. Exopolysaccharide (EPS) degradation by human fecal microbiota<ol><li>Load 0.2 &#956;L of fermented supernatants onto pre-coated silica gel-60 thin-layer chromatography (TLC) aluminum plates, then dry using a hair dryer.</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 dryer.</li><li>Soak the plates in the orcinol reagent to dye, then dry using a hair dryer.<ol style='list-style-type:decimal;'><li>Prepare orcinol reagents by dissolving 900 mg of Lichenol in 25 mL of distilled water, then adding 375 mL of ethanol. As a result, 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>

<table class='ck-table-resized'><tbody><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>Ethanol</td><td>Sigma-Aldrich</td><td>E7023</td><td>Use to prepare vitamin K1</td></tr><tr><td>Formic acid</td><td>Sigma-Aldrich</td><td>399388</td><td>Used for TLC</td></tr><tr><td>Lichenol</td><td>Solarbio</td><td>C8380</td><td>Use to prepare color plate</td></tr><tr><td>n-Butanol</td><td>ChemSpider</td><td>71-36-3</td><td>Used for TLC</td></tr><tr><td>Orcinol</td><td>Sigma-Aldrich</td><td>447420</td><td>Used to prepare orcinol reagents</td></tr><tr><td>Sulfuric acid</td><td>Sigma-Aldrich</td><td>150692</td><td>Used to prepare orcinol reagents</td></tr><tr><td>TLC aluminium sheets</td><td>MerckMillipore</td><td>116835</td><td>Used for TLC</td></tr></tbody></table>

performing thin-layer chromatography to assess bacterial exopolysaccharide degradation

Performing Thin-Layer Chromatography to Assess Bacterial Exopolysaccharide Degradation

Source: <a href='https://app.jove.com/v/59725/analysis-interactions-between-endobiotics-human-gut-microbiota-using'>Hu, Y., et al. Analysis of Interactions between Endobiotics and Human Gut Microbiota Using In Vitro Bath Fermentation Systems. J. Vis. Exp. (2019)</a>This video demonstrates the use of thin-layer chromatography (TLC) to assess the degradation of exopolysaccharides (EPS) by human fecal microbiota. EPS fragments in fermentation supernatants are separated based on size and polarity using TLC, then visualized as colored bands following carbohydrate-specific staining.

Source: Hu, Y., et al. Analysis of Interactions between Endobiotics and Human Gut Microbiota Using In Vitro Bath Fermentation Systems. J. Vis. Exp. ...

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Start with fermented supernatants containing degraded and undegraded exopolysaccharides, or EPS.These EPS are obtained during fermentation, where distinct fecal bacterial populations in each supernatant act on EPS differently.Spot the supernatants near the base of a thin-layer chromatography plate pre-coated with a polar stationary phase.Air-dry the plate, then place it in a chamber containing a mobile phase with lower polarity than the stationary phase.As the mobile phase rises, EPS migrate based on their polarity and size.Large, undegraded EPS interact strongly with the stationary phase and remain near the origin.Smaller degraded fragments bind weakly and migrate upward with the mobile phase.Remove the plate and air-dry it.Immerse the plate in a carbohydrate-staining reagent that reacts with EPS.Air-dry the plate, then heat it to dehydrate the EPS and form colored bands.A single band near the origin confirms undegraded EPS, while multiple bands confirm EPS degradation by bacteria.Load 0.2 microliters of the fermented supernatants onto each pre-coated silica gel 60 TLC aluminum plate and swirl to spread. Then, use a hair dryer with hot air to dry.Immerse one end of the sampled plates in 20 milliliters of formic acid:n-butanol:water solution for several minutes, until the electrophoresis extends almost to the other end. Then take out the plates with forceps, and dry with a hair dryer. Then, soak the plates in the orcinol reagent for two minutes to dye and dry again.Transfer the plates to a baking oven at 120 degrees Celsius to heat for three minutes. Then take pictures of the plate to evaluate degradation of EPS by measuring TLC bands.

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138 Views. Start with fermented supernatants containing degraded and undegraded exopolysaccharides, or EPS.These EPS are obtained during fermentation, where distinct fecal bacterial populations in each supernatant act on EPS differently.Spot the supernatants near the base of a thin-layer chromatography plate pre-coated with a polar stationary phase.Air-dry the plate, then place it in a chamber containing a mobile phase with lower polarity than the stationary phase.As the mobi...

Performing Thin-Layer Chromatography to Assess Bacterial Exopolysaccharide Degradation

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Name	Company	Catalog Number	Comments
Baking oven	Shanghai Yiheng Scientific Instruments Co., Ltd	DHG-9240A	Use to heat and bake
Ethanol	Sigma-Aldrich	E7023	Use to prepare vitamin K1
Formic acid	Sigma-Aldrich	399388	Used for TLC
Lichenol	Solarbio	C8380	Use to prepare color plate
n-Butanol	ChemSpider	71-36-3	Used for TLC
Orcinol	Sigma-Aldrich	447420	Used to prepare orcinol reagents
Sulfuric acid	Sigma-Aldrich	150692	Used to prepare orcinol reagents
TLC aluminium sheets	MerckMillipore	116835	Used for TLC