Investigating the Human Gut Microbial Activity on Exopolysaccharides

Samenvatting

Source: Hu, Y. et.al., Analysis of Interactions between Endobiotics and Human Gut Microbiota Using In Vitro Bath Fermentation Systems. J. Vis. Exp. (2019)

This video demonstrates the analysis of short-chain fatty acid production by human gut microbiota in response to exopolysaccharides. This highlights microbial fermentation products separation and quantification using gas chromatography. The results reveal that exopolysaccharides modulate microbial metabolites that influence host-pathogen dynamics in the gut.

Protocol

1. Effects of exopolysaccharide (EPS) on short-chain fatty acid (SCFA) production by human intestinal microbiota
   1. Add fermented supernatants (1 mL) prepared in step 5.3 to 2 mL centrifuge tubes.
   2. Add 0.2 mL of 25% (w/v) metaphosphoric acid to each of the samples and thoroughly mix the solutions by vortexing.
   3. Centrifuge the mixtures at 13,000 x g for 20 min and transfer the supernatants to fresh tubes.
   4. 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.
   5. Filter the samples using a 0.22 μM membrane.
   6. Detect SCFA concentrations using high-performance gas chromatography.
      NOTE: An InertCap FFAP column (0.25 mM x 30 m x 0.25 μM) 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.

Materialen

Lijst van materialen gebruikt in dit artikel

Naam	Bedrijf	Catalogusnummer	Opmerkingen
0.22 µm membrane filters	Millipore	SLGP033RB	Use to filter samples
0.4-mm Sieve	Thermo Fischer	308080-99-1	Use to prepare human fecal samples
Acetic	Sigma-Aldrich	71251	Standard sample for SCFA
Butyric	Sigma-Aldrich	19215	Standard sample for SCFA
Capillary column	SHIMADZU-GL	InertCap FFAP (0.25 mm × 30 m × 0.25 μm)	Used to SCFA detection
Gas chromatography	Shimadzu Corporation	GC-2010 Plus	Used to SCFA detection
Isobutyric	Sigma-Aldrich	46935-U	Standard sample for SCFA
Isovaleric Acids	Sigma-Aldrich	129542	Standard sample for SCFA
Metaphosphoric Acid	Sigma-Aldrich	B7350	Standard sample for SCFA
NaOH	Sigma-Aldrich	30620	Basic solution used to adjust the pH of the buffers
Propionic	Sigma-Aldrich	94425	Standard sample for SCFA
Valeric	Sigma-Aldrich	75054	Standard sample for SCFA