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

Our protocol can be widely used for high-throughput and rapid screening of the interactions between endobiotics and human gut microbiota. Low cost and avoidance of interference effects from the host's metabolism. This procedure has the potential to be used in diagnosis.

First, dissolve BIF EPS and starch individually in hot water on a magnetic agitator to make both the concentration of eight grams per liter. In the prepared basic culture medium, VI, add 100 milliliters of the BIF EPS solution to make culture VI BIF solution. Add 100 milliliters of the starch solution to make culture VI starch solution.

Autoclave the two media with carbon source, and the medium without carbon source as a control, at 121 degrees Celsius for 15 minutes. Allow them to cool to room temperature and add haymen and cysteine. Transfer a sub-sample of five milliliters of each medium to culture tubes.

And store in an anaerobic incubator. Store the remaining media at four degrees Celsius. To prepare human fecal sample, transfer one gram of fresh fecal sample into 10 milliliters of 0.1 molar anaerobic PBS at pH seven in a glass beaker.

Then use a glass rod to stir it to achieve a slurry. In an anaerobic chamber, add 500 microliters of the filtered fecal slurry into the three prepared fermentation media, individually. Then, incubate at 37 degrees Celsius.

After 24 hours and 48 hours, collect two milliliters of fermented samples in the anaerobic chamber. And then centrifuge outside of the chamber at 6000 times G, for three minutes. Carefully transfer the supernatants to new centrifuge tubes that will be used for polysaccharide degradation analysis and short-chain fatty acid measurements.

Store the centrifugation pellets at 80 degrees Celsius. 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.

To study the effects of EPS on SCFA production, add one milliliter of the fermented supernatants to two-milliliter centrifuge tubes and add 0.2 milliliters of 25 weight/volume percent of metaphosphoric acid to each sample. Vortex to thoroughly mix the solutions. Centrifuge the mixtures at 13, 000 times G for 20 minutes.

In the meantime, prepare solutions of 120 millimolar acetic, propionic, butyric, isobutyric, valeric, and isovaleric acids in tubes. Then add one milliliter of each prepared acid to a tube containing 1.2 milliliters of 25 weight/volume percent metaphosphoric acid. And load them into the sample bottle of the gas-chromatography instrument as the standard cocktails.

Take the tubes out of the centrifuge, and transfer the supernatants to fresh tubes. Use a syringe equipped with 0.22 micron filter to filter the supernatants into new tubes for gas-chromatography analysis. In this study, the production of mucoid EPS was observed in bifidobacterium longum cultures on PYG plates, after anaerobic incubation for 72 hours.

Centrifugation of culture scrapes, followed by ethanol precipitation and drying, resulted in collection of cellulose-like EPS. TLC analysis revealed that the degradation rate of starch by human fecal microbiota was faster than that of BIF EPS. Principal Coordinate Analysis shows culture VI BIF, and culture VI starch groups, clustered separately from each other, indicating that EPS and starch availability differentially shape human fecal bacterial communities.

Linear Discriminant Analysis on effect size shows the genera collinsella, coprococcus, parabacteroides, and rhodopseudomonas were significantly more abundant in the culture VI BIF samples than in the culture VI starch samples. Furthermore, GC measurements show SCFAs that were measured from fermentation cultures included acetic, propionic, isobutyric, butyric, isovaleric, and valeric acids. Following fermentation for 24 hours and 48 hours, five of the six SCFA concentrations were similar and not statistically different between the culture VI BIF, culture VI starch, and culture VI groups.

However, proprionic acid concentrations were significantly higher in the culture VI BIF group than in the culture VI starch group, after 48 hours of fermentation. Do this experiment under anaerobic conditions, and then transfer the fixed samples into an anaerobic incubator as soon as possible. Some of the TLC reagents are hazardous.

You should be careful to use them.