This study investigates the survival of a synthetic bacterial community through simulated oral, gastric, and intestinal digestion. The effects of mucin and α-amylase on bacterial viability are assessed under varying pH conditions.
Begin with a tube containing a synthetic bacterial community commonly found in the human mouth.
Add simulated salivary fluid containing mucin, a glycoprotein, and the enzyme α-amylase to mimic the oral environment, then incubate with shaking.
The bacteria interact with mucin, which promotes aggregation, while α-amylase replicates the enzymatic conditions of saliva.
Collect a sample.
Next, introduce simulated gastric fluid with mucin at an acidic pH to mimic the stomach environment, and incubate with shaking.
The acidic conditions kill acid-sensitive bacterial species, but acid-tolerant and mucin-coated, mildly acid-tolerant bacterial species survive.
Collect another sample.
Finally, add simulated intestinal fluid containing bicarbonate and physiological salts. Incubate with shaking.
The salt-rich environment induces ionic stress, which disrupts the bacterial membrane and reduces viability.
Collect a final sample.
All three post-simulation samples are now ready for bacterial viability assessment.
To simulate oral digestion, transfer 3 milliliters of the bacterial community in a 50 milliliter sterile tube, and mix with 3 milliliters of simulated saliva fluid or SSF.
Add 75 units per milliliter of alpha-amylase from human saliva type 9A and two grams per liter of mucin from porcine stomach type 2. Incubate the mixture at 37 degrees celsius and 100 RPM for two minutes. Collect a 2 milliliter sample for DNA extraction and flow cytometry quantification.
To simulate gastric digestion, add 4 milliliters of simulated gastric fluid or SGF to 3 milliliters of the bacteria. Use one molar HCl to adjust the pH to 3 if necessary. Incubate the sample at 37 degrees Celsius and 100 RPM for two minutes.
Then collect a 2 milliliter sample. For small intestine digestions, add 4 milliliters of simulated intestinal fluid or SIF to the bacteria. Use sodium hydroxide to adjust the pH if necessary.
Then incubate the sample at 37 degrees celsius and 100 RPM for two minutes, and collect a 2 milliliter sample.
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