Exposing a Synthetic Oral Bacterial Community to Simulated Phases of Digestion

Samenvatting

Source: Arroyo, M. C., et al. Assessing the Viability of a Synthetic Bacterial Consortium on the In Vitro Gut Host-microbe Interface. J. Vis. Exp. (2018)

This video demonstrates the viability assessment of a synthetic oral microbial community during simulated oral, gastric, and intestinal digestion. Bacterial survival is quantified after each phase, revealing a progressive decline due to digestive stress.

Protocol

1. Bacterial Survival Following In Vitro Gastrointestinal Transit Conditions

NOTE: Prepare simulated digestion fluids with the modifications described below. Prepare all the dilutions with ultrapure water. Filter-sterilize all solutions through a 0.22 µm filter and perform the digestion procedure under sterile conditions.

1. Oral digestion:
   1. Place 3 mL of the bacterial community in a 50 mL sterile tube, mix with 3 mL of simulated saliva fluid (SSF), containing in millimoles per liter (mmol L⁻¹): Potassium chloride (KCl), 15.1; Monopotassium phosphate (KH₂PO₄), 3.7; Sodium bicarbonate (NaHCO₃), 6.8; Magnesium chloride hexahydrate (MgCl₂·6H₂O), 0.5, Ammonium carbonate [(NH₄)₂CO₃], 0.06; Hydrochloric acid (HCl), 1.1; Calcium chloride dihydrate (CaCl₂·2H₂O), 0.75. Add 75 U/mL of α-amylase from human saliva Type IX-A and 2 g/L mucin from porcine stomach (Type II) to the SSF.
   2. Incubate the mixture for 2 min at 37 °C and 100 rpm. Collect a 2 mL sample for DNA extraction and flow cytometry quantification (S1).

2. Gastric digestion:

1. Add 4 mL of simulated gastric fluid (SGF), containing (in mmol L-1): KCl, 6.9; KH₂PO₄, 0.9; NaHCO₃, 25; Sodium chloride (NaCl), 47.2; MgCl₂·6H₂O, 0.1, (NH₄)₂CO₃, 0.5; HCl, 15.6; CaCl₂·2H₂O, 0.075. In addition, the SGF contained 2 g/L of mucin from porcine stomach (Type II).
2. Measure the pH and adjust to pH 3 with 1 molar (1 M) HCl, if needed. Incubate at 37 °C and 100 rpm. Collect a 2 mL sample (S2).

3. Small intestine digestion:

1. Add 4 mL of simulated intestinal fluid (SIF) containing (in mmol L-1): KCl, 6.8; KH₂PO₄, 0.8; NaHCO₃, 85; NaCl, 38.4; MgCl₂·6H₂O, 0.33; HCl, 8.4; CaCl₂·2H₂O, 0.3, to the digestion tube.
2. Adjust to pH 7 with 1 M sodium hydroxide (NaOH), if needed. Incubate at 37 °C and 100 rpm. Collect a 2 mL sample (S3).

Materialen

Lijst van materialen gebruikt in dit artikel

Naam	Bedrijf	Catalogusnummer	Opmerkingen
Mucin from porcine stomach Type II	Sigma-Aldrich	M2378
PBS	Gibco	14190250
Millipore Milli-Q academic, ultra pure water system	Millipore, Merck KGaA	-
InnOva 4080 incubator shaker	New Brunswick Scientific	8261-30-1007
Nuclease-free water	Serva Electrophoresis	28539010
BD Accuri C6 flow cytometer	BD Biosciences	653118
InnuPREP PCRpure kit	Analytik Jena	845-KS-5010250	PCR purification kit