Gastrointestinal Digestion In Vitro

1. Inoculate four flasks containing 50 mL of lactose broth, at a level of 2% with a midexponential phase grown culture (24-30 h) of propionibacteria, and incubate statically at 37°C for 24 h.

2. Harvest the cells by centrifugation (20,000g, 10 min, 4°C), wash the pellet once, and resuspend each pellet in the 10th part of the original volume in sterile saline solution.

3. Add these cell suspensions to four flasks containing 50 mL of artificial gastric juice adjusted to pH 2.0, 3.0, 4.0, and 7, respectively. Mix with vortex and incubate the bacterial suspensions at 37°C with agitation at 200 opm to simulate peristalsis and the normal temperature of the human body.

4. Take samples of 5 mL in sterile screw-top glass tubes, for enumeration of viable microorganisms and P-galactosidase activity assay at 0, 30, 60, and 180 min (see Note 4).

5. After 180 min of gastric digestion, centrifuge 25 mL of each bacterial suspension at 20,000g, 10 min. Suspend the pellets to the original volume in intestinal fluid and incubate as before.

6. Take samples of 5 mL for enumeration of viable microorganisms and |3-galactosidase activity at 0, 30, 60, and 180 min (see Note 4).

7. For survival of propionibacteria: Take 100 ^L of each sample and dilute with 900 ^L of sterile peptone water. Make serial 10-fold dilutions and inoculate 0.5 mL of each dilution in a Petri dish. Add melted lactose agar medium by homogenizing properly.

8. Once they are solidified, invert the plates and incubate at 35°C for 5 d in an anaerobic chamber (Forma Scientifica Anaerobic System, model 1024) with a gas mixture of 90% N2 and 10% CO2 (see Note 5).

9. For determination of |3-galactosidase activity, see Subheading 3.7.

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