Porcine stomach

Suspensions of 12.2% w/w nanocrystalline cellulose (NCC) and 3% w/w nanofibrillated cellulose (NFC) were purchased from Cellulose Lab Company, Fredericton, NB, Canada. Tween 20 and β-carotene powder (≤95% purity) were purchased from Sigma-Aldrich Company (Sigma-Aldrich, Inc., St. Louis, MO, USA). Soybean oil without any purification was purchased from a local supermarket in Nakhon Pathom, Thailand. Pepsin from porcine gastric mucosa, porcine lipase, mucin from porcine stomach, porcine bile extract, and Nile Red were purchased from Sigma-Aldrich Company (Sigma-Aldrich, Inc., St. Louis, MO, USA). Sodium azide and chloroform were purchased from Ajax Finechem (Ajax Finechem Pty., Ltd., New SouthWales, Australia). Sodium chloride, calcium chloride, monobasic phosphate, and all chemicals used in this study were of analytical grade. Double distilled water was used for the preparation of all solutions.

Bacterial adhesion to mucus was determined according to a previously described procedure [14 (link)]. Briefly, human intestinal mucus extract and porcine mucin (porcine stomach, Sigma-Aldrich, St. Louis, MO, US) were passively immobilized on 96-well MaxiSorp plates (Nalge Nunc, Rochester, NY, US) by 50 mM carbonate/bicarbonate buffer at 4 °C overnight. The wells were washed three times with PBS and blocked for 1 h with PBS plus 1% Tween 20. Overnight cultured bacterial cells were adjusted to O.D._{600 nm} of 0.6 ± 0.03 (approximately 10⁸–10⁹ CFU/mL) with PBS. A 100 μL aliquot of bacterial cells was added to each well of the plates, which were then incubated at 37 °C for 2 h. Non-adhered bacterial cells were removed by washing three times with PBS plus 0.05% Tween 20, and plates were dried at 55 °C. Adhered cells were stained with crystal violet (1 mg/mL) for 45 min. After six washes with PBS, the colorant was liberated with 50 mM citrate buffer for 45 min and absorbance at 560 nm was determined using an ARVO™ MX plate reader (PerkinElmer Japan, Kanagawa, Japan). Adjusted bacterial cells without mucus were run as controls in all experiments. The human mucus binding assay was performed for each fecal mucus sample, and the porcine mucus binding assay was conducted in a quintuple well in triplicate using cells from independent cultures.

Small intestines were obtained from a freshly slaughtered adult pig at the Meat Lab at Michigan State University (USDA permit number 137 from establishment number 10053). The animal was slaughtered as part of the normal work of the abattoir according to the rules set by the Michigan State University Institutional Animal Care and Use Committee (IACUC). The small intestines were acquired from the abattoir with prior consent. The mucosa was gently scraped from the medial part of small intestine and frozen in liquid nitrogen before storage at –80°C. For each experiment, mucus samples were warmed to 37°C and equilibrated for 1 h in a 10-volume excess of M9 salts buffered to the desired pH. Bovine submaxillary gland mucin (M3895; Sigma-Aldrich) solution was prepared at 3% (wt/vol) in LB medium adjusted to pH 8.0 with sodium hydroxide. Nonsoluble particles were separated from the preparation by centrifugation at 21,130 relative centrifugal force (rcf) for 10 min. Porcine stomach (M2378; Sigma-Aldrich) mucin solution was prepared at 3% (wt/vol) in M9 salts at pH 8.0. The survival rate of V. cholerae in Bovine submaxillary gland mucin was calculated by enumerating colonies on LB agar plates supplemented with 50 μg/ml kanamycin. Fluorescent beads were added to the samples at 0.15% (wt/vol) and gently mixed.

The anticancer protein P8 was identified from Lactobacillus rhamnosus CBT LR5 (= KCTC 12202BP) isolated from the human intestine. P. pentosaceus SL4(-7) that was used as a drug delivery vehicle is a derivative of P. pentosaceus SL4 (= KCTC 10297BP) isolated from the traditional Korean fermented vegetable kimchi. These strains were derived from the culture collection of Cell Biotech Co., Ltd., Gimpo, Korea, and routinely statically cultured at 37 °C for 18–24 h in Man, Rogosa and Sharpe broth (Difco, Detroit, MI, USA) or M9 broth with 1% glucose for protein expression. Escherichia coli DH5α was cultured for 18–24 h in Lysogeny broth (Difco) at 37 °C. Under the strictly anaerobic condition, A. muciniphila KCTC 15667 was statically cultured for 48 h in brain heart infusion broth (Becton, Dickinson and Company, Bergen County, NJ, USA) with 3% of mucin from the porcine stomach (Sigma-Aldrich, St. Louis, MO, USA) at 37 °C. T. sanguinis DSM 14220 was strictly anaerobically cultured for 48 h in chopped meat broth (Becton, Dickinson and Company, Bergen County, NJ, USA) at 37 °C.

The ability of S. maltophilia strains to adhere to mucin was tested as described by Muñoz-Provencio et al. [21 (link)] with modifications. Flat-bottomed polystyrene 96-well plates (Sarstedt, Newton, USA) were covered with mucin (porcine stomach, Sigma, Germany) in 50 mM carbonate buffer pH 9.6 (mucin) at a concentration of 30 mg/ml, while the wells of control plates were filled with the same volume of 50 mM carbonate buffer (200 μl). Plates were incubated overnight at 4°C. After immobilization, wells were washed three times with PBS and blocked for 1 h with PBS plus 1% Tween 20. After washing, 200 μl aliquots of bacterial suspension adjusted to the turbidity of a 0.5 McFarland were added and plates were incubated for 2h at 37°C. Non-adherent cells were removed by washing three times with PBS plus 0.05% Tween 20 and the plates were dried at 65°C. Adhered cells were stained with 0.1 mg/ml of crystal violet (100μl/well) for 45 min. After six washes with PBS, the colorant was liberated with 50 mM citrate buffer pH 4.0 (100 μl/well) for 1 h and the absorbance was measured at 595 nm.