P7545

The available carbohydrate content of the meals was measured by digesting the carbohydrates to reducing sugars with a combination of pancreatic amylase (P-7545, Sigma-Aldrich, Darmstadt, Germany), amyloglucosidase (E-AMG, Megazyme, Bray, Ireland) and invertase at 37 °C for 120 min.

In vitro starch digestibility assay was conducted following the methods described by Englyst et al. (1992) and Chung et al. (2009) (link) with modifications. 450 mg of porcine pancreatic alpha-amylase (P-7545; Sigma-Aldrich Co., MO, United States) was dispersed in 4 ml of distilled water in the 50 ml of conical tube (SPL Life Science, Pocheon, Korea) and centrifuged at 1,500 g for 10 min. A 2.7 ml aliquot of the supernatant was transferred to a new 50-ml tube and 0.3 ml of amyloglucosidase (A-9913; Sigma-Aldrich Co., MO, United States) and 0.2 ml of invertase (E-INVPD; Megazyme Co., Wicklow, Ireland; 250 mg/ml) were added to the solution. Starch (100 mg) and 4 ml of 0.5 M sodium acetate buffer (pH 5.2) were added to each glass test tube (15 mm × 120 mm). One milliliter of enzyme solution and five glass beads (4 mm diameter) were added to each glass tube which were then incubated in a shaking water bath (37°C, 200 rpm). Aliquots (0.1 ml) were collected at each six time points (0, 15, 30, 60, 120, and 240 min) and 1 ml of 80% ethanol was added to stop the reactions. Tubes were centrifuged for 10 min with 2,000 rpm, and the glucose content was measured by supernatant of each sample. GOPOD kit (K-GLUC; Megazyme Co., Wicklow, Ireland) was used for determination of glucose content.

A model simulating the gastrointestinal tract (GIT) in humans consisting of three digestive compartments divided into the mouth, stomach, and small intestine was applied. The simulated juices were prepared from previous works^{47 (link),48 (link)}. The chemical composition and pH of simulated salivary fluid (SSF), simulated gastric juice (SGJ), and simulated intestinal fluid (SIF) are presented in Supplementary Table 1. The mouth step was defined as 5 min at 37 °C in the SSF adjusted to pH 7 and containing 1500 U mL⁻¹ of α-amylase (A1031, Sigma). The gastric step was then conducted at 37 °C for 2 h in the SGJ containing 3 g L⁻¹ of porcine pepsin (P7000, Sigma) and adjusted to pH 2. Finally, the duodenum step was carried out over 2 h in the SIF at pH 7 and included the addition of pancreatic enzymes at 1 g L⁻¹ (P7545, Sigma) and bile fluid containing 45 g L⁻¹ of bile salts (B8381, Sigma). After each step of the simulated digestion process, samples of each condition (Planktonic, Biofilm and CPB_Biofilm) were collected and the viable log (CFU/g) was enumerated by spotting onto MRS agar pH 5.8 and incubating at 37 °C for 48 h.

Milk aliquots were subjected to standardized static in vitro digestion [23 (link)]. Briefly, gastric digestion was carried out with porcine pepsin (2000 U/mg protein) (P7000; Sigma, Poznań, Poland) for 1 h at pH 3 (adjusted with 0.1 M HCl). This was followed by intestinal digestion with pancreatin (100 U/mL) (P7545; Sigma) and porcine bile salt (48305; Sigma, Poznań, Poland) to 10 mM in the final mixture at pH adjusted to 7.0 with 0.1 M NaHCO₃, with constant stirring for 2 h at 37 °C. Samples after the intestinal step were submitted to dialysis through a 15 kDa molecular mass cut-off dialysis membrane (Spectra/Por 2.1, Spectrum Medical, Gardena, CA). All other reagents except enzymes were obtained from POCH (Poland). After digestion, the samples were immediately collected and stored at −20 °C for further analyses. Aliquots for spectral analysis were freeze-dried.

The fermentation involved two probiotic strains, Lactobacillus fermentum (LMG 6902) and Lactobacillus rhamnosus (LMG 25626), which were obtained from BCCM/LMG Bacteria Collection. Dried de Man, Rogosa, and Sharpe (MRS) growth media were acquired from HIMEDIA (Einhausen, Germany).
The commercial soy beverage originated by Dennree (Allemagne, Germany), was purchased from a food store (Cluj-Napoca, Romania). The raw materials of the soy beverage were water and 8% soy from ecologic farming, having 1.5% fat, 0.9% carbohydrates, and 3% proteins. The product was UHT pasteurized. Xylitol, and inactivated dried organic Chlorella vulgaris powder were acquired from specialized stores in Cluj-Napoca, Romania. The country of origin this powder was China, having 60% proteins, 12% fibers, 9.5% carbohydrates, and 7.9% fat.
The enzymes used for the simulation of gastrointestinal digestion, pepsin from porcine gastric mucosa (P6887), pancreatin from porcine pancreas (P7545), and bovine bile extract (B8631), were acquired from Sigma-Aldrich (Taufkirchen, Germany). The chemicals for the biological characterization, DPPH (1,1-diphenyl-2-picrylhydrazyl), Trolox, and Folin–Ciocâlteu reagent were also purchased from Sigma-Aldrich. All the materials and chemicals used in the experiment were of analytical grade.

Normal corn starch (~25% amylose) was obtained from Samyang Genex (Incheon, South Korea). DL-malic acid was purchased from Sigma (M1210, Sigma-Aldrich, St. Louis, MO, USA). The enzymes used in the starch digestion were porcine pancreatin (P7545, activity 8 × USP/g, Sigma, St. Louis, MO, USA) and amyloglucosidase (AMG 300L, activity 300 AGU/mL, Novozymes Inc., Bagsvaerd, Denmark), where USP and AGU stand for United States Pharmacopia and amyloglucosidase activity, respectively. All chemicals and reagents used were of analytical grade.

The high protease assay described by Mandalari et·al.^{37 (link)} was performed. Briefly, 30 µL of 2.5 µg/µL BSA as control protein or 30 µL of PmPV1 (2.7 µg/µL in distilled water) were added to vials containing standard intestinal fluid (150 µL), which consisted of pancreatin (10 mg/mL, Sigma, P7545) in 67 mM K₂HPO₄ pH 7.6 with a final pancreatin:substrate ratio of 1:9.6. In parallel, standard intestinal fluid and PmPV1 (without pancreatin) were run as controls. Samples were incubated in a water bath at 37 °C and aliquots were taken at intervals of 20 min. Aliquots were analysed by SDS-PAGE as described above.
Proteinase K treatment was performed following Kim et al.^{61 (link)} using the concentrations modified by Frassa et al.^{17 (link)}. PmPV1 (1 mg/mL) was incubated with proteinase K (1, 10 and 100 µg/mL) in 50 mM Tris/HCl buffer (pH 8.0) containing 10 mM CaCl₂ at 37 °C for 30 min. Digestion was ended by boiling samples in SDS sample buffer, and products were analysed by SDS-PAGE as above.