α amylase solution

The digestive stability of LAB strains was assessed by measuring their viability under simulated human gastrointestinal conditions. This experiment was performed with slight modifications to a previously described method (Kim et al., 2022 ). First, each strain suspended in saline (1.0 × 10⁹ CFU/mL) was poured into a 50-mL conical tube, and 26 μL of 0.3 M CaCl₂ solution (Sigma-Aldrich) and 4 mL of 6.55 mg/mL α-amylase solution (Sigma-Aldrich) were added. The suspension was then adjusted to pH 7.0 with 1 M NaOH (Sigma-Aldrich) and incubated at 37 °C for 5 min to simulate oral conditions. To simulate gastrointestinal conditions, 6 μL of 0.3 M CaCl₂, 694 μL of distilled water, and 9.1 mL of 0.07 mg/mL pepsin were added. The pH was adjusted to 3 with 1 M HCl (Sigma-Aldrich) and the mixture was incubated at 37 °C for 1 h. Finally, intestinal conditions were simulated by adding 40 mL of 0.3 M CaCl₂, 1.31 mL of distilled water, 2.5 mL of 160 mM bile extract, and 16 mL of 22 mg/mL pancreatic solution to the mixture, followed by incubation at 37 °C for 2 h. The survival rate of the strains at each digestion step was determined by measuring the number of viable cells on the MRS plate after aliquoting the mixture of each step.

All of the solvents were analytical grade, of high-performance liquid chromatography-mass spectrometry (HPLC-MS) quality (>99%) and were obtained from Merck (Darmstadt, Germany). Deionized water (DIW) was used for the extractions and was of Milli-Q quality for liquid chromatography (LC) and UV-VIS spectrophotometric measurements. The delphinidin-3-O-rutinoside was purchased from Phytolab (Vestenbergsgreuth, Germany). The phenolic acid standards (protocatechuic acid, p-hydroxybenzoic acid, gallic acid, gentisic acid, vanillic acid, salicylic acid, syringic acid, caffeic acid, p-coumaric acid, m-coumaric acid, ferulic acid, o-coumaric acid and chlorogenic acid) were purchased from Sigma-Aldrich (Poole, Dorset, UK). The acarbose was purchased from J&K (Beijing, China), α-Amylase solution from Sigma Aldrich (Steinheim, Germany), 3,5-dinitrosalicylic acid (DNS) from Fluorochem (Hadfield, UK) and Na-K tartrate tetrahydrate (p. A.) from Chemsolute (Roskilde, Denmark). All of these pure standards were used for identification and quantification purposes.

PGN was isolated essentially following a published procedure [29 ]. Briefly, biomass was harvested from 1 l of culture grown to the stationary phase by centrifugation (5000 g, 30 min, 4 °C), resuspended in 60 ml distilled water and transferred drop-wise into 65 ml of boiling 8% sodium dodecyl sulfate (SDS; Sigma) under constant stirring to lyse cells. The suspension was further boiled for 1 h, reduced to the former volume using a rotary evaporator, and stirred overnight. SDS was removed by several washing steps with distilled water, 60 ml, each, using an Optima L-100XP ultracentrifuge from Beckman Coulter (rotor Ti70, 35,000 rpm, 30 min, 40 °C) followed by dialysis against distilled water for 4 days at room temperature. For the total volume of 12 ml of that PGN solution, 200 μl of an α-amylase solution (24 mg ml^− 1; Sigma) were added and the mixture was incubated at 37 °C for 2 h under constant shaking. Further, 320 μl of pre-incubated Pronase E solution (10 mg ml^− 1 in 10 mM Tris-HCl, pH 7.5; Sigma) was added and incubated at 60 °C for 1.5 h. Preparations were washed, boiled for 1 h, washed again, and dried in a Speed Vac vacuum centrifuge (Thermo Fisher Scientific, Vienna, Austria).