Spectronic 200

The concentration of the total phenolic compounds in the PAC was quantified via microscale Folin–Ciocalteu colorimetry [101 ]. The procedure was scaled down to 1.5-mL cuvettes by halving the reaction volume. Hence, 10 µL of sample was added to a mixture of 0.79 mL of ultrapure water and 50 µL FC reagent. After vortexing the mixture was incubated for 5 min and 150 µL of 25% (w/v) sodium carbonate were added into the cuvettes. The solution was thoroughly mixed by vortexing again and incubated for 1 h at room temperature. The measurement was performed at 765 nm using a spectrophotometer (Spectronic 200, Thermo Scientific) and the amount of total phenolics was determined as gallic acid equivalents via a gallic acid calibration curve ranging from 0.05 to 1 g/L. Like the laccase samples, the single calibration points were prepared as 1:10 dilutions in 10% acetic acid to compensate for possible side reactions of the acetic acid with the FC reagent.

The production of indolic compounds was determined by growing the strains overnight in 2 mL of TSB medium and incubating them at 30 °C [76 (link)]. The strains were inoculated in tubes containing 5 mL of TSB medium supplemented (150, 300, and 600 μg mL⁻¹) or not supplemented with tryptophan (HI-media^®, Maharashtra, India). The tubes were incubated at 30 °C at 180 rpm for 24 h. The fermented broth was centrifuged at 13,000 rpm for 3 min (Mikro200 Hettich, Tuttlingen, Germany). The supernatant was used to quantify the production of indolic compounds, mixing in triplicate in 0.5 mL of Salkowsky’s reagent [77 (link)] and 0.5 mL of supernatant, which were mixed by inversion and incubated in the dark for 20 min at room temperature. Following this, the absorbance at 535 nm was determined using a spectrophotometer (Thermofisher, Spectronic 200, Suwa, Japan), with a standard curve configured using indole acetic acid (50–1000 µg L⁻¹) (Hi-media^®, Maharashtra, India) and 0.5 mL of culture medium plus 0.5 mL of Salkowski’s reagent as a control. The results are expressed as μg IAA mL⁻¹.

The Folin–Ciocâlteu (F–C) method was utilized to determine the total polyphenols content [12 (link)]. The dried material was grounded in an analytical mill (IKA A11 basic, IKA Werke GmbH & Co. KG, Staufen, Germany), and 0.3 g of the powder was weighed into glass beakers, adding 20 mL of 80% ethanol solution (Avantor Performance Materials, Gliwice, Poland) [23 (link)]. The solution was slightly heated for 3 min, not allowing it to boil, filtered and made up to 50 mL. 0.18 mL of extract was placed in glass tubes and diluted with 4.92 mL of distilled water; afterward, 0.3 mL of F–C reagent (Merck, Darmstadt, Germany) was added and mixed. After 3 min, the pH of the solution was changed by adding and mixing 0.6 mL of supersaturated sodium carbonate solution (Avantor Performance Materials, Gliwice, Poland). The incubation was carried out in the darkness at 25 °C for an hour. The absorbance of the solutions was measured using a spectrophotometer (Spectronic 200; Thermo Fisher Scientific Inc., Waltham, MA, USA) at a wavelength of 750 nm against a blank sample. The content of polyphenols was calculated as gallic acid equivalent (mg GAE/100 g dm) with the calibration curve in the range of 1–5 mg/mL. The analysis was conducted three times.

Antioxidant capacities were determined using the 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) and 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging assays [16 (link),17 (link)]. Briefly, the ABTS radical solution was adjusted to an absorbance of 0.650 ± 0.020 at 734 nm. Reactions between the ABTS radical solution and the diluted compound 1–7 (satisfying the standard curve range) were allowed to proceed at 37 °C for 10 min, and decreases in the absorbance of the resulting solution were measured using a spectrophotometer (SPECTRONIC 200; Thermo Fisher Scientific Inc., Waltham, MA, USA). For the DPPH assay, the absorbance of DPPH radicals in 80% (v/v) aqueous methanol was set to 0.650 ± 0.020 at 517 nm. Reactions between the DPPH radical solution and the diluted compound 1–7 (satisfying the standard curve range) were allowed to proceed at ambient temperature for 30 min. Decreases in the absorbance of the resulting solution were monitored at 517 nm using a spectrophotometer (SPECTRONIC 200). Antioxidant capacities were expressed as mg vitamin C equivalent (VCE)/100 mg.

The commercial strains BB12 and LA5 were obtained from ATCC. Both probiotic strains Bifidobacterium animalis ssp. lactis ATCC 27536 (BB12) and Lactobacillus acidophilus ATCC 4356 (LA5) were cultured in MRS (De Man, Rogosa, and Sharpe) broth (BD Difco^TM, Fisher Scientific, Waltham, MA, USA) with the incubation conditions of 37 ℃, 72 h in an anaerobic jar using gaspak sachets (BD^TM Fisher Scientific, USA). After the activation and three sub-culturing steps, the cells were collected using centrifugation (1000× g for 15 min at 4 °C). The supernatant was removed, followed by 2–3 washings with the isotonic PBS solution (pH 7.2) to get the bacterial cells of BB12 and LA5. The cell concentration was further optimized at 0.8–0.9 OD, 600 nm using a spectrophotometer (Spectronic 200, ThermoFisher Scientific, Madison, WI, USA) to spike the conjugated solution at a concentration of 9–10 logs CFU/mL. The cell suspensions of BB12 and LA5 were held at 4 °C for inoculation in the conjugated whey protein hydrolysate solution.