Acetic acid

Milk samples were thoroughly mixed, followed by transferring 5 mL of homogenate in 50 mL PFTE tube containing a mixture of 10 mL deionized water and 10 mL acidified acetonitrile with acetic acid (Scharlau, Barcelona, Spain). The mixture was shaken manually, then added 4.0 g of anhydrous Mg sulfate, 1.0 g of Na Cl, 1 g of Na citrate, and 0.5 g Na hydrogen citrate sesquihydrate (Merck, Darmstadt, Germany). Finally, the content was mixed by shaking for 1 min, followed by centrifugation at 1792 × g for 3 min. Subsequently, 1 mL of the supernatant clear solution was transferred into 15 mL of polyethylene tube containing 25 mg amine sorbent (Supelco, Bellefonte, USA) and 150 mg anhydrous Mg sulfate (Merck). Then the tube was shaken vigorously manually for 1 min then centrifuged (Sigma, Darmstadt, Germany) for 1 min. at 2800 ×g. Finally, 0.5 mL of supernatant was taken into a glass vial, evaporated to dryness, and re-dissolved in 0.5 mL n-hexane for gas chromatograph-microelectron capture detector (GC-μECD) analysis (Agilent Technologies, Santa Clara, CA, USA).

All reagents were of analytical grade. Water was obtained with a Milli-Q system from Millipore (Bedford, MA, USA). Acetone, hexane, hydrochloric acid (HCl), acetic acid (AA), acetonitrile (ACN), and methanol (MeOH) were from Scharlau (Barcelona, Spain). Tris(hydroxymethyl)aminomethane (Tris), sodium dodecyl sulfate (SDS), DL-dithiothreitol (DTT), sodium chloride (NaCl), urea, sodium hydroxide (NaOH), trifluoroacetic acid (TFA), β-mercaptoethanol (β-M), bovine serum albumin (BSA), lysozyme from chicken eggs (LYS), myoglobin from equine heart (MYO), and concanavalin (CONC) were from Merck (Darmstadt, Germany). Laemmli buffer, Mini-Protean precast gels, Tris/glycine/SDS running buffer, Bio-Safe Coomassie stain, and Bradford reagent (Coomassie Blue G-250) were acquired at Bio-Rad (Hercules, CA, USA).
Raw olives of Picual variety were kindly donated by the World Olive Germplasm Bank of IFAPA (Córdoba, Spain) and cheese whey was kindly donated by a cheese factory.

The sulforhodamine B (SRB) cytotoxicity assay was employed to investigate the antiproliferative potential of Lactobacillus AGR 4 against HT-29 and A375 cells. Cells were seeded in 96-well plates at a density of 7000 cells per well and were incubated overnight. The next day, the wells were washed with PBS and the bacteria were added in the wells at a density of 10⁶ or 10⁷ CFU/mL. After 24 or 48 h incubation the cells were washed with PBS, fixed by 50% (w/v) cold trichloroacetic acid (TCA) (Fluka, Buchs, Switzerland) and stained with 0.4% (w/v) SRB (Sigma-Aldrich) diluted in 1% (v/v) acetic acid (Scharlau, Barcelona, Spain). Removal of the excessive dye was achieved by washing with 1% (v/v) acetic acid. The bound dye was dissolved in 10 mM Tris base (Sigma-Aldrich). For the measurement of the absorbance at 570 nm a multiplate reader was used (Tecan, Männedorf, Switzerland). For the calculation of the cellular survival the following formula: ((sample OD₅₇₀-media blank OD₅₇₀)/(mean control OD₅₇₀-media blank OD₅₇₀)) × 100 was applied.

The viability of cancer cells after treatment with the juice was determined using the Sulforhodamine B (SRB) assay, as previously described with few modifications [14 (link)]. SRB is a dye that binds to basic amino acids of cellular proteins and, then, the number of viable cells is estimated with colorimetric evaluation [15 (link)]. Cells were plated in 96-well plates and treated with different concentrations of the juice (0.0007–1% v/v). Then, the cells were fixed with the addition of 25 μL of 50% (w/v) cold trichloroacetic acid (TCA) (Applichem, Darmstadt, Germany) to the growth medium and incubation of the plates at 4 °C for 1 h. The cells were washed five times with tap water and then stained with 50 μL of 0.4% (w/v) SRB dye (Sigma-Aldrich, St Louis, MO, USA) in 1% (v/v) acetic acid (Scharlau, Barcelona, Spain) for 30 min at room temperature. Next, the cells were rinsed five times with 1% (v/v) acetic acid to remove the unbound dye. The fixed, stained plates were allowed to air-dry and afterwards the bound dye was solubilized by adding 100 μL of 10 mM Trizma base (Sigma-Aldrich, St Louis, MO, USA) for at least 5 minutes. Absorbance was measured at 570 nm using an ELISA plate reader (Sunrise, Tecan, Männedorf, Switzerland) and the percent cellular survival was calculated using the formula:

HPLC grade acetonitrile, 2-propanol, n-hexane, methanol, formic and acetic acid were purchased from Scharlau (Scharlab SL, Spain). Reference standards for the phenolic compounds analysis: Ferulic acid, caffeic acid, coumaric acid, vanillic acid, syringic acid and apigenin-7-O-glucoside were obtained from Extrasynthese (Genay, France), while p-hydroxybenzoic acid, sinapic acid, catechin, and procyanidin B2 were purchased from Sigma Aldrich (Steinheim, Germany). 2,2′-azobis(2,4-dimethylvaleronytril) and 6-hydroxy-2- 5,7,8-tetramethylchroman-2-carboxylic acid used in ORAC assay were obtained from Sigma Aldrich (Steinheim, Germany) and 2,2-diphenyl-1-picrylhydrazyl reagent were purchased from Alfa Aesar (Ward Hill, MA, USA) to DPPH test. All other reagents used were of analytical grade from Scharlau (Scharlab SL, Spain). Deionized water was obtained from a Milli-Q water purification system (Millipore, Bedford, MA, USA).

Medium molecular weight chitosan with a deacetylation degree of 75–85% (448877) and high molecular weight chitosan with a >75% deacetylation degree (419419) were purchased from Sigma-Aldrich (Burlington, MA, USA)and used as received. Ascorbic acid (AC05150) and acetic acid (AC0344) were purchased from Scharlau (Spain), and Tween^® 20 (Polysorbate) (97062-332) was purchased from VWR (Radnor, PA, USA) and used as received. Grape seed essential oil was purchased from Herbavit (Bucharest, Romania), and sea buckthorn essential oil was purchased from Hofigal (Bucharest, Romania) and used as received. Folin–Ciocalteu reagent (F9252), xylene (214736), sodium bicarbonate (S6014), sodium acetate (S8750), 2,2-diphenyl-1-picrylhydrazyl (DPPH) (247642), and potassium chloride (P3911) were purchased from Merck (Darmstadt, Germany).