Trichloroacetic acid

Cell viability after genistein treatments was evaluated by the Sulforhodamine B (SRB) assay. A colorimetric assay consisted of staining the cellular protein content of adherent cells. Cell lines were seeded in 96-well plates at a density of 20,000 cells per well at 37 °C with 5% CO₂. They were then incubated for 24 h to allow their adherence, and subsequently, they were treated with different concentrations of genistein (3.7–740 µM), as well as with the vehicle used (Dimethyl sulfoxide DMSO at 1%) as growth control. After each treatment for 24 and 48 h, cells were fixed with trichloroacetic acid (PanReac AppliChem, Barcelona, Spain) for one hour at 4 °C. Cellular proteins were determined by staining with 0.4% SRB (Sigma-Aldrich) for 30 min at room temperature. Subsequently, 5 washes with 1% acetic acid were performed. For these latter procedures, the plate was allowed to dry at room temperature. SRB-bound proteins were solubilized with 10 mM Tris-base and the reading was performed by absorbance at 490 nm in a microplate reader (Bio-Rad iMark^TM, Hercules, CA, USA).

The reagents sodium carbonate, di-sodium hydrogen phosphate, sodium carbonate, potassium ferricyanide, potassium persulfate, ethylenediamine tetra acetic acid (EDTA) and gallic acid were purchased from Scharlau (Barcelona, Spain). Folin–Ciocalteu’s phenol reagent, absolute ethanol and trichloroacetic acid (TCA) were purchased from Panreac (Castellar del Vallès, Barcelona, Spain). 2,2-diphenyl-1-picrylhydrazyl (DPPH•) free radical, Iron (II) Chloride 4-hydrate, 6-hydroxy-2,5,7,8-tetramethylchromane-2-carboxylacid (Trolox), (L)-Dehydroascorbic acid and malondialdehyde (MDA) were purchased from Sigma Chemical Co. (Steinheim, Germany, and St. Louis, MO, USA). Propylgallate (PG) was purchased from Acrōs Organics (Fair Lawn, NJ, USA). 2-Thiobarbituric acid (TBA) was purchased from Merck KGaA (Darmstadt, Germany). The following standards were used: phenolic acids—hydroxybenzoic acid, vanillic acid, protocatechuic acid; cinnamic acids—chlorogenic acid, 4-coumaric acid; flavanols—catechin; flavonols—kaempferol, quercetin, kaempferol-3-O-rutinoside, kaempferol-3-O-glucoside, isorhamnetin-3-O-rutinoside, rutin or quercetin-3-O-rutinoside; flavanones—naringenin, eriodictyol, eriodictyol-7-O-glucoside (Merck KGaA, Darmstadt, Germany).

The three oils used in this study were coconut oil (Organic extra virgin coconut oil from PLANTIS^®, Barcelona, Spain), rapeseed oil (Organic cold pressed rapeseed oil Terpenic lab S.L., Barcelona, Spain) and grape seed oil (Refined grape seed Terpenic lab S.L., Barcelona, Spain). Three different batches of each type of oil were purchased in a local market.
2-thiobarbituric acid, and fatty acid methyl esters were obtained from Sigma-Aldrich Chemical (Steinheim, Germany). Boron/methanol trifluoride and heptane were purchased from Merck (Whitehouse Station, NJ, USA). Potassium hydroxide, hexane, cyclohexanone, hydrochloric acid, trichloroacetic acid and ammonium sulfate were from Panreac (Barcelona, Spain).
Salmonella typhimurium strains TA97a and TA98 and the Mutazime S9 mix at 10% from livers of Aroclor 1254-induced rats were purchased from Moltox (Boone, NC, USA). Nutrient broth and Phosphate Buffered Saline (PBS) tablets were obtained from Oxoid (Basingtone, UK). 4-nitro-o-phenylenediamine (NPD), 2-aminoanthracene (AA) and 2-aminofluorene (AF), histidine and biotin were obtained from Sigma-Aldrich.

Lipid peroxide assay was modified from the TBARS protocol (thiobarbituric acid reactive substance) (Uchiyama and Mihara 1978 (link)). In a microtube, 45 μL of PBS (pH 7–7.4) was added to 5 μL of each sample. Each microtube was then filled with 12.5 μL of SDS (Sigma-Aldrich, Germany) at a concentration of 8.1% (w/v), 93.5 μL of trichloroacetic acid (Panreac, Spain) at a concentration of 20% (w/v), and 93.5 μL of thiobarbituric acid (Sigma-Aldrich, Germany) at a concentration of 1% (w/v). Each microtube was then filled with 50.5 μL of MQ-grade ultrapure water before being stirred in a vortex for 30 s. The microtubes’ lids were pierced with a needle, and after 10 min in boiling water, they were immediately put on ice for a short while to cool. Then, each microtube received 62.5 μL of MQ-grade ultrapure water. After that, the microtubes were mixed for a minute. Each well of a 96-well microplate received a duplicate 150 μL of each microtube, and each well’s absorbance was measured at 530 nm using a microplate reader. Malondialdehyde bis(dimethylacetal) (MDA) (Merck) was used as the standard to create a ten-point calibration curve (0–0.1 μM TBARS) to quantify lipid peroxides. The results are represented in relation to the total cytosolic protein concentration of the sample (pmol.mg⁻¹ total cytosolic protein).

The method proposed by Celletti et al. [74 (link)] was used to assess the level of lipid peroxidation, expressed as malondialdehyde (MDA) content, as MDA is considered to be a biomarker of oxidative stress. An amount of 0.5 g FW of lettuce leaves were homogenized in 5 mL of a pre-chilled reagent prepared by dissolving 0.25 g of 2-thiobarbituric acid (TBA) (Merck KGaA, Darmstadt, Germany) in 100 mL of 10% (w/v) trichloroacetic acid (Panreac, Castellar del Vallès, Barcelona, Spain). The samples were first incubated at 95 °C for 30 min and then ice-cooled to stop the reaction. After centrifugation at 5000 rpm for 20 min, the absorbance of the supernatants was measured at 532 nm and 600 nm using a UV-Vis spectrophotometer (8453, Agilent, Santa Clara, CA, USA). The non-specific turbidity adjustment was derived by subtracting the absorbance value observed at 600 nm. The lipid peroxidation level was estimated using the molar extinction coefficient (155 mM⁻¹ cm⁻¹) of the MDA–TBA complex.

Pork lard with a fat content of 99.9% was obtained after a clarification process from Iberian pork fat (local supermarket, Madrid, Spain). SPC with 72% protein, was kindly donated by Lactotecnia S.L. Ingredientes Alimentarios, Barcelona, Spain. A pork rind protein extract (PRP with 90% protein of which 70% is collagen and 10% fat) was obtained from Prosur (Murcia, Spain). к-carrageenan was supplied by Tradissimo, Trades S.A. (Barcelona, Spain). MTG with 50 U/g transglutaminase activity (Activa^® EB) was donated by Ajinomoto Foods Europe (Paris, France). The food grade preparation (diatomaceous earth powder (DP); Tierra de Diatomeas^®) with a SiO₂ richness of 85%, thus containing 40% Si, was kindly donated by Vitality Gest S.L. (Valencia, Spain). Pepsin (≥2500 U/mg protein, P7012), pancreatin from porcine pancreas (8 x USP, P7545), and bile extract porcine (B8631), Fast Green FCF (F7252), Red Nile (72,485), dehydrate calcium chloride, potassium chloride, sodium bicarbonate, and sodium hydrogen carbonate were supplied by Sigma Aldrich Chemie GmbH (Steinheim, Germany). Sodium chloride, hydrochloric acid 37%, sodium hydroxide pellets, trichloroacetic acid, sulfate ammonium, and hexane were supplied by Panreac (Barcelona, Spain).