Maxq orbital shaker thermo scientific

WL were kindly provided by the cellar Grandes Vinos y Viñedos S.A (Cariñena PDO area, Cariñena, Spain). They were collected after racking the red wine (first-fermentation WL), which was made from grapes of Cabernet variety. The preparation of the new PWL was as follows: 10 mL of WL were mixed with the commercial enzyme solution Flavourzyme^® (enzyme/substrate ratio, 80 LAPU/g protein). Hydrolysis was carried out at 25 °C for 2 h at pH 4.0 and 250 rpm in a MaxQ Orbital Shaker Thermo Scientific (Thermo Fisher Scientific, Waltham, MA, USA). These enzymatic conditions were set according to a previous study focus on selecting the most appropriate enzymatic preparation and choosing process conditions easily applicable on an industrial scale (data not shown). Reaction was finished adding 1 M HCl to decrease the solution pH to 3. The final volume of the hydrolysate was 11.25 mL. Subsequently, the hydrolysate was centrifuged at 3000× g for 15 min at 4 °C to eliminate non-soluble particles and the supernatant (PWL) was collected for analysis. In addition, no-hydrolysis control WL (0 WL) was also subjected to the same procedure but replacing the enzyme volume by Milli-Q water.

The chicken foot protein hydrolysate Hpp11 was elaborated using the commercial enzymatic solution protamex as previously was described.20 Briefly, chicken feet were cleaned, crushed, lyophilized, milled, and sieved using a 2‐mm pore size sieve to obtain a fine chicken foot powder. Chicken foot powder (20 mg mL⁻¹, w/v) was resuspended in distilled water and incubated for 90 min in a water bath set at 100 °C at 100 rpm. Subsequently, the protamex enzymatic solution was added at a final concentration of 2.67 µg mL⁻¹ (enzyme/substrate ratio, 0.4 AU g⁻¹ protein). Hydrolysis was carried out at 50 °C for 2 h at pH 7.0 in a MaxQ Orbital Shaker Thermo Scientific (Thermo Fisher Scientific, Waltham, MA, USA). At the end of the reaction, the enzyme was heat‐inactivated (80 °C, 10 min) in a water bath. Then, the hydrolysate was centrifuged at 10 000 × g for 20 min at 4 °C and the supernatant was filtered through a 0.45‐µm membrane; finally, the filtrate was collected for analysis.