Uv vis spectrophotometer 1204

The DH of casein was measured by the o-phthaldialdehyde (OPA) method as described by Church et al. (1983) , with some modifications. The OPA reagent was prepared as following: 25 mL of 0.1 M borate buffer (pH 9.3), 2.5 mL of 20% (w/v) SDS solution, 100 µL of N,N-dimethyl-2-mercaptoethylammonium chloride, 40 mg of OPA dissolved in 1 mL methanol and brought to 50 mL of total volume with distilled water. For the assay, 100 µL of the each sample was mixed with 1900 µL of the OPA reagent and incubated at 25 °C for 2 min; then, the absorbance was read at 340 nm (UV-VIS Spectrophotometer 1204; Shimadzu, Kyoto, Japan). Results were expressed as µg of lysine per mg of CN, using a standard calibration curve obtained with different concentrations of lysine. Moreover, during enzymatic hydrolysis, for each considered CN haplotype, the percentage variation (D%) of proteolytic activity was determined as:

The number of free thiol groups of raw, cooked and digestedcooked meat was determined according to Ellman's method (1959) , with some modifications. Two hundred fifty microliter of each samples were mixed with 2.5 mL of 0.1 M sodium phosphate buffer (containing 1 mM ethylenediaminetetraacetic acid (EDTA); pH 8.0, reaction buffer; Sigma-Aldrich, Milan, Italy) and 50 lL of 5-5 0 -dithiobis(2-nitrobenzoic acid) (DTNB) reagent solution (4 mg in 1 mL of sodium phosphate buffer; Sigma-Aldrich, Milan, Italy). After the solution was mixed and allowed to stand at room temperature (25 °C) for 30 min, absorbance was read at 412 nm, using UV-vis Spectrophotometer 1204 (Shimadzu, Kyoto, Japan). Reaction buffer was used instead of sample, as a reagent blank. A molar extinction coefficient of 14.150 M À1 cm À1 was used to calculate moles of thiol groups. Thiol groups are expressed as nanomoles of free thiol groups per milligram of protein. Each determination and measurement was made in triplicate.

The FRAP assay was performed according to the procedure described by Benzie and Strain (1996) , with some modifications. The FRAP reagent was prepared by mixing 10 mL of 300 mM acetate buffer (pH 3.6), 1 mL of 10 mM TPTZ in 40 mM HCl, and 1 mL of 20 mM FeCl3 (in the ratio 10:1:1, v/v/v). It was daily prepared and warmed to 37 °C before use. Aliquots of 100 μL of water-soluble extracts of samples were mixed with 2.9 mL of FRAP reagent, and incubated at 37 °C for 30 min. The increase in absorbance was measured at 593 nm against acetate buffer (pH 3.6), using a UV-VIS Spectrophotometer 1204 (Shimadzu, Kyoto, Japan). The blank reagent was prepared by adding distilled water instead of the sample. Aqueous solutions of FeSO4 7H2O (100 to 1000 μM) 166 were used for the calibration and the results were expressed as FRAP value (μM Fe (II)) of the sample. Each determination and measurement was made in triplicate.

The number of free thiol groups was determined according to Ellman's method (1959) , with some modifications. Two hundred fifty μL of water-soluble extract of samples were mixed with 2.5 mL of 0.1 M sodium phosphate buffer (containing 1 mM EDTA; pH 8.0, reaction buffer) and 50 μL of DTNB reagent solution (4 mg in 1 mL of sodium phosphate buffer). After the solution was mixed and allowed to stand at room temperature (25 °C) for 30 min, absorbance was read at 412 nm, using UV-VIS Spectrophotometer 1204 (Shimadzu, Kyoto, Japan). Reaction buffer was used instead of sample, as a reagent blank. A molar extinction coefficient of 14.150 M -1 cm -1 was used to calculate moles of thiol groups. Each determination and measurement was made in triplicate.

A modification of the original method of Re and others (1999) was applied to assess the scavenging capacity of yogurt samples in a reaction with the ABTS radical cation (ABTS•+), generated by oxidation of ABTS diammonium salt stock solution with potassium persulfate (K2S2O8). Stock solutions of ABTS (7 mM) and potassium persulfate (140 mM) were prepared in water, and ABTS•+ radical solution was produced by reacting 10 mL of the Donkey's yogurt as novel food . . . ABTS stock solution with 175 μL of potassium persulfate solution. The mixture was left in the dark at room temperature for 12 to 16 h before use. For the evaluation of antioxidant capacity, the ABTS•+ solution was diluted with ethanol (96%) to obtain the absorbance of 0.700 ± 0.020 at 734 nm. Two milliliters of ABTS•+ 150 solution was mixed with 100 μL of the water-soluble extracts of samples in a cuvette and the decrease in the absorbance was measured after 30 min, using a UV-VIS Spectrophotometer 1204 (Shimadzu, Kyoto, Japan). The reagent blank was prepared by adding 100 μL of ethanol instead of the sample. Antioxidant activity was expressed as a percentage inhibition (I) of ABTS•+ radical and calculated by the equation: