Protein isolate of α-lactalbumin (Biopure-lactoalbumin^TM) was provided by Davisco Food International Inc. (Le Sueur, MN, USA). Alcalase was from Novozymes Biopharma US Inc. Buffer salts were NaHPO₄ (Mallinckrodt) and NaH₂PO₄ (J. T. Baker). Folin reagent and 1-anilin-8-naftensulfide (ANS) were purchased from Sigma Aldrich (St. Louis, MO). For electrophoresis analysis: acrylamide, bis-acrylamide, tricine, sodium dodecyl sulphate (SDS) and Tris were purchased from MP Biomedicals; 2-mer- captoethanol and the molecular weight marker (MWM) with 7 bands from 2.5 to 17 kDa (MW-SDS-17S) were purchased from Sigma Aldrich (St. Louis, MO); bromophenol blue (J. T. Baker), HCl (Dorwill), acetic acid (VETEC). For ABTS assays: 2,20- azinobis-(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS) and 6-hy- droxy-2,5,7,8-tetramethylchroman-2-acid (Trolox) were purchased from Sigma Aldrich, and potassium persulphate was from J. T. Baker. For ORAC (Oxygen Radical Antioxidant Capacity)-FL assay: Fluorescein (FL) disodium salt and 2,20-azobis (2- methylpropionamidine) dihydrochloride (AAPH) were obtained from Sigma Aldrich (St. Louis, MO). For antiglycant assays: methylglyoxal (MGO) and aminoguanidine (AG) were purchased from Sigma Aldrich (St. Louis, MO); bovine serum albumin (BSA) (GIBCO BRL) and glucose (Glc) (Fluka). For angiotensin converting enzyme (ACE)-inhibition assay: histidil-hipuril-leucine (HHL) was purchased from Sigma Aldrich (St. Louis, MO).

The hydrolysis reaction was carried out using Alcalase enzyme with 8% (w/V) of protein isolate in buffer phosphate solution 100 mM pH 7 and incubation temperature of water bath 30˚C with agitation of 150 rpm. After incubation time (as in Section 2.3), reaction was stopped by heating at 100˚C for 10 minutes. Samples were freezed, lyophilized and stored at −20˚C for subsequent analysis.

For the optimization of hydrolysis process an experimental design [31] (compose central design) was used based on a response surface model, full factorial design. The hydrolysis reaction was evaluated by determining ABTS and ORAC-FL values (response variables) with the variation of two factors, enzyme:substrate ratio (r) (% w/w) and time (t) (minutes). Seven samples were prepared: four assays, two factors (r and t) with two levels (0.0050 and 0.1000% w/w, 0 and 60 minutes, respectively) and three central points (0.0525% w/w and 30 minutes, for r and t factors, respectively) in order to estimate the experimental error (sample conditions are listed in Table 1). The equation for the proposed model of response variables ABTS and ORAC-FL (Y_i) is shown in Equation (1):

where β₀ is the intersection point; β₁ and β₂ are de linear coefficients; β_1,2 is the coefficient which represents the interaction between the independent variables (factors r and t); and ε is the variable error. Model parameters where calculated with Statgraphic Plus version 5.1 program by multiple linear regression (MLR).

Protein content was determined by Lowry method [32] preparing 0.3 mg/mL solutions of each hydrolysate in phosphate buffer 10 mM pH = 7.4. Surface hydrophobicity was determined with the method described by Hayakawa and Nakai [33] , based in spectrofluorimetry measurements with ANS (fluorescent probe). Hydrolysates solutions of 1 mg/mL were prepared in sodium phosphate buffer 100 mM pH = 7.0, agitated for 30 minutes at room temperature and centrifuged at 10.000 g and 4˚C for 10 minutes. 8 mM

Results are expressed as the means ± SD (n = 6). ANOVA analysis was made by column using Tukey test. Means values in the same column with different letters (a, b, c) state significant differences (p < 0.05).

ANS solution was prepared weighting 0.00128 g of ANS in 5 mL of buffer. Measurements were made in spectrofluorimeter microplate reader (Varioskan® Flash, Thermo Electron Corporation) by adding 250 μL of different concentration solutions and 3.75 μL of ANS in each well. Also, 250 μL of MeOH and 3.75 μL of ANS were added to determine maximum fluorescence. This probe was measured at 363 nm and 475 nm wave lengths of excitation and emission, respectively. According to Kato and Nakai [34] surface hydrophobicity index (S₀) corresponds to the initial slope of the curve of probe’s fluorescence intensity as a function of protein concentration. Equation (2) shows how to calculate surface hydrophobicity:

where IF_protein+ANS is the protein’s fluorescence intensity with ANS, IF_protein is protein’s fluorescence intensity without ANS, IF_maximum is the maximum fluorescence which corresponds to methanol with ANS.

Tricine-SDS-PAGE analyses were performed for the hydrolysates according to the method described by Schägger and von Jagow [35] . Electrophoresis gel contains three parts: stacking, spacer and separating gels with 4%, 10% and 16% of acrylamide, respectively. Sample solutions were prepared of 2 mg/mL in sample buffer containing 12% glycerol, 4% SDS, 2% 2-mercaptoethanol (v/v), 50 mMTris, 0.01% bromophenol blue and 0.1 M HCl for pH adjustment. Cathodic buffer was prepared with 100 mMTris, 0.1% SDS (w/V) and 100 mMTricine, pH 8.9. Anodic buffer was prepared with 200 mMTris and 0.1 M HCl for pH adjustment (pH 8.9). Electrophoresis conditions were as follows: 30 V and 30 mA until the front went through the stacking gel and then 90 V (Sigma Aldrich PS 250-2) for two gels of 1.0 mm thick. The apparatus used was an Amersham Biosciences SE 260. Gel fixation was performed by 50% methanol and 10% acetic acid solution during 30 minutes agitation and coloration was performed by a Coomasie Blue R250 0.1% solution in 30% methanol and 10% acetic acid over night. Decoloration was performed by 30% methanol and 10% acetic acid solution during 30 minutes agitation and several washes with 15% methanol and 5% acetic acid solution. Molecular weight marker with 7 bands from 2.5 to 17 kDa was used.

SE-HPLC analysis were carried out as described by Molina et al. [36] by the equipment Shimadzu, SPD-20A detector and LC-10AT pump detecting at 280 nm. Samples were eluted in a Molecular Exclusion Column BioSep-Sec 2000 with an isocratic flow of 1 mL/min and phosphate buffer 50 mM (pH 6.8) 0.5% of SDS as mobile fase. Samples were prepared in mobile fase in a concentration of 2 mg/mL. Hydrolysis percentage was determined by quantifying α-lactalbumin in each hydrolysate with anα-lactalbumin calibration curve.

Antioxidant capacity was determined by two methods: ABTS and ORAC-FL, as electron transfer (ET) and hydrogen atom transfer (HAT) methods, respectively. ABTS was performed by the method described by Re et al. [37] in which antioxidant capacity is determined by measuring ABTS reagent absorbance at 734 nm. All samples were compared with a calibration curve of trolox. Briefly, ABTS stock solution (7 mM) with potassium persulphate (2.45 mM) was mixed with phosphate buffer 5 mM pH 7.4 until reaching an absorbance of 0.7. Then, 30 μL of sample was mixed in tubes with 3 mL of ABTS in buffer and 10 minutes later absorbance was measured in a spectrophotometer. In addition, doses-response curves were done to calculate IC₅₀ by constructing the curve% Inhibition vs. [Protein] (mg/mL) (from 0.25 to 5 mg/mL of protein) and obtaining the logarithmic function in order to calculate the corresponding protein concentration to 50% of inhibition. Inhibition percentage is calculated according to Equation (3):

where A_control is the absorbance of 3 mL of ABTS in buffer with 30 μL of buffer and A_antioxidant is the absorbance of 3 mL of ABTS in buffer with 30 μL of trolox or sample.

ORAC-FL was performed as described by Ou et al. [38] modified by Dávalos et al. [39] in which a fluorescent probe (FL) is oxidized by an oxygen radical generator (AAPH) lowering its fluorescence intensity. Measurements were displayed at 485 nm and 520 nm of excitation and emission wave lengths, respectively, at 37˚C for 104 minutes in the equipment Varioskan® Flash. Briefly, samples and reagents were prepared in phosphate buffer 75 mM pH 7.4. Each well had a final volume of 200 μL: 120 μL of 1.17 mM fluorescein solution (70 nM final concentration), 60 μL of AAPH (12 mM final concentration) and 20 μL of antioxidant substance (trolox or sample). All samples were prepared in duplicate and each one of the preparations was tested at least in triplicate. Equation (4) shows calculation of the area under the curve (AUC) Fluorescence vs Time:

where f₀ is the fluorescence at 10 minutes of incubation at 37˚C and f_i is the fluorescence measured every minute. Curves of Fluorescence vs Time were normalized to the curve of the blank calculating the net AUC as the difference between AUC_{antioxidant (trolox or sample)} and AUC_blank. Trolox calibration curve (net AUC_trolox vs n_TE (μmol TE)) was constructed in order to calculate samples antioxidant capacity (μmol TE/mg of protein). Besides punctual measurements, IC₅₀ values were obtained by constructing the curve n_TE vs [Protein] (mg/mL) to obtain logarithmic function and the concentration of protein correspondent to 50% inhibition of peroxyl radicals.

Antiglycant activity was assessed using two methods: BSA-MGO and BSA-Glc.

In vitro glycation assay with BSA-MGO was determined as described by Wang et al. [28] with some modifications. Briefly, concentrations of 5 and 10 mM (final concentration) of methylglyoxal (MGO) were mixed with 2 mg/mL BSA (1 mg/mL final concentration) and sufficient volume of phosphate buffer 10 mM pH 7.4 with 0.02% sodium azide, in a final volume of 5 mL. Negative control was prepared with 2.5 mL of BSA and 2.5 mL of phosphate buffer, and positive control was prepared with BSA, MGO and sufficient volume of phosphate buffer. Aminoguanidine was used as the reference substance and it was mixed with BSA, MGO and phosphate buffer in three final concentrations: 1, 4 and 8 mM. Sample 4 was tested in concentrations of 1 and 10 mg/mL. All the tubes were incubated at 37˚C for 13 days measuring fluorescence at 340 nm and 420 nm of excitation and emission wave lengths, respectively. Inhibition percentage was calculated as fallows (Equation (5)):

In vitro glycation assay with BSA-Glc was determined as described by Mesías et al. (25) with some modifications. Glucose was prepared in concentrations of 20 and 40 mM (final concentration) in a final volume of 5 mL mixing it with BSA (1 mg/mL final concentration) and sufficient volume of the same phosphate buffer as BSA-MGO model. Negative control used was the same as BSA-MGO assay and positive control was prepared with Glc, BSA and phosphate buffer. Sample tested concentrations were the same, incubation and measurement conditions were the same as well.

Antihypertensive activity was determined as described by Cushman & Cheung [40] modified by Kim et al. [41] . In this assay, the inhibition of angiotensin converting enzyme (ACE) was evaluated. Briefly, samples concentration of 5 mg/mL were prepared in borate buffer 0.2 M pH 8.3 and agitated at 25˚C, 700 rpm, for 30 minutes to solubilize them. Then, samples were centrifuged at 9.500 rpm for 20 minutes at 20˚C. Assay buffer (borate buffer 0.2 M and NaCl 2M, pH 8.3) was added to the sample, mili Q water and HHL 5 mM. Eppendorf tubes were incubated at 37˚C, 700 rpm, for 5 minutes, and ACE was added in cold (0 - 10 mU). Same tubes were incubated at 37˚C, 700 rpm, for 30 minutes and after, tubes were incubated at 90˚C for 10 minutes. Finally, colour reagent and potassium phosphate buffer 0.2 M pH 8.3 were added, samples were centrifuged at 20˚C, 6000 rpm, for 10 minutes and supernatant absorbance was measured at 382 nm. ACE inhibition percentage was calculated according to equation 6:

where A_s and A_0s are the absorbance of sample in ACE’s presence and absence, respectively; A_max is the absorbance in the absence of sample and A_0max is the absorbance in the absence of sample and ACE.

All the measurements were determined at least in triplicate. Results were expressed as means values ± standard deviation. Analysis of variance (ANOVA) and Tukey test were applied to determine significant differences between values (p < 0.05). Statistical analysis was done using Infostat v. 2015 and Statgraphic Plus v. 5.1 programs.

Hydrolysis of α-lactalbumin with Alcalase enzyme preparation on buffer phosphate solution (pH 7, 100 mM) was studied as a strategy for the valorization of whey protein fractions, a by-product usually discarded by small and medium dairy enterprises. Initially, protein content and hydrophobicity were determined in the supernatants of the hydrolysates samples 1 to 7. Sample 3 formed a gel after heating at 100˚C for 10 minutes, probably because of the reaction conditions that favoured α-lactalbumin intermolecular interaction by disulfide bonds. For this reason, sample 3 was discarded for subsequent analysis.

It could be observed that protein content measurements tended to increase with factors time of hydrolysis and r (enzyme to substrate ratio, see Table 1). Conversely, surface hydrophobicity decreased after 60 min of hydrolysis at factor r = 0.1000.

Hydrolysis carried out by subtilisin-like enzymes (such as alcalase) might convert some hydrophobic groups to hydrophilic groups by generating two-end carbonyl and amino groups [42] [43] . Furthermore, enhanced protein hydrolysate content can be attributed to the release of small soluble peptides and new carboxylic and amine groups from aminoacids, increasing exposure of more polar and charged groups to surrounding water [44] , therefore reducing hydrophobicity values.

Hydrolysis percentages determined by SE-HPLC were 0 for samples with 0 minutes of reaction as expected. Samples 5, 6 and 7 have the same reaction conditions and presented no significant differences between them (p < 0.05) as expected. Sample 4 had the higher hydrolysis percentage of all samples related to greater protein hydrolysis.

To continue with the characterization of hydrolysates, electrophoresis gels were made (Figure 1). As it can be observed in Figure 1, samples 1 and 2 (lanes 1 and 2, respectively) show the same bands as α-lactalbumin (lane 8) confirming there was no hydrolysis at 0 minutes of reaction. As to samples 4, 5, 6 and 7, no band can be discriminated probably because of the extent of hydrolysis reaction (Table 1) forming small peptides which could have escaped the gel pore.

According to Peng et al. [45] , hydrolysis percentage of whey protein isolate after 5 hours of enzymatic hydrolysis with Alcalase was of 35% - 36%, which is a little bit higher than sample 4 percentage of hydrolysis. In such study, this percentage increased in the first 3 hours reaching a plateau at 5 hours. The value of 3 hours of hydrolysis would be comparable to the one of sample 4 (1 hour of hydrolysis) which could be explained because of being a mix of whey proteins in contrast with α-lactalbumin isolate, and because of different hydrolysis conditions. Adjonu et al. [3] treated whey protein isolate with chymotrypsin, pepsin and trypsin reaching lower percentage of hydrolysis. Alcalase hydrolysates appear to be more hydrolyzed than digestive enzymes, possibly because of its non-specificity [3] producing a great quantity of short peptides with bioactive properties [9] [46] .