The search for highly effective, available, and safe antioxidants from natural sources is a justifiable tendency of modern scientific research [1]. Cow’s milk is traditionally considered the best replacement for mother’s milk for children. It contains bioactive constituents that smooth the transition from intrauterine life to life under worldly conditions and stimulates development of the brain, digestive tract, and immune system [2]. Colostrum is a specific initial diet of newborn mammals that is rich in immunoglobulins, antimicrobial peptides, and other bioactive molecules, including growth factor [3]. Protein constituents of adult milk and colostrum can be divided into two main groups, i.e., serum proteins and caseins. The ratio of serum proteins to casein in colostrum is 80:20 while it is 60:40 in adult milk. The ratio is 50:50 in late stages of breast feeding [4]. Bovine milk serum and colostrum serum especially contain >200 different proteins, the dominant constituents of which are α- and β-lactalbumins, bovine serum albumin (BSA), and immunoglobulins [5]. Proteome methodology was successfully used in research and characterization of proteins in milk and milk products [6,7,8].

The goal of the present work was to study proteolytic hydrolysates of bovine colostrum serum proteins as a source of antioxidant peptides. Purified peptides derived from colostrum serum proteins could find applications as a biologically compatible agent of human functional food.

Bovine colostrum was defatted by centrifugation as reported before [9]. Casein was precipitated by acidification of defatted colostrum to pH 4.3 (addition of 1M HCl). The precipitate of casein was removed after holding the suspension for 12 h at 4°C and centrifuging under the above conditions. The supernatant containing serum proteins was hydrolyzed for 4 h by three different types of proteolytic enzymes, i.e., papain (pH 5.8, 50°C), alkalase (pH 9.0, 45°C), and trypsin (pH 7.8, 37°C). The molecular masses of the bovine colostrum serum proteins (BCP) and peptides from enzymatic hydrolysis were analyzed comparatively using 15% polyacrylamide gel-electrophoresis in the presence of sodium dodecyl sulfate (SDS-PAGE).

Figure 1 shows that the selected enzymes effectively hydrolyzed colostrum serum proteins into peptides. However, the alkalase hydrolysate contained more fractions of peptides with molecular masses 4–15 kDa than the two other enzyme hydrolysates.

Fig. 1
figure 1

SDS-PAGE of bovine colostrum serum proteins and their proteolytic hydrolysates: M, marker molecular masses; PBC, proteins of bovine colostrum serum; TH, AH, and PH, hydrolysates obtained by treatment with trypsin, alkalase, and papain, respectively.

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The degree of hydrolysis (DH) characterized the efficiency of the degradation of polypeptide bonds and a numerical index for the number of peptides changing to a soluble phase during the hydrolysis and responsible for the antioxidant properties of the protein hydrolysis products [10].

Figure 2 illustrates the hydrolysis kinetics of BCP. The DH was observed to increase sharply during the initial 2.5 h and then grew slowly and even stabilized. The DH of the alkalase reaction mixture was greater than those of the proteolytic mixtures with papain and trypsin. This result showed that the DH was significantly higher for treatment of BCP with alkalase than with the other two proteases after hydrolysis for 4 h. The DH decreased with long reaction times. This was explained by a decrease in the number of peptide bonds in the starting protein substrate that was available for hydrolysis. The higher DH led to short peptides that could exhibit more effective biological activity, including antioxidant [11].

Fig. 2
figure 2

Degree of hydrolysis of colostrum serum proteins without casein.

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Total peptides from alkalase hydrolysis were next subjected to centrifugal ultrafiltration to determine the most active antioxidant (for neutralization of OH•– and ABTS•+). This produced three fractions (PBC-a >10 kDa, PBC-b 3–10 kDa, and PBC-c <3 kDa).

Table 1 presents the antioxidant activities of the obtained peptide fractions. The highest activity was observed for fraction PBC-c with EC50 = 132.92 ± 0.06 μg/mL (for ABTS•+) and 405.96 ± 0.11 μg/mL (for OH•–). Our research results agreed with the literature, according to which low-molecular-mass products from protein hydrolysis were more active antioxidants than their high-molecular-mass precursors [12, 13].

Table 1 Radical-neutralizing Activity of Peptide Fractions PBC-a, PBC-b, and PBC-c
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The amino-acid composition of peptides is known to play an important role in their antioxidant properties [14, 15]. All 17 amino acids except Trp (which decomposed under the acid-hydrolysis conditions) were identified in all three fractions from centrifugal membrane ultrafiltration in the present study. Table 2 shows that the peptide fractions had equilibrated and rich amino-acid compositions and good food value.

Table 2 Amino-acid Composition of Alkalase Hydrolysate of Three Peptide Fractions, %
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According to one report [13], several amino acids, e.g., Pro, Ala, Gly, and Glu, can improve radical-neutralizing activity. The present study showed that the alkalase hydrolysate fraction PBC-c (MM ≤3 kDa) had the highest contents of Pro (12.18%), Ala (9.31), Gly (21.75), and Glu (11.75). Correspondingly, this same fraction had the highest antioxidant activity of the obtained peptide fractions.

Our results showed that the proteolytic enzyme alkalase more efficiently formed peptides from casein-free colostrum serum than papain. The alkalase enzyme hydrolysate had a relatively higher DH value, total yield of peptides, and antioxidant and radical-neutralizing activity. The most active alkalase hydrolysate fraction contained peptides with molecular masses of 1000–3000 Da and exhibited potent antioxidant activity with EC50 values of 132.92 ± 0.06 μg/mL (for ABTS•+) and 405.96 ± 0.11 μg/mL (for OH•–). The amino-acid compositions after acid hydrolysis were determined for the three peptide fractions.

Experimental

Colostrum samples (collected up to 48 h after birth) were acquired at the market in Turpan (Xinjiang, PRC). Defatted colostrum was produced by centrifugation in a CR22N high-speed refrigerated centrifuge (Hitachi Koki Co., Ltd., Japan) at 10,000 rpm for 15 min. Defatted milk was acidified with HCl (1M) to pH 4.3. The precipitate of casein was removed by centrifugation as above. The pH of the defatted and casein-free colostrum obtained in this manner was adjusted (1M NaOH or HCl) according to the optimal values of the proteolytic enzymes before hydrolysis using trypsin (pancreatic, porcine), alkalase (from Bacillus licheniformis), and papain (from papaya latex) (Beijing Solarbio Science & Technology (PRC) at the optimal pH values and temperatures keeping the enzyme-to-substrate ratio at 3:97.

Determination of Total Protein Content. The total protein content was measured using a Pierce Albumin Kit for protein analysis (Thermo Scientific, USA) with bovine serum albumin (BSA) as a standard.

Electrophoretic Analysis. Electrophoresis used polyacrylamide gel (T = 15%, C = 3.4%) containing Na-SDS (0.1%) and Laemmli buffer system [16]. The electrophoresis was performed at 75 V potential in concentration mode (30 min) and 150 V in separation mode. Proteins in the gel were stained by the usual method using Coomassie Brilliant Blue R-250.

Determination of DH. The DH values (%) of products from enzymatic hydrolysis of colostrum serum proteins were calculated using o-phthalaldehyde (OPA) as before [17] with slight modifications. Each peptide fraction (10 μL each) was mixed with OPA reagent (1.0 mL) [a solution of sodium tetraborate decahydrate (7.62 g) and Na-SDS (0.2 g) in H2O (150 mL) was treated with a solution of OPA (160 mg) in EtOH (4 mL), adding before use dithiothreitol (176 mg) and adjusting the total reagent volume to 200 mL]. The mixture was shaken for 2 min at room temperature and centrifuged at 4500 rpm for 15 min. Absorption by the enzyme reaction mixture and the same mixture with enzyme inactivated by boiling was measured at 340 nm using a UV2550 spectrophotometer (Shimadzu, Japan). The DH (%) was calculated as before [18] using the formula

$$ \mathrm{DH},\%=\mathrm{n}/{\mathrm{n}}_0\bullet 100\%=,\mathrm{n}=\left({\mathrm{A}}_{340\ \mathrm{nm}\ \mathrm{after}}-{\mathrm{A}}_{340\ \mathrm{nm}\ \mathrm{before}}\right)/\varepsilon \mathrm{MF}, $$

where n0 is the total number of peptide bonds, n, the number of peptide bonds after hydrolysis; A340 nm after, absorption of the mixture at 340 nm after hydrolysis; A340 nm before, absorption of the mixture at 340 nm before hydrolysis; and ε, the molar absorption coefficient.

Isolation and Purification of Antioxidant Peptides. The hydrolysate obtained using alkalase that showed the highest antioxidant activity was fractionated using centrifugal ultrafiltration membranes (Millipore, USA) with molecular mass exclusion values of 3 and 10 kDa. First, the alkalase hydrolysate was processed through the 10-kDa membrane by centrifugation (5417R centrifuge, Eppendorf, Germany) at 3500 rpm for 30 min. This stage produced fractions held on the membrane (≥10 kDa, PBC-a) and passing through it (<10 kDa). The fraction passing through this membrane (<10 kDa) was diluted to 30 mL and filtered through the 3-kDa membrane under the same centrifugation conditions to produce two fractions, one held on the membrane (≥3 kDa, PBC-b) and passing through it (<3 kDa, PBC-c).

Analysis of Amino-acid Compositions of Peptide Fractions. The amino-acid compositions of the peptide fractions were analyzed after hydrolysis by a mixture of HCl (6M) and anhydrous trifluoroacetic acid (TFA) in a 2:1 ratio for 24 h at 110°C. The amino acids were determined after hydrolysis as their phenylthiocarbamoyl derivatives (PTC) using an Agilent 1100 HPLC chromatograph (Agilent, USA) and a Diamonsil AAA column (250 × 4.9 mm, 5 μm). Preparation of the PTC-amino acids and elution of the column by an MeCN gradient in NaOAc buffer (10 mM, pH 6.4) used the literature methods [19].

Radical-neutralizing Activity of Peptides for OH •–. The neutralizing activity for OH•– radicals was evaluated as before [19] with modifications. A reaction mixture consisting of a peptide fraction (in the concentration range 0.1–2.0 mg/mL), EDTA-Fe2+ (0.99 μM), safranin O (1.049 × 10–3 μM), and H2O2 (0.27 μM) in K-phosphate buffer solution (150 mM, pH 7.4) with a total volume of 4.5 mL was thermostatted at 37°C for 30 min. The absorption of the mixture was measured at 520 nm. The ability to neutralize OH•– radicals was determined using the following equation:

$$ \mathrm{Neutralization}\ \mathrm{of}\ {\mathrm{OH}}^{\bullet \hbox{--} }\ \mathrm{radicals},\%=\left[\left({\mathrm{A}}_{\mathrm{sample}\ 520\ \mathrm{nm}}\hbox{--} {\mathrm{A}}_{\mathrm{blank}\ 520\ \mathrm{nm}}\right)/\left({\mathrm{A}}_{\mathrm{control}\ 520\ \mathrm{nm}}\hbox{--} {\mathrm{A}}_{\mathrm{blank}\ 520\ \mathrm{nm}}\right)\right]\cdotp 100\%, $$

The radical-neutralizing activity of ascorbic acid (Wuhan Biocar Pharmacy Co., Ltd., PRC) was measured under the above conditions in the concentration range 0.010–0.500 mg/mL as a comparative control of the antioxidant activity.

The percent neutralization of the radical was plotted as a function of the antioxidant concentration using the experimental data. EC50 values were determined from the plot.

Radical-neutralizing Activity of Peptides for ABTS •+ Cation-radical. Peptide fractions were analyzed for the ability to neutralize ABTS•+ cation-radicals using the published method [3] with a slight modification. ABTS•+ (7 mM) and potassium persulfate (2.45 mM) solutions were mixed for 16 h in the dark at room temperature. Then, the mixture was diluted with an equal volume of K-phosphate buffer solution (5 mM, pH 7.4). The test for ABTS•+ neutralization was performed by mixing this diluted mixture (1.0 mL) and an equal volume of protein hydrolysate fraction (with peptide concentrations of 2–10 mg/mL) and incubating the reaction mixture for 15 min. Absorption of the reaction mixture of the peptide fractions and ABTS•+ was measured at 734 nm. The ABTS•+ neutralizing ability was calculated using the formula

$$ \mathrm{Neutralization}\ \mathrm{of}\ {\mathrm{A}\mathrm{BTS}}^{\bullet +}\ \mathrm{radicals},\%=\left({\mathrm{A}}_{\mathrm{control}}\hbox{--} {\mathrm{A}}_{\mathrm{sample}}/{\mathrm{A}}_{\mathrm{control}}\right)\times 100\%, $$

where Acontrol is the absorption at 734 nm of the control mixture; Asample, absorption at 734 nm of the mixture with the peptide fraction sample.

EC50 values for peptide fractions and ascorbic acid as an antioxidant standard were determined graphically as described above for neutralization of hydroxide anion-radical.