Original papers
Two-step Enzymatic Modification Method to Enhance the Zn2+-chelating Activity and Antioxidant Activity of Zein
2020 Volume 26 Issue 2 Pages 299-311
Details
2020 Volume 26 Issue 2 Pages 299-311
Zein is a major by-product of corn processing and has a wide range of applications in many fields due to its unique solubility and specific amino acid composition. The aim of this study is to hydrolyze zein stepwise using alcalase and proteinase K while considering the enzymatic hydrolysis sequence and assessing the zein-chelating ability, solubility, foaming, and emulsification properties of the zein hydrolysate. The antioxidant activity of Zn2+ chelating peptide was determined. Zinc-chelating ability was used as the evaluation index, and proteinase K was further hydrolyzed on the basis of alcalase hydrolysis. The optimal hydrolysis conditions were reaction time of 2 h, substrate concentration of 10 mg mL−1, enzyme addition amount of 2.8 U mg−1, reaction temperature of 60 °C, and hydrolysate chelation ability of 12.16 mg g−1, thereby corresponding to a degree of hydrolysis (DH) of 35.30%. On the basis of the hydrolysis by proteinase K, alcalase was further hydrolyzed, and the optimal hydrolysis conditions were reaction time of 3 h, substrate concentration of 10 mg mL−1, enzyme addition amount of 8.0×105 U mg−1, reaction temperature of 50 °C, and hydrolysate chelation ability of 13.72 mg g−1, thereby corresponding to a DH of 29.32%. Zein Zn2+-chelating peptide is significant for 1,1-diphenyl-2-picrylhydrazyl free radicals, hydroxyl radicals, and ABTS·+. The clearance effect is positively correlated with the mass concentration of the polypeptide. Zein Zn2+-chelating peptide has good Zn2+ chelating ability and antioxidant activity. Hence, it has broad research prospects for new dietary supplements.
Proteins, especially water-insoluble ones, can be chemically modified to improve their functional properties and for their effective use as food ingredients. Their functional properties are important in food applications. However, most native proteins do not have desirable functional properties. Thus, protein modification for improvement of properties, especially protein solubility, must be addressed (Andrés et al., 2006). Enzymatic hydrolysis is a common approach to modify proteins because of its mild reaction conditions and ease of control. This approach can disrupt the 3D protein structure, reduce molecular weight (MW), and consequently alter functional properties (Kristinsson and Rasco, 2000). Enzymatic hydrolysis has been applied successfully for improvement of functional properties by cleaving proteins into peptides of desired sizes, charges, and surface properties (Andrés et al., 2006). These peptides exert different physiological functions after enzymatic hydrolysis, but they are inactive within the parent protein (Sarmadi and Ismail, 2010). Hydrolysates have excellent solubility values at high degrees of hydrolysis (DH). However, excessive hydrolysis negatively affects the functional properties, e.g., foaming and emulsification (Klompong et al., 2007).
Protein hydrolysates from food sources, such as rice endosperm (Zhang et al., 2010) and rapeseed (Pan et al., 2011), possess antioxidant activities besides functional properties. Enzymatic hydrolysis affects the molecular size, hydrophobicity, as well as polar and ionizable groups of protein hydrolysates (Klompong et al., 2007). Zn2+-chelating peptides from wheat germ protein hydrolysates by alcalase have high Zn2+-chelating capacity and possess higher zinc bioavailability than ZnSO4 (Zhu et al., 2015).
Zein, a major corn protein, is a heterogeneous mixture of polypeptides. It is rich in glutamine that could be hydrolyzed by specific enzymes and chelated with metal ions. All zein peptides are amphiphilic; they contain both hydrophobic and hydrophilic amino acid residues. As a water-insoluble, hydrophobic prolamin, zein has been studied for numerous applications. Its ability to form films and coatings is well-known (Dawson et al., 2003; Wang et al., 2005). In addition, some biological activity of hydrolysates of zein was also researched by other researchers. Wang et al. (2015) investigated zein hydrolyzed by double enzymes immobilized with calcium alginate-chitosan beadsand the product accepted had the functions of antioxidant and antihypertensive activity. However, given its water insolubility property, zein is rarely used as food ingredient.
In the present work, alcalase and proteinase K were applied to hydrolyze zein. The main aim is to investigate the Zn2+- chelating activities and functional properties of zein hydrolysates using different DH values by two-step enzyme hydrolysis. In addition, the antioxidant activities of zein hydrolysates were also evaluated and compared to investigate the effects of two-step hydrolysis on selected functional properties.
Materials and chemicals Zein (86.41% ± 0.5% on dry basis) and Folin-Ciocalteu's phenol reagent (suitable for total protein determination by Lowry's method, 1N) were provided by Shanghai Macklin Biochemical Technology Co., Ltd. (Shanghai, China). Alcalase (from Bacillus licheniformis, activity of 2×105 units·g−1 protein) and proteinase K (from Tritirachium album limber, activity of 40 units·mg−1 protein) were purchased from Beijing Solarbio Science & Technology Co., Ltd (Beijing, China). 2,2-Diphenyl-1-picrylhydrazyl (DPPH) and 2,2′-azinobis (3-ethylbenzothiazoline sulfonic acid) (ABTS) were purchased from Sigma Chemical Co (St. Louis. MO, USA). All other chemicals and regents used were of analytical grade.
Preparation of zein hydrolysates via two-step enzyme hydrolysis Zein was dissolved in phosphate buffer solution at pH 12 to give an original protein solution. Alcalase and proteinase K were used stepwise based on the previous results of single-enzyme hydrolysis. In view of the effect on zinc-chelating activity and DH, the order of enzyme addition was discussed in the present study.
According to the previous results, zein solution (10 mg·mL−1) was mixed with alcalase (2.0×105 U·mg−1) at 50 °C with stirring for 3 h, and then, the substrate for the following hydrolysis was obtained. The substrate formed from the abovementioned process was mixed with water and different volumes of proteinase K solution to give substrate concentrations of 6 mg·mL−1 to 16 mg·mL−1 and proteinase K addition levels of 2.0, 2.4, 2.8, 3.2, 3.6, and 4.0 U·mg−1. The enzymatic reaction was carried out at different temperatures (from 45 °C to 70 °C) with stirring for various time periods (from 1 h to 6 h). Some portions were separated from the reaction mixture as analysis samples for Zn2+-chelating activity and DH. A control sample was treated under the same conditions, but without reaction.
Otherwise, transpose experiment was investigated. According to the previous results, a zein solution of 10 mg·mL−1 was mixed with proteinase K of 2.4 U·mg−1 at 60 °C with stirring for 2 h, and then, the substrate for the following hydrolysis was obtained. The formed substrate was mixed with water and different volumes of alcalase solution to obtain substrate concentrations of 6 mg·mL−1 to 16 mg·mL−1 and alcalase addition levels of 2.0×105, 8.0×105, 1.4×106, 2.0×106, 2.6×106, and 3.2×106 U·mg−1. The enzymatic reaction was carried out at different temperatures from 40 °C to 65 °C with stirring at various time periods from 1 h to 6 h. Some portions were separated from the reaction mixture as analysis samples for zinc-chelating activity and DH. A control sample was treated under the same conditions but without reaction.
Zn2+-chelating ability A reference procedure with some modification was applied to analyze Zn2+-chelating ability (Wang et al., 2012). Zinc peptide complexes were obtained by adding 1 mL of 0.05 M ZnSO4 to 10 mL of ethanol solution containing 10% (W/V) of zein hydrolysate. The reaction mixture was continuously stirred for 1 h at 30 °C until white crystals were formed.
For the total amount of zinc, 10 mL of the above mentioned reaction mixture was precisely obtained and fixed to 50 mL of distilled water. Then, 20 mL was obtained to measure the total amount of Zn2+ by EDTA complexing titration. After adding two drops of xylenol orange and 4 mL of hexamethylenetetramine, the solution was titrated with 0.001 M EDTA until the color changed from purple to yellow.
For the amount of Zn2+ chelating, the above mentioned reaction solution was concentrated by gradual evaporation, and the white solid was washed with absolute ethanol and separated via frozen centrifugation (4 °C, 5 000 r·min−1, and 20 min). The obtained solid was dissolved in distilled water and fixed to 50 mL, and the chelated zinc ion was assayed by EDTA complexing titration, as mentioned above. Zinc-chelating ability was calculated using Equation (1). Each evaluation was carried out in triplicate, and the final results were subjected to statistical analysis.
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where AP is zinc content (mg), and P is sample protein content (g).
Determination of DH value The DH value was determined using the method of o-Phthalaldehyde (OPA) (Rutherfurd, 2010; Anzani et al., 2017) with slight modification. This value was used to monitor the hydrolysis reaction as the number of hydrolyzed peptide bonds related to the total number of peptide bonds of the protein. A total of 2 g of sodium dodecyl sulfonate (SDS) was added to 30 mL of 0.4 M boric acid buffer (pH 9.5), heated in a water bath to completely dissolve, and cooled to room temperature. Then, 1 mL of 80 mg·mL−1 OPA solution (in absolute ethanol) and 200 µL of β-mercaptoethanol were added. Finally, boric acid buffer solution was used to fix the volume to 100 mL. The reagent was covered with aluminum foil to protect from light. The sample was mixed with an equal volume of OPA reagent and accurately timed for 5 min. The absorbance of this solution was measured at 340 nm with SPECORD 210 Plus UV-Vis-spectrophotometer (Analytikjena Company, Germany). A standard curve was prepared using L-Leucine (0–36 µg·mL−1). DH was calculated using Equation (2), and each evaluation was carried out in triplicate.
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where A is sample concentration (mg·mL−1), and hT is the total peptide number of the sample protein (7.8 mmol·g−1).
Determination of solubility Zein and zein hydrolysates were diluted using 0.2 mol·L−1 sodium phosphate buffers at pH 3.0 to 10.0, and filtrate aliquots were used. The soluble protein contents in the filtrates were determined according to Lowry et al. (1951) as modified by Hartree (1972) using bovine serum albumin (Sigma Chemical Co., St. Louis, MO, USA) as standard. The absorbance was read at 660 nm with a UV-2401 PC spectrophotometer (Shimadzu Corporation, Kyoto, Japan), and the solubility was calculated in terms of the percentage of total nitrogen and expressed in grams of soluble protein per 100 mL of solution.
Separation of hydrolysates by Sephadex G-15 Sephadex G-15 powder was immersed in deionize water for at least 24 h at room temperature, with constant stirring with a glass rod to ensure swelling of the gel. According to the requirements of the packed column, the swelling gel should be put into the column at the same time, and attention should be paid to keeping the column wet to avoid air bubbles or stratification in the column loading. 3–5 column volumes of deionized water at least were used to balance gel column before loading until the baseline of the recorder was stable.
The sample of hydrolysate was prepared at the concentration of 1 mg·mL−1 and passed through the membrane of 0.22 µm for later use. The sample solution was loaded and eluted several times, using 5 mL of sample each time, 0.3 mL·min−1, at 220 nm. A tube fraction was collected by automatic collector every 10 min and the fractions with the same maximum absorption peak were combined and lyophilized for subsequent detection of amino acid composition.
Analysis of amino acid composition The amino acid composition of zein and its hydrolysate components separated by Sephadex G-15 were determined according to GB 5009.124-2016.
Foaming property The foaming ability and foam stability of zein hydrolysates were determined according to the method of Latorres J. M. et al. (2017) with modification. Samples were dispersed in 100 mL of phosphate buffer solution at pH 12, placed in a filling bottle, and homogenized at a speed of 10 000 r·min−1 for 1 min using a homogenizer. The dispersions formed were transferred to a 500 mL graduated beaker. The foaming ability was expressed as foam expansion at 0 min, whereas the foam stability was obtained as foam expansion after 10 min at 25 °C. The foaming ability and foam stability were calculated using Equations (3) and (4). The evaluation was carried out in triplicates, and the final results were subjected to statistical analysis.
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where A is the foam volume after homogenization (mL), and B is the foam volume after 10 min (mL).
Evaluation of emulsifying indices The emulsifying activities of the samples were evaluated via the turbidimetric method of Pearce and Kinsella (1978) and Tang CH et al. (2005) to obtain the emulsifying activity index (EAI) and emulsion stability index (ESI). For the emulsion formation, 10 mL of 10 mg·mL−1 protein solution (0.02 M phosphate buffer, pH 12) and 2.5 mL of soybean oil were homogenized in a high-speed homogenizer (FJ-200, Shanghai Specimen Model Co., Shanghai, China) at 12 000 r·min−1 for 1 min. A 50 µL emulsion was obtained at different times from the bottom of the homogenized emulsion and diluted (1:100) in 0.1% (w/w) SDS solution. The absorbance values of these samples were also measured at 500 nm against 0.1% (w/w) SDS solution, as described above. The EAI (m2·g−1) and ESI (%) of zein or zein hydrolysates were calculated using Equations (5) and (6). Each EAI and ESI evaluation was carried out in triplicate, and final results were subjected to statistical analysis.
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where A500 represents the absorbance of the analyzed sample at 500 nm, DF is the dilution factor (100), C is the initial concentration of protein (10 g·mL−1), φ is the fraction of oil used to form the emulsion (0.25), L is the optical path of the cuvette (0.01 m), EAImax is the highest value of EAI obtained for the diluted emulsions right after their formation, and EAImin is the value obtained from the emulsion after storage for 10 min.
DPPH-radical-scavenging activity The DPPH-radical-scavenging activity of zein hydrolysates with optimal zinc-chelating activity and zinc-chelating compound of zein was evaluated by the method of ÖZTÜRK M et al. (2007) with some modifications. A total of 2 mL of sample solution at different concentrations (0.0, 2.0, 4.0, 6.0, 8.0, and 10.0 mg·mL−1) was added successively to 1 mL of 0.2 M phosphate buffer at pH 6.6 and 2 mL of 0.1 mmol·L−1 DPPH in absolute ethanol. The mixture was mixed vigorously using vortex mixer and incubated for 30 min in the dark at room temperature. The absorbance of the resulting mixture was measured at 517 nm with a UV-spectrophotometer (UV-1750; Shimadzu, Kyoto, Japan). A blank was prepared in the same manner, except that distilled water was used instead of the sample. Meanwhile, Vc solutions at different concentrations (0.0, 0.2, 0.4, 0.6, 0.8, and 1.0 mg·mL−1) were used as positive controls. The DPPH radical scavenging activity was calculated using Equation (7). Each evaluation was carried out in triplicate, and final results were subjected to statistical analysis.
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where A0 is the absorbance of the sample or Vc without DPPH solution, A1 is the absorbance of the sample or Vc, and A2 is the absorbance of a blank.
Assay of hydroxyl radicals scavenging activity
The scavenging capacity for hydroxyl radical of the protein hydrolysates with optimal zinc-chelating activity and Zn2+-chelating compound of zein were measured according to the modified method of Tian Y T et al. (2012) Samples of 2 mL of protein hydrolysates with optimal Zn2+-chelating activity and Zn2+-chelating compound of zein with different concentrations (0.0, 2.0, 4.0, 6.0, 8.0 and 10.0 mg mL−1) were added to 3 mL distilled water, 0.5 mL of 6 mmol L−1 salicylic acid solution, 0.5 mL of 6 mmol L−1 FeSO4, 1 mL of 6 mmol L−1 H2O2. The mixture was homogenized in vortex, and kept standing in the absence of light for 30 min. After standing, the absorbance of the solution was measured at 517 nm, three parallel experiments per sample, distilled water as a blank control. Meanwhile, Vc solutions with different concentrations (0.0, 0.2, 0.4, 0.6, 0.8 and 1.0 mg mL−1) were used as positive controls. The hydroxyl radical-scavenging activity was calculated using Equation (8). Each evaluation was carried out in triplicate, and final results were subjected to statistical analysis.
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Where A0 is the absorbance of sample or Vc were added to distilled water, A1 is the absorbance of sample or Vc, A2 is the absorbance of the sample solution was changed to an equal amount of distilled water.
Scavenging activity of ABTS·+ radical The ability to capture the ABTS·+ cationic radical by hydrolysates was measured according to the method described by Chi et al. (2015) and Latorres J M et al. (2017) The ABTS radical cation (ABTS·+) solution was produced by reacting 0.1 g ABTS solution and 0.029 g K2SO4 with 100 mL 0.02 M phosphate buffered saline and allowing the mixture to stand in the dark at room temperature for 12–16 h before use. For the assay, the ABTS·+ solution was diluted with 0.02 M phosphate buffered saline to an absorbance of 0.85 (±0.02) at 743 nm. In the assay, a 1 mL of sample was mixed with 3.9 mL of diluted ABTS·+ solution and an absorbance (734 nm) reading was taken at 30 °C exactly after 10 min. All determinations were carried out in triplicate, at each separate concentration of the sample. Aqueous phosphate buffer (without ABTS-solution) was used as the blank. Meanwhile, Vc solutions with different concentrations (0.0, 0.2, 0.4, 0.6, 0.8 and 1.0 mg mL−1) were used as positive controls. The scavenging activity of ABTS radicals (%) was calculated using Equation (9). Each evaluation was carried out in triplicate, and final results were subjected to statistical analysis.
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Where A0 is the absorbance of sample solvent instead of sample solution, A1 is the absorbance of sample or Vc, A2 is the absorbance of phosphate buffered saline instead of ABTS·+ solution.
Statistical analysis All data were expressed as means ± standard deviation from at least three independent experiments. MS Excel 2007 program (Microsoft Corporation, Redmond, WA, USA), Origin Pro 8.0 statistics program (Origin Lab Corporation, Northampton, MA, USA), and SPSS 22.0 software (SPSS Inc., Chicago, IL, USA) were used to analyze and report the data.
Enzymatic hydrolysis Zein was subjected to hydrolysis using alcalase and proteinase K stepwise. Alcalase is a well-known endopeptidase that can hydrolyze proteins with broad specificity for peptide bonds and has a preference for large uncharged residues. Proteinase K is a broad-cut serine proteinase with a wide range of activity at a pH range of 4–12.5 and optimal temperature of 50 °C–70 °C. Hydrolysis was monitored based on zinc-chelating activity, and DH values were also determined.
First, under the optimum reaction conditions obtained from previous studies, zein was hydrolyzed by alcalase to obtain the substrate (named ZA in the succeeding experiments) for subsequent reaction by proteinase K. The effects of reaction time, substrate concentration, addition level of proteinase K, and reaction temperature on the Zn2+-chelating activity and DH of zein were determined and shown in Fig. 1.
Effects of reaction time (A), substrate concentration (B), enzyme addition (C), and reaction temperature (D) on the zinc-ion-chelating abilities and DH of zein using alcalase and proteinase K in sequentially.
Substrate was the product obtained by previous results of alcalase hydrolysis reaction. The optimal reaction conditions for alcalase hydrolysis were 10 mg·mL−1 of zein solution, 2.0×105 U·mg−1 of alcalase and 50 °C of reaction temperature with stirring for 3 h.
The Zn2+-chelating ability and DH of ZA increased with increasing reaction time, addition level of proteinase K and substrate, and reaction temperature. The Zn2+-chelating ability reached 11.05 mg·g−1 at 2 h and achieved a higher level compared with other reaction times. However, when using only alcalase for 2 h at the same concentration of zein, only 3.51 mg·g−1 was achieved. Hence, the Zn2+-chelating ability was increased by 68.24%. Therefore, 2 h was the optimum enzymatic hydrolysis time for using proteinase K (Fig. 1A). When the concentration of ZA was 6–10 mg·mL−1, the Zn2+-chelating ability gradually increased and reached the highest level of 11.03 mg·g−1 at the zein concentration of 10 mg·mL−1 (Fig. 1B). Then, the zinc-chelating ability sharply decreased subsequently along with increasing ZA concentration. The main reason for this was that a considerably high concentration of ZA impeded the contact of the zein molecule with the zinc ion. On the basis of hydrolysis by alcalase, the result was affected by the addition of proteinase K, as shown in Fig. 1C. With the addition of proteinase K in the range of 2.0–2.8 U·mg−1, the zinc-chelating ability of ZA gradually increased and reached the highest level of 12.16 mg·g−1. It increased by 23.03% compared with 9.36 mg·g−1 by only alcalase, and the corresponding DH was 35.30%. With increasing amount of the added proteinase K, the zinc-chelating ability decreased first and then increased slightly. Therefore, 2.8 U·mg−1 proteinase K was the optimum enzyme addition amount. With increasing proteinase K addition, macromolecular ZA was rapidly hydrolyzed into polypeptides. If a high amount of proteinase K was added, the more soluble polypeptides were hydrolyzed into free amino acids, thereby resulting in the decline of the zinc-chelating ability. ZA was incubated with proteinase K at 2.8 U·mg−1 at five reaction temperatures, and the evaluation results indicated that 60 °C was the most suitable temperature, as shown in Fig. 1D.
Based on the selections above, zein hydrolysates (named ZAK in the succeeding experiments) were prepared under the following conditions: ZA concentration of 10 mg·mL−1, addition level of proteinase K of 2.8 U·mg−1, reaction temperature of 60 °C, and reaction time of 2 h.
The hydrolysis of zein by proteinase K and alcalase was also studied. Similar to the abovementioned experiment, under the optimum reaction conditions in the previous work, zein was hydrolyzed by proteinase K to obtain the substrate (named ZK in the succeeding experiments) for subsequent reaction by alcalase. The effects of reaction time, substrate concentration, addition level of alcalase, and reaction temperature on the zinc-chelating ability and DH of ZK were determined. The results are given in Fig. 2. Both zinc-chelating ability and DH of ZA were enhanced with prolonged reaction time, increased addition level of alcalase and substrate, and increased reaction temperature. The zinc-chelating ability achieved the highest level at 12.68 mg·g−1 at 3 h, but a fluctuating trend was observed with increasing reaction time (Fig. 2A). When the concentration of ZK was 6–16 mg·mL−1, the zinc-chelating ability showed a gradually rising trend and reached the highest level of 12.70 mg·g−1 at the zein concentration of 10 mg·mL−1 (Fig. 2B). Then, it sharply decreased subsequently with increasing ZK concentration. The main reason was that considerably high concentration of ZK hindered the contact of the molecule substrate with the zinc ion. On the basis of hydrolysis using proteinase K, the result of hydrolysis was affected by the addition of alcalase for ZK, as shown in Fig. 2C. When the alcalase added was 8.0×105 U·mg−1, zinc-chelating ability reached an optimum level of 13.72 mg·g−1, and the corresponding DH was 29.32%. When ZK was incubated with 8.0×105 U·mg−1 alcalase at five reaction temperatures, evaluation results indicated that 50 °C was the most suitable temperature, as shown in Fig. 2D. The effect of temperature on the chelation reaction was remarkable. With increasing reaction temperature, the energy of the reactants and the effective collision frequency between the molecules also increased. When the temperature exceeded a certain value, the spatial structure of the enzyme molecule began to change and the zinc-chelating ability began to decrease at a very high temperature (>50 °C in this study). Hence, 50 °C was the most suitable temperature in the present work.
Effects of reaction time (A), substrate concentration (B), addition of enzyme (C), and reaction temperature (D) on the zinc-chelating ability and DH of zein using proteinase K and alcalase sequentially.
Substrate was the product obtained by previous results of proteinase K hydrolysis reaction. The optimal reaction conditions for proteinase K were 10 mg·mL−1 of zein solution, 2.4 U·mg−1 of proteinase K and 60 °C of reaction temperature with stirring for 2 h.
Based on the selections above, zein hydrolysates (named ZKA in the succeeding experiments) were prepared under the following conditions: ZK concentration of 10 mg·mL−1, alcalase addition level of 8.0×105 U·mg−1, reaction temperature of 50 °C, and reaction time of 3 h.
Separation by Sephadex G-15 According to the chromatogram (Fig. 3), two peaks appeared in ZAK-Zn2+ sample at 85 min and 128 min, respectively, which were named ZAK-Zn2+-F1 and ZAK-Zn2+-F2, and the peak areas were 170.98 and 578.69, respectively. There were also two peaks in ZKA-Zn2+ sample at 64 min and 108 min, respectively, which were named ZKA-Zn2+-F1 and ZKA-Zn2+-F2, and the peak areas were 107.94 and 346.57, respectively. Collect the eluent of each component, rotate, evaporate, freeze dry, and store at -20 °C for later use.
Separation of Sephadex G-15
Both ZAK-Zn2+ and ZKA-Zn2+ were separated into two Fractions eluted by Sephadex G-15, respectively. The Fractions were named ZAK-Zn2+-F1, ZAK-Zn2+-F2, ZKA-Zn2+-F1 and ZKA-Zn2+-F2, respectively.
Amino acid composition The amino acid composition of food proteins and peptides is a major determinant of their functionality and bioactivity. Zein contained significantly (P < 0.05) higher amounts of Glu, Leu, and Ala. However, after its hydrolysis by different enzymes, the content of Leu was greatly reduced, whereas those of Asp, Glu and Pro were greatly increased. The hydrolysate mainly contained acidic and nonpolar amino acids. The acidic and nonpolar amino acid contents of the hydrolysate ZKA-Zn2+ increased and were remarkably higher than those of ZAK-Zn2+ (Table 1). This result is similar to that reported by Guo et al. (2014). The amino acid residue side chains of whey protein hydrolysates, including Asp, Glu, His, Ser, Cys, Asn, and Gln, have been reported to bind divalent metals. Peptides with more acidic amino acid residues could bind more Zn2+ than those with fewer acidic amino acid residues (Jiang et al., 2014). ZKA-Zn2+, which has a higher acidic amino acid content (51.67%), showed better Zn2+ chelation ability (13.72 mg·g−1) in this study. Analysis of amino acid components found that acidic amino acids, such as Glu and Asp, remarkably contributed to the chelation reaction. ZKA-Zn2+ had more Zn2+-chelating amino acids (58%) than ZAK-Zn2+ (38%), and the samples had similar total amounts of cationic amino acids (5% and 2%, respectively), which impeded Zn2+ chelation due to electrostatic repulsion. Based on the amino acid profiles, both hydrolysates can chelate Zn2+, but ZKA-Zn2+ has higher Zn2+-chelating capacity.
| Amino acid | Zein | ZAK-Zn2+-F1* | ZAK-Zn2+-F2* | ZKA-Zn2+-F1* | ZKA-Zn2+-F2* |
|---|---|---|---|---|---|
| Phe | 7.04±0.08 | 2.11±0.06 | 3.73±0.33 | 2.17±0.11 | 4.40±0.07 |
| Ala | 9.93±0.12 | 5.65±0.04 | 4.71±0.18 | 6.89±0.27 | 7.05±0.13 |
| Met | 1.63±0.06 | 1.05±0.11 | 0.96±0.06 | 1.24±0.13 | 1.25±0.14 |
| Pro | 7.78±0.15 | 7.29±0.13 | 3.69±0.12 | 9.90±0.09 | 5.95±0.21 |
| Gly | 1.16±0.22 | 0.84±0.08 | 0.63±0.05 | 1.17±0.13 | 0.93±0.12 |
| Glu | 24.4±0.13 | 13.8±0.16 | 11.5±0.36 | 16.7±0.24 | 16.7±0.18 |
| Lys | 0.18±0.20 | 0.14±0.03 | 0.057±0.09 | 0.11±0.06 | 0.87±0.21 |
| Ser | 5.03±0.11 | 3.93±0.15 | 2.31±0.13 | 5.16±0.31 | 0.077±0.06 |
| Thr | 2.81±0.08 | 1.61±0.06 | 1.40±0.06 | 2.13±0.22 | 2.73±0.13 |
| Asp | 4.98±0.07 | 3.65±0.15 | 2.29±0.17 | 4.77±0.26 | 13.5±0.27 |
| Val | 3.57±0.11 | 2.56±0.07 | 1.77±0.21 | 3.40±0.30 | 3.65±0.31 |
| Arg | 1.52±0.23 | 0.20±0.01 | 0.49±0.04 | 0.26±0.11 | 2.08±0.16 |
| Tyr | 5.10±0.09 | 2.27±0.11 | 2.43±0.22 | 2.56±0.19 | 3.70±0.28 |
| Leu | 19.4±0.13 | 8.58±0.23 | 9.65±0.24 | 10.0±0.26 | 2.78±0.32 |
| Ile | 3.89±0.18 | 3.20±0.15 | 1.82±0.08 | 4.56±0.35 | 2.83±0.11 |
| His | 1.23±0.05 | 0.56±0.06 | 0.52±0.11 | 0.64±0.11 | 0.74±0.06 |
Solubility The solubilities of zein and some products were determined under acidic (pH 3 to 6), neutral (pH 7), and alkaline (pH 8 to 10) conditions, as shown in Fig. 4. The lowest solubilities of control and prepared zein hydrolysates (ZAK and ZKA) occurred at pH 4. Obviously, for controlZAK and controlZKA at a nonhydrolysated state, solubility was lower than that of zein hydrolysates (ZAK and ZKA) under all specified pH conditions. The solubility of zein hydrolysates changed with the addition sequence of alcalase and proteinase K. In general, hydrolysis of protein with high MW produced a higher number of small peptides, which in turn led to the production of more polar residues that can form hydrogen bonds and increase solubility (Gbogouri et al., 2004). The solubilities of ZAK and ZKA at pH 4 increased remarkably to 66.7% and 55.6%, respectively, compared with the control zein.
Effect of pH on the solubility of zein hydrolysates obtained from alcalase and proteinase K with different Zn2+-chelating abilities as influenced by pH.
S1: means the hydrolysate obtained under the optimum conditions of the double enzymatic hydrolysis reaction of adding alcalase first and then proteinase K, and ControlS1 means control of above reaction.
S2: means the hydrolysate obtained under the optimum conditions of the double enzymatic hydrolysis reaction of adding proteinase K first and then alcalase and ControlS2 means control of above reaction.
Foaming properties The foaming ability and foam stability of zein and its hydrolysates at pH 7 are presented in Fig. 5. Both properties of S1 and S2 were higher than those of the original zein. S1 with a DH value of 27.0% DH showed the highest foaming ability, followed by S2 with 36.0%.
Foam and emulsifying properties of zein hydrolysates obtained from alkaline proteinase and proteinase K with different zinc-chelating abilities. Zein was dissolved in phosphoric acid buffer only at pH 12.
Control S1: control of S1, no reaction of zein with alkaline protease first and then proteinase K.
S1: the sample obtained under the double enzymatic hydrolysis reaction of adding alkaline protease first and then proteinase K. DH of S1 was 35.30%.
Control S2: control of S2, no reaction of zein with proteinase K first and then alkaline protease.
S2: obtained under the double enzymatic hydrolysis reaction of adding proteinase K first and then alkaline protease. DH of S2 was 29.32%.
Fig. 5 A shows the foam capacity and foam stability of zein hydrolysates obtained from alcalase and proteinase K with different Zn2+-chelating abilities. A significant difference (P < 0.05) was observed between the foam capacities and foam stabilities of zein and its hydrolysates. The foam capacity and foam stability of zein were significantly lower than those of its hydrolysates, whereas the pH of zein solution was higher than that of its hydrolysates. The foaming properties of the hydrolysates were also affected by pH value. The ability of a protein to be quickly absorbed at the air-water interface is required for foam formation, as reported by Razali et al. (2015). According to Naqash and Nazeer (2016), the foam capacity decreases with increasing pH.
The foam capacity of control under enzyme conditions with consideration of the order of enzyme addition was significantly lower than those with reaction under enzyme conditions with consideration of the order of enzyme addition. In addition, the former pH is higher than the latter. The ionic repulsion of peptides at the air-water interface reduced foam capacity under highly alkaline condition (Klompong et al., 2007). The hydrolysate has better foam capacity and foam stability in the S1 with 35.30% DH compared with that in S2 with 29.32% DH. Smaller peptides can immigrate more quickly to the air-water interface (Intarasirisawat et al., 2012). The pepsin hydrolysate with 44.08% DH showed higher foam capacity compared with the alcalase with 27.62% DH, according to Mahsa et al. (2019).
Emulsifying properties Fig. 5 B shows the emulsifying properties of the zein and its hydrolysates for EAI (m2·g−1) and ESI (%). For the zein and other hydrolysates, the highest emulsifying activity was observed in zein (dissolved in phosphoric acid buffer only at pH 12). According to Taheri et al. (2013), alkaline pH enhanced the emulsifying properties because of the unfolding of the polypeptides due to the negative charges in this pH range. The repulsion resulting from this change allows greater orientation at the interface, thereby exposing the hydrophilic and hydrophobic peptide residues that promote important interactions in the emulsion properties. Furthermore, more hydrophobic amino acids resulted in higher EAI according to Samsudin et al. (2018). The emulsion stability of zein hydrolysates obtained from S1 with 35.30% DH and S2 with 29.32% DH was significantly better than that of untreated zein. An inverse relationship between the extent of the hydrolysis and the emulsifying properties was reported by Witono et al. (2016).
At a concentration of 10 mg·mL−1, both hydrolysates showed high EAI [1.5×107 m2·g−1 for S1 and S2] compared with those under unreaction conditions. Reaction hydrolysate with high DH [35.30% for S1 and 29.32% for S2] exhibited higher EAI than the unreacted hydrolysates under enzyme conditions in consideration of the order of enzyme addition. The unreacted hydrolates with 1.1×107 and 1.2×107 m2·g−1 EAI likely contained peptides with more amphiphilic characteristics, which might favor the migration of peptides to oil-in-water interface and stable emulsion. The hydrolysates formed from the reaction with high DH [35.30% for S1 and 29.32% for S2] have higher emulsion stability. According to some studies, high DH increases emulsion stability (Chalamaiah et al., 2015; Zhao et al., 2012).
DPPH-radical-scavenging activity Fig. 6 A and B show the results of the DPPH-radical-scavenging activity of the hydrolysates and Zn2+-chelating ability of zein. All the samples presented antioxidant activity against radical sequestration, indicating hydrolysates and Zn2+-chelating ability of zein as electron donors, as well as the capability to stabilize the DPPH free radical (Chi et al., 2015; Rabeb et al., 2017). An increase in the concentration of the samples resulted in a higher antioxidant activity, corroborating with the results found by Jemil et al. (2014). Among the different hydrolysates and Zn2+-chelating ability of zein, the samples with higher concentration presented a greater sequestration capacity for the DPPH radical compared with those with lower concentration. This behavior is associated with the peptide length and composition of protein hydrolysates (Jemil et al., 2017). According to Shavandi et al. (2017), the capturing ability of the DPPH radical depends on the enzyme used in the hydrolysis and the protein concentration.
DPPH-radical-scavenging capacity and zinc-chelating zein hydrolysates, as well as Vc, where zein is not a reaction protein, but alkaline proteinase and proteinase K are under consideration of the enzyme sequence. ZAK refers to the zinc-chelating zein hydrolysates with alkaline proteinase and proteinase K; ZKA refers to the zinc-chelating zein hydrolysates with proteinase K and alkaline proteinase.
The Zn2+-chelating ability of zein improved radical-scavenging activity by formation of shorter chain peptides and blind zinc. The antioxidant activity also increased with increasing concentration (0–10 mg·mL−1). The DPPH activities of the two produced samples by alcalase and proteinase K (reaction 91.58%>unreaction 60.66%) demonstrated a remarkable difference. The DPPH scavenging activity was increased by increasing the concentration of Zn2+-chelating zein (P < 0.05). The hydrolysates ZKA at all concentrations demonstrated higher DPPH scavenging activity compared with that of ZAK. Higher DH in ZAK (35.30%) had a negative effect on the bioactivity and extremely short peptides while having less hydrophobicity, which is probably due to the differences in the sequences of amino acids and size of peptides (Chalamaiah et al., 2013). The hydrolysates of zein probably have peptides or amino acids with electron-donating properties and can stop radical chain reactions (Jemi et al., 2014). The amino acids, such as His, Met, Cys, and Phe, probably play roles in DPPH (Chalamaiah et al., 2015; Jemil et al., 2014). At a concentration of 8 mg mL−1, both hydrolysates represented higher radical-scavenging activity than Vc. Intarasirisawat et al. (2012) and Chalamaiah et al. (2013) have also reported an increase in DPPH-scavenging activity by increasing the concentration of the hydrolysates.
Hydroxyl-radical-scavenging activity Fig. 7 A and B present the analysis of the hydroxyl-radical-scavenging of zinc-chelating zein hydrolysates with Vc as positive control. Zn2+-chelating zein hydrolysates exhibited scavenging activity towards hydroxyl radicals in a concentration-dependent manner, and the scavenging effect increased based on the concentration of the Zn2+-chelating zein hydrolysates. When the concentration was above 8 mg mL−1, the Zn2+-chelating zein hydrolysates were observed to possess obviously greater free-radical-scavenging activity than that of Vc, thereby suggesting that the concentration of the former had a noticeable effect on hydroxyl-radical-scavenging activity. Kanbargi et al. (2016) determined the antioxidant activity, metal ion chelation, and reducing power of the protein hydrolysate derived from Ziziphus jujuba seeds and observed that the papain hydrolysate from these seeds are a good source of natural antioxidants. Most antioxidant peptides have small sizes, i.e., less than 1 kDa and contain high proportions of hydrophobic amino acid. Particularly, Tyr, Leu, Ala, Ile, Val, Lys, Phe, Cys, Met, and His exhibited high antioxidant activities. Sonklin et al. (2018) produced mungbean meal protein hydrolysate by bromelain and were fractionated using ultrafiltration membranes with different MW distributions; that with the lowest MW fraction (less than 1 kDa) showed the highest DPPH activity, superoxide-and hydroxyl-scavenging activities, and metal chelation activity. This fraction had poor ferric reducing power, thereby indicating that low MW has an important effect on antioxidant activities.
Hydroxyl-radical-scavenging capacity of zein hydrolysates, zinc-chelating zein hydrolysates, and Vc.
ABTS·+-scavenging activity ABTS radical capture is a method used to measure the antioxidant activity of hydrophilic and lipophilic compounds (Centenaro et al., 2014). ABTS·+ is widely used to determine the antioxidant capacity of samples. The protein extract reacts with ABTS·+ radicals to make it fade, lower its absorbance, and strengthen its antioxidant capacity. Fig. 8 A and B show the results of the ABTS·+-scavenging activity by the hydrolysates and Zn2+-chelating zein hydrolysates. All samples demonstrated ABTS·+-scavenging activity, and zein Zn2+-chelating peptides had significant ABTS·+-scavenging activity compared with unreacted zein (P < 0.05). However, the ABTS·+-scavenging activity of Vc was significantly better than that of zein Zn2+-chelating peptide. According to Alema´n et al. (2011), the capacity of a radical exerted by hydrolysates is related to the size of the chain and to the specificity of the breakage of the enzyme used in hydrolysis. The ABTS·+-scavenging activity of all samples increased gradually with increasing sample concentration, thereby showing a significant dose-effect relationship. Memarpoor-Yazdi et al. (2012) isolated NYDGSTDYGILQINSR antioxidant polypeptide from egg whites and demonstrated significant ABTS·+-scavenging activity. A novel antioxidant peptide from the protein hydrolysate of Allium tuberosum Rottler was identified as Phe-Pro-Leu-Pro-Ser-Phe (FF-6), which could quench multiple radicals efficiently and promote cell survival by protecting against oxidative damage. The Leu residue played a vital role in the radical-scavenging process and interaction with ABTS·+ radicals according to Chen et al. (2018).
ABTS·+-scavenging activity of zein hydrolysates, zinc-chelating zein hydrolysates, and Vc.
In this study, alcalase and proteinase K were selected for the hydrolysis of zein, respectively. The effect of double-enzymatic Zn2+-chelating ability is better than that of single-enzyme hydrolysis. The Zn2+-chelating ability of ZKA is better than that of ZAK, but the DH of the former is lower than that of the latter. An extremely high or low DH was not conductive to chelation with zein. In addition, Zn2+-chelating peptide of zein has significant DPPH-, hydroxyl-radical-, and ABTS·+-scavenging abilities, thereby providing a theoretical basis for new dietary supplements. However, given the complexity of the enzymatic hydrolysis reaction, the mechanism of zein and Zn2+ binding remains unclear, and the Zn2+-chelating peptide of zein requires further separation, purification, and structural identification investigation.
Acknowledgements This work was supported by National Key Research and Development Plan (2017YFD0400201), Science Foundation Project of Heilongjiang Province (QC2015028), Heilongjiang Bayi Agricultural University Support Program for San Heng San Zong (TDJH201906), and Science and Technology Research Project of Heilongjiang Education Department (12541595).
