Assessment of antioxidant properties of membrane ultrafiltration peptides from mungbean meal protein hydrolysates

Materials

Mungbean meal and bromelain was provided from Sittinun Co., Ltd., Thailand and K-Much-Industry Co., Ltd. (Bangkok, Thailand). Mungbean meal was defatted using hexane based on a previously described method (Sonklin, Laohakunjit & Kerdchoechuen, 2011). All chemical reagents were of analytical grade. 2,2-diphenyl-1-picrylhydrazyl (DPPH), glutathione reduced (GSH), 1-10-Phenanthroline, 2,4,6-tris(2-pyridyl)-s-triazine (TPTZ), 3-(2-pyridyl)-5,6-diphenyl-1,2,4-triazine-p,p′-disulfonic acid monosodium salt hydrate (ferrozine) and pyrogallol were purchased from Sigma Aldrich (MO, USA). All other reagents were purchased from Fisher Scientific (Hampton, NH, USA) except stated otherwise.

Screening conditions to produce enzymatic MMPH using Response Surface Methodology (RSM)

Response Surface Methodology (RSM) was used for screening the optimal conditions used to produce mungbean protein hydrolysate according to the method described by Sonklin et al. (2018). The optimal condition was selected based on enzyme content and hydrolysis time which contributed to the highest degree of hydrolysis, DPPH and ABTS scavenging activities. Defatted mungbean meal 10 g was mixed in 100 mL of distilled water, and acidified to pH 6.0 using 2 M HCl. Bromelain was added to samples at 5, 10, 15 and 20% (w/w). All samples were hydrolysed for 6, 12, 18 and 24 h, respectively. After complete reaction at each hydrolysis time, enzymatic reaction was terminated by heating at 95 °C for 15 min, cooled in a water bath, filtered, freeze dried and analysed for degree of hydrolysis (DH) (Flavia & Maria, 1998), DPPH and ABTS (Sonklin et al., 2018) as bellowed.

The degree of hydrolysis (DH): The DH was determined using trichloroacetic acid (TCA), as described by Flavia & Maria (1998). Total protein was measured by the Kjeldahl method. The DH values were calculated with the following equation. ${\displaystyle \text{\%}DH=\left(\text{Soluble protein in TCA}∕\text{Total protein}\right)\times 100.}$ DPPH scavenging activity: The scavenging activity of peptide samples against DPPH radicals was determined using a previously described method (Girgih et al., 2011) with slight modifications for a 96-well microplate. Samples were dissolved in 0.1 M sodium phosphate buffer, pH 7.0 containing 1% (w/v) Triton X-100. DPPH was dissolved in methanol to a final concentration of 100 µM. Peptide samples (100 µL) were mixed with 100 µL of the DPPH solution in the 96-well microplate to a final assay concentration of 0.5 mg/mL and incubated at room temperature in the dark for 30 min. The absorbance values of the blank (A_b) and samples (A_s) were measured at 517 nm. The blank well contained sodium phosphate buffer instead of the peptide sample. The percentage of DPPH radical scavenging activity of the samples was determined using the following equation: ${\displaystyle \text{DPPH radical scavenging activity}\left(\text{\%}\right)=\frac{\left({\mathrm{A}}_{\mathrm{b}}-{\mathrm{A}}_{\mathrm{s}}\right)}{{\mathrm{A}}_{\mathrm{b}}}\times 100.}$ The ABTS radical-scavenging activity: The ABTS activity was determined with an assay, modified from Binsan and co-workers (2008). The stock solutions were 7.4 mM 2,2′-azino-bis (3-ethylbenzthiazoline-6-sulfonic acid) (ABTS) solution and 2.6 mM potassium persulfate solution. The working solution was prepared by mixing the two stock solutions in equal quantities and allowing them to react for 12 h at room temperature in the dark. The solution was then diluted by mixing 1 mL ABTS solution with 50 mL ethanol to obtain an absorbance of 1.1 ± 0.02 units at 734 nm using a spectrophotometer. Fresh ABTS solution was prepared for each assay. The sample (50 µL) was mixed with 950 µL of ABTS solution, and the mixture was left at 25 °C for 2 s in the dark. The absorbance was then measured at 734 nm using a spectrophotometer. The control was prepared in the same manner, except distilled water was used instead of the sample. The blank was 50 µL of sample solution mixed with 950 µL ethanol. The ABTS radical-scavenging activity was calculated as: ${\displaystyle \text{ABTS radical scavenging activity}\left(\text{\%}\right)=\frac{\left(A_{\mathrm{control}}+A_{\mathrm{blank}}\right)-A_{\mathrm{sample}}}{A_{\mathrm{control}}}\times 100.}$ For the RSM step, enzyme concentration (x₁) and hydrolysis time (x₂) were independent variables. Three responses (y) were analysed DH, DPPH and ABTS. Each response can be described by the equation below (Khuri & Cornell, 1987). ${\displaystyle y=b_0+b_1{x}_1+b_2{x}_2+b_{12}{x}_1{x}_2+b_{11}{x}_1^2+b_{22}{x}_2^2}$ Where: b₀ was the constant regression coefficients of the model. b₁ and b₂ were the linear regression coefficients of the model. b₁₁ and b₂₂ were the quadratic regression coefficients of the model. b₁₂ was the interaction regression coefficients.

The experiment plan was designed and statistically analysed for the effects of the independent variables using the SPSS 22.0 program. A p-value less than 0.05 was considered statistically significant (p < 0.05). Statistica program 5.0 (StatSoft Inc., 1995, USA) was used to build the surface plots of each variables.

Preparation of peptide fractions with different molecular weight distributions

MMPH (crude-MMPH) from the optimized hydrolysis condition was selected for the ultrafiltration step. The crude-MMPH was fractionated through a series of ultrafiltration membranes with molecular weight cut-off (MWCO) of 10, 5 and 1 kDa (Millipore, Darmstadt, Germany). This process yielded four fractions: F1 (MW > 10 kDa), F2 (MW 5–10 kDa), F3 (MW 1–5 kDa) and F4 (MW <1 kDa). The fractions were freeze-dried and stored at −20°C until used.

Determination of the amino acid compositions

Total and free amino acid of crude-MMPH and free amino acid of peptide fractions (F1, F2, F3, and F4) were analysed according to the method described by Li et al. (2008). Pre-treatment of total amino acid analysis, sample was carried out by hydrolysing samples using 6 M HCl at 110 °C for 22 h, samples were vigorously shaken and filtered. The acid in the permeate dried using a desiccator and re-dissolved using 1 mL of 0.02 HCl. The dried samples were kept at 4 °C until it was injected into the HPLC. For the pre-treatment of free amino acid analysis, trichloroacetic acid (TCA) was added to samples at ratio 1:1 and incubated for 2 h. After incubation, samples were filtered and centrifuged at 6,000 g for 10 min. Supernatant was collected and stored at 4 °C before injection. After pre-treatment step, each samples were injected into reversed-phase, high performance liquid chromatography (RP-HPLC) (Agilent 1100; Agilent Technologies, Santa Clara, CA, USA). The following conditions were used for analysis. O-phthaldialdehyde (OPA) was added into column (Zorbax 80 A C18 column (4.6 i.d. × 180 mm; Agilent Technologies, Palo Alto, CA, USA)) for pre-column derivatization. Samples (1 µL) were injected into the column using two mobile phases. Mobile phase A was 7.35 mM of sodium acetate : triethylamine : tetrahydrofuran at ratio 500 : 0.12 : 2.5 (v/v/v) and acidified to pH 7.2 with acetic acid. Mobile phase B was 7.35 mM sodium acetate : methanol : acetonitrile with ration 1:2:2 (v/v/v) and adjusted to pH 7.2. The system was operated at 40° C and peaks detected at two wave-lengths 338 and 262 nm. Eighteen amino acids (Ala, Arg, Asp, Cys, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Tyr, Val and Trp) were used as external standards.

Antioxidant properties of peptide fractions

Ferric reducing antioxidant power (FRAP)

The FRAP assay was performed according to the method of Benzie & Strain (1996) as described by Karamać and co-worker (2016). The FRAP reagent was prepared by mixing 0.3 M acetate buffer, 10 mM TPTZ at pH 3.6 in 40 mM HCl, 20 mM FeCl₃.6H₂O pH 3.6 at ratio of 5:1:1 (v/v/v). Samples were dissolved in deionized water at concentration of 2 mg/mL. The reaction was carried out in a 96-well microplate. Samples and FRAP reagent were incubated at 37 °C before adding 40 µL of samples to 200 µL of the FRAP reagent and absorbance was measured at 593 nm. Glutathione (GSH), which was the positive control, was reacted with FRAP reagent as same as sample. A standard curve was generated using FeSO_4.7H₂O (0.03–0.9 µmol/mL) and results were reported as mmole of Fe²⁺ reduced per g protein from the regression slope of the standard curve.

Statistical analysis

All assays were determined in triplicate. Results were subjected to one way analysis of variance (ANOVA) using SAS program Version 6.0 (SAS Institute, 1997, USA). Statistical significance of difference between samples was accepted at p-value less than 0.05 (p < 0.05) using the Duncan’s multiple range test (DMRT).

Screening of optimum conditions to produce MMPH by bromelain using RSM

The optimum bromelain treatment conditions used to produce MMPH was determined using the RSM. The effect of different enzyme treatment conditions on DH, DPPH and ABTS are reported using a quadratic equation. Enzyme concentration (x₁) and hydrolysis time (x₂) a statistically significant interaction (p < 0.05) on the DH, DPPH and ABTS value were shown in Table 1 and significant values of DH, DPPH and ABTS were performed in the equations for each variable are shown below: (1)${\displaystyle DH:y=8.401+3.305X_1+2.318X_2-0.0986X_1^2-0.0654X_2^2-0.0256X_1{X}_2}$ (2)${\displaystyle DPPH:y=-2.967+8.468X_1+3.429X_2-0.254X_1^2-0.0951X_2^2-0.0307X_1{X}_2}$ (3)${\displaystyle ABTS:y=0.238+9.255X_1+1.418X_2-0.310X_1^2-0.0296X_2^2--0.0007961X_1{X}_2.}$ The equations showed that enzyme concentration (X₁) was higher important factors affecting DH, DPPH and ABTS values than hydrolysis time (X₂) because an estimated regression coefficient of X₁ presented the higher value. The determination coefficients (R²) of the DH, DPPH and ABTS were 0.818, 0.925 and 0.969, respectively. The equations were used to build response surface plots to predict the critical points and effectiveness of DH (Fig. 1A), DPPH (Fig. 1B) and ABTS (Fig. 1C). For response surface plots, DH, DPPH, ABTS values increased with increasing enzyme concentration and hydrolysis time until the critical point, all of three variables were constant and slightly decreased. Results indicated that enzyme concentration and hydrolysis time had influenced on DH, DPPH, ABTS values of mungbean meal protein hydrolysate.

An optimum condition to produce MMPH was selected from an overlay of Figs. 1–3 and choosing the range of enzyme concentration and hydrolysis time which gave the maximum DH, DPPH and ABTS activity. Results showed that the optimal enzyme concentration was 10–18% and hydrolysis time 8–24 h. Therefore, 15% of bromelain and 12 h hydrolysis time were chosen to produce MMPH. The models used were confirmed by observed and predicted values as showed in Table 2. Results showed that the predicted values which were derived from the models were close to the observed value.

Amino acid composition of peptide fractions

The crude-MMPH was analysed for both total amino acids and free amino acids, but F1, F2, F3 and F4 were used to analyse only total amino acids. High content of Glu, Asp, Lys, Arg, Leu, Ser, Pro and Phe were found in the crude-MMPH (162.66, 104.39, 72.11, 67.00, 62.93, 54.27, 48.01 and 45.61 mg/g protein, respectively) (Table 3). The predominant amino acids were hydrophobic amino acids (HHA) group. The highly abundant in peptides were found in Crude-MMPH. Therefore, it can refer that F1-F4 had a plenty of peptides as well. Asp and Glu were the highest in all peptide fractions (Table 3). In particular, F4 had the highest content of Glu, Arg, Gly, Leu, Met, Tyr, Phe, Trp and Ser. All peptide fractions had high amounts of total HAA, and the contents were quite similar in each fraction. The proportion of aromatic amino acid contents (Tyr, Phe, Trp) in fraction F4 was also found to be the highest compared to the other peptide fractions.

DPPH radical scavenging activity

The activity of crude-MMPH and its ultrafiltrated peptide fractions to scavenge the DPPH radical is presented as the EC₅₀ value (Fig. 2). The R² of GSH, Crude-MMPH, F1, F2, F3 and F4 was 0.9922, 0.9771, 0.8888, 0.9805, 0.9619 and 0.7962, respectively. Crude-MMPH and its peptide fractions scavenged the radical to a 50% inhibition at a range of 0.4 –0.9 mg/mL. All peptide fractions had low EC₅₀ values of DPPH than the hydrolysate. EC₅₀ value of fraction 4 showed the lowest EC₅₀ as 0.53 mg/mL, followed by F1, F3 and F2 with EC₅₀ as 0.63, 0.68 and 0.69, respectively. EC₅₀ value of fraction 4 (0.53 mg/mL) was quite close to the EC₅₀ value of standard which was glutathione (0.41 mg/mL).

Ferric reducing activity power (FRAP)

FRAP is related to the generated Fe²⁺ as shown in mmole Fe²⁺/g protein. As depicted in Fig. 3C, FRAP of crude-MPH and its fraction increased significantly with increasing protein concentration from 0.2–1 mg/mL. Moreover, FRAP of peptide fractions in this study increased with increasing in the molecular weight of peptides. F1 (>10 kDa), which had a HMW, had the greatest reducing power at all concentrations. F1 generated Fe²⁺ as 0.061, 0.107, 0.118, 0.231 and 0.242 mmole Fe²⁺/g protein when used protein concentration at 0.2, 0.4, 0.6, 0.8 and 1.0 mg/mL protein, respectively.