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Effect of enzymolysis-assisted electron beam irradiation on structural characteristics and antioxidant activity of rice protein
Journal of Cereal Science 89 (2019) 102789
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Effect of enzymolysis-assisted electron beam irradiation on structural characteristics and antioxidant activity of rice protein
T
Ting Lia,b,d, Li Wanga,b,d,∗, Dongling Sunc, Yanan Lib,d, Zhengxing Chena,b,d,∗∗ a
State Key Laboratory of Food Science and Technology, Jiangnan University, Lihu Road 1800, Wuxi, 214122, China School of Food Science and Technology, National Engineering Laboratory for Cereal Fermentation Technology, Jiangnan University, Lihu Road 1800, Wuxi, 214122, China c Wuxi EL PONT Radiation Technology CO., Ltd, No. 8, Weiye Road, Qianqiao Industrial Park (Xi'nan), Huishan District, Wuxi, 214151, Jiangsu, China d Jiangsu Provincial Research Center for Bioactive Product Processing Technology, Jiangnan University, Lihu Road 1800, Wuxi, 214122, China b
ARTICLE INFO
ABSTRACT
Keywords: Electron beam irradiation Structural characteristics Antioxidant activity Rice protein
This research investigated the effect of enzymolysis-assisted electron beam irradiation (EBI) on the structural characteristics and antioxidant activity of rice protein (RP). The amino acid content of RP was increased by enzymatic hydrolysis, but the amino acid content of the rice protein hydrolysates (RPHs) was decreased gradually as the irradiation dose increased. Additionally, the secondary structural change of the irradiated hydrolysates revealed that a flexible and unfolded structure was induced by EBI. Irradiation increased the fluorescence intensity of the hydrolysates, which can probably be attributed to the exposure and changes in the surroundings of the hydrophobic region. Microscopic graphs showed that the tight conformation of the RP was destroyed by enzymolysis. Furthermore, the antioxidant activity of the RPHs was improved by EBI, with the highest 1, 1-Diphenyl-2-picrylhydrazyl (DPPH) and 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) radical scavenging activity of 96.81% and 92.04% at 50 kGy, respectively. This study will provide a feasible coupling technology for the food industry to improve the antioxidant ability of RP. Consequently, irradiated hydrolysates could be regarded as a promising functional food ingredient due to their higher antioxidant ability.
1. Introduction Rice protein (RP) is acknowledged as a hypoallergenic and high nutritional value protein compared to other major cereal proteins. However, the application of RP in the food industry is limited by the poor solubility induced by its compact conformation. To improve the properties of proteins, many methods to change the structure such as chemical modification, physical treatment and enzymatic treatment have recently been used (Amagliani et al., 2017). Among these methods, antioxidant peptides can be released by enzymatic hydrolysis (Rani et al., 2018). Dietary antioxidants play an important role in controlling oxidative stress-induced damage. Additionally, natural antioxidants can act as alternatives for synthetic antioxidants and then delay the oxidation of lipids in the food industry. Recently, the potential benefits of an antioxidant peptide derived from rice have attracted the attention of researchers. For example, rice protein hydrolysates (RPHs) with antioxidant ability could enhance the stability of emulsion-type
∗
food systems and extend the shelf life of foodstuffs (Rani et al., 2018). Therefore, higher antioxidant activity of proteins is regarded as an important parameter, which is related to the exploitation and application of food products. Food irradiation is a physical method to maintain food freshness and even improve food quality. Radiation, including ionizing and nonionizing radiation, has been expanded for the modification of proteins. Corresponding to the absorbed irradiation dose, chemical changes such as polymerization, cross-linking and depolymerization can be achieved via free radical formation (Kuan et al., 2013). Electron beam irradiation (EBI), an ionizing irradiation, has been widely used in the modification of food components and improvement of quality (Kuan et al., 2013). The thermal stability of gelatin film has been enhanced by irradiation (Benbettaieb et al., 2016). In addition, EBI is regarded as a nonthermal, economic, environmentally friendly and safe technique to improve the biochemical properties of proteins. Jin et al. (2017) investigated the quality characteristics of egg white protein (EWP) after
Corresponding author. State Key Laboratory of Food Science and Technology, Jiangnan University, Lihu Road 1800, Wuxi, 214122, China. Corresponding author. State Key Laboratory of Food Science and Technology, Jiangnan University, Lihu Road 1800, Wuxi, 214122, China. E-mail addresses: [email protected], [email protected] (L. Wang), [email protected] (Z. Chen).
∗∗
https://doi.org/10.1016/j.jcs.2019.102789 Received 22 February 2019; Received in revised form 17 June 2019; Accepted 18 June 2019 Available online 18 June 2019 0733-5210/ © 2019 Elsevier Ltd. All rights reserved.
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irradiation and found that the degree of hydrolysis of EWP was enhanced after EBI treatment due to structural changes. Additionally, the Ca2+-ATPase activity of T. granosa meat was decreased as the irradiation dose increased. The edible qualities such as ‘hardness’, ‘gumminess’, ‘chewiness’ and ‘resilience’ of T. granosa meat were improved after EBI treatment (Lv et al., 2018). In our previous study, the wheat germ protein was hydrolyzed by alcalase and then treated by EBI (Wang et al., 2019). It was found that the antioxidant ability and functional properties of wheat germ protein hydrolysates were increased by EBI. Currently, the food industry is developing innovative technology to meet the demand of consumers for safer and higher quality foods. As described above, methods of enzymolysis and EBI can improve the quality and antioxidant ability of protein. However, there are few reports concerning the effect of enzymolysis-assisted EBI on structural changes and the antioxidant activity of RP. In this study, the amino acid composition of rice protein under enzymolysis-assisted EBI was measured by high-performance liquid chromatography (HPLC). Additionally, the structural changes in the RP after enzymolysis-assisted EBI were determined via Fourier Transform Infrared (FTIR) spectroscopy, fluorescence spectroscopy and microscopy. This study also determined the antioxidant ability of rice protein and hydrolysates by the electron spin resonance (ESR) technique. Therefore, this research will provide a scientific basis for the application of a novel coupling method (i.e., enzymolysis-assisted EBI) in the modification of rice protein.
2.3. Determination of amino acid composition The amino acid composition was determined according to the method of Zhao et al. (2018) with some modification. Samples (intact protein and hydrolysates) were prepared after hydrolysis with 6 mol/L HCl (110 °C, 22 h). Then, the amino acid composition of the samples was measured by high-performance liquid chromatography (HPLC) (Agilent 1260, USA). The parameters of the experiment were as follows: ultraviolet detector (VWD, G1314A), Agilent Hypersil ODS column (5 μm, 4.0 × 250 mm), 40 °C column temperature. Additionally, the solvent A was composed of sodium acetate, triethylamine and tetrahydrofuran at a proportion of 500/0.11/2.5 (v/v). The solvent B was composed of sodium acetate, methanol and acetonitrile at a proportion of 1/2/2 (v/v). A gradient elution program with a flow rate of 1 mL/ min (0–17 min, 8% solvent B; 17–20 min, 50% solvent B; 20–24 min, 100% solvent B; 24–46 min, 0% solvent B) was used in this test. 2.4. Fourier transform infrared spectroscopy The Fourier transform infrared (FTIR) spectra were scanned by the FTIR spectrometer (Nicolet IS10, USA). The powdered samples (intact protein and hydrolysates) were mixed with potassium bromide (KBr) and pressed into a slice. The wavelength of the spectra ranged from 4000 to 400 cm−1 with 4 cm−1 resolution and 32 scans. All measurements were carried out in a dry atmosphere at room temperature (25 °C). The results were analyzed by the software of OMNIC 8.2 (a professional infrared spectral analysis software from Nicolet) and Peakfit 4.12 (Wang et al., 2015).
2. Materials and methods 2.1. Materials
2.5. Fluorescence spectroscopy
Rice was purchased from Jinwanxian Co., Ltd. (Anhui, China). Alcalase 2.4 L (2.4 AU/g) was obtained from Novozymes China Co., Ltd. (Beijing, China). 1,1-Diphenyl-2-picrylhydrazyl (DPPH), 2,2′-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) and 1-anilino-8-naphthalene sulfonate (ANS) were purchased from Sigma-Aldrich Corporation (St. Louis, MO, USA). Other chemicals were obtained from Sinopharm Chemical Co., Ltd. (Shanghai, China).
The fluorescence spectra were determined using the ANS fluorescence probe according to the method of Yang et al. (2017) with slight modification. The fluorescence spectra were measured by the Fluoro Max4 fluorescence spectrophotometer (Horiba JY Company, United States). The samples (intact protein and hydrolysates) were dispersed in 0.01 M phosphate buffer (1%, w/v) and then mixed with 50 μL ANS. Then, the mixture was kept in the dark for 5 min before being transferred into the cuvette of the spectrophotometer. Additionally, the emission spectra were scanned from 400 to 600 nm at the excitation wavelength of 390 nm.
2.2. Preparation of protein and hydrolysates RP was extracted by the method of alkaline (pH 12) extraction and acid (pH 4.8) precipitation. The protein content in RP (96.1%) was determined by the Kjeldahl method (AOAC, 2010). Then, protein was dispersed in distilled water (1%, w/v) at 55 °C and incubated with alcalase for 2 h. The enzyme to substrate ratio was 1:50 (w/w). The reaction mixture was kept at pH of 8.0 by consecutive additions of 1 mol/ L NaOH during hydrolysis. The degree of hydrolysis (DH) was measured by the pH-stat method and calculated using equation (1) (Xu et al., 2016). Reactions of enzymatic hydrolysis were stopped by immersion in a boiling water bath for 10 min. Then, hydrolysates were adjusted to pH 7.0 by dilute acid. Next, 100-mL RPHs suspensions (1%, w/v) were irradiated with various doses (5 kGy, 10 kGy, 25 kGy, and 50 kGy) at 5.0 MeV energy level. The duration of the EBI treatment was 0.86 s, 1.72 s, 4.3 s, and 8.6 s, respectively. Irradiation processing was conducted at room temperature (25 °C). EBI treatment employed a highenergy linear accelerator (AB type high frequency and high voltage) of Wuxi EI Pont Radiation Technology Co., Ltd. Finally, all samples (intact protein and hydrolysates) were lyophilized by freeze drying (Christ Beta 2–8 LD plus, Germany) and stored at −20 °C until further use.
DH =
B × Nb × 100% × Mp × htot
2.6. Microscopy studies The lyophilized samples (including intact protein and hydrolysates) were milled through a 100-mesh sieve. The microstructure of the lyophilized samples coated with gold and platinum was measured by scanning electron microscopy (SEM) (Quanta 200, FEI Co., Holland) with magnification of 1000 and an accelerating voltage of 10.0 kV. The fluorescent dye, 20 mM ANS (10 μL), was added to the 1% sample solutions (including intact protein and hydrolysates) that were dispersed in distilled water (w/v). Then, the mixture was placed on the glass slide. The shape of the sample in solution was observed by inverted fluorescence microscopy (IFM) using bright and fluorescent fields. 2.7. Antioxidant ability measurement DPPH and ABTS radical scavenging activity are two of the most common and efficient methods to test the antioxidant ability of intact protein and hydrolysates. The results of free radical scavenging activity determined by DPPH and ABTS are partly different due to their different mechanisms. The DPPH radical scavenging assay is based on the reduction of 1,1-Diphenyl-2-picrylhydrazyl by accepting an electron or a hydrogen from the antioxidant compounds. The ABTS radical scavenging assay is based on the ability to eliminate green radicals (2,2azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)). The ABTS radical is
(1)
where B is the volume of NaOH to maintain a constant pH of 8.0, Nb is the molar concentration of NaOH, α is the degree of dissociation of αNH2 groups, Mp is the total mass of the substrate protein, and htot is the number of peptide bonds in the substrate protein. 2
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more likely to react with antioxidants than the DPPH radical. However, the preparation of DPPH in solvent is easier than preparation of ABTS reagent in solvent. In addition, DPPH radical has higher stability compared to ABTS (Mareček et al., 2017). Therefore, two different methods for measuring free radical scavenging activity of intact protein and hydrolysates can lead to the impartial results in this study.
Table 1 Amino acids composition of rice protein and hydrolysates. Amino acids/(g/100 g)
Asp Glu Ser His Gly Thr Arg Ala Tyr Cys-s Val Met Phe Ile Leu Lys Pro TAAs/(g/100 g) TEAAs/(g/100 g) THAAs/(g/100 g)
2.7.1. Scavenging activity of DPPH radical DPPH radical scavenging activity of samples was measured by the method of Liu et al. (2009) with some modification. A 100-μL sample (including intact protein and hydrolysates) was mixed with 100 μL DPPH (2 mg/mL ethanol). The mixture of sample and DPPH was transferred into a quartz capillary tube after 1 min. Then, the quartz capillary tube was transferred into a nuclear magnetic resonance tube. Then, the nuclear magnetic resonance tube was put into the resonant cavity, and the spectrum was then determined by the electron spin resonance (ESR) spectrometer (EMXplus-10/12, Bruker, Germany). The experimental parameters were the following: microwave power, attenuation and frequency were 20 mW, 10 dB, 9.44 GHz, respectively; modulation amplitude was 6 G; scan time was 20 s; central field was 3350 G and the sweep width was 150 G. Each sample was scanned three times to acquire a spectra with a suitable signal-to-noise ratio. The DPPH radical scavenging activity was calculated by the following equation (2).
Radical scavenging activity (RSA) =
H0
H H0
× 100%
Rice protein
6.35 14.3 2.82 1.53 3.21 2.12 6.42 3.95 3.12 0.26 5.04 1.87 3.95 3.43 6.02 2.19 4.71 71.3 24.6 30.2
Rice protein hydrolysates 0 kGy
5 kGy
10 kGy
25 kGy
50 kGy
8.65 18.6 3.77 2.46 4.64 2.73 8.57 5.22 3.90 0.29 7.78 1.68 5.45 4.62 7.75 3.14 4.50 93.8 33.2 39.2
8.01 17.1 3.25 2.22 4.19 2.41 7.75 4.74 3.34 0.21 7.10 1.48 4.72 4.27 7.11 2.85 4.67 85.4 29.9 36.0
8.32 17.7 3.36 1.84 4.39 2.44 7.84 4.95 3.42 0.16 7.25 1.41 3.86 4.28 7.10 2.83 4.42 85.6 29.2 35.3
7.20 15.1 2.85 1.28 3.71 2.04 6.43 4.24 2.30 0.10 6.10 0.96 2.66 3.56 5.81 2.40 4.17 70.9 23.5 28.8
7.34 15.0 2.60 0.47 3.53 1.83 6.00 4.25 2.03 0.09 6.29 0.94 2.16 3.47 5.44 2.19 4.79 68.4 22.3 28.4
Note: TAAs, Total amino acids; Total essential amino acids (TEAAs) = Thr + Val + Met + Phe + Ile + Leu + Lys; Total hydrophobic amino acids (THAAs) = Ala + Tyr + Val + Phe + Ile + Leu + Pro.
(2)
100 g, respectively. After hydrolysis, TAAs, TEAAs and THAAs of RPHs were higher than RP, with the TAAs of 93.8 g/100 g, the TEAAs of 33.2 g/100 g and the THAAs of 39.2 g/100 g. Consistently, Zhang et al. (2017) found that hydrophobic and essential amino acid contents of soy protein were increased after hydrolysis by alcalase. Shazly et al. (2019) observed more hydrophobic amino acids in buffalo casein and bovine casein hydrolysates compared to the original protein. As is well known, alcalase was regarded as a commercial serine proteases with an optimum temperature of 55 °C and pH of 8.0, which could cleave the peptide bonds of proteins. The DH is defined as the proportion of the cleavage of peptide bonds of total peptide bonds in a protein substrate. In this study, the DH of RP after hydrolysis was 11.8%. Additionally, in Table S1 (Supplementary file), the solubility of intact protein was increased by enzymatic hydrolysis (from 3.94% to 15.0%). Therefore, it was assumed that the cleavage of peptide bonds and the increased solubility increased the acid-accessibility of hydrolysates when the amino acid composition was measured after acid hydrolysis, resulting in an increased final determined content of TAAs, TEAAs and THAAs. However, the amino acid content of RPHs decreased as the irradiation dose increased, reaching the minimum value at 50 kGy. Compared with 0 kGy, TAAs and TEAAs of the irradiated RPHs were reduced by 27.05% and 32.67% at 50 kGy, respectively. Nevertheless, both TAAs and TEAAs of the hydrolysates were higher than the intact protein with the irradiation dose below 10 kGy. Zhao et al. (2018) also found that TAAs and TEAAs of spicy yak jerky decreased at an irradiation dose of 9 kGy when compared with 0 kGy. However, the opposite result was reported by (Olotu et al., 2014). In their study, some amino acid contents (lysine, threonine, etc) of the irradiated samples increased at 10 kGy, which could be related to the deamination and decarboxylation of the amino acids. According to the research of Elias and Cohen (1977), sulfur-containing amino acids were most sensitive to irradiation. Consistently, in present results, cystine and methionine declined gradually as the irradiation dose increased. Additionally, the cysteine and methionine of RPHs at 50 kGy decreased drastically by 68.97% and 44.05%, respectively, compared with native RPHs. As discussed above, the amino acid content of the intact protein was increased by enzymatic hydrolysis, possible attributed to the increased acid-accessibility of the protein after enzymolysis. Therefore, the increased content of TEAAs indicated that hydrolysates had a better
where the H and H0 were the third resonance peak for DPPH with and without samples (including intact protein and hydrolysates). 2.7.2. Scavenging activity of ABTS radical The ABTS radical scavenging activity was determined by the method of our previous study (Wang et al., 2017). The ABTS stock solution (7 mM) was mixed with potassium persulfate solution (2.45 mM) to prepare the ABTS working solution. Then, the working solution sat for 12 h at room temperature in the dark. Then, a 100-μL sample (including intact protein and hydrolysates) was mixed with ABTS working solution (100 μL) and reacted for 1 min. The spectrum was determined by the ESR spectrometer as mentioned above (Section 2.7.1). The ABTS radical scavenging activity was calculated using equation (2). The H and H0 were the first resonance peak for ABTS with and without samples (including intact protein and hydrolysates). 2.8. Statistical analysis The experiment was measured in triplicate. Data were analyzed by the Analysis of Variance (ANOVA) in SPSS statistical software (version 20.0). The differences between samples were determined using the Duncan's multiple range test at a level of 0.05. 3. Results and discussion 3.1. Amino acid composition The amino acid composition is an important index to evaluate the quality of the protein (Olotu et al., 2014). RP has a reasonable amino acid composition, and it is rich in limited essential amino acids such as lysine and threonine (Amagliani et al., 2017). The chemical composition (i.e., protein, lipid, ash) of the intact protein was roughly unchanged after enzymolysis (data not shown). However, the determination of the amino acid composition was influenced mainly by the content of moisture due to the conditions of the environment. Thus, the amino acid composition was calculated on the basis of dry basis weight of the samples in this study. As shown in Table 1, the total amino acids (TAAs), the total essential amino acids (TEAAs) and the hydrophobic amino acids (THAAs) of RP were 71.3 g/100 g, 24.6 g/100 g and 30.2 g/ 3
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available amino acid profile than the intact protein, which proved that enzymolysis promote the digestion and utilization of rice protein. However, a negative trend of amino acid content of hydrolysates after irradiation was shown in our test. Kuan et al. (2013) suggested that some free radicals generated by EBI resulted in amino acid (i.e., histidine, cysteine and methionine) loss through oxidation and molecular degradation. In addition, as shown in Table S1, the solubility of hydrolysates was increased due to the structural changes and the exposure of amino acids, thus probably promoting the process of amino acid oxidation. Nevertheless, the amino acid content of irradiated hydrolysates with lower doses (< 10 kGy) was still superior to the intact protein. Therefore, to maintain the nutritional value of RPHs, the irradiation dose should not exceed 10 kGy, which is in agreement with the required dose of WHO.
observed that irradiation decreased the α-helix content and increased the β-turn content. Lin et al. (2015) found that EBI reduced the contents of the α-helix and β-sheet of samples, indicating that the ordered structure of the protein was obviously destroyed by EBI. Combining the present study and the previous study by the researchers, the conformation of the intact protein was altered by irradiation. As described above, the decreased content of the β-sheet showed that a more flexible and unfolded structure of RPHs was induced by irradiation. Combined with the results of Table 1, the content of THAAs of the intact protein was increased from 30.2 g/100 g–39.2 g/100 g after enzymatic hydrolysis. Therefore, it was supposed that some amino acids of the RP were exposed by enzymatic hydrolysis from the buried hydrophobic core of the protein (Li et al., 2016a). Then, the exposed protein construction was destroyed by EBI. Therefore, a more flexible and extended conformation of rice protein was formed through enzymolysis-assisted EBI.
3.2. Fourier transform infrared spectroscopy The information of the functional groups of the protein can be provided from the FTIR spectra, and the analysis of the amide I band (1600-1700 cm−1) has been widely used to calculate the content of the secondary structure owing to its sensitivity to protein conformation changes. Data processing of the amide I band from FT-IR spectra was conducted according to the method of Haque et al. (2010). The amide I region of the spectra was baseline-corrected by OMINC 8.2 and deconvolved by PeakFit 4.12. The secondary structure content of each component was calculated by a peak area that was fitted by PeakFit 4.12. The peak area of 1600–1640 cm−1 represented the β-sheet structure, 1640-1650 cm−1 belonged to the random coil structure, 1650-1660 cm−1 corresponded to the structure of α-helix, and β-turn content was speculated to be 1660-1700 cm−1 (Lin et al., 2015). Fig. 1A shows the FTIR spectra of 400–4000 cm−1, and the content of the secondary structure is observed in Table 2. As seen in Fig. 1A, the peak strength (amide I band at 1600-1700 cm−1) of RPHs was lower than the peak strength of RP, and the absorption band of C]O was shifted from 1650 cm−1 to 1654 cm−1 after enzymolysis. This result was derived from the decreased content of the β-sheet (Malik et al., 2017), which is consistent with the results from Table 2. Additionally, EBI-treated hydrolysates exhibited spectral profiles similar to untreated hydrolysates because the basic structure of the hydrolysates was not significantly destroyed by EBI (Jin et al., 2017). In Table 2, compared to the control (RP), the α-helix content of RPHs increased from 12.50% to 14.38%, and the β-turns content of RPHs increased from 36.75% to 48.61%. The structure of the β-sheet was a stable and folded structure, whereas the β-turn was unfolding with a flexible conformation (Wang et al., 2015). Xu et al. (2016) found that the decreased content of the βsheet as well as the increased content of the α-helix appeared in rice glutelin hydrolysates compared with native rice glutelin, which revealed that extended and flexible structures were unfolded by enzyme hydrolysis. Additionally, the β-sheet and random coil contents of irradiated RPHs decreased, and the β-turn content increased with the increased dose of irradiation. There was no obvious change in the α-helix content of the hydrolysates with different doses. Malik et al. (2017)
3.3. Fluorescence spectroscopy Fluorescence emission spectroscopy using ANS as a fluorescence probe is a sensitive way to characterize the surface hydrophobicity of a protein during processing. The surface hydrophobicity is associated with the content of hydrophobic amino acids such as Trp, Tyr and Phe (Li et al., 2016b). The emission fluorescence spectra of RP and RPHs are shown in Fig. 1B, indicating the hydrophobic bonding region with ANS and the surrounding changes. The fluorescence spectral profile of RP was similar to the fluorescence spectral profile of hydrolysates with an irradiation dose of 0 kGy. However, the fluorescence intensity of the peak at 500–550 nm of hydrolysates was slightly increased when compared with the protein, indicating that the hydrophobic binding domains of protein cores were slightly exposed after hydrolysis. Additionally, the fluorescence intensity of the peak at 500–550 nm of RPHs increased intensely with the increasing irradiation dose, revealing that the binding ability between the ANS and the hydrophobic patches of the hydrolysates was enhanced (Li et al., 2018). Interestingly, a new peak (400–450 nm) of irradiated hydrolysates was enhanced as irradiation dose increased. The maximum peak of the irradiated hydrolysates was blue-shifted, possibly owing to the surrounding changes of hydrophobic amino acids. The hydrophobic zones of the hydrolysates were exposed to a polar environment due to their flexible and extended structure, resulting in the surrounding changes of binding areas (Misra, 2019; Wang et al., 2015). In addition, the surface hydrophobicity index through the initial slope of fluorescence intensity versus protein concentration was measured to confirm the result of emission spectra. As shown in Table S1 (Supplementary file), the surface hydrophobicity index of the intact protein was increased by enzymolysis-assisted EBI, which proved the results shown in Fig. 1B. Wang et al. (2017) also found that the surface hydrophobicity of irradiated pea protein hydrolysates increased significantly (P < 0.05) when compared to nonirradiated hydrolysates. Previous researchers have reported that the content of THAAs was positively related to the surface hydrophobicity of proteins (Li and Jiang, 2015; Wang et al., 2015). In the present
Table 2 Content of secondary structure of rice protein and hydrolysates. Content of secondary structure
Rice protein
Rice protein hydrolysates 0 kGy
α-helix β-sheet β-turns random coil
12.5 37.4 36.8 13.4
± ± ± ±
0.72 0.29 0.63 0.20
a c a c
14.4 24.0 48.6 13.0
± ± ± ±
5 kGy 0.09 b 0.4 b 0.34 b 0.03 bc
14.1 24.7 48.3 13.0
± ± ± ±
10 kGy 0.05 ab 0.21 b 0.11 b 0.17 bc
14.4 22.3 50.4 12.9
± ± ± ±
25 kGy 0.02 b 0.21 a 0.18 c 0.01 b
14.4 22.7 50.1 12.8
± ± ± ±
50 kGy 0.01 b 0.29 a 0.31 c 0.02 ab
13.4 22.9 51.2 12.4
± ± ± ±
1.38 0.70 0.97 0.29
ab a c a
Note: Values are shown as the mean ± SD of three determinations (different samples); Superscript letters (a, b, c, d) in the same row indicate significant differences (P < 0.05). 4
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Fig. 1. Fourier transform infrared spectroscopy spectra (A) and fluorescence emission spectra (B) of rice protein and hydrolysates (a. rice protein, b-f. rice protein hydrolysates with irradiation dose of 0, 5, 10, 25, 50 kGy).
research, the higher hydrophobic amino acid content of hydrolysates was consistent with the increased surface hydrophobicity. However, the content of THAAs of irradiated hydrolysates was decreased in an unexpected way, probably owing to the oxidation of the amino acids (Kuan et al., 2013) and the structural unfolding of peptide (Yuan et al., 2009).
surface of the irradiated samples (Fig. 2C1- F1) became smoother than the surface of the nonirradiated samples (Fig. 2B1). Meanwhile, some punctured pores occurred after irradiation (Fig. 2C1, D1, E1, red arrow). Additionally, more lamellar structures of hydrolysates were formed under irradiation (Fig. 2D1). Similarly, some researchers observed that punctured pores appeared on the surface structure of proteins with the EBI treatment (Jin et al., 2017). Malik et al. (2017) also described that some cracks on the surface of sunflower protein isolates appeared after gamma irradiation. The results of Fig. 2 and other researchers proved that irradiation damaged the microstructure of hydrolysates. Furthermore, in images of the fluorescent field from IFM, the fluorescent region and signal of the intact protein particles were decreased after treatment by enzymolysis and EBI, revealing that particles of RP in solution were degraded by hydrolysis and irradiation (Fig. 2A3-F3). Consistently, the same trend was observed in the graphs from the bright field of IFM
3.4. Morphological studies The surface morphology of the hydrolysates was analyzed by SEM. Additionally, the shape and size of the hydrolysates in solution were studied using IFM (bright and fluorescent fields). The images of SEM and IFM are shown in Fig. 2. In Fig. 2A1, the surface structure of RP was compact and cross-linked. However, smaller pieces of RP were formed by enzymatic hydrolysis (Fig. 2B1). With regard to hydrolysates, the
Fig. 2. Microstructure of rice protein and hydrolysates (A. rice protein, B–F. rice protein hydrolysates with irradiation dose of 0, 5, 10, 25, 50 kGy; 1. scanning electron microscopy (SEM), 2. inverted fluorescence microscopy (IFM) with bright field, 3. IFM with fluorescent field). 5
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Fig. 3. Free radicals scavenging capacity of DPPH and ABTS of rice protein and hydrolysates (A. DPPH radical scavenging capacity, B. ABTS radical scavenging capacity; a. rice protein, b-f. rice protein hydrolysates with irradiation dose of 0, 5, 10, 25, 50 kGy).
Table 3 DPPH and ABTS radical scavenging activity (RSA) of rice protein hydrolysates. RSA
DPPH/% ABTS/%
Rice protein
46.0 ± 1.00 48.6 ± 0.27
Rice protein hydrolysates
a a
0 kGy
5 kGy
10 kGy
25 kGy
50 kGy
66.7 ± 0.51b 71.1 ± 0.25b
65.8 ± 0.27b 87.7 ± 0.11c
82.5 ± 2.17c 87.9 ± 0.17c
86.8 ± 0.14d 88.9 ± 0.46c
96.8 ± 0.94e 92.0 ± 1.16d
Note: Values are shown as the mean ± SD of three determinations (different samples); Superscript letters (a, b, c, d, e) in the same row indicate significant differences (P < 0.05).
(Fig. 2A2-F2). As discussed above, it was concluded that the compact structure of the intact protein was degraded by the enzyme. Meanwhile, enzymolysis-assisted EBI accelerated the degree of denaturation. Furthermore, more fragmentations of hydrolysates resulted from bombardment by high energy electrons during irradiation.
increasing the accessibility to the free radicals (Wang et al., 2019). Furthermore, the increased solubility might contribute to the higher antioxidant ability of hydrolysates. Considering the present study and other research, enzymolysis-assisted EBI is an effective technology to develop products with higher antioxidant activity.
3.5. Antioxidant activity
4. Conclusions
Recently, some studies have reported that RP exhibited antioxidant activity by observing the ability of reducing the free radicals (Wang et al., 2016). This study used the ESR technique to detect free radicals that had unpaired electrons in the magnetic field (Liu et al., 2009). DPPH and ABTS radical were relatively stable and were widely used to test the ability of the antioxidant of protein and hydrolysates (Mareček et al., 2017). DPPH and ABTS radical scavenging activities of hydrolysates are illustrated in Fig. 3 and Table 3. RP exhibited antioxidant activity, with the DPPH radical scavenging activity of 46.00% and ABTS radical scavenging activity of 48.57%, respectively (Table 3). Additionally, DPPH and ABTS radical scavenging activities of hydrolysates were higher than the radical scavenging activities of the intact protein, indicating that the antioxidant activity of the protein was increased by enzymatic hydrolysis. In Fig. 3A, the height of the third peak showed an obvious decrease in irradiated hydrolysates at 50 kGy, and the scavenging activity of DPPH was 96.81% (Table 3). In Fig. 3B, a marked reduction of the first peak of the hydrolysates was observed after irradiation, with the 92.04% ABTS scavenging activity at 50 kGy (Table 3). The results of Fig. 3 and Table 3 revealed that antioxidant activity of hydrolysates increased significantly (P < 0.05) after irradiation. According to the results of increased content of amino acids and smaller peptide pieces, the antioxidant activity of the hydrolysates was higher than the antioxidant activity of the nonhydrolyzed protein, probably due to proteolysis producing smaller peptides (Zhou and Zhang, 2012) and exposing hydrophobic groups (Shazly et al., 2019). Additionally, irradiated hydrolysates exhibited better ability than nonirradiated hydrolysates, possible relating to unfolded secondary structure and changes of hydrophobic regions in protein cores. Therefore, irradiated hydrolysates achieved great antioxidant ability by
As mentioned above, it was concluded that EBI induced the amino acid oxidation, as well as it destroyed the secondary structure and microscopic morphology of the hydrolysates. Interestingly, the amino acid content of RP increased with enzymatic hydrolysis, but irradiation reduced the TAAs and TEAAs of the hydrolysates. Nevertheless, the amino acid content of the irradiated hydrolysates was higher than the amino acid content of the intact protein at a lower irradiation dose (< 10 kGy). Furthermore, the antioxidant activity of the irradiated samples was significantly improved, possibly due to changes in the secondary structure and hydrophobic regions. Overall, enzymolysisassisted EBI caused permanent damage to the protein structure and increased the antioxidant ability of rice protein. This research will provide guidance for the application of electron beam irradiation in the food protein industry. Conflicts of interest The authors declare no competing financial interest. Acknowledgment This research was supported by the National Natural Science Foundation of China (31471616), the National Key R&D Program of China (2017YDF0401100), and National Top Youth Talent for Grain Industry (LQ2016301), Natural Science Foundation of Jiangsu Province (BK20171137), National first-class discipline program of Food Science and Technology (JUFSTR20180203) and Postgraduate Research & Practice Innovation Program of Jiangnan University (JNKY19_004). Notes: The authors declare no competing financial interest. 6
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Appendix A. Supplementary data
Malik, M.A., Sharma, H.K., Saini, C.S., 2017. Effect of gamma irradiation on structural, molecular, thermal and rheological properties of sunflower protein isolate. Food Hydrocolloids 72, 312–322. Mareček, V., Mikyška, A., Hampel, D., Čejka, P., Neuwirthová, J., Malachová, A., Cerkal, R., 2017. ABTS and DPPH methods as a tool for studying antioxidant capacity of spring barley and malt. J. Cereal Sci. 73, 40–45. Misra, G., 2019. Chapter 3 - fluorescence spectroscopy. In: Misra, G. (Ed.), Data Processing Handbook for Complex Biological Data Sources. Academic Press, pp. 31–37. Olotu, I.O., Enujiugha, V., Obadina, A.O., 2014. The effect of γ-irradiation and cooking on the amino acid profile of african oil bean seed ( P entaclethra macrophylla benth). J. Food Process. Preserv. 38, 2020–2026. Rani, S., Pooja, K., Pal, G.K., 2018. Exploration of rice protein hydrolysates and peptides with special reference to antioxidant potential: computational derived approaches for bio-activity determination. Trends Food Sci. Technol. 80, 61–70. Shazly, A.B., Mu, H., Liu, Z., El-Aziz, M.A., Zeng, M., Qin, F., Zhang, S., He, Z., Chen, J., 2019. Release of antioxidant peptides from buffalo and bovine caseins: influence of proteases on antioxidant capacities. Food Chem. 274, 261–267. Wang, L., Li, T., Sun, D., Tang, M., Sun, Z., Chen, L., Luo, X., Li, Y., Wang, R., Li, Y., Li, J., Chen, Z., 2019. Effect of electron beam irradiation on the functional properties and antioxidant activity of wheat germ protein hydrolysates. Innov. Food Sci. Emerg. Technol. 54, 192–199. Wang, L., Zhang, X., Liu, F., Ding, Y., Wang, R., Luo, X., Li, Y., Chen, Z., 2017. Study of the functional properties and anti-oxidant activity of pea protein irradiated by electron beam. Innov. Food Sci. Emerg. Technol. 41, 124–129. Wang, T., Liu, F., Wang, R., Wang, L., Zhang, H., Chen, Z., 2015. Solubilization by freezemilling of water-insoluble subunits in rice proteins. Food Funct. 6, 423–430. Wang, Z., Li, H., Liang, M., Yang, L., 2016. Glutelin and prolamin, different components of rice protein, exert differently in vitro antioxidant activities. J. Cereal Sci. 72, 108–116. Xu, X., Liu, W., Liu, C., Luo, L., Chen, J., Luo, S., McClements, D.J., Wu, L., 2016. Effect of limited enzymatic hydrolysis on structure and emulsifying properties of rice glutelin. Food Hydrocolloids 61, 251–260. Yang, X., Li, Y., Li, S., Oladejo, A.O., Ruan, S., Wang, Y., Huang, S., Ma, H., 2017. Effects of ultrasound pretreatment with different frequencies and working modes on the enzymolysis and the structure characterization of rice protein. Ultrason. Sonochem. 38, 19–28. Yuan, D.B., Yang, X.Q., Tang, C.-H., Zheng, Z.X., Wei, M., Ahmad, I., Yin, S.-W., 2009. Physicochemical and functional properties of acidic and basic polypeptides of soy glycinin. Food Res. Int. 42, 700–706. Zhang, Y., Zhou, F., Zhao, M., Ning, Z., Sun-Waterhouse, D., Sun, B., 2017. Soy peptide aggregates formed during hydrolysis reduced protein extraction without decreasing their nutritional value. Food Funct. 8, 4384–4395. Zhao, L., Zhang, Y., Pan, Z., Venkitasamy, C., Zhang, L., Xiong, W., Guo, S., Xia, H., Liu, W., 2018. Effect of electron beam irradiation on quality and protein nutrition values of spicy yak jerky. LWT - Food Sci. Technol. 87, 1–7. Zhou, M., Zhang, M., 2012. Preparation of anti-oxidation bioactive peptide of rice bran protein. Natural Product R & D 24, 793–799.
Supplementary data to this article can be found online at https:// doi.org/10.1016/j.jcs.2019.102789. References Amagliani, L., O'Regan, J., Kelly, A.L., O'Mahony, J.A., 2017. The composition, extraction, functionality and applications of rice proteins: a review. Trends Food Sci. Technol. 64, 1–12. AOAC, 2010. AOAC Offcial Method 979.09: Protein in Grains. Association of Analytical Communities International, Gaithersburg. Benbettaieb, N., Karbowiak, T., Brachais, C.-H., Debeaufort, F., 2016. Impact of electron beam irradiation on fish gelatin film properties. Food Chem. 195, 11–18. Elias, P.S., Cohen, A.J., 1977. Radiation Chemistry of Major Food Components. Elsevier Scientific Pub. Co. Haque, E., Bhandari, B.R., Gidley, M.J., Deeth, H.C., Møller, S.M., Whittaker, A.K., 2010. Protein conformational modifications and kinetics of Water−Protein interactions in milk protein concentrate powder upon aging: effect on solubility. J. Agric. Food Chem. 58, 7748–7755. Jin, Y., Liang, R., Liu, J., Lin, S., Yu, Y., Cheng, S., 2017. Effect of structure changes on hydrolysis degree, moisture state, and thermal denaturation of egg white protein treated by electron beam irradiation. LWT - Food Sci. Technol. 77, 134–141. Kuan, Y.-H., Bhat, R., Patras, A., Karim, A.A., 2013. Radiation processing of food proteins - a review on the recent developments. Trends Food Sci. Technol. 30, 105–120. Li, D., Jiang, L., 2015. Correlation between Amino Acid Composition and Surface Hydrophobicity of Soybean 7S and 11S Proteins. China Oils & Fats. vol. 40. pp. 25–29. Li, J., Xu, X., Chen, Z., Wang, T., Wang, L., Zhong, Q., 2018. Biological macromolecule delivery system fabricated using zein and gum Arabic to control the release rate of encapsulated tocopherol during in vitro digestion. Food Res. Int. 114, 251–257. Li, S., Yang, X., Zhang, Y., Ma, H., Liang, Q., Qu, W., He, R., Zhou, C., Mahunu, G.K., 2016a. Effects of ultrasound and ultrasound assisted alkaline pretreatments on the enzymolysis and structural characteristics of rice protein. Ultrason. Sonochem. 31, 20–28. Li, S., Yang, X., Zhang, Y., Ma, H., Qu, W., Ye, X., Muatasim, R., Oladejo, A.O., 2016b. Enzymolysis kinetics and structural characteristics of rice protein with energy-gathered ultrasound and ultrasound assisted alkali pretreatments. Ultrason. Sonochem. 31, 85–92. Lin, X., Yang, W., Xu, D., Wang, L., 2015. Effect of electron irradiation and heat on the structure of hairtail surimi. Radiat. Phys. Chem. 114, 50–54. Liu, Q., Kong, B., Jiang, L., Cui, X., Liu, J., 2009. Free radical scavenging activity of porcine plasma protein hydrolysates determined by electron spin resonance spectrometer. LWT - Food Sci. Technol. 42, 956–962. Lv, M., Mei, K., Zhang, H., Xu, D., Yang, W., 2018. Effects of electron beam irradiation on the biochemical properties and structure of myofibrillar protein from Tegillarca granosa meat. Food Chem. 254, 64–69.
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Effect of enzymolysis-assisted electron beam irradiation on structural characteristics and antioxidant activity of rice protein
T
Ting Lia,b,d, Li Wanga,b,d,∗, Dongling Sunc, Yanan Lib,d, Zhengxing Chena,b,d,∗∗ a
State Key Laboratory of Food Science and Technology, Jiangnan University, Lihu Road 1800, Wuxi, 214122, China School of Food Science and Technology, National Engineering Laboratory for Cereal Fermentation Technology, Jiangnan University, Lihu Road 1800, Wuxi, 214122, China c Wuxi EL PONT Radiation Technology CO., Ltd, No. 8, Weiye Road, Qianqiao Industrial Park (Xi'nan), Huishan District, Wuxi, 214151, Jiangsu, China d Jiangsu Provincial Research Center for Bioactive Product Processing Technology, Jiangnan University, Lihu Road 1800, Wuxi, 214122, China b
ARTICLE INFO
ABSTRACT
Keywords: Electron beam irradiation Structural characteristics Antioxidant activity Rice protein
This research investigated the effect of enzymolysis-assisted electron beam irradiation (EBI) on the structural characteristics and antioxidant activity of rice protein (RP). The amino acid content of RP was increased by enzymatic hydrolysis, but the amino acid content of the rice protein hydrolysates (RPHs) was decreased gradually as the irradiation dose increased. Additionally, the secondary structural change of the irradiated hydrolysates revealed that a flexible and unfolded structure was induced by EBI. Irradiation increased the fluorescence intensity of the hydrolysates, which can probably be attributed to the exposure and changes in the surroundings of the hydrophobic region. Microscopic graphs showed that the tight conformation of the RP was destroyed by enzymolysis. Furthermore, the antioxidant activity of the RPHs was improved by EBI, with the highest 1, 1-Diphenyl-2-picrylhydrazyl (DPPH) and 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) radical scavenging activity of 96.81% and 92.04% at 50 kGy, respectively. This study will provide a feasible coupling technology for the food industry to improve the antioxidant ability of RP. Consequently, irradiated hydrolysates could be regarded as a promising functional food ingredient due to their higher antioxidant ability.
1. Introduction Rice protein (RP) is acknowledged as a hypoallergenic and high nutritional value protein compared to other major cereal proteins. However, the application of RP in the food industry is limited by the poor solubility induced by its compact conformation. To improve the properties of proteins, many methods to change the structure such as chemical modification, physical treatment and enzymatic treatment have recently been used (Amagliani et al., 2017). Among these methods, antioxidant peptides can be released by enzymatic hydrolysis (Rani et al., 2018). Dietary antioxidants play an important role in controlling oxidative stress-induced damage. Additionally, natural antioxidants can act as alternatives for synthetic antioxidants and then delay the oxidation of lipids in the food industry. Recently, the potential benefits of an antioxidant peptide derived from rice have attracted the attention of researchers. For example, rice protein hydrolysates (RPHs) with antioxidant ability could enhance the stability of emulsion-type
∗
food systems and extend the shelf life of foodstuffs (Rani et al., 2018). Therefore, higher antioxidant activity of proteins is regarded as an important parameter, which is related to the exploitation and application of food products. Food irradiation is a physical method to maintain food freshness and even improve food quality. Radiation, including ionizing and nonionizing radiation, has been expanded for the modification of proteins. Corresponding to the absorbed irradiation dose, chemical changes such as polymerization, cross-linking and depolymerization can be achieved via free radical formation (Kuan et al., 2013). Electron beam irradiation (EBI), an ionizing irradiation, has been widely used in the modification of food components and improvement of quality (Kuan et al., 2013). The thermal stability of gelatin film has been enhanced by irradiation (Benbettaieb et al., 2016). In addition, EBI is regarded as a nonthermal, economic, environmentally friendly and safe technique to improve the biochemical properties of proteins. Jin et al. (2017) investigated the quality characteristics of egg white protein (EWP) after
Corresponding author. State Key Laboratory of Food Science and Technology, Jiangnan University, Lihu Road 1800, Wuxi, 214122, China. Corresponding author. State Key Laboratory of Food Science and Technology, Jiangnan University, Lihu Road 1800, Wuxi, 214122, China. E-mail addresses: [email protected], [email protected] (L. Wang), [email protected] (Z. Chen).
∗∗
https://doi.org/10.1016/j.jcs.2019.102789 Received 22 February 2019; Received in revised form 17 June 2019; Accepted 18 June 2019 Available online 18 June 2019 0733-5210/ © 2019 Elsevier Ltd. All rights reserved.
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irradiation and found that the degree of hydrolysis of EWP was enhanced after EBI treatment due to structural changes. Additionally, the Ca2+-ATPase activity of T. granosa meat was decreased as the irradiation dose increased. The edible qualities such as ‘hardness’, ‘gumminess’, ‘chewiness’ and ‘resilience’ of T. granosa meat were improved after EBI treatment (Lv et al., 2018). In our previous study, the wheat germ protein was hydrolyzed by alcalase and then treated by EBI (Wang et al., 2019). It was found that the antioxidant ability and functional properties of wheat germ protein hydrolysates were increased by EBI. Currently, the food industry is developing innovative technology to meet the demand of consumers for safer and higher quality foods. As described above, methods of enzymolysis and EBI can improve the quality and antioxidant ability of protein. However, there are few reports concerning the effect of enzymolysis-assisted EBI on structural changes and the antioxidant activity of RP. In this study, the amino acid composition of rice protein under enzymolysis-assisted EBI was measured by high-performance liquid chromatography (HPLC). Additionally, the structural changes in the RP after enzymolysis-assisted EBI were determined via Fourier Transform Infrared (FTIR) spectroscopy, fluorescence spectroscopy and microscopy. This study also determined the antioxidant ability of rice protein and hydrolysates by the electron spin resonance (ESR) technique. Therefore, this research will provide a scientific basis for the application of a novel coupling method (i.e., enzymolysis-assisted EBI) in the modification of rice protein.
2.3. Determination of amino acid composition The amino acid composition was determined according to the method of Zhao et al. (2018) with some modification. Samples (intact protein and hydrolysates) were prepared after hydrolysis with 6 mol/L HCl (110 °C, 22 h). Then, the amino acid composition of the samples was measured by high-performance liquid chromatography (HPLC) (Agilent 1260, USA). The parameters of the experiment were as follows: ultraviolet detector (VWD, G1314A), Agilent Hypersil ODS column (5 μm, 4.0 × 250 mm), 40 °C column temperature. Additionally, the solvent A was composed of sodium acetate, triethylamine and tetrahydrofuran at a proportion of 500/0.11/2.5 (v/v). The solvent B was composed of sodium acetate, methanol and acetonitrile at a proportion of 1/2/2 (v/v). A gradient elution program with a flow rate of 1 mL/ min (0–17 min, 8% solvent B; 17–20 min, 50% solvent B; 20–24 min, 100% solvent B; 24–46 min, 0% solvent B) was used in this test. 2.4. Fourier transform infrared spectroscopy The Fourier transform infrared (FTIR) spectra were scanned by the FTIR spectrometer (Nicolet IS10, USA). The powdered samples (intact protein and hydrolysates) were mixed with potassium bromide (KBr) and pressed into a slice. The wavelength of the spectra ranged from 4000 to 400 cm−1 with 4 cm−1 resolution and 32 scans. All measurements were carried out in a dry atmosphere at room temperature (25 °C). The results were analyzed by the software of OMNIC 8.2 (a professional infrared spectral analysis software from Nicolet) and Peakfit 4.12 (Wang et al., 2015).
2. Materials and methods 2.1. Materials
2.5. Fluorescence spectroscopy
Rice was purchased from Jinwanxian Co., Ltd. (Anhui, China). Alcalase 2.4 L (2.4 AU/g) was obtained from Novozymes China Co., Ltd. (Beijing, China). 1,1-Diphenyl-2-picrylhydrazyl (DPPH), 2,2′-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) and 1-anilino-8-naphthalene sulfonate (ANS) were purchased from Sigma-Aldrich Corporation (St. Louis, MO, USA). Other chemicals were obtained from Sinopharm Chemical Co., Ltd. (Shanghai, China).
The fluorescence spectra were determined using the ANS fluorescence probe according to the method of Yang et al. (2017) with slight modification. The fluorescence spectra were measured by the Fluoro Max4 fluorescence spectrophotometer (Horiba JY Company, United States). The samples (intact protein and hydrolysates) were dispersed in 0.01 M phosphate buffer (1%, w/v) and then mixed with 50 μL ANS. Then, the mixture was kept in the dark for 5 min before being transferred into the cuvette of the spectrophotometer. Additionally, the emission spectra were scanned from 400 to 600 nm at the excitation wavelength of 390 nm.
2.2. Preparation of protein and hydrolysates RP was extracted by the method of alkaline (pH 12) extraction and acid (pH 4.8) precipitation. The protein content in RP (96.1%) was determined by the Kjeldahl method (AOAC, 2010). Then, protein was dispersed in distilled water (1%, w/v) at 55 °C and incubated with alcalase for 2 h. The enzyme to substrate ratio was 1:50 (w/w). The reaction mixture was kept at pH of 8.0 by consecutive additions of 1 mol/ L NaOH during hydrolysis. The degree of hydrolysis (DH) was measured by the pH-stat method and calculated using equation (1) (Xu et al., 2016). Reactions of enzymatic hydrolysis were stopped by immersion in a boiling water bath for 10 min. Then, hydrolysates were adjusted to pH 7.0 by dilute acid. Next, 100-mL RPHs suspensions (1%, w/v) were irradiated with various doses (5 kGy, 10 kGy, 25 kGy, and 50 kGy) at 5.0 MeV energy level. The duration of the EBI treatment was 0.86 s, 1.72 s, 4.3 s, and 8.6 s, respectively. Irradiation processing was conducted at room temperature (25 °C). EBI treatment employed a highenergy linear accelerator (AB type high frequency and high voltage) of Wuxi EI Pont Radiation Technology Co., Ltd. Finally, all samples (intact protein and hydrolysates) were lyophilized by freeze drying (Christ Beta 2–8 LD plus, Germany) and stored at −20 °C until further use.
DH =
B × Nb × 100% × Mp × htot
2.6. Microscopy studies The lyophilized samples (including intact protein and hydrolysates) were milled through a 100-mesh sieve. The microstructure of the lyophilized samples coated with gold and platinum was measured by scanning electron microscopy (SEM) (Quanta 200, FEI Co., Holland) with magnification of 1000 and an accelerating voltage of 10.0 kV. The fluorescent dye, 20 mM ANS (10 μL), was added to the 1% sample solutions (including intact protein and hydrolysates) that were dispersed in distilled water (w/v). Then, the mixture was placed on the glass slide. The shape of the sample in solution was observed by inverted fluorescence microscopy (IFM) using bright and fluorescent fields. 2.7. Antioxidant ability measurement DPPH and ABTS radical scavenging activity are two of the most common and efficient methods to test the antioxidant ability of intact protein and hydrolysates. The results of free radical scavenging activity determined by DPPH and ABTS are partly different due to their different mechanisms. The DPPH radical scavenging assay is based on the reduction of 1,1-Diphenyl-2-picrylhydrazyl by accepting an electron or a hydrogen from the antioxidant compounds. The ABTS radical scavenging assay is based on the ability to eliminate green radicals (2,2azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)). The ABTS radical is
(1)
where B is the volume of NaOH to maintain a constant pH of 8.0, Nb is the molar concentration of NaOH, α is the degree of dissociation of αNH2 groups, Mp is the total mass of the substrate protein, and htot is the number of peptide bonds in the substrate protein. 2
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more likely to react with antioxidants than the DPPH radical. However, the preparation of DPPH in solvent is easier than preparation of ABTS reagent in solvent. In addition, DPPH radical has higher stability compared to ABTS (Mareček et al., 2017). Therefore, two different methods for measuring free radical scavenging activity of intact protein and hydrolysates can lead to the impartial results in this study.
Table 1 Amino acids composition of rice protein and hydrolysates. Amino acids/(g/100 g)
Asp Glu Ser His Gly Thr Arg Ala Tyr Cys-s Val Met Phe Ile Leu Lys Pro TAAs/(g/100 g) TEAAs/(g/100 g) THAAs/(g/100 g)
2.7.1. Scavenging activity of DPPH radical DPPH radical scavenging activity of samples was measured by the method of Liu et al. (2009) with some modification. A 100-μL sample (including intact protein and hydrolysates) was mixed with 100 μL DPPH (2 mg/mL ethanol). The mixture of sample and DPPH was transferred into a quartz capillary tube after 1 min. Then, the quartz capillary tube was transferred into a nuclear magnetic resonance tube. Then, the nuclear magnetic resonance tube was put into the resonant cavity, and the spectrum was then determined by the electron spin resonance (ESR) spectrometer (EMXplus-10/12, Bruker, Germany). The experimental parameters were the following: microwave power, attenuation and frequency were 20 mW, 10 dB, 9.44 GHz, respectively; modulation amplitude was 6 G; scan time was 20 s; central field was 3350 G and the sweep width was 150 G. Each sample was scanned three times to acquire a spectra with a suitable signal-to-noise ratio. The DPPH radical scavenging activity was calculated by the following equation (2).
Radical scavenging activity (RSA) =
H0
H H0
× 100%
Rice protein
6.35 14.3 2.82 1.53 3.21 2.12 6.42 3.95 3.12 0.26 5.04 1.87 3.95 3.43 6.02 2.19 4.71 71.3 24.6 30.2
Rice protein hydrolysates 0 kGy
5 kGy
10 kGy
25 kGy
50 kGy
8.65 18.6 3.77 2.46 4.64 2.73 8.57 5.22 3.90 0.29 7.78 1.68 5.45 4.62 7.75 3.14 4.50 93.8 33.2 39.2
8.01 17.1 3.25 2.22 4.19 2.41 7.75 4.74 3.34 0.21 7.10 1.48 4.72 4.27 7.11 2.85 4.67 85.4 29.9 36.0
8.32 17.7 3.36 1.84 4.39 2.44 7.84 4.95 3.42 0.16 7.25 1.41 3.86 4.28 7.10 2.83 4.42 85.6 29.2 35.3
7.20 15.1 2.85 1.28 3.71 2.04 6.43 4.24 2.30 0.10 6.10 0.96 2.66 3.56 5.81 2.40 4.17 70.9 23.5 28.8
7.34 15.0 2.60 0.47 3.53 1.83 6.00 4.25 2.03 0.09 6.29 0.94 2.16 3.47 5.44 2.19 4.79 68.4 22.3 28.4
Note: TAAs, Total amino acids; Total essential amino acids (TEAAs) = Thr + Val + Met + Phe + Ile + Leu + Lys; Total hydrophobic amino acids (THAAs) = Ala + Tyr + Val + Phe + Ile + Leu + Pro.
(2)
100 g, respectively. After hydrolysis, TAAs, TEAAs and THAAs of RPHs were higher than RP, with the TAAs of 93.8 g/100 g, the TEAAs of 33.2 g/100 g and the THAAs of 39.2 g/100 g. Consistently, Zhang et al. (2017) found that hydrophobic and essential amino acid contents of soy protein were increased after hydrolysis by alcalase. Shazly et al. (2019) observed more hydrophobic amino acids in buffalo casein and bovine casein hydrolysates compared to the original protein. As is well known, alcalase was regarded as a commercial serine proteases with an optimum temperature of 55 °C and pH of 8.0, which could cleave the peptide bonds of proteins. The DH is defined as the proportion of the cleavage of peptide bonds of total peptide bonds in a protein substrate. In this study, the DH of RP after hydrolysis was 11.8%. Additionally, in Table S1 (Supplementary file), the solubility of intact protein was increased by enzymatic hydrolysis (from 3.94% to 15.0%). Therefore, it was assumed that the cleavage of peptide bonds and the increased solubility increased the acid-accessibility of hydrolysates when the amino acid composition was measured after acid hydrolysis, resulting in an increased final determined content of TAAs, TEAAs and THAAs. However, the amino acid content of RPHs decreased as the irradiation dose increased, reaching the minimum value at 50 kGy. Compared with 0 kGy, TAAs and TEAAs of the irradiated RPHs were reduced by 27.05% and 32.67% at 50 kGy, respectively. Nevertheless, both TAAs and TEAAs of the hydrolysates were higher than the intact protein with the irradiation dose below 10 kGy. Zhao et al. (2018) also found that TAAs and TEAAs of spicy yak jerky decreased at an irradiation dose of 9 kGy when compared with 0 kGy. However, the opposite result was reported by (Olotu et al., 2014). In their study, some amino acid contents (lysine, threonine, etc) of the irradiated samples increased at 10 kGy, which could be related to the deamination and decarboxylation of the amino acids. According to the research of Elias and Cohen (1977), sulfur-containing amino acids were most sensitive to irradiation. Consistently, in present results, cystine and methionine declined gradually as the irradiation dose increased. Additionally, the cysteine and methionine of RPHs at 50 kGy decreased drastically by 68.97% and 44.05%, respectively, compared with native RPHs. As discussed above, the amino acid content of the intact protein was increased by enzymatic hydrolysis, possible attributed to the increased acid-accessibility of the protein after enzymolysis. Therefore, the increased content of TEAAs indicated that hydrolysates had a better
where the H and H0 were the third resonance peak for DPPH with and without samples (including intact protein and hydrolysates). 2.7.2. Scavenging activity of ABTS radical The ABTS radical scavenging activity was determined by the method of our previous study (Wang et al., 2017). The ABTS stock solution (7 mM) was mixed with potassium persulfate solution (2.45 mM) to prepare the ABTS working solution. Then, the working solution sat for 12 h at room temperature in the dark. Then, a 100-μL sample (including intact protein and hydrolysates) was mixed with ABTS working solution (100 μL) and reacted for 1 min. The spectrum was determined by the ESR spectrometer as mentioned above (Section 2.7.1). The ABTS radical scavenging activity was calculated using equation (2). The H and H0 were the first resonance peak for ABTS with and without samples (including intact protein and hydrolysates). 2.8. Statistical analysis The experiment was measured in triplicate. Data were analyzed by the Analysis of Variance (ANOVA) in SPSS statistical software (version 20.0). The differences between samples were determined using the Duncan's multiple range test at a level of 0.05. 3. Results and discussion 3.1. Amino acid composition The amino acid composition is an important index to evaluate the quality of the protein (Olotu et al., 2014). RP has a reasonable amino acid composition, and it is rich in limited essential amino acids such as lysine and threonine (Amagliani et al., 2017). The chemical composition (i.e., protein, lipid, ash) of the intact protein was roughly unchanged after enzymolysis (data not shown). However, the determination of the amino acid composition was influenced mainly by the content of moisture due to the conditions of the environment. Thus, the amino acid composition was calculated on the basis of dry basis weight of the samples in this study. As shown in Table 1, the total amino acids (TAAs), the total essential amino acids (TEAAs) and the hydrophobic amino acids (THAAs) of RP were 71.3 g/100 g, 24.6 g/100 g and 30.2 g/ 3
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available amino acid profile than the intact protein, which proved that enzymolysis promote the digestion and utilization of rice protein. However, a negative trend of amino acid content of hydrolysates after irradiation was shown in our test. Kuan et al. (2013) suggested that some free radicals generated by EBI resulted in amino acid (i.e., histidine, cysteine and methionine) loss through oxidation and molecular degradation. In addition, as shown in Table S1, the solubility of hydrolysates was increased due to the structural changes and the exposure of amino acids, thus probably promoting the process of amino acid oxidation. Nevertheless, the amino acid content of irradiated hydrolysates with lower doses (< 10 kGy) was still superior to the intact protein. Therefore, to maintain the nutritional value of RPHs, the irradiation dose should not exceed 10 kGy, which is in agreement with the required dose of WHO.
observed that irradiation decreased the α-helix content and increased the β-turn content. Lin et al. (2015) found that EBI reduced the contents of the α-helix and β-sheet of samples, indicating that the ordered structure of the protein was obviously destroyed by EBI. Combining the present study and the previous study by the researchers, the conformation of the intact protein was altered by irradiation. As described above, the decreased content of the β-sheet showed that a more flexible and unfolded structure of RPHs was induced by irradiation. Combined with the results of Table 1, the content of THAAs of the intact protein was increased from 30.2 g/100 g–39.2 g/100 g after enzymatic hydrolysis. Therefore, it was supposed that some amino acids of the RP were exposed by enzymatic hydrolysis from the buried hydrophobic core of the protein (Li et al., 2016a). Then, the exposed protein construction was destroyed by EBI. Therefore, a more flexible and extended conformation of rice protein was formed through enzymolysis-assisted EBI.
3.2. Fourier transform infrared spectroscopy The information of the functional groups of the protein can be provided from the FTIR spectra, and the analysis of the amide I band (1600-1700 cm−1) has been widely used to calculate the content of the secondary structure owing to its sensitivity to protein conformation changes. Data processing of the amide I band from FT-IR spectra was conducted according to the method of Haque et al. (2010). The amide I region of the spectra was baseline-corrected by OMINC 8.2 and deconvolved by PeakFit 4.12. The secondary structure content of each component was calculated by a peak area that was fitted by PeakFit 4.12. The peak area of 1600–1640 cm−1 represented the β-sheet structure, 1640-1650 cm−1 belonged to the random coil structure, 1650-1660 cm−1 corresponded to the structure of α-helix, and β-turn content was speculated to be 1660-1700 cm−1 (Lin et al., 2015). Fig. 1A shows the FTIR spectra of 400–4000 cm−1, and the content of the secondary structure is observed in Table 2. As seen in Fig. 1A, the peak strength (amide I band at 1600-1700 cm−1) of RPHs was lower than the peak strength of RP, and the absorption band of C]O was shifted from 1650 cm−1 to 1654 cm−1 after enzymolysis. This result was derived from the decreased content of the β-sheet (Malik et al., 2017), which is consistent with the results from Table 2. Additionally, EBI-treated hydrolysates exhibited spectral profiles similar to untreated hydrolysates because the basic structure of the hydrolysates was not significantly destroyed by EBI (Jin et al., 2017). In Table 2, compared to the control (RP), the α-helix content of RPHs increased from 12.50% to 14.38%, and the β-turns content of RPHs increased from 36.75% to 48.61%. The structure of the β-sheet was a stable and folded structure, whereas the β-turn was unfolding with a flexible conformation (Wang et al., 2015). Xu et al. (2016) found that the decreased content of the βsheet as well as the increased content of the α-helix appeared in rice glutelin hydrolysates compared with native rice glutelin, which revealed that extended and flexible structures were unfolded by enzyme hydrolysis. Additionally, the β-sheet and random coil contents of irradiated RPHs decreased, and the β-turn content increased with the increased dose of irradiation. There was no obvious change in the α-helix content of the hydrolysates with different doses. Malik et al. (2017)
3.3. Fluorescence spectroscopy Fluorescence emission spectroscopy using ANS as a fluorescence probe is a sensitive way to characterize the surface hydrophobicity of a protein during processing. The surface hydrophobicity is associated with the content of hydrophobic amino acids such as Trp, Tyr and Phe (Li et al., 2016b). The emission fluorescence spectra of RP and RPHs are shown in Fig. 1B, indicating the hydrophobic bonding region with ANS and the surrounding changes. The fluorescence spectral profile of RP was similar to the fluorescence spectral profile of hydrolysates with an irradiation dose of 0 kGy. However, the fluorescence intensity of the peak at 500–550 nm of hydrolysates was slightly increased when compared with the protein, indicating that the hydrophobic binding domains of protein cores were slightly exposed after hydrolysis. Additionally, the fluorescence intensity of the peak at 500–550 nm of RPHs increased intensely with the increasing irradiation dose, revealing that the binding ability between the ANS and the hydrophobic patches of the hydrolysates was enhanced (Li et al., 2018). Interestingly, a new peak (400–450 nm) of irradiated hydrolysates was enhanced as irradiation dose increased. The maximum peak of the irradiated hydrolysates was blue-shifted, possibly owing to the surrounding changes of hydrophobic amino acids. The hydrophobic zones of the hydrolysates were exposed to a polar environment due to their flexible and extended structure, resulting in the surrounding changes of binding areas (Misra, 2019; Wang et al., 2015). In addition, the surface hydrophobicity index through the initial slope of fluorescence intensity versus protein concentration was measured to confirm the result of emission spectra. As shown in Table S1 (Supplementary file), the surface hydrophobicity index of the intact protein was increased by enzymolysis-assisted EBI, which proved the results shown in Fig. 1B. Wang et al. (2017) also found that the surface hydrophobicity of irradiated pea protein hydrolysates increased significantly (P < 0.05) when compared to nonirradiated hydrolysates. Previous researchers have reported that the content of THAAs was positively related to the surface hydrophobicity of proteins (Li and Jiang, 2015; Wang et al., 2015). In the present
Table 2 Content of secondary structure of rice protein and hydrolysates. Content of secondary structure
Rice protein
Rice protein hydrolysates 0 kGy
α-helix β-sheet β-turns random coil
12.5 37.4 36.8 13.4
± ± ± ±
0.72 0.29 0.63 0.20
a c a c
14.4 24.0 48.6 13.0
± ± ± ±
5 kGy 0.09 b 0.4 b 0.34 b 0.03 bc
14.1 24.7 48.3 13.0
± ± ± ±
10 kGy 0.05 ab 0.21 b 0.11 b 0.17 bc
14.4 22.3 50.4 12.9
± ± ± ±
25 kGy 0.02 b 0.21 a 0.18 c 0.01 b
14.4 22.7 50.1 12.8
± ± ± ±
50 kGy 0.01 b 0.29 a 0.31 c 0.02 ab
13.4 22.9 51.2 12.4
± ± ± ±
1.38 0.70 0.97 0.29
ab a c a
Note: Values are shown as the mean ± SD of three determinations (different samples); Superscript letters (a, b, c, d) in the same row indicate significant differences (P < 0.05). 4
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Fig. 1. Fourier transform infrared spectroscopy spectra (A) and fluorescence emission spectra (B) of rice protein and hydrolysates (a. rice protein, b-f. rice protein hydrolysates with irradiation dose of 0, 5, 10, 25, 50 kGy).
research, the higher hydrophobic amino acid content of hydrolysates was consistent with the increased surface hydrophobicity. However, the content of THAAs of irradiated hydrolysates was decreased in an unexpected way, probably owing to the oxidation of the amino acids (Kuan et al., 2013) and the structural unfolding of peptide (Yuan et al., 2009).
surface of the irradiated samples (Fig. 2C1- F1) became smoother than the surface of the nonirradiated samples (Fig. 2B1). Meanwhile, some punctured pores occurred after irradiation (Fig. 2C1, D1, E1, red arrow). Additionally, more lamellar structures of hydrolysates were formed under irradiation (Fig. 2D1). Similarly, some researchers observed that punctured pores appeared on the surface structure of proteins with the EBI treatment (Jin et al., 2017). Malik et al. (2017) also described that some cracks on the surface of sunflower protein isolates appeared after gamma irradiation. The results of Fig. 2 and other researchers proved that irradiation damaged the microstructure of hydrolysates. Furthermore, in images of the fluorescent field from IFM, the fluorescent region and signal of the intact protein particles were decreased after treatment by enzymolysis and EBI, revealing that particles of RP in solution were degraded by hydrolysis and irradiation (Fig. 2A3-F3). Consistently, the same trend was observed in the graphs from the bright field of IFM
3.4. Morphological studies The surface morphology of the hydrolysates was analyzed by SEM. Additionally, the shape and size of the hydrolysates in solution were studied using IFM (bright and fluorescent fields). The images of SEM and IFM are shown in Fig. 2. In Fig. 2A1, the surface structure of RP was compact and cross-linked. However, smaller pieces of RP were formed by enzymatic hydrolysis (Fig. 2B1). With regard to hydrolysates, the
Fig. 2. Microstructure of rice protein and hydrolysates (A. rice protein, B–F. rice protein hydrolysates with irradiation dose of 0, 5, 10, 25, 50 kGy; 1. scanning electron microscopy (SEM), 2. inverted fluorescence microscopy (IFM) with bright field, 3. IFM with fluorescent field). 5
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Fig. 3. Free radicals scavenging capacity of DPPH and ABTS of rice protein and hydrolysates (A. DPPH radical scavenging capacity, B. ABTS radical scavenging capacity; a. rice protein, b-f. rice protein hydrolysates with irradiation dose of 0, 5, 10, 25, 50 kGy).
Table 3 DPPH and ABTS radical scavenging activity (RSA) of rice protein hydrolysates. RSA
DPPH/% ABTS/%
Rice protein
46.0 ± 1.00 48.6 ± 0.27
Rice protein hydrolysates
a a
0 kGy
5 kGy
10 kGy
25 kGy
50 kGy
66.7 ± 0.51b 71.1 ± 0.25b
65.8 ± 0.27b 87.7 ± 0.11c
82.5 ± 2.17c 87.9 ± 0.17c
86.8 ± 0.14d 88.9 ± 0.46c
96.8 ± 0.94e 92.0 ± 1.16d
Note: Values are shown as the mean ± SD of three determinations (different samples); Superscript letters (a, b, c, d, e) in the same row indicate significant differences (P < 0.05).
(Fig. 2A2-F2). As discussed above, it was concluded that the compact structure of the intact protein was degraded by the enzyme. Meanwhile, enzymolysis-assisted EBI accelerated the degree of denaturation. Furthermore, more fragmentations of hydrolysates resulted from bombardment by high energy electrons during irradiation.
increasing the accessibility to the free radicals (Wang et al., 2019). Furthermore, the increased solubility might contribute to the higher antioxidant ability of hydrolysates. Considering the present study and other research, enzymolysis-assisted EBI is an effective technology to develop products with higher antioxidant activity.
3.5. Antioxidant activity
4. Conclusions
Recently, some studies have reported that RP exhibited antioxidant activity by observing the ability of reducing the free radicals (Wang et al., 2016). This study used the ESR technique to detect free radicals that had unpaired electrons in the magnetic field (Liu et al., 2009). DPPH and ABTS radical were relatively stable and were widely used to test the ability of the antioxidant of protein and hydrolysates (Mareček et al., 2017). DPPH and ABTS radical scavenging activities of hydrolysates are illustrated in Fig. 3 and Table 3. RP exhibited antioxidant activity, with the DPPH radical scavenging activity of 46.00% and ABTS radical scavenging activity of 48.57%, respectively (Table 3). Additionally, DPPH and ABTS radical scavenging activities of hydrolysates were higher than the radical scavenging activities of the intact protein, indicating that the antioxidant activity of the protein was increased by enzymatic hydrolysis. In Fig. 3A, the height of the third peak showed an obvious decrease in irradiated hydrolysates at 50 kGy, and the scavenging activity of DPPH was 96.81% (Table 3). In Fig. 3B, a marked reduction of the first peak of the hydrolysates was observed after irradiation, with the 92.04% ABTS scavenging activity at 50 kGy (Table 3). The results of Fig. 3 and Table 3 revealed that antioxidant activity of hydrolysates increased significantly (P < 0.05) after irradiation. According to the results of increased content of amino acids and smaller peptide pieces, the antioxidant activity of the hydrolysates was higher than the antioxidant activity of the nonhydrolyzed protein, probably due to proteolysis producing smaller peptides (Zhou and Zhang, 2012) and exposing hydrophobic groups (Shazly et al., 2019). Additionally, irradiated hydrolysates exhibited better ability than nonirradiated hydrolysates, possible relating to unfolded secondary structure and changes of hydrophobic regions in protein cores. Therefore, irradiated hydrolysates achieved great antioxidant ability by
As mentioned above, it was concluded that EBI induced the amino acid oxidation, as well as it destroyed the secondary structure and microscopic morphology of the hydrolysates. Interestingly, the amino acid content of RP increased with enzymatic hydrolysis, but irradiation reduced the TAAs and TEAAs of the hydrolysates. Nevertheless, the amino acid content of the irradiated hydrolysates was higher than the amino acid content of the intact protein at a lower irradiation dose (< 10 kGy). Furthermore, the antioxidant activity of the irradiated samples was significantly improved, possibly due to changes in the secondary structure and hydrophobic regions. Overall, enzymolysisassisted EBI caused permanent damage to the protein structure and increased the antioxidant ability of rice protein. This research will provide guidance for the application of electron beam irradiation in the food protein industry. Conflicts of interest The authors declare no competing financial interest. Acknowledgment This research was supported by the National Natural Science Foundation of China (31471616), the National Key R&D Program of China (2017YDF0401100), and National Top Youth Talent for Grain Industry (LQ2016301), Natural Science Foundation of Jiangsu Province (BK20171137), National first-class discipline program of Food Science and Technology (JUFSTR20180203) and Postgraduate Research & Practice Innovation Program of Jiangnan University (JNKY19_004). Notes: The authors declare no competing financial interest. 6
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Appendix A. Supplementary data
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