Hydrolysis of rapeseed meal protein under simulated duodenum digestion: Kinetic modeling and antioxidant activity

LWT - Food Science and Technology 68 (2016) 523e531

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Hydrolysis of rapeseed meal protein under simulated duodenum digestion: Kinetic modeling and antioxidant activity Cunshan Zhou a, b, c, Xiaojie Yu a, Xiaopei Qin a, Haile Ma a, b, c, *, Abu ElGasim A. Yagoub d, Jiali Hu a a

School of Food and Biological Engineering, Jiangsu University, No.301 Xuefu Road, Zhenjiang 212013, China Jiangsu Provincial Key Laboratory for Physical Processing of Agricultural Products, Jiangsu University, No.301 Xuefu Road, Zhenjiang 212013, China Jiangsu Provincial Research Center of Bio-process and Separation Engineering of Agri-products, Jiangsu University, No.301 Xuefu Road, Zhenjiang 212013, China d Faculty of Agriculture, University of Zalingie, PO Box 6, Zalingie, Sudan b c

a r t i c l e i n f o

a b s t r a c t

Article history: Received 14 October 2015 Received in revised form 25 November 2015 Accepted 27 November 2015 Available online 9 December 2015

Kinetics modelling, DPPH radical scavenging activity and reducing power of rapeseed meal protein were investigated under simulated duodenum digestion. Kinetics of the reaction was considered in relation to initial substrate concentration, initial enzyme concentration, and hydrolysis time. Optimum hydrolysis conditions were trypsin concentration of 10 g/L, substrate concentration of 4 g/L, time of 40 min and temperature of 37
C. From the results a general kinetic enzymolysis equation was suggested, providing a rational theoretical basis for determining the parameters of the reaction. The highest antioxidant activity was in accordance with the optimum reaction conditions. Antioxidant free amino acids increased after hydrolysis. Antioxidant oligopeptides of molecular weights of 2608, 1695 and 211 Da were identified. At a concentration of 2 mg/mL, the peptide fraction of CS-F2-CC2; with molecular weight of 1000e1500 Da; showed the highest DPPH radical scavenging activity and reducing power, accounting for 70.32% and 0.538, respectively. The trypsin-hydrolysate displayed a potential capacity to scavenge free radicals and binding irons after duodenal digestion, appearing as promising ingredient to formulate functional foods with antioxidant activity. © 2015 Elsevier Ltd. All rights reserved.

Keywords: Rapeseed meal protein Simulated duodenum digestion Kinetics model Peptides Antioxidant activity Chemical compounds studied in this article: Cytochrome C (PubChem CID: 439171) Aprotinin (PubChem CID: 16197280) L-leucine (PubChem CID: 6106) L-tryptophan (PubChem CID: 6305) Ferric chloride (PubChem CID: 24380) Trichloroacetic acid (PubChem CID: 6421) Potassium ferricyanide (PubChem CID: 26250) Sulfosalicylic acid (PubChem CID: 7322) 1,1-Diphenyl-2-picrylhydrazyl (PubChem CID: 2735032) 2,4,6-Trinitrobenzenesulfonic acid (PubChem CID: 498085)

1. Introduction Rapeseed is one of the most important oilseed crops worldwide and also it is the world's second leading source of protein meal (World Health Organization, 2002). The protein content of defatted rapeseed meal, a by-product of oil industry, is high (35e45%). The * Corresponding author. School of Food and Biological Engineering, Jiangsu University, 301 Xuefu Road, Zhenjiang 212013, China. E-mail address: [email protected] (H. Ma). http://dx.doi.org/10.1016/j.lwt.2015.11.057 0023-6438/© 2015 Elsevier Ltd. All rights reserved.

nutritive and functional properties of the rapeseed are characterized by two main protein families; cruciferin (12S globulin) and napin (2S albumin), with molecular weights around 300 kDa and 14 kDa, respectively (Bos et al., 2007). It is, thus, a kind of ideal and high quality protein resource, with good utilization value. Therefore, efforts have been made to develop efficient methods to prepare acceptable products from rapeseed meal for human benefit (Pan, Jiang, & Pan, 2011). Food proteins may act as sources of bioactive peptides. These peptides can be released from proteins in vitro during food processing or in vivo by the gastrointestinal

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digestion. Peptides with low molecular weights, which easily absorbed in the gastrointestinal tract, have been reported displaying potential antioxidative, antihypertensive and immune effects (Byun, Lee, & Park, 2009; Monchi & Rat, 1993; Grimble, Rees, & Keohane, 1987). Antioxidative peptides derived from soy proteins (Chen, Muramoto, Yamauchi, & Nokihara, 1996), soy milk whey ~ a-Ramos & Xiong, 2003), egg-yolk proteins (Sakanaba & (Pen Tachibana, 2006), amaranth protein or their hydrolysates (Orsini ~o n 2011), and buck wheat proteins (Ma, Delgado, Tironi, and An Xiong, Zhai, Zhu, & Dziubla, 2010) were monitored. Yet, there are only few studies examining the rapeseed meal as a source of bioactive peptides to enhance the value of this industry by-product. These studies were focused on production of antihypertensive peptides (Marczak, Ohinata, Lipkowski, & Yoshikawa, 2006; Marczak et al., 2003), HIV protease inhibitory hydrolysate (Yust €kinen, Johannson, et al., 2004), and ACE-inhibitory peptides (Ma Gerd, Pihlava, & Pihlanto, 2012). Recently, it is found that digestion of food proteins by human gastrointestinal tract can approximately be simulated in an in vitro environment. However, different food proteins under simulated gastrointestinal digestion showed different digestive characteristics. To render the enzymatic process much more close to the human digestion, maximum utilization of trypsin is indispensable. In respect to the release of potential bioactive peptides of low molecular weights, trypsin is reported to be more efficient than pepsin (Wang, Ma, Ma, & Fan, 2014). Moreover, trypsin-catalysed canola protein digestion produce high yields of bioactive hydrolysates, and less amounts of free amino acids compared to pepsin/trypsin digestion. (Alshai et al., 2014). Toward this aim, many kinetic models including empirical and mechanistic models have been proposed (Bansal, Hall, & Realff, 2009; Xu & Ding, 2007). It has been generally accepted that enzyme deactivation during the enzymatic hydrolysis process results in some hydrolytic rate slowdown (Gan, Allen, & Taylor, 2003; Ghadge, Patwardhan, & Sawant, 2005). However, many models have been proposed without considering enzyme deactivation (Shen & Agblevor, 2008). Peptides presenting radical scavenging capacity have been released by simulated gastrointestinal digestion from diverse food sources (Ma et al., 2010). Despite that, gastrointestinal digestion of proteins may increase probability of release of more amounts of free amino acids; and simultaneously a decrease in amounts of the bioactive peptides, produced by pancreatic enzymes, due to low s, Ramírez-Moreno, Sa nchez-Mata, and Gon ~i acidity. Díez Marque (2011) reported that gastrointestinal digestion starts at acidic medium (pH 1.3 to 3) and then followed by intestinal digestion, starting at duodenum (fasting pH 6.5) where gastric acid lowers the pH to 5.4 before increasing to 5.8 at the end of duodenal digestion In this respect, simulated duodenal digestion may be a perspective method of preparation of bioactive peptides. Accordingly, no research has been reported on the simulated duodenum digestive conditions and kinetics for producing antioxidant peptides from rapeseed protein. So that, we deduced a simple mathematical equation that directly described the relationship between the degree of hydrolysis and two reaction conditions (initial enzyme concentration and initial substrate concentration). Antioxidant activity of rapeseed protein under the simulated duodenum digestion was also investigated. This study may help providing antioxidant peptides that conform to the human physiological environment, hence increasing their efficacy when incorporated into food and pharmaceutical systems.

2. Material and methods 2.1. Material and reagents Rapeseed meal with 42.03% protein (Kjeldahl, AOAC, 2000), was purchased from Hubei Weipu Biologic Technology Company (Hubei, China). The meal was ground to pass 80 mesh screen and kept until use. Trypsin (EC 3.4.21.4) with an activity of 50,000 U/g and L-leucine were purchased from Sinopharm Chemical Reagent Company (Zhenjiang, China). 2,4,6-trinitrobenzenesulfonic acid (TNBS), cytochrome C (12,500 Da), aprotonin (6500 Da), L-tryptophan (204 Da) were from Sigma Chemical Co. (St. Louis, MO, USA). 1,1-diphenyl-2-picrylhydrazyl (DPPH) was purchased from Aladdin Reagent Co. Ltd (Shanghai, China). Sephadex G-25 (Shanghai Yuanye Biotechnology Co. Ltd). All the other reagents were of analytical grade. 2.2. Simulated duodenum digestion of the rapeseed meal protein The method used in simulated duodenum digestion was an adaptation of published methods (Fallingborg, 1999; Emma, Hala, & Abdul, 2008). The digestion experiments were done at different trypsin/protein ratios and hydrolysis times. After pre-incubation of the simulated duodenum digestive fluid (0.04 M KH2PO4 buffer, pH 6.4) for 5 min at 37
C, solutions with different concentrations of rapeseed meal (1, 2, 4, 8 and 16 g/L) and trypsin (2, 4, 6, 10 and 12 g/ L) in the digestive fluid were prepared according to the method in our previous study (Zhou, Qin, Yu, Yang, Hu, & Ma, 2015). Digestion experiments were done at different times (0e120 min) and at 37
C with continuous agitation. In the end, reactions were terminated by boiling the mixtures for 10 min. Finally, the digests were cooled and centrifuged at 4360  g for 20 min. The centrifuge supernatant (CS) was collected, lyophilized and kept at e20
c until analysis. Experiments were replicated three times. 2.3. Determination of degree of hydrolysis of the rapeseed meal protein The degree of hydrolysis (DH) was measured by the reaction of free amino groups with 2,4,6-trinitrobenzenesulfonic acid (TNBS) as described by Orsini Delgado et al. (2011).The DH was calculated using Eq. (1):

DHð%Þ ¼

½NH2 h  ½NH2 0  100 ½NH2 ∞  ½NH2 0

(1)

Where, [NH2] indicates the concentration of free amino groups in the non-hydrolysed samples (0), or in the hydrolysed samples (h), [NH2]∞ indicates the concentration of total free amino groups. 2.4. Enzymolysis kinetics of the rapeseed meal protein Kinetics of enzyme-catalysed reactions is the science of studying enzymatic reaction velocity and its influence factors. The kinetic equation, however, is explained by modeling the hydrolysis as a zero order reaction and inactivation of the enzyme as a second lez, Pa ez, Ma rquez, & Fern order reaction (Camacho, Gonza andez, 1993; Zhou et al., 2013; Zhou, Yu, Zhang, He, & Ma, 2012). According to the enzyme reaction intermediate complex theory, the process of enzymatic hydrolysis of a protein is expressed as follows: k1

ƒ! E þ S ƒƒƒƒƒ ƒƒƒƒƒ ƒ ESƒƒƒƒ!E þ P k1

k2

(2)

where, S is the concentration of substrate (g/L), E is the

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concentration of enzyme (g/L), P is the product (g/L), ES is the concentration of enzymeesubstrate complex (g/L), k1, k-1,k2 are the reaction rate constants of enzyme adsorption (L/U min), enzyme desorption (g/U min), and product formation (g/U min), respectively. At a constant pH and temperature, the hydrolysis velocity can be determined by the irreversible stage (Marquez & Vazquez, 1999):

V ¼ S0

dðDHÞ ¼ k3 ½ES dt

(3)

Where, V is the hydrolysis velocity (g/L min), S0 is the initial substrate concentration (g/L), DH is the degree of hydrolysis (%), and k3 is the reaction rate constant for inactivation (L/U min). According to the mechanism and kinetic model of enzymatic hydrolysis of protein, the hydrolysis velocity also can be expressed by the following equation:

V ¼ aS0 exp½bðDHÞ k2 E0 S0 ;

(4) k3 KM k2

k3 ðk1 þk2 Þ Where, k2 k1

In Eq. (4), a ¼ b¼ ¼ a is the kinetic parameter (min1); b is the kinetic parameter, dimensionless and KM is the MichaeliseMenten constant (g/L). The relationship between the hydrolysis velocity and DH is expressed in Eq. (5) that derived from Eq. (3) and Eq. (4):

dðDHÞ ¼ a exp½bðDHÞ dt

(5)

Eq. (6), which expresses the relationship between the degree of hydrolysis and the hydrolysis time, is obtained by integrating Eq. (5):

DH ¼

1 lnð1 þ abtÞ b

(6)

The kinetic parameters a and b can be obtained by linear regression to fit Eq. (6). Therefore, am (the fitted value for a), V and DH will be determined, and accordingly enzymolysis kinetic equation can be obtained under certain conditions. 2.5. Determination of antioxidant activity of the hydrolysates of rapeseed meal protein 2.5.1. DPPH radical scavenging activity The DPPH radical scavenging activity of the digested rapeseed meal (i.e. centrifuge supernatant; CS) was determined by a method described by Shimada, Fujikawa, Yahara, and Namkamura (1992) with some modifications. Two millilitres of a sample was mixed with 2 mL of DPPH (0.2 mM in 95% ethanol). After shaking vigorously, the absorbance was read immediately at 517 nm. As a control, distilled water was used instead of the sample. The scavenging activity was calculated as follows:

  A517sample DPPH,scavenging rateð%Þ ¼ 100  1  A517control

(7)

Where, A517control is the absorbance of the control and A517sample is the absorbance of the sample. 2.5.2. Reducing power The reducing power of the rapeseed meal protein hydrolysates was measured according to the method of Oyaizu (1986) with some modification. One millilitre of the hydrolysate was mixed with 1 mL of 0.2 M phosphate buffer (pH 6.6) and 1 mL of 0.01% potassium ferricyanide and then the mixture was incubated for 20 min at 50
C. After incubation, 1 mL of 10% TCA was added. An aliquot of

525

2 mL from this mixture was added to 2 mL of distilled water and 0.5 mL of 0.01% ferric chloride. After standing at room temperature for 10 min, the absorbance of the resulting solution was measured at 700 nm using a spectrophotometer (VIS-7220N, Beijing Beifen Ruili analytical instrument company). Ascorbic acid (0.02 mg/mL) was used as positive control and used for comparison. An increase in the absorbance (A700 nm) of the reaction mixture indicates an increase in the reducing power. 2.6. Molecular weight distribution The molecular weights of the digested rapeseed meal protein were measured using HPLC (High Performance Liquid Chromatography) according to Kotzamanis, Gisbert, and Gatesoupe (2007) with some modification. The digested supernatants were filtered through a 0.22 mm pore filter and analysed in a Agilent 1260 (Agilent Technology Trading Co., USA) system equipped with a TSKgel-G2000 SWXL(7.8 mm  30 cm, Tosoh) molecular exclusion column. The phosphate buffer (0.1 mol/L, pH ¼ 6.7) was used as the mobile phase at a flow rate of 0.5 mL/min. Column effluent was monitored for UV light absorption at 220 nm. System control and data processing were performed using the Chromeleon software (Chromeleon6.8; Dionex Corp., Sunnyvale, CA, USA). A molecular weight (MW) calibration curve was prepared using the following standards: cytochrome C (12500 Da), aprotinin (6500 Da), and Ltryptophan (204 Da). A relationship between the retention time and the logarithm of the MW of standards was obtained (R2 ¼ 0.999). The total surface area of the chromatogram was separated into the following fractions, according to IUPAC nomenclature; MW > 2500 Da (polypeptides), 500e2500 Da (oligopeptides) and 200e500 Da (dipeptides and tripeptides) (Zhou, Hu, et al., 2015; Zhou, Qin, et al., 2015). 2.7. Isolation of antioxidant peptide from CS 2.7.1. Separation of rapeseed meal protein hydrolysates by ultrafiltration The ultrafiltration was performed on a Millipore Pellicon system (Millipore Co. America) equipped with a membrane with the nominal molecular cut-off weight of 3 kDa. The protein hydrolysate was pumped to the membrane surface and the filtrate was collected, while the retentate was recirculated until the absorbance of the filtrate at 220 nm was close to zero. The retentate (CS-F1, molecular weight > 3000 Da), and the filtrate (CS-F2, molecular weight<3000 Da) were concentrated and then lyophilized. Reducing power and DPPH scavenging rate of CS-F1 and CS-F2 were determined in solutions of concentrations of 2 mg/mL distilled water. 2.7.2. Gel filtration chromatography of CS-F2 The fraction of CS-F2 was purified by SephadexG-25 gel filtration column (2.6 cm  60 cm), which had been equilibrated previously with distilled water. The column was eluted with distilled water at a fiow rate of 0.4 mL/min, and the elution solution was collected with the peaked samples and two sub-fractions (CS-F2-CC1 and CS-F2CC2) were obtained and then lyophilised. The reducing power and DPPH scavenging rate of CS-F2-CC1 and CS-F2-CC2 were determined in solutions of concentration of 2 mg/mL distilled water. 2.8. Free amino acids analysis The free amino acids content of the hydrolysates was obtained after hydrolysis with sulfosalicylic acid. Briefly, hydrolysate samples were mixed with equal volumes of 3 g/100 g (w/w) sulfosalicylic

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acid, and left to stand at 4
C for 1 h. Samples were then centrifuged (4
C, 15 min, 4360  g), and precipitated protein was removed. The amino acids concentrations were measured by an automatic amino acid analyser (Sykam S-433 D, Germany). 2.9. Statistical analysis All experiments were performed in triplicate and data were expressed as mean ± SD. Statistical analysis was performed using Origin 8.0, and comparisons among all groups were done with oneway ANOVA. Differences among means were considered significant at p < 0.05. 3. Results and discussion 3.1. Effect of substrate and enzyme concentrations on the degree of hydrolysis of the rapeseed meal protein As mentioned previously, this work was designed to release active peptides from rapeseed meal proteins by simulating in vitro duodenal digestion process. Thus, these proteins were subjected to the action of trypsin at 37
C and pH of 6.4. Based on preliminary study on DH, 10 g/L of trypsin is selected as the best concentration; resulting in the highest DH. This enzyme concentration is involved as a constant factor on studying the effect of different levels of substrate concentrations on DH. Similarly, 4 g/L of substrate is selected as a constant factor on studying the effect of substrate concentrations on the DH. 3.1.1. Substrate concentration At a constant level of trypsin (10 g/L), low concentrations of the substrate (<4 g/L) increased the DH, while substrate concentrations higher than 4 g/L decreased the DH (Fig. 1A). After 10 min of hydrolysis, the DH increased steadily, with significant magnitudes (p < 0.05), as the substrate concentration increased from 1 g/L to 2 g/L, and then to a maximum value at 4 g/L. This increasing order in the DH of samples with concentrations <4 g/L continued as the hydrolysis time prolonged to 40 min. Beyond 40 mine90 min, the DH of these samples did not vary significantly and thereafter the DH of the substrates with concentrations of 1 g/L and 2 g/L surpassed that of 4 g/L. Accordingly, the substrate with concentration of 2 g/L resulted in the highest DH (21.71%) at a reaction time of 120 min. As can be seen, the enzymolysis reaction rate decreased under certain conditions of the substrate and enzyme concentrations. At higher substrate concentration than the optimal, the reaction rate decreased, suggesting an inhibitory effect on enzyme activity. 3.1.2. Enzyme concentration In Fig. 1B, at the initial stage of the reaction, the DH of the substrate (constant concentration ¼ 4 g/L) increased rapidly for all trypsin enzyme concentrations (2 g/L to 12 g/L) and subsequently the rate of increase in the DH decreased as the hydrolysis time prolonged. Moreover, the DH increased with increasing the concentration of the enzyme from 2 g/L (lowest DH) to 10 g/L (highest DH). At enzyme concentration >10 g/L, the DH dropped sharply to the lower value. According to these findings, it can be suggested that the hydrolysis rate showed increasing trend at a limited range of enzyme concentration. Therefore, at a concentration higher than the critical the hydrolysis process would be inhibited, and as a consequence the DH decreased rapidly. This might be related to the decrease in the number of peptide bonds of the substrate proteins apted to be hydrolysed and then reflected in reduced enzyme activity.

3.2. Kinetic model of enzymolysis of rapeseed meal protein From the mechanism and the kinetic equation the proposed kinetic parameters for enzymatic hydrolysis of the rapeseed meal can be calculated. The experimental data in Fig. 1A and B were fitted into Eq. (6) using linear regression analysis, and the kinetic data are shown in Table 1. The influence of the initial substrate concentration (S0) and the initial enzyme (E0) on a and b parameters was checked. However, parameter b changed when S0 and E0 changed. The parameter b values lies within a very small range and therefore an average value of 0.103 (averages of S0 þ E0) was used for further analysis. Moreover, parameter a values calculated from this average value (Table 1) showed a clear dependence on S0. Whereas, the values of a increased with increasing S0, reaching its highest level (9.81 min1) at 4 g/L for S0. Thereafter, the a values decreased as S0 increased. Similarly, the a values (Average b ¼ 0.103) increased when the initial enzyme concentration increased. The average value of b was brought into Eq. (6) and yields the expression DH ¼ 9.67 ln (1 þ 0.103 a t), which applied in calculation of am values (Table 1). A plot (Fig. 2) of am values and [E0]/[S0]; with R2 ¼ 0.926; led to Eq. (8):

am ¼ 1:26½E0 =S0  þ 5:62

(8)

The hydrolysis velocity was obtained from Eqs. (5), (6) and (8), and expressed as:

V ¼ 1:26 ½E0  ð5:62E0 =1:26Þ exp ½  0:103 ðDHÞ

(9)

In Eq. (8), the critical enzyme concentration was 5.62S0/1.26. However, when the enzyme concentration above or below the critical value, the hydrolysis reaction did not occur. Since the total hydrolysis rate was negative, the substrate or the product of hydrolysis may have irreversible inhibition to the enzymatic reaction. Similarly, the critical substrate concentration was 1.26E0/5.62, and above or below this concentration the hydrolysis reaction also did not occur. The kinetic parameters b from Table 1 and am from Eq. (8) were brought into Eq. (6), and hence we obtained the kinetic equation of enzymatic hydrolysis of the rapeseed protein under simulated duodenum digestion at 37
C and pH 6.4 [Eq. (10)]:

DH ¼ 9:67 ln ½1 þ ð0:13E0 =S0 þ 0:58Þt

(10)

Eq. (10) showed that the degree of hydrolysis is related to the initial enzyme concentration, E0, initial substrate concentration, S0, and hydrolysis time, t. That is, the DH increased with increasing the E0, under a constant substrate concentration. In contrast, the DH decreased with increasing the S0, under a constant enzyme concentration. These facts agreed with the results depicted in Fig. 1A and B, indicating adequacy between the calculated values of the DH versus time and the experimental conditions. 3.3. Antioxidant activity of rapeseed protein during hydrolysis process Antioxidant activities, as determined by DPPH and iron chelating assays, of the rapeseed meal protein during hydrolysis are shown in Fig. 1CeF. 3.3.1. Effect of substrate concentration The free radical scavenging capacity of the rapeseed meal protein during trypsin enzymolysis was examined by its ability to scavenge the stable DPPH radical. The DPPH radical scavenging activity showed a concentration dependency (Fig. 1C). However, an

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527

Fig. 1. The effect of initial substrate concentration (S0) and initial trypsin concentration (E0) on the degree of hydrolysis (DH), DPPH radical scavenging rate and reducing power of the rapeseed meal protein subjected to simulated duodenum digestion. Plots of DH, DPPH radical scavenging rate and reducing power versus S0 (A, C and E) and E0 (B, D and F), respectively. A700 nm, Absorbance at 700 nm. A, C, and E: 1 g/L, 2 g/L, 4 g/L, 8 g/L, 16 g/L B, D, and F: 2 g/L, 4 g/L, 8 g/L, 10 g/L, 12 g/L.

Table 1 Kinetics parameters of rapeseed protein enzymatic hydrolysis by trypsin. [S0] (g/L)

[E0] (g/L)

[E0]/[S0]

a (min1)

b (min1)

am (min1)

1 2 4 8 16 4 4 4 4 4

10 10 10 10 10 2 4 8 10 12

10.00 5.00 2.50 1.25 0.63 0.50 1.00 2.00 2.50 3.00

5.440 7.238 9.805 8.773 8.318 6.811 7.007 7.681 9.693 9.447

0.153 0.140 0.105 0.094 0.081 0.102 0.093 0.089 0.090 0.086

17.178 13.758 8.274 6.659 6.402 6.066 6.502 7.308 8.375 10.855

S0, Initial substrate concentration; E0, Initial enzyme concentration; a, The kinetic parameter; b, The kinetic parameter, dimensionless; am, The fitted value for a.

inversely proportional relationship between the DPPH and protein concentration was apparent. The higher the substrate concentration means the lower the DPPH radical scavenging rate. Whereas, the DPPH increased significantly (p < 0.05) in the manner of 1 g/ L > 2 g/L > 4 g/L > 8 g/L > 16 g/L. For the samples with different substrate concentrations, the increasing order in the DPPH with hydrolysis time was kept constant until 30 min. At 40 min, the DPPH radical scavenging activity was at its maxima for all the substrate concentrations, and the sample of 4 g/L resulted in the highest DPPH value. On the other hand, the presence of antioxidants in the tested samples resulted in reducing the Fe3þ/ferricyanide complex to the ferrous form. As seen in Fig. 1E, the reducing power increased significantly (p < 0.05) with increasing substrate concentration, and hence a positive correlation was noticed. The substrate concentration of 16 g/L resulted in the highest reducing power values throughout the hydrolysis period. In respect to the

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16

hydrolysates obtained in this study was in accordance with some results reported in the literature (Nalinanon et al., 2011). The changes in the DPPH and the power reducing activities agreed with the changes in free amino acids described in Section 3.4.

14

3.4. Free amino acids content

12

The amino acids (AAs) contents of the rapeseed meal protein and its hydrolysate at 40 min are shown in Table 2. The profile showed 28 AA residues of proteinogenic (20 AAs for human protein) and non-proteinogenic origins, in addition to a dipeptide residue, carnosine. Native rapeseed meal protein was rich in luecine, glutamic acid, tryptophan, b-Alanine and b-Aminoisobutyric acid, which accounted for 40.58, 28.06, 20.13, 103.11 and 132.93 mg/ mg. Except for alanine, proline, serine, Threonine, tyrosine, lysine, glutamic acid and asparagine, trypsin enzymolysis of the rapeseed meal protein for 40 min, increased significantly (p < 0.05) the contents of all AAs. The essential AAs in the 40 min-hydrolysate are higher than in the original protein, which indicates better nutritional value of the hydrolysate. Several AAs, such as methionine, histidine, tryptophan, lysine and tyrosine, were reported having antioxidant properties (Chen et al., 1996; Taheri, Sabeena Farvin, & Jacobsen, 2014). As presented in Table 2, the initial total content of methionine, histidine, and tryptophan increased significantly (p < 0.05) by 6.2% after hydrolysis time of 40 min, which agreed with the results of radical scavenging activity and reducing power depicted in Fig. 1CeF. In general, the hydrolysis of rapeseed meal protein for 40 min increased significantly (p < 0.05) the initial amounts of hydrophobic amino acids (HAA), aromatic amino acids (AAA) and positively charged amino acids (PCAA). You, Zhao, Cui, Zhao, and Yang (2009) stated that histidine and tryptophan could contribute significantly to the antioxidant activity of the hydrolysates. Due to presence of the imidazole ring in their structure, histidine or histidine-containing peptides, however, have the chelating radical trapping ability (Murase, Nagao, & Terao, 1993; Je, Park, & Kim, 2005). Additionally, aromatic amino acids can easily donate protons to electron-deficient radicals, so that they exhibit antioxidant activity. Moreover, they can retain their antioxidant stability via resonance structures (Rajapakse, Mendis, Jung, Je, & Kim, 2005; Hu, Xu, & Chen, 2004).

-1

am(min )

18

10 8 6 0

2

4

6

8

10

[E0]/[S0] Fig. 2. The linear relationship between am and [E0]/[S0]. Regression equation: am ¼ 1.26E0/S0 þ 5.62, R2 ¼ 0.926.

hydrolysis time, the chelating ability towards iron in the samples with substrate concentrations of 4e16 g/L increased steadily with increasing time; from zero to 30 min. At 40 min, a sharp increase in the reducing power rate was found and thereafter the rate decreased as the hydrolysis time prolonged. For substrate concentrations <4 g/L, the hydrolysis time had no affect on the reducing power. The results of the DPPH and the reducing power in relation to the substrate concentration were in accordance with the results observed in Fig. 1A. Similar relationship between the DH of proteins and the DPPH and the reducing power was reported in the literature (Nalinanon, Benjakul, Kishimura, & Shahidi, 2011). In general, hydrolysates contain peptides or proteins, which are hydrogen donors, could react with radicals to convert them into more stable products; thereby terminating the radical chain reaction (Khantaphant & Benjakul, 2008). This study suggests that the hydrolysates obtained from the duodenal digestion of rapeseed meal protein have potential to scavenge free radicals and to chelate ions. The DH, however, is considered the important parameter that controls the physicochemical and functional properties, which depend on the nature of peptides present in the hydrolysates. 3.3.2. Effect of enzyme concentration In Fig. 1D, the DPPH radical scavenging rate of the rapeseed meal protein hydrolysate increased with increasing concentrations of the enzyme. Nevertheless, the differences in the DPPH radical scavenging rate were not significant (p > 0.05) at the enzyme concentration range of 2 g/L to 10 g/L. Moreover, the DPPH radical scavenging activity in the all samples with different enzyme concentrations increased sharply when rising the hydrolysis time to 40 min and thereafter it decreased as the hydrolysis time prolonged. The highest value of DPPH radical scavenging rate was 48.91% for the enzyme concentration of 10 g/L and hydrolysis time of 40 min. These results suggested the dependency of the radicalscavenging activity on the DH. Similar to the effect on the DPPH radical-scavenging activity, the enzyme concentration followed the same trend with respect to the iron chelating ability of the protein hydrolysates (Fig. 1F). Results indicated that ion-binding capacity of the rapeseed meal protein hydrolysates was enhanced by trypsin hydrolysis. However, the hydroxyl groups of the hydrolysates play an important role in power reducing activity (Yoshimura, Iijima, Watanabe, & Nakasawa, 1997). The finding of chelating activity on Fe2þ of protein

3.5. Molecular weight distribution of rapeseed meal protein digests The molecular weight (MW) distribution of the simulated duodenal fluid and the rapeseed meal protein hydrolysate prepared by trypsin hydrolysis for 40 min is presented in Fig. 3. A regression plot of log MW against retention times (t), with good linearization, was obtained (log MW ¼ 0.203 t þ 7.779, R2 ¼ 0.999). Compared with simulated duodenal digestion fluid, the protein hydrolysate plot revealed unique three peaks, designated as 1, 2, and 3 with retention times of 21.49, 22.41, 26.87 min, respectively. The crude peptide content of the rapeseed meal protein represented 84.97%, and the peptides molecular weights were found in the range of 200 Dae5000 Da. The major peptides identified in the rapeseed meal protein digest were of MWs of 2608 Da, 1695 Da and 211 Da, corresponding mainly to polypeptides, oligopeptides and di- and tripeptides, respectively. 3.6. Fractionation of CS by ultrafiltration Most of the bioactive peptides from rapeseed meal hydrolysates have MWs < 3 kDa; representing the highest yield (Alshai et al., 2014). The chromatographic analysis indicates presence of peptides in the MW range of 200 Dae5000 Da (Section 3.5). Accordingly, MW cut-off membrane of 3 kDa was used to fractionate CS; in

C. Zhou et al. / LWT - Food Science and Technology 68 (2016) 523e531

529

Table 2 Changes in the free amino acids content in rapeseed meal protein during in vitro simulated duodenal digestion. Class

Content (mg/mg, dw)*

Amino acids

Rapeseed meal protein Nonpolar

-S Alkaline

Acidic Derivative

Special

14.28a ± (3.13) 17.53b ± (2.00) 46.25b ± (2.90) 22.15b ± (2.80) 2.39a ± (1.17) 18.34b ± (2.88) 8.25a ± (1.93) 21.20b ± (3.77) 11.09a ± (3.38) 4.30a ± (2.00) 18.11a ± (4.10) 11.74b ± (2.67) 14.00b ± (2.30) 8.36a ± (2.21) 10.41b ± (3.30) 14.07b ± (4.44) 3.01b ± (0.83) 28.41a ± (5.28) 8.74b ± (2.62) 9.42b ± (5.45) 152.58b ± (9.36) 13.64b ± (4.12) 14.83a ± (3.74) 122.92b ± (11.00) 15.04b ± (3.33) 26.33b ± (5.15) 8.390b ± (2.11) 12.98b ± (3.16) 166.92b ± (12.59) 185.99b ± (13.87) 57.65b ± (6.16) 32.84b ± (3.20) 14.83a ± (2.17)

14.36 ± (2.10) 15.74a ± (1.36) 40.58a ± (4.12) 14.28a ± (3.00) 2.09a ± (2.10) 16.87a ± (4.32) 7.88a ± (2.12) 20.13a ± (5.10) 10.79a ± (2.12) 4.03a ± (1.14) 17.75a ± (4.64) 10.77a ± (3.18) 12.68a ± (2.47) 8.03a ± (1.50) 9.14a ± (3.17) 13.37a ± (6.04) 0.00a ± (0.00) 28.06a ± (7.13) 7.90a ± (2.10) 0.00a ± (0.00) 132.93a ± (11.41) 12.61a ± (2.90) 14.67a ± (4.12) 103.11a ± (10.42) 10.31a ± (5.13) 19.84a ± (4.46) 7.210a ± (2.10) 11.09a ± (3.40) 146.37a ± (15.80) 165.25a ± (10.20) 54.75a ± (5.54) 30.54a ± (3.90) 14.67a ± (3.50)

Alanine Valine** Leucine** Isoleucine** Proline Phenyalanine Glycine Tryptophan Serine Threonine** Tyrosine** Methione** Cysteine** Lysine** Arginine** Histidine** Aspartic acid Glutamic acid a-Aminoadipic acid a-Aminobutyric acid b-Aminoisobutyric acid g-Aminobutyric acid Asparagine b-Alanine Phosphoserine Carnosine# Ornithine Citrulline EAA HAA AAA PCAA NCAA

-OH

40 min-Rapeseed meal protein hydrolysate

a

*

Mean value (±SD) from three separate samples. Within a row, means not sharing a common letter are significantly different according to DMRT at p < 0.05. EAA: essential amino acids (**). HAA: Hydrophobic amino acids (alanine, valine, isoleucine, leucine, tyrosine, phenylalanine, tryptophan, proline, methionine and cysteine). AAA: aromatic amino acids (phenylalanine, tryptophan and tyrosine). PCAA: positively charged amino acids (arginine, histidine, lysine). NCAA: negatively charged amino acids (aspartic, asparagine, and glutamic). #b-Alanyl-L-Histidine, is a dipeptide of the amino acids b-alanine and histidine.

term of separating peptides into two fractions, with MWs < 3 kDa and >3 kDa. The two fractions obtained are designated as CS-F1 (MW > 3 kDa), and CS-F2 (MW < 3 kDa). The yields of lyophilised centrifuge pellet and the ultrafiltrated fractions; CS-F1 and CS-F2;

3.7. In vitro antioxidant activity of the isolated fractions The rapeseed protein hydrolysate after digestion for 40 min was separated by ultrafiltration into two fractions, CS-F1 and CS-F2. The CS-F1 fraction was identified as a high MW fraction of >3000 Da, while CS-F2 as a low MW fraction of <3000 Da. As shown in Table 3, the CS-F2 fraction was separated into two sub-fractions; CS-F2-CC1 (MW ¼ 1500e3000 Da) and CS-F2-CC2 (MW ¼ 1000e1500 Da).

50

2 40

Area (mAU)

were 64.17%, 12.40% and 23.43%, respectively.

1 30

Table 3 Molecular weights and antioxidant activities of peptides from rapeseed meal protein.

3 20

Fraction

MW (Da)

CS CS-F1 CS-F2 CS-F2-CC1 CS-F2-CC2

e >3000 <3000 1500 - 3000 1000 - 1500

MW 5000 Da

10

0 10

15

20

25

30

35

Retention time (min) Fig. 3. Chromatographic profile of the simulated duodenal digestion (hydrolysis time of 40 min) product from rapeseed meal protein using TSKgel-G2000 SWXL column. Molecular weights of the marked peaks: 1, 2608 Da; 2, 1695 Da; 3, 211 Da. Simulated duodenal fluid, 40 min-rapeseed meal protein hydrolysate.

Reducing power (A700)*#

DPPH radical scavenging activity (%)*

0.213a ± 0.03 0.201a ± 0.01 0.335b ± 0.02 0.343b ± 0.02 0.538c ± 0.01

37.25a ± 0.81 32.16b ± 1.26 44.63c ± 0.93 45.59c ± 0.62 70.32d ± 0.67

* Mean value (±SD) from three separate samples. Within a column, means not sharing a common letter are significantly different according to DMRT at p < 0.05. MW, Average molecular weight. d No molecular weight distribution. #A700, Absorbance at 700 nm. CS, Centrifuge supernatant of rapeseed meal digest; CS-F1, Ultrafiltration retentate of CS; CS-F2, Ultrafiltration filtrate of CS; CS-F2-CC1 and CS-F2-CC2, Purified sub-fractions of CS-F2.

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C. Zhou et al. / LWT - Food Science and Technology 68 (2016) 523e531

According to International Union of Pure and Applied Chemistry (IUPAC), oligopeptides characterized by MWs ranged between 500 and 2500 Da (Kotzamanis et al., 2007). The low MW fraction, CS-F2CC2, showed a stronger DPPH radial scavenging activity (highest value of 70.32%) than the high MWs fractions, CS-F2-CC1 and CS-F1. Moreover, CS-F2-CC2 showed a significantly (p < 0.05) higher iron chelating ability (highest value of A700 ¼ 0.538) compared with CSF2-CC1. Different chain lengths and exposure of terminal amino groups, led to variances in antioxidant activities of peptides (You et al., 2009). Wu, Chen, and Shiau (2003) reported that the peptides with a molecular weight of approximately 1400 Da showed a stronger in vitro antioxidant activity. Previous works have demonstrated that antioxidant protein compounds are small peptides, containing between 2 and 20 amino acids (Orsini Delgado ~ a-Ramos, Xiong, & Arteaga, 2004). et al., 2011; Pen 4. Conclusion In this study, the best hydrolysis conditions of the rapeseed meal protein subjected to simulated duodenum digestion were trypsin concentration of 10 g/L, substrate concentration of 4 g/L, temperature of 37
C and pH 6.4. A mathematical model of the hydrolysis kinetics of the rapeseed meal protein (Eq. (10)) based on the initial trypsin concentration, initial substrate concentration and hydrolysis time was established numerically. The hydrolysis kinetic equation was obtained to guide and optimize the hydrolysis reaction process. Thus it gives the possibility of estimation of the critical concentrations of the enzyme and substrate, the reaction rate and the degree of hydrolysis. Antioxidant AAs (methionine, histidine, tryptophan, lysine and tyrosine) contents were improved by hydrolysis. Moreover, 3 oligopeptides with antioxidant activities were determined. Acknowledgement The work was funded by National Natural Science Foundation of China (No. 31471698, 31271966, 31170672), the National High-tech Research and Development Program (2013AA102203), China Postdoctoral Science Foundation (2014T70489, 2013M541621, 1302152C), Jiangsu Government Scholarship Fund for Study Abroad, the Foundation for the Eminent Talents and Research Foundation for Advanced Talents of Priority Academic Program DevelopmentJiangsu University (12JDG074), Six talent peaks project in Jiangsu Province (2015-NY-016) and the Project Funded by the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD) PAPD-2013. References Alshai, A. M., Blanchard, C. L., Mailer, R. J., Agboola, S. O., Mawson, A. J., He, R., et al. (2014). Antioxidant properties of Australian canola meal protein hydrolysates. Food Chemistry, 146, 500e506. AOAC. (2000). Official methods of analysis of AOAC Intl. (17th ed.). Washington DC, USA: Association of the Official Analytical Chemists. Bansal, P., Hall, M., & Realff, M. J. (2009). Modeling cellulase kinetics on lignocellulosic substrates. Biotechnology advances, 27(6), 833e848. Bos, C., Airinei, G., Mariotti, F., Benamouzig, R., Berot, S., Evrard, J., et al. (2007). The poor digestibility of rapeseed protein is balanced by its very high metabolic utilization in humans. Journal of Nutrition, 137, 594e600. Byun, H.-G., Lee, J. K., & Park, H. G. (2009). Antioxidant peptides isolated from the marine rotifer Brachionus rotundiformis. Process Biochemistry, 44(8), 842e846. rquez, M. C., & Fern lisis Camacho, F., Gonz alez, P., P aez, M., Ma andez, V. (1993). Hidro ~ ola de Cienciay Tecnología de Alimentos, 33, de caseína con Alcalasa. Revista Espan 59e70. Chen, H., Muramoto, K., Yamauchi, F., & Nokihara, K. (1996). Antioxidant activity of designed peptides based on the antioxidative peptide derived from digests of a soybean peptide. Journal of Agricultural and Food Chemistry, 44, 2619e2623. s, C., Ramírez-Moreno, E., S ~ i, I. (September Díez Marqua anchez-Mata, M. C., & Gon 2011). LWT e Food Science and Technology. 44(7), 1611e1615. Emma, L., Hala, M., & Abdul, W. (2008). Gut instincts: Explorations in intestinal

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