Optimization of enzymatic hydrolysis of palm kernel cake protein (PKCP) for producing hydrolysates with antiradical capacity

Industrial Crops and Products 43 (2013) 725–731

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Optimization of enzymatic hydrolysis of palm kernel cake protein (PKCP) for producing hydrolysates with antiradical capacity Khar Ling Ng a , Mohd Khan Ayob a,∗ , Mamot Said a , Md Anuar Osman b , Amin Ismail c a b c

School of Chemical Sciences and Food Technology, Faculty of Science and Technology, Universiti Kebangsaan Malaysia, 43600 UKM-Bangi, Selangor, Malaysia Biocompatibility Laboratory, Faculty of Science and Technology, Universiti Kebangsaan Malaysia, 43600 UKM-Bangi, Selangor, Malaysia Department of Nutrition and Dietetics, Faculty of Medicine and Health Sciences, Universiti Putra Malaysia, 43400 UPM-Serdang, Selangor, Malaysia

a r t i c l e

i n f o

Article history: Received 15 June 2012 Received in revised form 2 August 2012 Accepted 6 August 2012 Keywords: Antioxidant Antiradical Hydrolysate Optimization PKCP (palm kernel cake protein) Trypsin

a b s t r a c t The enzymatic hydrolysis of palm kernel cake protein (PKCP) with trypsin to obtain PKCP hydrolysates (PKCPH) was optimized using response surface methodology (RSM). A central composite design (CCD) was used to study the influence of four independent variables, namely pH, hydrolysis temperature (◦ C), substrate concentration (w/v) and enzyme/substrate (w/w) ratio on the degree of hydrolysis (DH%). The hydrolysis was carried out using different combinations of four hydrolytic parameters at five levels for 6 h. The CCD consisted of 24 experimental points and six replicates of the central points. The data were analyzed using Design-Expert Software. The results showed that all of the variables evaluated significantly influenced the DH% in a second polynomial model, and different combinations of parameters were generated to obtain three different levels of DH (30%, 40% and 50%), namely PKCPH 30, PKCPH 40 and PKCPH 50. The PKCPH with different DH% showed significantly different antiradical properties (p < 0.05). The PKCPH 50 preparation had the lowest EC50 value for DPPH radical scavenging capacity (0.14 mg/ml). In the ABTS• + radical scavenging capacity and PCL-ACW (photo chemiluminescence-antiradical capacity of water soluble substances) assays, PKCPH 50 showed the highest Trolox equivalent antioxidant capacity value (326.67 ± 5.77 ␮mol TEAC/g) and ascorbic acid equivalent value (11.43 ± 0.03 ␮g AAE/mg) of the preparations tested. Moreover, the protein hydrolysates also exhibited a notable reducing effect in a dose-dependent manner. Optimum conditions for enzymatic hydrolysis of PKCP were established in this study to produce an antiradical agent. © 2012 Elsevier B.V. All rights reserved.

1. Introduction Due to the rapid growth of the palm oil industry in Malaysia, palm kernel cake (PKC) has become one of the abundant byproducts produced. Malaysia is the world’s second largest palm oil-producing country (after Indonesia), and it has been reported that approximately 1.4 million tons of PKC are produced by

Abbreviations: AAE, ascorbic acid equivalents; ABTS, 2,2 -azino-bis(3ethylbenzthiazoline-6S); CCD, central composite design; DH, degree of hydrolysis; DPPH, 2,2-diphenyl-1-picrylhydrazl; EC50 , half-maximal effective concentration; FCR, Folin–Ciocalteu reagent; GAE, gallic acid equivalents; HCl, hydrochloric acid; NaOH, sodium hydroxide; PCL-ACW, photo chemiluminescence-antiradical capacity of water soluble substances; PKC, palm kernel cake; PKCP, palm kernel cake protein; PKCPH, palm kernel cake protein hydrolysates; PKCPH 30, palm kernel cake protein hydrolysates with 30% of DH; PKCPH 40, palm kernel cake protein hydrolysates with 40% of DH; PKCPH 50, palm kernel cake protein hydrolysates with 50% of DH; RSM, response surface methodology; TEAC, Trolox® equivalent antioxidant capacity; TPC, total phenolic content; Trolox® , ((±)-6-hydroxy-2,5,7,8-tetramethylchromane-2carboxylic acid); RP-HPLC, reverse phase-high performance liquid chromatography. ∗ Corresponding author. Tel.: +60 3 89215963; fax: +60 3 89213232. E-mail address: [email protected] (M.K. Ayob). 0926-6690/$ – see front matter © 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.indcrop.2012.08.017

Malaysian palm oil millers annually (Chin, 2008). Several byproducts are produced in the oil milling process. PKC is one of the most abundant byproducts of expeller processing. The solids remaining after the expeller processing are called PKC or palm kernel expeller (PKE). Due to the sustainable growth of palm oil in the world market, the amount of PKC produced is increasing annually. Therefore, the full utilization of PKC as a source of valuable ingredients, such as protein, represents a significant economic and social benefit. Several attempts have been made to bio-convert palm kernel waste (such as black liquor) into useful resources, especially antioxidant sources (Bhat et al., 2009). To date, PKC has been used as a bio-resource for the production of cellulose, hemicellulose and lignin (Yan Yan et al., 2009) or as feed for cattle, swine, ruminants and aquacultured fish (Zahari and Alimon, 2003). PKC may be considered as a medium grade protein feed because it contains 14.6–16.0% crude protein (Chin, 2008). Due to its protein content and low cost, PKC should be a valuable raw material for the extraction of high-value plant protein or peptides. Fatah Yah (2008) and Arifin et al. (2009) reported limited success in attempts to extract protein from PKC. However, Ng and Mohd Khan (2012) described an alkaline extract of PKC with a protein

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content of 68.50%. This encouraging result prompted further studies of the extraction and quality of PKCP. If this extracted protein can be transformed or modified into a bioactive plant protein, the uses of PKC may be expanded to include the production of highended products. Hannu and Anne (2006) suggested that the protein might be modified through hydrolysis to provide mixed bioactive peptides. Moreover, as reported by Aluko and Monu (2003) and by Korhonen and Pihlanton (2006), enzymatic hydrolysis can produce peptides with biological activity and reduce the allergenic potential of some intact plant proteins (Tsumura et al., 1999). According to Wu et al. (2008), smaller molecular weight proteins or peptides exhibit better biological effects because they are generally well absorbed through the intestinal mucous membrane. Thus, the aims of the present study were to: (1) optimize hydrolysis condition of PKCP with trypsin using RSM; (2) calculate the conditions for different degree of hydrolysis using fitted RSM model; and (3) determine the effect of the degree of hydrolysis on its antiradical capacity and reducing power. 2. Materials and methods 2.1. Materials Palm (Elaeis guineensis) kernel cake (PKC) was kindly supplied by FELDA Kernel Products Sdn. Bhd., Malaysia. The PKC was collected and stored at 4 ◦ C until further treatment. 2.2. Chemicals All chemicals used in this study were of analytical grade, including trypsin (Sigma Aldrich, St. Louis, Missouri, USA), 2,2-diphenyl-1-picrylhydrazl (DPPH) (Aldrich, D9132), 2,2 azino-bis(3-ethylbenzthiazoline-6S) (ABTS) (Sigma–Aldrich, A1888), gallic acid monohydrate (Sigma–Aldrich, 398225, 98%), Trolox ((±)-6-hydroxy-2,5,7,8-tetramethylchromane-2-carboxylic acid) (Aldrich, 238133) and Folin–Ciocalteu’s phenol reagent (Sigma–Aldrich, F9252). An analytical kit was used for the photochemiluminescence-antiradical capacity of water soluble substances (PCL-ACW) (Analytik Jena AG) assay. 2.3. Preparation of palm kernel cake protein (PKCP) PKCP was prepared as previously described (Ng and Mohd Khan, 2012). Briefly, pulverized PKC was initially dispersed into an alkaline solution at a ratio of 1:10 pulverized PKC:1.0 M NaOH. The PKCP was extracted by shaking the suspension in a water-bath shaker (902/OVT 27-2176, Hotech Instruments Corp., Taiwan) at 50 ◦ C and 150 rpm for 30 min. This step was followed by centrifugation at room temperature at 4000 rpm for 20 min to obtain the supernatant. Next, the supernatant was precipitated using 3.0 M HCl at pH 4.3–4.5. The precipitate obtained was defatted by resuspension in absolute ethanol and centrifuged (10,000 rpm) for 20 min. Finally, the pellet (PKCP) was collected and lyophilized using a laboratory freeze dryer (Alpha 1-4 LD Plus, Model HO27080, Christ, Germany) at −40 ◦ C and with vacuum pressure, 0.010 mbar for 24 h. 2.4. Experimental design The parameters for PKCP hydrolysis were optimized using RSM. To optimize the DH% of PKCP, four independent variables at five levels were employed in a central composite experimental design (CCD). The variables were pH (A: 5.5–11.5), hydrolytic temperature (B: 20–60 ◦ C), substrate (PKCP) concentration (C: 0.5–2.5, w/v) and enzyme/substrate ratio (D: 0.5–6.5, w/w). The range of variables of this study was chosen according to Ng and Mohd Khan (2012)

study. From the study, the highest DH (47.95%) using trypsin was obtained when 0.1 g PKCP in 10 ml of PBS was hydrolyzed with 2% of trypsin (w/w) at pH 8.5; 40 ◦ C for 6 h. Thus, the combinations of variables, as shown in Table 1 are in the desirable range to achieve optimum DH. The experimental design consisted of 24 experimental points and six replicates of the central points. The DH% value was selected as the experimental response Y (Table 2). This experimental design was also used to estimate the interactions of the response functions and factors. The data obtained from the CCD (Table 2) were analyzed by multiple regressions through the least squares method to fit the second order polynomial model shown in Eq. (1) below: Y = ˇ0 +

3  i=1

ˇi Xi +

3  i=1

ˇii2 Xi2 +

3 

ˇij Xi Xj

(1)

j=i+1

where Y is the predicted response (degree of hydrolysis, DH%), ˇ0 is the constant coefficient, ˇi is the linear coefficient, ˇii is the quadratic coefficient and ˇij is the cross-product coefficient of the model. Xi and Xj represent the independent variables (Obero et al., 2011). Design-Expert Software (Version 6.0.10) was used to analyze and calculate the predicted responses and experimental design for the desired DH%. 2.5. Preparation of PKCP hydrolysates (PKCPH) The PKCPH was prepared according to the parameters calculated for the desired DH (30%, 40% and 50%) using fitted model equation obtained by RSM as shown in Table 3. The freeze-dried PKCP was re-suspended in phosphate buffer at the desired pH and then subjected to trypsin hydrolysis in a water bath shaker. For enzymatic hydrolysis, all factors were controlled according to the experimental design (Table 3) for 6 h. The hydrolysis reaction was terminated by immediately boiling the product in a water bath for 10 min to inactivate the trypsin. The mixtures were then centrifuged at 2600 × g for 20 min (Kubota, Model 2420, Japan), and the supernatant was collected and lyophilized as PKCPH for further analysis. 2.6. Determination of the degree of hydrolysis (DH%) The degree of hydrolysis (DH%) of PKCPH was determined based on the method described by Sathivel et al. (2003). A 5 ml aliquot of PKCPH was added to 5 ml of 20% trichloroacetic acid (TCA) to prepare 10% TCA-soluble and TCA-insoluble mixtures. Each mixture was centrifuged, and the 10% TCA-soluble nitrogen of the supernatant was measured using a Kjeltec 2200 Analyzer unit (Foss, Denmark) based on the standard Kjeldahl method (AOAC, 2000). A conversion factor of 6.25 was used to calculate the total protein content. The DH% was calculated using Eq. (2). DH% =

 10% TCA soluble nitrogen in the sample  total nitrogen in sample

× 100

(2)

2.7. Antiradical assay 2.7.1. Determination of EC50 The 2,2-diphenyl-1-picryhydrazyl (DPPH) radical scavenging capacity of PKCPH was determined as described by Yu (2008) with minor modifications. Briefly, 100 ␮l of DPPH solution (0.15 mM in ethanol) was reacted with 100 ␮l of PKCPH dissolved in deionized water. The reaction was allowed to proceed at room temperature for 20 min, and the absorbance was read at 517 nm against a blank (DPPH solution in ethanol). The inhibition of the DPPH radical was

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Table 1 Actual and coded analytical parameters at five levels in the RSM constructed to optimize the hydrolysis of PKCP by trypsin. Analytical parameters

Unit

Code

pH Temperature Substrate (PKCP) concentration (w/v) Enzyme (trypsin)/PKCP (w/w)

pH ◦ C g/100 ml g/100 g

A B C D

Levels −2

−1

0

+1

+2

5.5 20 0.5 0.5

7.0 30 1.0 2.0

8.5 40 1.5 3.5

10.0 50 2.0 5.0

11.5 60 2.5 6.5

Table 2 Central composite experimental design (CCD) of the independent variables (analytical factors/parameters) and the corresponding response value Y (DH%) for the hydrolysis of PKCP with trypsin. Run 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30

Factor 1 (A) pH

Factor 2 (B) Temperature (◦ C)

Factor 3 (C) Substrate concentration (g/100 ml, w/v)

Factor 4 (D) Enzyme/substrate ratio (g/100 g, w/w)

Response, Y DH (%)

7.00 10.00 7.00 10.00 7.00 10.00 7.00 10.00 7.00 10.00 7.00 10.00 7.00 10.00 7.00 10.00 5.50 11.50 8.50 8.50 8.50 8.50 8.50 8.50 8.50 8.50 8.50 8.50 8.50 8.50

30 30 50 50 30 30 50 50 30 30 50 50 30 30 50 50 40 40 20 60 40 40 40 40 40 40 40 40 40 40

1.00 1.00 1.00 1.00 2.00 2.00 2.00 2.00 1.00 1.00 1.00 1.00 2.00 2.00 2.00 2.00 1.50 1.50 1.50 1.50 0.50 2.50 1.50 1.50 1.50 1.50 1.50 1.50 1.50 1.50

2.00 2.00 2.00 2.00 2.00 2.00 2.00 2.00 5.00 5.00 5.00 5.00 5.00 5.00 5.00 5.00 3.50 3.50 3.50 3.50 3.50 3.50 0.50 6.50 3.50 3.50 3.50 3.50 3.50 3.50

32.19 42.29 41.75 45.52 26.67 34.07 35.83 40.67 38.79 49.43 45.39 49.16 29.90 38.65 41.89 46.53 24.51 22.45 33.94 33.85 57.91 36.74 29.63 48.93 46.69 44.89 46.69 47.05 41.30 42.02

Enzyme: trypsin; substrate: PKCP.

calculated according to Eq. (3). DPPH radical quenched capacity (%)



= 1−

 Abs. of sample  Abs. of control

× 100

(3)

where Abs is the absorbance. To obtain the EC50 (half-maximal effective concentration) value, PKCPH was dissolved in deionized water at different concentrations (0.05–0.9 mg/ml) to construct a linear curve, and the EC50 of the sample was determined based on this curve. 2.7.2. ABTS radical scavenging capacity assay (TEAC) The radical scavenging capacity of the PKCPH was also determined by the ABTS (2,2 -azino-bis(3-ethylbenzthiazoline-6S)) cationic radical assay as described by Yu (2008). The preparation

of the ABTS•+ working solution is critical for the accuracy of this assay. First, 5 mM ABTS (in water) was prepared, and 5 g of manganese dioxide was added to oxidize ABTS to ABTS•+ . The ABTS•+ working solution was then filtered through Whatman No. 1 filter paper. The filtered solution was diluted with 0.5 M phosphate buffer (pH 7.4) until the absorbance at 734 nm was exactly 0.70. To generate a standard curve, 0.5 mM of Trolox® stock solution was prepared, and Trolox® working standards of 1–120 ␮M were prepared by diluting the stock solution with phosphate buffer. To carry out the assay, 1 ml of ABTS•+ working solution was mixed with 80 ␮l Trolox® working standard/sample extract and allowed to stand for 30 s. Then, the mixture was vortexed for 1 min, and the absorbance at 734 nm was read immediately. The results were expressed in units of Trolox® equivalent antioxidant capacity (␮mol TEAC/g).

Table 3 Parameters for PKCPH with the desired DH% obtained by using fitted RSM model equation. DH%

pH

Temperature (◦ C)

Substrate concentration (w/v)

Enzyme/substrate concentration (w/w)

Desirability

30 40 50

7.06 7.18 8.36

30.92 40.76 46.24

1.67 1.44 1.09

2.62 3.53 3.84

1.00 1.00 1.00

Enzyme: trypsin; substrate: PKCP.

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Table 4 Analysis of variance for the RSM quadratic model. Source

Sum of squares

Mean square

F value

Prob > F

Model

1629.99

8

203.75

11.78

<0.0001**

A B C D A2 B2 C2 D2

103.29 124.08 357.67 262.35 597.95 117.00 45.80 14.18

1 1 1 1 1 1 1 1

103.29 124.08 357.67 262.35 597.95 117.00 45.80 14.18

5.97 7.17 20.68 15.17 34.57 6.76 2.65 0.82

0.0234* 0.0141* 0.0002** 0.0008** <0.0001** 0.0167* 0.1186 0.3755

Residual Lack of fit Pure error Total

363.20 331.01 32.19 1993.19

21 16 5 29

17.30 20.69 6.44

3.21

R2 2 Radj

0.8178 0.7484

* **

DF

0.1010

Significant at 5%. Significant at 1%.

2.7.3. Photochemiluminescence-antiradical capacity of water soluble substances (PCL-ACW) assay The PCL-ACW assay was carried out as recommended by the kit manufacturer (Analytik Jena AG Protocol ACW-Kit, 2007) to measure the antioxidant activity at an appropriate concentration of PKCPH (1 mg in 1000 ␮l of deionized water; dilution 1:1000). A standard curve was constructed using ascorbic acid with concentrations ranging between 0.02 and 1.0 nmol. The measurement time for the PCL-ACW assay was 250 s. The results were expressed as ascorbic acid equivalents (␮g AAE/mg). 2.7.4. Determination of reducing power The reducing power of PKCPH was determined based on the method described by Moure et al. (2006) with minor modifications. A series of PKCPH solutions with concentrations ranging between 0.01 and 0.10 mg/ml were prepared, and 1 ml of each concentration was mixed with 2.5 ml of 0.2 M phosphate buffer (pH 6.6) and 2.5 ml of 1% potassium ferricyanide. The mixtures were then incubated in a water bath at 50 ◦ C for 20 min. Then, 2.5 ml of TCA was added to the mixture, and the mixture was centrifuged for 10 min at 3000 rpm. The supernatants were collected, and 2.5 ml of each supernatant was diluted in 2.5 ml of distilled water and then mixed with 0.5 ml of 0.1% ferric chloride. The absorbance of the resulting solution was measured at 700 nm (UV 2450, Shidmazu, Japan). An increase in the absorbance of the mixture indicates an increase in the reducing power as measured by the reduction of ferric ions. Ascorbic acid was used as a positive control. 2.7.5. Determination of the total phenolic content of PKCPH The total phenolic content (TPC) of PKCPH was determined using the Folin–Ciocalteu reagent (FCR) assay as described by Singleton et al. (1999). A 40 ␮l aliquot of PKCPH (1 mg/ml) was dissolved in 1.16 ml of deionized water, followed by the addition of 200 ␮l of FCR and thorough mixing. The mixture was incubated at room temperature for at least 1 min but no longer than 8 min. Then, 600 ␮l of 20% sodium carbonate solution was added, and the reaction

mixture was incubated in the dark at room temperature for 2 h. The absorbance of the resulting solution was read at 765 nm. The phenolic content was expressed in mg gallic acid equivalents/g (mg GAE/g). 2.8. Amino acid profile determination The amino acid profile of PKCPH was determined using reverse phase-high performance liquid chromatography (RP-HPLC) (Galdón et al., 2010). The lyophilized PKCPH was digested with 6 M HCl at 110 ◦ C for 24 h. The amino acid profile was reported as g amino acid/100 g protein. 2.9. Statistical analysis All the antiradical assay results obtained were subjected to ANOVA and Duncan’s multiple ranges tests to determine whether the differences between samples were significant (p < 0.05) using the SAS package (SAS Institute Inc.). The ANOVA and 3D surface plots of the experimental model were generated using DesignExpert Software Version 6.0.10. 3. Results and discussion The extraction of protein from PKC usually involves the use of acid, alkaline and saline solutions (Eromosele et al., 2008). However, Arifin et al. (2009) indicated that an alkaline solvent is the most efficient solution for the extraction of protein from PKC. Thus, an alkaline extraction method was adopted to extract protein from PKC. The effects of pH (A), hydrolysis temperature (B), substrate (PKCP) concentration (C) and enzyme/substrate ratio (D) on the DH% of enzymatic hydrolysis were studied. The data from 24 experimental runs and six central point runs (pH 8.5, 40 ◦ C, 1.5 g PKCP/100 ml and 3.5 g trypsin/100 g PKCP) using CCD are presented in Table 2. The central point values ranged between 41.30% and 47.05%. The maximum DH% (57.91%) was found with

Table 5 Antiradical activity of palm kernel cake protein hydrolysates (PKCPH). PKCPH PKCPH 30 PKCPH 40 PKCPH 50

EC50 a b

0.26 0.34a 0.14c

% ABTS (␮mol TEAC/g)

PCL-ACW (␮g AAE/mg)

Total phenolic content (mg GAE/g)

70.00 ± 0.00 106.67 ± 5.77b 326.67 ± 5.77a

10.92 ± 0.01 10.38 ± 0.02c 11.43 ± 0.03a

50.36 ± 0.75a 45.94 ± 0.58c 47.72 ± 0.84b

c

Value within a column followed by the different letters (a–c) are significantly different (p < 0.05). a EC50 is measured using DPPH radical scavenging capacity.

b

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729

Fig. 1. Surface response plots showing the combined effects of (a) pH and concentration of substrate (PKCP), (b) pH and temperature, (c) pH and concentration of enzyme (trypsin)/PKCP, (d) concentration of PKC and trypsin/PKCP, (e) concentration of PKCP and temperature and (f) temperature and concentration of trypsin/PKCP.

the combination of pH 8.5, 40 ◦ C, 0.5 g of PKCP/100 ml and 3.5 g trypsin/100 g PKCP. However, the substrate concentration in the optimal DH% was rather low, which might lead to a low yield of PKC protein hydrolysate. Furthermore, the value of desirability to achieve 57.91% of DH was relatively low (0.860). Therefore, 50% of DH was set as an optimum DH in this enzymatic hydrolysis procedure. The combination of analytical parameters is shown in Table 3. The apparent value of desirability of this experimental design was 1.00.

p-value becomes smaller. From Eq. (4), the variable with the greatest effect on the linear term was the enzyme/substrate ratio (D), followed by the quadratic term, which was the concentration of PKCP (C2 ). There was no significant interaction among the four variables. Therefore, the interaction factors were eliminated from the model equation. DH% = 44.77 + 2.07A + 2.27B − 3.86C + 3.31D − 4.67A2 − 2.07B2 + 1.29C 2 − 0.72D2

(4)

3.1. Model fitting Table 4 presents the data fitting of the RSM quadratic model obtained using Design-Expert Software. The fitted model in coded variables is given in Eq. (4). As shown in Table 4, the significance of each coefficient was determined using the F-test and p-value. Atkinson and Doney (1992) stated that the corresponding variables are more significant when the F-value becomes greater and the

The model has shown a good fit with the experimental data, with an R2 value of 0.8178. The quality of the model is shown in Table 4, where the probability was greater than the F of the quadratic model (significant at p < 0.05) and the lack of fit was not significant relative to pure error. In other words, the observed mathematical model (Eq. (4)) significantly represented the actual relationship among

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Fig. 2. Radical (DPPH• + ) scavenging capacity of PKCPH 30, 40 and 50. The EC50 of each preparation is indicated.

hydrolysis parameters and was useful to monitor and optimize the hydrolysis of PKCP using trypsin. 3.2. Influence of hydrolytic parameters on the degree of hydrolysis (DH%) of PKCPH As shown in Table 2, 30 different combinations of experimental conditions could be generated by the compilation of RSM to optimize the degree of hydrolysis. Obvious changes in DH% were observed with changes in the experimental parameters. A statistical analysis was performed on the experimental DH% results to obtain a regression model, which is shown in Eq. (4). The three-dimensional response surface plots shown in Fig. 1 illustrate the influence of the independent variables on the DH%. The effects of pH (A) and temperature (B) on the DH% when the concentration of substrate; and trypsin enzyme to substrate ratio are held at an optimum level are illustrated in Fig. 1(b). As shown in Fig. 1(a)–(c), the optimal pH of the extraction solution was approximately 8.5. The concentration of substrate and the enzyme to substrate ratio (Fig. 1(d)) were not optimized in the provided range; thus, the response surface plot does not show the optimized level. Meanwhile, the response surface plot depicted in Fig. 1(e) is slightly saddle shaped, suggesting that the DH% begins to be optimized at an elevated temperature. The quadratic effect of pH and temperature was manifested, and it is obvious that they are the most influential parameters in obtaining the desired DH%. The different combinations of analytical parameters shown in Table 4 were applied, and the achieved DH% was near the predicted value in all cases. These results strongly validate the mathematical model that was used. 3.3. Influence of DH% on the antiradical capacity of PKCPH To validate the influence of DH% on the antiradical activity of PKCPH, three assays were carried out to determine the antiradical capacity, reducing power and total phenolic contents of different PKCPH preparations. The DPPH•+ radical scavenging capacity assay was chosen to determine the EC50 of PKCPH. This is the most commonly used antioxidant assay, and it is a reliable method for investigating the antioxidant potential of different natural components in vitro, especially plant protein hydrolysates (Vastag et al., 2010). As shown in Fig. 2, PKCPH was able to scavenge DPPH•+ radicals, and the PKCPH 50 showed the lowest EC50 value (0.14 mg/ml, p < 0.05) (Table 5) among the hydrolysate preparations tested. Based on the ABTS•+ radical scavenging capacity and PCL-ACW assays, PKCPH 50 showed the highest TEAC (326.67 ± 5.77 ␮mol TEAC/g) and AAE (11.43 ± 0.03 ␮g AAE/mg). Based on its mode of action, antioxidative protein hydrolysate is considered either a chain-breaking antioxidant or a preventative antioxidant (Moure et al., 2006). Generally, preventative antioxidants inhibit or reduce the rate of the free radical chain reaction, while chain-breaking

Fig. 3. The reducing power of (a) ascorbic acid (positive control) and (b) PKCPH 30, 40 and 50.

antioxidants interfere with chain propagation to scavenge free radicals (Somogyi et al., 2007). As described above, PKCPH with high DH% may be considered as a potential antioxidant with the ability to inhibit free radicals in its environment. To further support this conclusion, the reducing power of PKCPH was measured because reducing power has been reported to be associated with antioxidant activity (Juntachote and Berghofer, 2005). As shown in Fig. 3(b), the reducing power of PKCPH increased, and each preparation displayed a positive linear regression curve similar to that of the control (ascorbic acid) (shown in Fig. 3(a)). The assay demonstrated that at a concentration of 0.1 mg/ml, the reducing power of all PKCPH was equally high (shown in Fig. 3(b)). However, a 10-fold concentration of PKCPH was required to produce a reducing power equivalent to that of ascorbic acid, which is well known as a potent antioxidant. Liu et al. (2010) reported that the DH% is related to antioxidant activity for most protein hydrolysates. Higher DH% values may be related to the production of smaller or shorter protein chains and thus greater antioxidant activity. In addition, as shown in Table 5, a significant total phenolic content (TPC) of PKCPH (45.94–50.36 mg GAE/g) was observed. Phenolic compounds have been associated directly and indirectly with antiradical activity (Balogh et al., 2010). The phenolic compounds detected in the PKCPH may be associated with bound phenolic acids expressed in various plant components, typically the cell walls (Pericin et al., 2009). During the initial extraction steps (the alkaline treatment), the bound phenolic compounds may be released from the cell matrix. However, further study is needed to fully evaluate the impact of phenolic compounds on PKCPH. 3.4. Influence of PKCPH amino acid profile on antiradical capacity Table 6 presents the amino acid profiles of PKCPH with DH values of 30%, 40% and 50%. In general, the amino acid profile of protein hydrolysates is considered important in contribution of antioxidant/antiradical capacity. The result in Table 6 indicated that free amino acids are released differently at different level of DH. At the highest DH (50%), the amount of antioxidative amino acids (Tyr, Met, His and Lys) was different significantly (p < 0.05) compared to

K.L. Ng et al. / Industrial Crops and Products 43 (2013) 725–731 Table 6 Amino acid profiles of palm kernel cake protein hydrolysates (PKCPH). Amino acid a

Asp Ser Glub Gly His NH3 Arg Thr Ala Pro Tyr Val Met Lys Ile Leu Phe

Total amino acid c

Total hydrophobic amino acid Antioxidant amino acidsd a b c d

References

PKCPH 30

PKCPH 40

PKCPH 50

7.95 3.46 14.58 3.99 2.11 0.27 9.82 2.26 4.10 3.24 2.91 5.57 2.49 2.41 4.12 6.79 4.53

7.03 3.06 12.59 3.62 1.81 0.26 8.72 2.01 3.67 2.95 2.61 4.83 2.12 2.19 3.59 5.98 3.97

9.91 4.31 17.74 5.11 2.55 0.37 12.28 2.84 5.18 4.16 3.68 6.80 2.99 3.09 5.05 8.42 5.59

79.59

71.01

99.06

b

34.83

9.92b

c

47.46a

8.73c

12.31a

30.73

731

Aspartic acid + asparagine. Glutamic acid + glutamine. Gly, Ala, Val, Leu, Pro, Met, Phe and Ile. Tyr, Met, His and Lys.

PKCPH 30 and PKCPH 40. His, Tyr, Met, Cys and Lys are all examples of amino acids that have been reported to show antioxidant activity. In particular, histidine exhibited strong radical scavenging activity due to the decomposition of its imidazole ring (Wang et al., 2007). Amino acids with aromatic residues can easily donate protons to electron-deficient radicals and participate directly in radical scavenging mechanisms; one example is the hydrogen-donating, lipid-peroxyl radical trapping and metal ion-chelating imidazole group (Sarmadi and Ismail, 2010). Moreover, during enzymatic hydrolysis, the hydrophobic group may be decreased or increased. It is depends on the exposure of intact protein to enzyme. According to Sarmadi and Ismail (2010), peptides with different compositions, structures and hydrophobicities will have different antioxidant properties. The total amount of hydrophobic amino acids of PKCPH 50 was 47.46 g/99.06 g protein was different significantly (p < 0.05). Thus the PKCPH 50 exhibited greater antiradical capacity. However, the positioning of the amino acids in the peptide sequence also will modulate their antiradical effects. Therefore, DH of protein hydrolysates has to be monitored to obtain antiradical capacity with the correct composition and positioning of amino acids. 4. Conclusions In this study, PKCP was extracted by alkaline treatment and then subjected to enzymatic hydrolysis. PKCPH with the desired DH% was successfully produced by trypsin-assisted procedures based on optimized extraction parameters established from an experimental RSM. Based on results of several antiradical assays, the DH% strongly influenced the antiradical activity of PKCPH. The PKCPH 50 exhibited the most promising antioxidant properties. Acknowledgments This study was sponsored by an MOA Science Fund Grant (0501-02-SF1006). The authors kindly acknowledge MARDI for the use of their laboratory facilities and FELDA Kernel Products Sdn. Bhd., Malaysia for supplying PKC.

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