EFFECTS OF pH, TEMPERATURE AND ENZYME TO SUBSTRATE RATIO ON THE ANTIOXIDANT ACTIVITY OF PORCINE HEMOGLOBIN HYDROLYSATE PREPARED WITH PEPSIN

jfbc_365 44..61

EFFECTS OF pH, TEMPERATURE AND ENZYME TO SUBSTRATERATIO ON THE ANTIOXIDANT ACTIVITY OF PORCINE

HEMOGLOBIN HYDROLYSATE PREPARED WITH PEPSIN

QIAN SUN1, YONGKANG LUO1,3, HUIXING SHEN2 and XIN HU1

1College of Food Science and Nutritional Engineering

2College of ScienceChina Agricultural University

Beijing 100083, China

Accepted for Publication March 4, 2009

ABSTRACT

The effects of pepsin-assisted hydrolysis conditions on the antioxidantactivity (AA) of porcine hemoglobin hydrolysate (PHH) were investigated inthis study. Effects of temperature, pH and enzyme to substrate ratio on the AAwere evaluated and compared using response surface methodology. Tempera-ture and pH were the major factors affecting the AA. Enzyme to substrate ratioinfluenced AA to a lesser extent. The model for the AA of PHH was establishedand it showed a good fit with the experimental data. The PHH (1 mg/mL)showed the strongest AA (65.43%) at 40.4C, pH 1.6, enzyme to substrateratio = 1.6% (w/w). The degree of hydrolysis (DH) and AA of pepsin-assistedhydrolyzed PHH were significant higher (P < 0.05) than chemical hydrolyzedPHH whatever the hydrolysis time. There was no correlation between DHand AA.

PRACTICAL APPLICATIONS

Blood protein is a by-product of the meat industry with high nutritionaland functional value but high pollution power. The proportion of hemoglobinin blood protein is 60–70%. Enzymatic hydrolysis of food proteins generallyresults in profound changes in the bioactivities of proteins treated. The porcinehemoglobin hydrolysate prepared with pepsin at the optimal hydrolysis con-ditions was a potent natural free radical scavenger, which would be expected

3 Corresponding author. TEL: 86-10-62737385; FAX: 86-10-62737385; EMAIL: [email protected], [email protected]

DOI: 10.1111/j.1745-4514.2010.00365.x

Journal of Food Biochemistry 35 (2011) 44–61.© 2010 Wiley Periodicals, Inc.44

to protect against oxidative damage in living systems and might be potentialanti-oxidant for application in food, cosmetic and medicine industries.

INTRODUCTION

Free radical-mediated reactive oxygen species (ROS), oxidative stressand lipid peroxidation have gained considerable attention nowadays (Athuko-rala et al. 2006; Manso et al. 2008). Major species of ROS with free radicalsare unstable and react readily with other groups or substances in living organ-isms during metabolism, resulting in cell damage and disease (Cavas andYurdakoc 2005). They are thought to accelerate aging and to play a causativerole in many degenerative diseases such as atherosclerosis, cancer, pancreatitisand inflammatory diseases (Butterfield et al. 2002). ROS also take part inantoxidation occurring in food materials because the autoxidation of lipids iskept on by a free radical mechanism (Je et al. 2007). Lipid oxidation contrib-utes to the subsequent development of unpleasant off-flavors and may alsogenerate potentially toxic end products (Thiansilakul et al. 2007). In the recentyears, interest in utilizing antioxidants from natural sources is considerablyenhanced by consumer preference for natural products. Various proteinhydrolysate have been found to exhibit good antioxidant activity (AA), such asthose in porcine myofibrillar (Saiga et al. 2003), yellowfin sole frame (Junet al. 2004), Alaska Pollack frame (Je et al. 2005), egg-yolk (Sakanaka andTachibana 2006) and yellow stripe trevally (Klompong et al. 2007).

Pork is an important protein source but porcine blood is usually dis-carded. About one million tons of porcine blood generated in slaughterhouseannually in China and the availability is less than 10%. Except some porcineblood utilized as “blood tofu” and blood powder, quite a few are discharged assewage. The lack of effective use of porcine blood caused severe environmen-tal problems because of the associated high organic pollutant and microbialloads (Fontes et al. 2004). Blood protein is nutritional and functional, and theproportion of hemoglobin in blood protein is 60–70%. Over the past severalyears, hemoglobin have been studied as novel source that have been hydro-lyzed to produce a variety of hydrolysates with biological activity of potentialmedicinal value (Nyberg et al. 1997; Lignot et al. 1999; Daoud et al. 2005;Nedjar-Arroume et al. 2006; Yu et al. 2006). Recently, hydrolysate with AAfrom porcine hemoglobin was produced using alcalase and flavourzyme(Chang et al. 2007). Nevertheless, little information assessing the effects ofenzymatic hydrolysis conditions on the AA of hemoglobin hydrolysate andlarge number of studies had not demonstrated clear evidence or rationale forselection of hydrolysis conditions on the bioactivity of hydrolysate. Adler-Nissen (1977) indicated that the variables affecting the enzymatic process

45ANTIOXIDANT ACTIVITY OF HEMOGLOBIN HYDROLYSATE

were protease specificity and concentration, temperature, pH, nature of theproteinaceous substrate and degree of hydrolysis (DH) attained. Jun et al.(2004) reported proteinases can affect the functional properties and antioxi-dative activity of the protein hydrolysate.

Optimization through response surface methodology (RSM) is a commonpractice in biotechnology for the optimization of enzymatic hydrolysis condi-tions. The objectives of this study were to determine and evaluate the AA(DPPH free radical scavenging activity) of PHH, by assessing the effects of (1)temperature, pH and enzyme to substrate ratio (using RSM); (2) DH; and (3)pepsin-assisted hydrolysis and chemical hydrolysis, to develop an innovativeprocessing strategy to convert porcine hemoglobin into antioxidanthydrolysates.

MATERIALS AND METHODS

Chemicals and Enzyme

2, 4, 6-trinitrobenzenesulphonic acid (TNBS, P2297), 2, 2-diphenyl-1-picrylhydrazyl (DPPH, D9132), were purchased from Sigma-Aldrich (St.Louis, MO). All chemicals were of analytical grade. Pepsin (from porcinestomach mucosa) used for protein hydrolysis with declared activities of1:3,000 U/g was purchased from Amresco (Solon, OH).

Preparation of Hemoglobin Sample

The fresh blood of porcine species was obtained from Shunxin agricul-tural Co. Ltd. (Shun-Yi District, Beijing). The porcine blood was collected asthe animal was bled and sodium citrate (3 g/L blood) was quickly added toprevent clotting, and kept below 4C to avoid bacterial proliferation. The bloodwas centrifuged at 9,000 ¥ g and 4C for 10 min to obtain the blood cells. Theblood cells were then added with an equal volume of water to burst, andcentrifuged at 4,000 ¥ g and 4C to remove the stroma and obtain the hemo-globin. The hemoglobin was freeze-dried and stored at 4C until use.

Preparation of PHH with Pepsin

The method developed by In et al. (2002) for the preparation of hydroly-sate was adopted with some modifications. Five grams of dried hemoglobinwere dissolved in 100 mL distilled water at room temperature. The hydrolysisexperiments were carried out in a 400-mL glass reactor under controlledconditions (pH, temperature, enzyme to substrate ratio and stirring speed).

46 Q. SUN ET AL.

During hydrolysis, pH was maintained at the desired value by addition of1 mol/L HCl. The time of hydrolysis for the RSM design was 60 min throughpreliminary trials.

Determination of DH

The cleavage of peptide bonds during hydrolysis was quantified by theTNBS method (Adler-Nissen 1979). After hydrolysis, test samples dilutedwith deionized water (0.25 mL) were mixed with 2 mL of sodium phosphatebuffer (0.2 mol/L, pH 8.2). TNBS reagent (2 mL) was then added to eachtube followed by mixing and incubation at 50C for 60 min in a coveredwater bath to exclude light. After incubation, the reaction was quenched bythe addition of 0.1 mol/L HCl (4.0 mL) to each tube. Samples were thenallowed to cool at room temperature for 30 min before the absorbancewere measured at 340 nm (model UV-2600A, UNICO, Shanghai, China).L-Leucine (0–2.0 mmol/L) was used to generate a standard curve and freeamino groups were expressed in terms of L-Leucine. DH was calculatedusing the following formula:

DH L L L L= −( ) −( )[ ] ×1 0 0 100max

where L1 is the amount of free amino groups released after hydrolysis, L0 is theamount of free amino groups in the original porcine hemoglobin, and Lmax istotal amount of free amino groups in the original porcine hemoglobin obtainedafter acid hydrolysis (6 mol/L HCl at 100C for 24 h).

Determination of AA

The AA was estimated according to the procedure reported by Shimadaet al. (1992). Test samples in 1.5 mL of water were mixed with 1.5 mL of1 mmol/L DPPH ethanol solution. This mixture was shaken and kept at 25Cfor 30 min, and then, the absorbance of the mixture was spectrophotometri-cally measured at 517 nm. The DPPH scavenging activity as a percentage iscalculated as follows:

DPPH blank control sample DPPH sample

DPPH blank

+( ) − ×100

where the DPPH blank is 1.5 mL of water/1.5 mL of 1 mmol/L DPPH ethanolsolution, the DPPH sample is 1.5 mL of sample solution/1.5 mL of 1 mmol/L


DPPH ethanol solution and the control sample is 1.5 mL of sample solution/1.5 mL of ethanol.

Experimental Design for Optimization

A three-factor, five-level central composite design (CCD) was used forthe optimization procedure with temperature(X1), pH (X2) and enzyme–substrate ratio (E/S) (w/w) (X3) as the independent variables to optimize theinfluence of these three variables (Table 1). In the following RSM design, atotal of 23 hydrolysis trials were conducted. The Ranges of these three param-eters and the central point were determined from the preliminary trials. Thedesign with three variables including nine replicates at the centre point wasused for fitting the second-order response surface. The measured variableresponses were: DH and AA. The order of the experiments has been fullyrandomized. Data were analyzed by multiple regressions through the least-square method, to fit the following second-order equation:

Y X X X Xi ii

ii ii

ij ij

ji

= + + += = ==∑ ∑ ∑∑β β β β0

1

3

1

3

2

3

1

2

where Y is the measured response variable, b0, bibii and bij, the constant, linear,quadratic and cross-product regression coefficients of the model, respectively;and where Xi and Xj represent the hydrolysis parameters.

All assays were performed in at least triplicate, and results are expressedas the mean � standard deviation of measured values. Design-Expert software(v. 7.1.3, Stat-Ease Inc., Madison Heights, MI) was used for the generation andevaluation of the statistical experimental design. Statistical significance ofdifferences between means was evaluated by analysis of variance using ageneral linear model with pairwise comparisons by Tukey’s method(P < 0.05).

TABLE 1.RANGE OF VARIABLES USED FOR RSM

Range and levels

Formulation variables -1.682 -1 0 1 1.682X1 = temperature (C) 32 34 37 40 42X2 = pH 1.3 1.6 2 2.4 2.7X3 = E/S (%, w/w) 0.7 1 1.4 1.8 2.1

E/S, enzyme/substrate.

48 Q. SUN ET AL.

RESULTS AND DISCUSSION

Optimization AA of PHH Prepared with Pepsin by CentralComposite Design

The crude protein content of the porcine hemoglobin powder preparedwas 92%, which indicated that the porcine hemoglobin is very suitable as aprotein source for high-valued functional products. When DPPH encounters aproton-donating substance such as an antioxidant, the radical would be scav-enged and the absorbance is reduced. Hence the AA of the PHH was expressedas its ability in scavenging the DPPH radical in this study.

The influence of temperature (X1), pH (X2) and E/S (X3) on the hydrolysisby pepsin was determined using CCD as mentioned in Table 1. The CCD ofthree variables in coded along with DH and AA as responses was presented inTable 2. After the analysis of variance, the values of coefficient of determina-tion (R2) for DH and AA were 63.8% and 97.2%, respectively. Figure 1 showedno significant correlation (P > 0.05) between DH and AA. The result was alsofound in the process of alcalase-hydrolyzed silver carp protein (Dong et al.2008), protease P-hydrolyzed sericin (Wu et al. 2008) and five enzyme includ-ing pepsin-hydrolyzed rice bran protein (Adebiyi et al. 2007). This suggeststhat the specific protease and cleavage sites are important for the resultingfunctionality of the hydrolysate.

A response surface quadratic model was drawn and the statistic analysisfor the linear, the quadratic and the interaction of the three variables (X1, X2

and X3) on the response values (AA) was presented in Table 3. The model Fvalue of 50.03 implies the model is significant. The high coefficient of deter-mination value (R2 = 0.9719) demonstrated that the model can explain 97.2%variation in the response. The adjusted R2 and predicted R2 values are 0.9525and 0.8496, respectively. The adjusted R2 value is particularly useful whencomparing models with different number of terms. The predicted R2 is inreasonable agreement with the adjusted R2. The “adequate precision value”is an index of the signal to noise ratio and a value >4 is an essential pre-requisite for a model to be a good fit. Also, the model has an “adequateprecision value” of 20.52 that indicates an adequate signal. This model canbe used to navigate the design space. A non-significant lack of fit F value of0.09 was also observed, which indicated that the quadratic model was validto fit the spatial influence of the variables to the response (Zulkali et al.2006).

The independent variables considered had a high significant effect(P < 0.01) except E/S. The linear terms of temperature and pH, all the inter-actions and second order terms were significant within a 99% confidenceinterval. Bhaskar et al. (2008) reported that pH had relatively higher


significant effect (P < 0.01) in enzymatic hydrolysis of visceral waste proteinsof Catla (Catla catla) for preparing protein hydrolysate using alcalase.However, in this study, as shown by the F value of the three dependentvariables, temperature played dominant roles in the process, followed by pHon the basis of the AA increase (P < 0.01).

By applying multiple regression analysis methods, the best explanatoryregression model equation for the % AA (in the presence of 1mg of PHHsolids/mL assay) (Y) of PHH as a function of temperature (X1), pH (X2) and E/S(X3) and their interactions, using the constant, linear and quadratic regressioncoefficients of main factors and linear by linear regression coefficients ofinteractions was derived and is as follows:

TABLE 2.CENTRAL COMPOSITE DESIGN USED IN RESPONSE SURFACE METHODOLOGY FOR 23

HYDROLYSIS TRIALS AND THEIR CORRESPONDING RESPONSES FOR DEGREE OFHYDROLYSIS AND ANTIOXIDANT ACTIVITY (AA)

Run no. Coded level of variables Response

X1 X2 X3 Y1 (DH, %) Y2 (AA, %)

Corner points1 -1 -1 -1 6.30 � 0.67 48.24 � 2.772 -1 -1 1 5.49 � 0.23 48.89 � 3.553 -1 1 -1 4.91 � 0.04 54.05 � 2.694 -1 1 1 4.86 � 0.17 45.45 � 1.635 1 -1 -1 5.91 � 0.77 55.99 � 1.946 1 -1 1 7.05 � 0.20 66.74 � 1.407 1 1 -1 5.08 � 0.24 52.26 � 2.818 1 1 1 5.58 � 0.42 51.47 � 1.64

Axial points9 -1.682 0 0 4.41 � 0.26 49.39 � 4.18

10 1.682 0 0 4.75 � 0.27 58.85 � 4.0011 0 -1.682 0 5.22 � 0.22 56.92 � 4.4312 0 1.682 0 2.78 � 0.33 51.68 � 3.6113 0 0 -1.682 4.50 � 0.42 52.83 � 6.1414 0 0 1.682 5.74 � 0.22 47.17 � 1.40Center point replicates15 0 0 0 5.22 � 0.41 62.72 � 2.2016 0 0 0 5.28 � 0.24 62.44 � 2.8017 0 0 0 5.27 � 0.23 61.29 � 1.8418 0 0 0 5.23 � 0.09 63.73 � 1.4319 0 0 0 5.41 � 0.14 63.87 � 2.1820 0 0 0 5.34 � 0.16 64.01 � 2.2421 0 0 0 5.29 � 0.21 64.66 � 0.7622 0 0 0 5.30 � 0.12 63.51 � 3.2323 0 0 0 5.21 � 0.26 64.87 � 4.09

50 Q. SUN ET AL.

Y X X X X X X XX X

= − + + + − + −−

692 9 28 9 177 6 42 7 2 2 1 916 2 0

1 2 3 1 2 1 3

2 3

. . . . . .. .44 19 3 28 81

22

23

2X X X− −. .

Although E/S (X3) was non significant, it was still considered in Eq. (3)because the model is hierarchical. The negative coefficients of X1

2, X22 and X3

2

indicated that the elliptical contours were modelling a maximum. The actualand the predicted AA are shown in Fig. 2. The figure proves that the predicteddata of the response from the empirical model is in good agreement with theobserved ones in the range of the operating variables. Also Fig. 2 demonstratedthat the model is adequate and there is no reason to suspect any violation of theindependence or constant variation assumption.

Effect of Temperature and pH on AA

The effect of temperature and pH was illustrated in the three-dimensional(3D) response surface curve and two-dimensional (2D) contour plot (Fig. 3),where the E/S was constant at 1.4%. As shown in Fig. 3, The 3D responsesurface curve was convex which suggesting that there were well-defined tem-perature and pH, and the 2D contour plot of temperature and pH had remark-able interaction. The maximum AA value was observed at around 36–42C and

FIG. 1. LINEAR CORRELATION BETWEEN AA AND DH, BOTH ALIGNEDAt the 95% confidence level, |r| < r0.05, indicating the absence of a statistically

significant correlation.AA, antioxidant activity; DH, degree of hydrolysis.


pH 1.4–2.0. The AA increased with the temperature increasing from 34C to39C and decreased afterwards. The AA increased with pH increased initiallyand peaked on 1.6, followed by a decline with the pH increasing from 1.6–2.4.Therefore, at around 39C, pH1.6, the porcine hemoglobin may be easier tounfold, exposing the hydrophobic or proton-donating residues buried insidethe molecular and facilitate cleavage of the protein molecular, and contributesto the AA of the hydrolysates. High temperature (42C) might cause the activityof pepsin decrease. Similarly, Ren et al. (2008) also reported that the increaseof temperature results in an increase in inhibitory activity of the grass carpsarcoplasmic protein hydrolysates prepared using papain.

Effect of Temperature and E/S on AA

The 3D response surface curve and the 2D contour plot for the interactionof temperature and E/S were represented in Fig. 4, when the pH was kept at2.0. The shape of the contour showed a significant interaction between the twovariables. The response value reached its highest level at around 39C whereasE/S at around 1.4%. The AA increased with E/S increased initially and peakedon E/S = 1.4%, followed by a slight decline with the E/S increasing from 1.4

TABLE 3.ANALYSIS OF VARIANCE TABLE FOR ANTIOXIDANT ACTIVITY OF PORCINE

HEMOGLOBIN HYDROLYSATE PREPARED WITH PEPSIN AS AFFECTED BYINDEPENDENT VARIABLES DURING OPTIMIZATION EXPERIMENTS

Factors SS df MS F value Prob > F

Model 993.98 9 110.44 50.03 <0.0001*LinearTemperature (X1) 153.16 1 153.16 69.39 <0.0001*pH (X2) 47.36 1 47.36 21.45 0.0005*E/S (X3) 4.14 1 4.14 1.87 0.1942InteractionsX1 ¥ X2 57.04 1 57.04 25.84 0.0002*X1 ¥ X3 40.15 1 40.15 18.19 0.0009*X2 ¥ X3 54.02 1 54.02 24.47 0.0003*QuadraticTemperature (X1) 157.70 1 157.70 71.44 <0.0001*pH (X2) 151.42 1 151.42 68.60 <0.0001*E/S (X3) 337.34 1 337.34 152.83 <0.0001*Statistic analysis for the modelLack of fit 18.41 5 3.68 2.86 0.0899Pure error 10.29 8 1.29R2 0.9719Adj R2 0.9525

* Significant within a 99% confidence interval.

52 Q. SUN ET AL.

FIG. 2. PLOT OF RESIDUALS VERSUS PREDICTED RESPONSE FOR ANTIOXIDANTACTIVITY DATA

FIG. 3. THREE-DIMENSIONAL RESPONSE SURFACE CURVE AND TWO-DIMENSIONALCONTOUR PLOT FOR THE EFFECT OF TEMPERATURE (X1) AND PH (X2) ON THE AA (Y)

OF PHHHydrolysis time = 60 min.

AA, antioxidant activity; PHH, porcine hemoglobin hydrolysate.


to 1.7%. Ren et al. (2008) also reported that the increase of E/S results in aincrease in AA of the grass carp sarcoplasmic protein hydrolysates preparedusing papain. The pepsin at low concentration was not enough to hydrolyze allthe hemoglobin and liberate high antioxidant peptides.

Effect of pH and E/S on AA

Figure 5 depicted the interaction of pH and E/S when the temperature wasconstant at 37C. The shape of the response surface indicated there was sig-nificant interaction between these two factors, similarly, Bhaskar et al. (2008)showed that the interaction between E/S level and pH were significant in theprocess of preparing visceral waste proteins of Catla (Catla catla) hydrolysateusing Alcalase. The AA increased with the pH increasing from 1.4 to 1.9 anddecreased afterwards. It is possible that the lower pH contributes to that thesequence with more hydrophobic or proton-donating residues in the PHH canbe easier to expose and release.

Model Confirmation for AA of PHH Prepared with Pepsin

Although the actual situation may be more complicated than that we havereported here, an attempt for the optimization has been made by RSM. Canoni-cal analysis revealed that the experimental conditions for the production of thehydrolysate with the highest AA were pH 1.6, T = 40.4C and E/S = 1.6%. The

FIG. 4. THREE-DIMENSIONAL RESPONSE SURFACE CURVE AND TWO-DIMENSIONALCONTOUR PLOT FOR THE EFFECT OF TEMPERATURE (X1) AND E/S RATIO (X3) ON THE

AA (Y) OF PHHHydrolysis time = 60 min.

AA, antioxidant activity; PHH, porcine hemoglobin hydrolysate.

54 Q. SUN ET AL.

estimated AA for these conditions was 66.13 � 1.06%. The accuracy of themodel was further tested by conducting hydrolysis experiments using thecritical values for the optimum AA. Under the optimal conditions, the experi-mental AA was 65.43 � 2.01%. This result was very closed to the predictedvalue for AA. Therefore, the mathematical model could be effectively used topredict the AA.

Comparison of DH and AA between Chemical Hydrolysis andPepsin-assisted Hydrolysis

Because of the drastic experimental conditions fixed for hydrolysisaccording to the experimental design, a chemical hydrolysis could occursimultaneously with the enzymatic hydrolysis. A new set of experiments wereperformed under the same conditions (pH 1.6, 40.4C): a pepsin-assistedhydrolysis (E/S = 1.6%) and a control without pepsin added in order to evalu-ate the DH and AA of chemical hydrolysis and enzymatic hydrolysis (Fig. 6).Figure 6(a) showed that the DH (%) obtained for both experiments increasedwith increasing hydrolysis time. The significant difference (P < 0.05) of DHwas observed between the two hydrolysates whatever the hydrolysis time.When using pepsin, the reaction indicated a high hydrolysis rate, whichshowed 7.6% at 60 min. Lebrun et al. (1998) reported that the DH of bovinehemoglobin was 11% after 24 h hydrolysis giving conditions (temperature40C, pH 4.0 and E/S 1.4%) for the digestion by pepsin. In this study, the lower

FIG. 5. THREE-DIMENSIONAL RESPONSE SURFACE CURVE AND 2D CONTOUR PLOTFOR THE EFFECT OF PH (X2) AND E/S RATIO (X3) ON THE AA (Y) OF PHH

Hydrolysis time = 60 min.AA, antioxidant activity; PHH, porcine hemoglobin hydrolysate; E/S, enzyme/substrate.


pH (1.6) may contribute to increasing the DH of porcine hemoglobin. Thecontrol without pepsin added showed a lower hydrolysis rate and it did notobtain a DH higher than 4% after 240 min of hydrolysis.

Mainly of hydrophobic amino acid residues in peptides such as Leu, Ile,Val, Phe, Tyr and Trp are responsible for AA. Pepsin mainly cleaves thepeptide bonds adjacent to the amino-terminal side of Phe, Trp and Tyr. Thephenyl groups of aromatic amino residues at peptide ends were likely toscavenging the free radical to prevent DNA damages. Aubes-Dufau et al.(1996) found that the highest concentrations of peptides composing mainly of

FIG. 6. DH (%) AND AA (%) OF PHH PREPARED USING PEPSIN (E/S = 1.6%) OR WITHOUTENZYME ADDITION

Experimental conditions: pH = 1.6, T = 40.4C., hydrolysis with no enzyme added, , pepsin-assisted hydrolysis; E/S, enzyme/substrate.

56 Q. SUN ET AL.

hydrophobic amino acid residues were reached in pepsin-derived bovinehemoglobin hydrolysates with 8–16% DH. The DH was 7.6% at 60 min in thiswork, which was very close to 8%. However, Fig. 6b showed that the AA (%)reached maximum at 60 min of pepsin-assisted hydrolysis, which sloweddown afterwards. Similarly, Megías et al. (2008) found that in hydrolysis ofsunflower protein with pepsin after 60 min, resulted maximum antioxidativeactivity. Beyond 60min, most of the antioxidant peptides were possibly beinghydrolyzed into the lower antioxidant ones. Therefore, the release of thesepeptides may not be necessarily directly a function of the DH. Thus it was alsoconcluded that the DH does not follow antioxidant response. Similar resultswere found by Kim et al. (2001) and Kim et al. (2004) in which prolongedhydrolysis of bovine blood plasma beyond 6 h with alcalase, neutrase, orpronase E and of corn gluten beyond 8 h with flavourzyme, respectively,resulted in a decrease in AA. Chemical hydrolysis shows that the maximumAA (%) at 120 min slowed down afterwards. It confirmed the occurrence ofchemical hydrolysis but it was inefficient during the hydrolysis process. Theseresults was agree with Guerard et al. (2007) who reported that the chemicalhydrolysis occured simultaneously with the enzymatic hydrolysis using alca-lase when the hydrolysis of shrimp processing discards were carried out in thedrastic conditions of pH (9.2, 9.9 and 10.4) and temperature (65 and 68.4C).Even if the AA of the control increased progressively from 0 min to 120 min,it remained lower than in pepsin-assisted hydrolysis. The significant difference(P < 0.05) of AA was observed between the two hydrolysates whatever thehydrolysis time. This difference may be caused by differences in the structureand length of the peptides in the hydrolysate.

CONCLUSIONS

Model for the AA of PHH was established by RSM and it showed a goodfit with the experimental data. The PHH (1 mg/ mL) showed the strongest AA(65.43%) under 40.4C, pH 1.6, E/S = 1.6% (w/w). There was no correlationbetween DH and AA. The analysis of variance showed that temperature andpH were the major factors affecting the AA. E/S influenced AA to a lesserextent. The DH and AA of pepsin-assisted hydrolyzed PHH were significanthigher (P < 0.05) than chemical hydrolyzed PHH whatever the hydrolysistime. Therefore, the pepsin-assisted hydrolysis was more efficient than chemi-cal hydrolysis.

This study showed that the hydrolysis of porcine hemoglobin with pepsinunder optimal hydrolysis conditions (pH, temperature and E/S) is a promisingprocedure for prepare PHH with high AA. The PHH was a potent free radicalscavenger, which would be expected to protect against oxidative damage in


living systems in relation to aging and carcinogenesisin living systems andmight be potential antioxidant for application in food, cosmetic and medicineindustries.

ACKNOWLEDGMENTS

This work was supported financially by National Science TechnologyMinister of China (award nr 2006BAD05A16) and Department of Science andTechnology of Zhejiang Province of China (award nr 2006C12082).

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EFFECTS OF pH, TEMPERATURE AND ENZYME TO SUBSTRATE RATIO ON THE ANTIOXIDANT ACTIVITY OF PORCINE HEMOGLOBIN HYDROLYSATE PREPARED WITH PEPSIN