Food Research International 44 (2011) 290–296

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Food Research International

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Antioxidant and angiotensin converting enzyme (ACE) inhibitory activities of cocoa(Theobroma cacao L.) autolysates

Bahareh Sarmadi a, Amin Ismail a,b,⁎, Muhajir Hamid c

a Department of Nutrition and Dietetics, Faculty of Medicine and Health Sciences, Universiti Putra Malaysia, 43400 Serdang, Selangor, Malaysiab Laboratory of Analysis and Authentication, Halal Products Research Institute, Universiti Putra Malaysia, 43400 Serdang, Selangor, Malaysiac Department of Microbiology, Faculty of Biotechnology and Biomolecular Sciences, Universiti Putra Malaysia, 43400 UPM Serdang, Selangor, Malaysia

⁎ Corresponding author. Department of Nutrition and DHealth Sciences, Universiti Putra Malaysia, 43400 Serdang,89472435; fax: +60 3 89472459.

E-mail address: amin@medic.upm.edu.my (A. Ismail

0963-9969/$ – see front matter © 2010 Elsevier Ltd. Aldoi:10.1016/j.foodres.2010.10.017

a b s t r a c t

a r t i c l e i n f o

Article history:Received 14 July 2010Accepted 8 October 2010

Keywords:Cocoa (Theobroma cacao L.)AutolysateAntioxidant activityACE inhibition

The present study investigated antioxidant and angiotensin converting enzyme (ACE) inhibitory activities ofcocoa autolysates. After removal of cocoa fat, alkaloids and polyphenols, the remaining proteinous powderwas autolyzed at pH 3.5 and 5.2. At similar concentrations, autolysates produced at pH 3.5 indicated thehighest reducing power and ACE inhibition activity. However, those generated at pH 5.2 showed the highestantioxidant activity based on β-carotene bleaching assay. The results displayed a dose-dependent trend.Based on amino acids composition, slight differences were detected between autolysates, and as it was found,they were rich in hydrophobic amino acids. Qualitative and quantitative tests were applied to assure that theresults from the assays were not due to the polyphenols of cocoa autolysates. Based on the results nopolyphenols could be detected from cocoa autolysates. It can be indicated that among other useful substancesof cocoa, its peptides and amino acids could contribute to its health-promoting properties. Furthermore, thesebioactive substances can be exploited into functional foods or used as a source of nutraceuticals.

ietetics, Faculty of Medicine andSelangor, Malaysia. Tel.: +60 3

).

l rights reserved.

© 2010 Elsevier Ltd. All rights reserved.

1. Introduction

Free radicals are generated through normal reactions within thebody during respiration in aerobic organisms. They can exert diversefunctions like signaling roles and providing defense against infections(Hancock, Desikan, & Neill, 2001). However, any excessive amount ofreactive radicals can cause cellular damage which, in turn, initiatesseveral diseases like atherosclerosis, arthritis, diabetes and cancer(Halliwell, 1994). Therefore, in certain circumstances that endoge-nous defense system fails to protect the body against reactive radicalson its own, external supply of antioxidant is needed.

Several peptides from protein ingredients have been found topossess antioxidant capacity. Bioactive peptides are regarded asspecific protein fragments which are inactive in the parent proteinsequence. They can exert several physiological functions after they arereleased by enzymatic hydrolysis (Korhonen & Pihlanto, 2003;Sarmadi & Amin, 2010). The amino acid composition and sequencesaffect the bioactive peptide activity (Chen, Muramoto, Yamauchi,Fujimoto, & Nokihara, 1998). Moreover, several peptides have beenfound to possess multifunctional properties such as antioxidant andACE inhibitory capacity (Pihlanto, Akkanen, & Korhonen, 2008).

Antihypertensive peptides derived from food proteins are the mostcomprehensively studied bioactive peptides. Angiotensin-convertingenzyme (ACE) is a dipeptidyl carboxypeptidase (EC 3.4.15.1) and iswidely distributed in mammalian tissues (Li, Le, Shi, & Shrestha, 2004).It converts angiotensin I to angiotensin II, a vasoconstrictor. It alsoinactivates bradykinin which is a vasodilator peptide. Therefore, thisenzyme plays an important role in the regulation of blood pressurethrough these two mechanisms (Unger, 2002). ACE inhibitors havebeen shown to be effective antihypertensive agents.

Further, in the conditions of hypertension, angiotensin II amplifiesthe oxidative stress as it intervenes many of its cellular functionsthrough stimulating the formation of intracellular reactive radicalspecies (ROS) (Schiffrin & Touyz, 2004). Therefore, in addition toblood pressure control, ACE inhibitors have been shown to intensifythe antioxidant defense system in animals and humans by inhibitionof angiotensin II formation (de Cavanagh, Inserra, Ferder, & Fraga,2000).

Antioxidant and ACE inhibitory activities have been reported forpeptides and hydrolysates from plant and animal sources includingpeanut protein hydrolysates (Jamdar et al., 2010), sardine by-productprotein hydrolysates (Bougatef et al., 2008), whey protein hydro-lysates (Peng, Xiong, & Kong, 2009), peptide from the algae proteinwaste (Sheih, Fang, & Wu, 2009) and peptide from soybean protein(Kuba, Tana, Tawata, & Yasuda, 2005) and potato hydrolysates(Pihlanto et al., 2008).

Theobroma cacao refers to a plant which yields cocoa fruits and itsunprocessed beans are referred to as cacao. Cocoa polyphenols have

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long been reported to have health benefits. As an example,supplementation of cocoa extracts to obese-diabetic rats decreasedplasma oxidative stress biomarker and increased superoxide dis-mutase activity, attributable to its polyphenols and methylxanthines(Abbe, Amin, Chong, Muhajir, & Hasbullah, 2008). Cacao beancontains four proteins including: albumin, globulin, prolamin andglutelin (Zak & Keeney, 1976a,b). According to previous studies cocoaprotein could be cleaved to hydrophilic and hydrophobic peptides andamino acids through autolysis under different pHs (Amin, Jinap,Jamilah, Harikrisna, & Biehl, 2002; Voigt et al., 1994). Among othercomponents of cocoa, these valuable compounds stand unexplored.This assumption leads us to hypothesize that besides cocoa poly-phenols, its peptides and amino acids can contribute to its healtheffects. To our knowledge, this is the first study reported onantioxidant and ACE inhibitory activities of cocoa autolysate. Itsfindings can provide fundamental information for further work in thisarea. Besides, the bioactive substances of cocoa autolysates can beused as ingredients of functional foods and dietary supplements aswell as a pharmaceuticals and nutraceuticals.

2. Materials and methods

2.1. Materials

β-carotene, linoleic acid, Tween 20, butylated hydroxytoluene(BHT), thioglycollic acid, ascorbic acid, 2,4,6-tripyridyl-s-triazine(TPTZ), ferric chloride (FeCl3·6H2O), ferrous sulphate (FeSO4·7H2O),ethyl acetate, sodium phosphate dibasic, sodium phosphate mono-basic, sodium acetate trihydrate, and angiotensin converting enzyme(ACE) from rabbit lung and hipouril histidine leucine (HHL) werepurchased from Sigma Chemical Co. (St. Louis, MO, USA).

2.2. Preparation of cocoa acetone dry powder (AcDP)

Fresh cocoa fruit of PBC 140 and UIT 1 clones were obtained fromMalaysian Cocoa Board, Jengka, Pahang, Malaysia. Unfermented andfresh beans were taken from the pods immediately after arrival andshock-frozen in liquid nitrogen. After removal of testae and radiculae,they were freeze-dried. The freeze-dried cotyledons were crushedwith mortar and pestle. They were defatted using a Soxhlet apparatuswith petroleum ether (bp 40–60 °C) for 8 h, twice. Purine alkaloidswere partially extracted with chloroform for 8 h in a Soxhletapparatus.

In order to remove the polyphenols a method described by Aminet al. (2002) was followed. After complete extraction of polyphenols,residual water was removed by dehydration with 100% cold acetone.The resulting polyphenol-free whitish powder (acetone dry powder,AcDP)was stored at−20 °C in anairtight glass container before use. Theefficiency of polyphenol extraction was checked by qualitative andquantitative tests. For qualitative test, a small portion of AcDP washeated with 2 ml of 5 MHC1 for a few seconds (appearance of red colorindicates the presence of residual polyphenols). Quantitatively, RP-HPLC analysis for detection of polyphenols in cocoa autolysates wascarried out following a method described by He and Xia (2007). Thephenolic compounds in cocoa autolysate were identified by comparingtheir retention time with an authentic standard. Detection of phenoliccompounds was carried out in a spectrum from λ=230–540 nm.

2.3. Preparation of cocoa autolysate

Cocoa cotyledons contain endogenous endopeptidase (asparticendoprotease, optimum pH 3.5) and exopeptidase (carboxypeptidase,optimum pH 5.8) both of which can be activated and cleave the proteinprecursor under optimum pH and temperature in vitro, in laboratorycondition, and during fermentation process (Voigt et al., 1994).Therefore, to obtain cocoa autolysates, a method was applied following

amethoddescribedbyVoigt et al. (1994) inwhich cocoaAcDP (1 g)wassuspended both in acetic acid pH 3.5 (100 ml) and in sodium acetatebuffer 10 mM, pH 5.2 (100 ml). The suspensions were incubated at50 °C, in a shaking water bath for 16 h for autolysis. Autolysis wasstopped by adding 70%methanol (v/v) to the suspensions. Subsequent-ly, the suspensions were stirred at room temperature for 1 h andcentrifuged at 20,000×g for 30 min. The supernatants were collected,and the methanol was removed under pressure at 40 °C by means of arotary evaporator. Finally, the aqueous solutions were freeze-dried andstored at−20 °C before use. In this article, autolysate of PBC 140 at pH3.5, PBC 140 at pH 5.2, UIT1 at pH 3.5 and UIT1 at pH 5.2will be referredto as P3, P5, U3 and U5, respectively.

2.4. Determination of protein content

The protein contents of autolysates were measured by themethods of Kjeldahl (AOAC, 2000).

2.5. Determination of amino acid composition

The Pico Tag method, with modification, was used for determiningthe amino acid composition of the autolysates (Khan, Kuo, Kebede, &Lambein, 1994). The dry sample (weight equivalent to 4% protein)was added with 6 N HCl (15 ml) and placed in the oven at 110 °C for24 h. Internal standard (α-aminobutyric acid, 10 ml) was added to themixture. Mobile phase A (0.1 M ammonium acetate, pH 6.5) andmobile phase B (0.1 M ammonium acetate containing acetonitrile andmethanol, 44:46:10, v/v, pH 6.5) were utilized. Sample (100 μl) wasinjected on a C18 reversed-phase column (250 mm×4 mm, i.d. andparticle size 5 μm, Merck KGaA, Darmstadt, Germany) and aminoacids were detected at 254 nm.

2.6. Determination of antioxidant capacity

2.6.1. Ferric reducing/antioxidant power (FRAP)FRAPassaywasdeterminedbasedon the reductionof Fe3+-TPTZ to a

blue colored Fe2+ TPTZ (Benzie & Strain, 1996). The FRAP reagent wasprepared by mixing 300 mM acetate buffer (pH 3.6), 10 mM TPTZ and20mM FeCl3·6H2O in a ratio of 10:1:1. Forty microlitres of the samplewas added to the test tubes containing 3 ml of freshly prepared FRAPreagent. Absorbancewasmeasured at 593 nmusing spectrophotometer(ShimadzuUV1601, Japan). In the FRAP assay, the antioxidant potentialof samplewas determined from a standard curve using FeSO4·7H2O at aconcentration range between 62.5 and 1000 μM.

2.6.2. β-carotene–linoleate bleaching assayβ-carotene bleaching assay was conducted using a method

developed by Velioglu, Mazza, Gao, and Oomah (1998) with slightmodification. β-carotene (0.2 mg in 1 ml chloroform), linoleic acid(0.02 ml) and Tween 20 (0.2 ml) were transferred into a roundbottomed flask. Chloroformwas removed using a rotary evaporator at50 °C. Following evaporation, 50 ml of distilled water was added tothe mixture and then shaken vigorously to form an emulsion. Next,2 ml of the emulsionwere pipetted into test tubes containing 0.2 ml ofcocoa autolysates or standard (BHT) or control (methanol 70%) andimmediately placed in a water bath at 50 °C. The absorbance was readat 20 min intervals for 100 min at 470 nm. Antioxidant activity (AA)was expressed as percent of inhibition relative to the control, usingthe following formula:

AA% = 1− A0−Atð Þ= A0c−Atcð Þ½ � × 100

where A0 is absorbance of sample/Std at t=0, At is absorbance ofsample/Std at t=100, A0c is absorbance of control at t=0 and Atc isabsorbance of control at t=100 min.

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2.7. Angiotensin converting enzyme (ACE) inhibition assay

The ability of the cocoa autolysates to inhibit the activity of ACE invitro was measured according to the spectrophotometric method ofCushman and Cheung (1971). Briefly, 100 μl of cocoa autolysate and100 μl of hippuryl-L-histidyl-L-leucine (HHL, 12.5 mM in 0.05 Msodium borate buffer containing NaCl 0.4 M, pH 8.3) were incubatedat 37 °C for 5 min. Then, 150 μl of ACE was added and the mixture wasincubated for an hour. The enzymatic reaction was stopped by adding250 μl of 0.5 N HCl. The hippuric acid formed by the action of the ACEon HHL was extracted from the acidified solution into 1.5 ml ethylacetate by vortexing for 15 s. The mixture was centrifuged at 3290×gfor 10 min at 4 °C, and a 0.5 ml aliquot of each ethyl acetate layer wastransferred to clean tubes and evaporated by heating at 120 °C for20 min on a heating plate. The hippuric acid was redissolved in 3 ml of1 M NaCl, and the amount formed was determined by its absorbanceat 228 nm. Assay mixture without cocoa autolysate was referred ascontrol. The IC50 value was defined as the concentration ofhydrolysate that inhibits 50% of the ACE activity. For calculating %ACE inhibition, the following formula was used:

%ACE Inhibition = Acontrol−Asample = Acontrol

� �× 100

2.8. Statistics

Experiments were performed 2 or 3 times and every singleexperiment was replicated 3 times. The results were expressed asmean±SD. Significant differences among means of samples wereevaluated by one-way analysis of variance (ANOVA) at Pb0.05, usingSPSS 15. Pearson's correlation was applied to find any significant(Pb0.05) correlation between means.

3. Results and discussion

3.1. Cocoa autolysate

Two clones of cocoa, namely PBC 140 and UIT 1, were used for thisstudy. These clones are commonly grown by Malaysian farmers.

Table 1Amino acids composition of cocoa autolysate based on their chemical properties.

Amino acid (%) PBC-140

pH 3.5 pH

Ser 6.7±0.01 6.Lys 7.7±0.1 8.Gly 5.4±0.03 4.His 2.2±0.009 2.Arg 5.2±0.01 7.Thr 5.9±0.03 4.Aspa1 10.4±0.06 11Glub1 15.9±0.03 20Val2,4 5.8±0.03 4.Ile2,4 3.7±0.12 3.Leu2,4 6.9±0.04 5.Ala4 5.8±0.08 4.Pro4 4.9±0.05 4.Phe3,4 7.3±0.03 6.Met4,5 1.4±0.02 1.Cys5 NDc NTyr3 4.4±0.01 4.1Acid/basic amino acid 26.3±0.1 312Branched chain amino acid 16.5±0.2 143Aromatic amino acid 11.8±0.02 114Hydrophobic amino acid 36±0.1 305S-containing amino acid 1.42±0.02 1.

Data correspond to the mean±SD of two experiments.aAspartic acid+Asparagine.bGlutamic acid+glutamine.cND: not detected.

However, the main objective of this study was to determinecontribution of cocoa peptide and amino acids in its antioxidant andACE inhibitory capacity; thus, selection of these clones was not basedon a specific rationale.

In order to avoid any intervening effects of cocoa polyphenols in theresults, its polyphenol contents were removed. The efficiency ofpolyphenol removal was checked using qualitative and quantitativetests. When the retention time of the autolysate was compared with anauthentic standard, nophenolic compounds couldbedetected. The resultsfrom both tests revealed that cocoa AcDP was free from polyphenols.

AcDP of both cocoa clones were autolyzed under optimum pHs andfour types of cocoa autolysates were produced. Our previous studydisplayed that during autolysis of AcDP at pH 3.5 mainly hydrophobicfractions were generated (Amin et al., 2002). In addition, it has beenreported that mostly hydrophilic fractions and hydrophobic free aminoacids were generated when AcDP was autolyzed at pH 5.2 (Voigt et al.,1994).

At pH 3.5, the aspartic endoprotease of cocoa cotyledons tends tohydrolyze substrates at their hydrophobic amino acid residues,resulting in formation of oligopeptides with hydrophobic aminoacid residues at C-terminal. Lower proportions of free amino acidswere generated compared to pH 5.2 (Amin et al., 2002; Voigt et al.,1994). Moreover, during fermentation-like incubation of cocoa AcDPat pH 5.2, carboxypeptidase of cocoa cotyledon prefers to cleavehydrophobic residues at their carboxyterminal ends (Voigt et al.,1994). Therefore, mostly hydrophilic peptides and hydrophobicamino acids were produced. Leucine, alanine, phenylalanine, andvaline were the predominant free amino acids accumulated duringautolysis of AcDP at pH 5.2 (Voigt et al., 1994). Hydrolysis of foodproteins results in production of a broad variety of peptides and freeamino acids. During hydrolysis, enzyme specificity affects the size andamino acid sequence of peptides as well as level and composition offree amino acids which subsequently could influence the antioxidant(Wu, Chen, & Shiau, 2003) and ACE inhibitory activity of hydrolysates(Vercruysse, Smagghe, Beckers, & Van Camp, 2009). Therefore,autolysis of cocoa under different pHs yielded a mixture of peptidesand amino acids with different characteristics. The yields ofcocoa autolysates were as follows: 9%±0.2 for P3, 10% for P5±0.3,

UIT-1

5.2 pH 3.5 pH 5.2

1±0.03 6.3±0.01 5.9±0.066±0.2 7.3±0.01 7.7±0.27±0.06 5.3±0.008 5.2±0.07±0.02 2.06±0.005 1.8±0.0204±0.005 5.6±0.21 6.4±0.0062±0.009 4.2±0.17 4.7±0.01.4±0.19 12.2±0.27 9.5±0.1.1±0.003 20.8±0.15 20.1±0.059±0.09 5.08±0.03 5.7±0.041±0.02 2.9±0.06 3.3±0.019±0.04 5.9±0.06 6.6±0.00021±0.1 4.6±0.17 4.5±0.082±0.003 4.4±0.03 4.7±0.023±0.05 6.9±0.004 7.3±0.066±0.04 1.3±0.001 1.5±0.004D 0.4±0.01 0.3±0.026±0.02 4.6±0.005 4.8±0.009.4±0.2 33±0.4 29.7±0.06±0.07 14±0.16 15.5±0.05±0.08 11.5±0.01 12.2±0.07.3±0.06 31.3±0.05 33.5±0.00361±0.04 1.7±0.02 1.77±0.3

Fig. 1. Antioxidant potential of cocoa autolysates using FRAP assay. Values are mean±SD(n=3).

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12%±0.12 for U3 and 17%±0.14 for U5±0.37. Protein content ofcocoa autolysate was high and included: 68±0.13 mg/100 mg ofautolysate for P3, 76±2 mg/100 mg of autolysate for P5, 65±3 mg/100 mg of autolysate for U3 and 54±0.7 mg/100 mg of autolysate forU5. A variation was observed among protein content of cocoaautolysates. Among them, autolysates produced from PBC 140 at pH5.2 contained the highest amount of protein. The high protein contentof autolysates can be attributed to the removal of fat from cocoapowder and elimination of insoluble undigested substances afterautolysis.

3.2. Amino acid composition

Analysis of the amino acid composition revealed that therewas notmuch difference between cocoa autolysates. In all autolysateshydrophobic amino acids outweighed the other amino acids(Table 1). Many food-derived peptides with hydrophobic aminoacids have been observed to exhibit antioxidant (Chen et al., 1998;Qian, Jung, & Kim, 2008) and ACE inhibitory (Byun & Kim, 2002)activities. Therefore, antioxidant and ACE inhibitory activities of cocoaautolysates may be related to the high amount of their hydrophobicamino acids.

Other amino acids in cocoa autolysates include acidic and basicamino acids which exist in high amounts followed by aromatic aminoacids.While carboxyl and amino groups in the side chains act as chelatorofmetal ions (Saiga, Tanabe, &Nishimura, 2003) and ashydrogendonor(Qian et al., 2008), ACE substrate or its competitive inhibitors with C-terminal dicarboxylic amino acids, like Glu, have little affinity for theenzyme (Cheung, Wang, Ondetti, Sabo, & Cushman, 1980). Aromaticresidues are effective radical scavengers since they can donate protonsto electron deficient radicals to make them stable and meanwhile, theycan keep the stability of molecule through resonance structure(Rajapakse, Mendis, Jung, Je, & Kim, 2005). It has also been reportedthat the peptide with comparatively low IC50 value contains a highcontent of branched and aromatic amino acids such as Ile, Val, Phe, andTyr in its peptide sequence (He et al., 2007).

Thus, it is very likely that aromatic amino acids along withhydrophobic amino acids contribute to antioxidant and ACE inhibitionactivity of cocoa autolysates. In addition, Glu and Asp may contributein antioxidant activity of cocoa autolysates, yet they do not seem toplay important roles in terms of their ACE inhibitory activity.

However, not only amino acid composition of peptides but theirstructure and amino acid sequence are also responsible for theiractivity (Chen et al., 1998). Our previous results have displayed thatthe oligopeptide pattern of cocoa autolysates from PBC 140 deviatedsignificantly from other clones with regard to the peak at 39–45 minretention time (Amin et al., 2002), indicating the discrepancybetween peptides in the autolysates. Thus, although the amino acidcomposition of cocoa from both clones was similar, the discrepancyamong fractions of the two clones could have affected autolysateproperties. Other factors can also affect antioxidant and ACE inhibitionactivities of cocoa autolysates that will be discussed in the followingsections.

3.3. Antioxidant capacity

The exact mechanism underlying the antioxidant activity ofpeptides has not fully been understood, yet various studies havedisplayed that they are inhibitors of lipid peroxidation (Qian et al.,2008; Wu et al., 2003), scavengers of free radicals (Qian et al., 2008;Rajapakse et al., 2005) and chelators of transition metal ions(Rajapakse et al., 2005; Saiga et al., 2003). In addition, it has beenreported that antioxidant peptides keep cells safe from damage byROS through the induction of genes (Erdmann, Grosser, Schipporeit, &Schroder, 2006). Tyr, Trp, Met, Lys, Cys, and His are examples of aminoacids that have antioxidant activity (Wang & De Mejia, 2005). In

addition to the presence of proper amino acids, their correctpositioning in peptide sequence plays an important role in antioxi-dant activity of peptides (Rajapakse et al., 2005).

Several methods have been developed to assess antioxidantcapacity. However, none of them can be used as an officialstandardized method. Hence, in the research, evaluation of antioxi-dant capacity is usually carried out by various methods of measure-ment in different oxidation conditions. The present work sought toinvestigate the antioxidant capacity of cocoa autolysates based on twodifferent reaction mechanisms, i.e., β-carotene–linoleate bleachingand FRAP assay.

3.3.1. Ferric reducing/antioxidant power (FRAP)At low pH (optimum pH 3.6) a ferric salt, Fe(III)(TPTZ)2Cl3 (TPTZ)

(as an oxidant), is reduced by antioxidants to its intense blue coloredform Fe2+-TPTZ with maximum absorbance at 593 nm (Benzie &Strain, 1996).

The reducing power results revealed that cocoa autolysates haveabilities to donate electron which is involved in the antioxidantactivity. The reducing power of cocoa autolysates augmented as theirconcentrations (1.25, 2.5, 5, 10 mg/ml) increased (Fig. 1). The effect ofconcentration on enhanced reducing power has been observed forchickpea protein hydrolysates (Li, Jiang, Zhang, Mu, & Liu, 2008).Antioxidant activity of U3 was significantly (Pb0.05) higher thanother autolysate following the order of P3NP5NU5 in similarconcentrations.

Whey protein hydrolysates (45 mg/ml) after 5 h hydrolysis hadthe FRAP value of about 1150 μM (Peng et al., 2009). In our study,FRAP value of U3 at 10 mg/ml was 723±5 μM which is morecomparable to FRAP results of plasma protein hydrolysates withFRAP value of 713.1±8.8 μM (Liu, Kong, Xiong, & Xia, 2010). Highreducing power ability of hydrolysates is possibly due to amplifiedlevels of hydrogen ions (protons and electrons) following hydrolysis(Liu et al., 2010). This could have been a possible explanation for thehigher reducing power of U3 in our study.

Reducing power has been reported for fraction IV from chickpeahydrolysates. This fraction had the highest amount of total hydro-phobic amino acid and high hydrophobicity (Li et al., 2008). Thereducing power of hemp protein hydrolysates was reported to beunrelated to the hydrophobic amino acids yet positively andsignificantly (Pb0.10) correlated with their surface hydrophobicity(Wang, Tang, Chen, & Yang, 2009). Antioxidant activity of proteinhydrolysates is affected by amount and composition of free aminoacid and peptides (Wu et al., 2003), the type of protease, degree ofhydrolysis (Liu et al., 2010) as well as size (Wu et al., 2003), structure,amino acid composition and amino acid sequences of peptides inhydrolysates (Chen et al., 1998).

In this study, the differences between cocoa autolysates regardingtheir amino acid composition are too little to be considered affecting

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factors. Saiga et al. (2003) have pointed out that a disparity in activityof two similar hydrolysates (regarding their amino acid compositions)can be related to the structure and length of the peptides in thehydrolysates. Correspondingly, various factors can be important inreducing power of cocoa autolysates. These can includemolecular sizeof peptides, their amino acid sequences, structural properties anddegree of hydrolysis, any of which can lead to differences in FRAPvalues of autolysates. A significant and high (r2=0.827) correlationwas observed between FRAP results and protein content ofautolysates.

3.3.2. β-carotene bleaching inhibition activityβ-carotene–linoleic bleaching inhibition assay simulates mem-

brane lipid oxidation and can be considered a good model formembrane based lipid peroxidation. In this oil–water emulsion-basedsystem, linoleic acid acts as a free radical generator that producesperoxyl radicals under thermally induced oxidation. The producedfree radicals attack the β-carotene chromophore resulting in bleach-ing effect, which can be inhibited by a free-radical scavenger.

Results showed that cocoa autolysates demonstrated an ability toinhibit the discoloration of β-carotene by scavenging linoleate-derived free radicals dose-dependently (Fig. 2). Concentrationdependency trend has also demonstrated for peanut protein hydro-lysates (Jamdar et al., 2010) and egg protein hydrolysates (Sakanaka &Tachibana, 2006). The most potent antioxidant autolysate was U5followed by P5, U3 and P3, respectively. Antioxidant activity ofautolysates that was produced under pH 5.2 was significantly(Pb0.05) higher than those produced at pH 3.5. Antioxidant activityof peptides or protein in the free radical-mediated lipid peroxidationsystem is influenced by molecular size, chemical properties andelectron transferring ability of amino acid residues in the sequence(Qian et al., 2008). Therefore, the discrepancy in antioxidant activityof cocoa autolysate may be related to differences in the sequences ofpeptides and/or their molecular size. Further, the difference can bedue to synergistic effect of Tween 20 with existing amino acids orpeptides. It has been stated that the antioxidant activity of aminoacids can be increased and their pro-oxidant activity can be lessenedor reverted to antioxidant activity by addition of an emulsifier orphosphate. Emulsifiers can reduce the particle size of peptides andenlarge the contact surface of the phases (Marcuse, 1962). Antioxi-dant activity of all extracts was relatively low, ranging from 28% to54% at 10 mg/ml. It can be due to the high amount of copper in cocoa(Joo, Kies, & Schnepf, 1995). Copper can bind to amino acids orpeptides and catalyze oxidation of linoleic acid strongly (Marcuse,1962). In addition, it is possible that cocoa autolysates could exerthigher antioxidant activity at higher concentrations. To examine

Fig. 2. Antioxidant activity of cocoa autolysate using β-carotene bleaching assay. Valuesare mean±SD (n=3).

whether increased doses of cocoa autolysates can enhance theirantioxidant activity, two more concentrations were added (20 and40 mg/ml). Antioxidant activity of P5, U3 and U5 rose significantly(Pb0.05). Under these concentrations all autolysates were signifi-cantly lower than BHT (a synthetic antioxidant). However, at highconcentrations autolysates did not differ significantly except P3whose antioxidant activity was significantly lower than all. Thisautolysate (P3) did not demonstrate a pronounced antioxidantactivity, which did not rise significantly even at higher concentrations.This could be due to the presence of peptides with weak antioxidantactivities, hindering effect of other compounds in autolysates or pro-oxidant effects of some amino acids (e.g., Cys, His) and peptides. Pro-oxidative effect was also observed for 10 mg/ml of seal proteinhydrolysates (Shahidi & Amarowicz, 1996). A significant and high(r2=0.762) correlation was observed between protein content andantioxidant activity of autolysates.

The decrease in the absorbance of β-carotene in the presence of10 mg/ml of cocoa autolysates and 70% methanol (as a control) wasrecorded as a function of time (Fig. 3). Cocoa autolysate suppressedbleaching of β-carotene compared with control in emulsion systemindicating their antioxidant activity. The absorbance of U5 droppedslowly during incubation with slight increase in rate after 60 min,while control dropped at a faster rate after 20 min.

3.4. Angiotensin converting enzyme (ACE) inhibition activity

ACE inhibitory activity of cocoa autolysate was reported as percentof ACE inhibition by samples. Based on the results, it was revealed thatall autolysates have the ability to inhibit ACE. Autolysates produced atpH 3.5 exerted more ACE inhibitory activity than autolysatesproduced at pH 5.2 (Pb0.05) (Fig. 4). At 2.5 mg/ml of U5 the ACE %inhibition was 44±1.6 which is comparable to 2 mg/ml of sardinehydrolysates produced by alcalase with ACE %inhibition of 43±2(Bougatef et al., 2008). The analysis of the data based on IC50(between 3 and 9.7 mg/ml) indicated a lower value than that ofoyster, scallop, codfish skin, and herring skin (more than 10 mg/ml)(He et al., 2007) but higher value than glycinin hydrolysates(0.148 mg/ml) (Kuba et al., 2005). The IC50 value for captopril (asynthetic ACE inhibitor) was 2±0.57 μM. The ACE inhibitory activityresults were in parallel with FRAP values where P3 and U3 displayedhigher reducing power. It seems that autolysates at pH 3.5 havemultifunctional roles. However, it remains to be elucidated whetherthe same peptide can have both functions in autolysates. Other studies

Fig. 3. Antioxidant activity of cocoa autolysates (10 mg/ml) as measured by changes inabsorbance values at 470 nm. Values are mean±SD (n=3).

Fig. 4. ACE inhibitory activity of cocoa autolysates. Values are mean±SD (n=3).

295B. Sarmadi et al. / Food Research International 44 (2011) 290–296

have also reported multifunctional roles for hydrolysates (Jamdar etal., 2010; Vercruysse et al., 2009).

It has been reported that ACE indicates a preference to substratesor competitive inhibitors containing branched chain amino acidresidues at the N-terminal position and hydrophobic amino acidresidues (aromatic or branched-side chains) at the C-terminalposition (Byun & Kim, 2002; Cheung et al., 1980). He et al. (2007)have suggested that high amounts of branched and aromatic aminoacids (Pro, Glu, Val, Phe and Tyr) in marine protein can be responsiblefor relatively low IC50 values of its hydrolysates. Similarly, highcontents of hydrophobic and aromatic amino acids in cocoaautolysates and high amounts of hydrophobic fractions in P3 and U3autolysates can contribute to their higher ACE inhibitory activity.

The hydrophilic amino acid residues in the peptide sequencedisrupt the access of the peptide to the active site of ACE affecting theinhibitory activity (Sheih et al., 2009). Therefore, the lower activity ofautolysates generated at pH 5.2 can be explained by their high contentof hydrophilic fractions. However, Li et al. (2004) have proposed thatthe hydrophilic–hydrophobic partitioning in the sequence can be acrucial factor in the inhibitory activity. Therefore, for a betterexplanation of autolysates activities, further work is required toidentify amino acid sequence of effective peptides. In addition,carboxypeptidase cleaves one or more amino acids from C-terminalpositions which can result in production of peptides with low or noACE inhibition activity. This can provide another probable reason forlower ACE inhibitory activity of P5 and U5. A significant andmoderatecorrelation was found between protein content and ACE inhibitoryactivity (r2=0.649).

It is also possible that other compounds like alkaloids oroligosaccharides contribute to ACE inhibitory activity of cocoaautolysates. However, the ACE inhibitory activity of autolysates variedusing different enzymes, suggesting that ACE inhibitor could be theproduct of cocoa protein hydrolysis.

4. Conclusion

Adverse effects of synthetic compounds lead to a growing interestfor extraction of natural compounds from food sources. In addition,there is an increasing tendency among public to choose them sincethese natural compounds are of no or little side effects. Accordingly,cocoa autolysates can be utilized as natural sources of peptides andamino acids with antioxidative and antihypertensive properties. Anadditional merit of bioactive peptides to other natural compounds isthat they have nutritional and functional advantages.

The present study indicated that cocoa autolysates exhibitantioxidant and ACE inhibitory activities, depending on their

concentrations. Cocoa autolysates produced at pH 3.5 containedmore reducing agents and ACE inhibitors than those formed under pH5.2. In addition, cocoa autolysates produced more antioxidants underpH 5.2 using the linoleic acid oxidation system. This indicates theeffect of various enzymes on formation of peptides with differentpotential activities. It can be suggested that besides other compoundsof cocoa, its peptides and amino acid compositions could contribute toits antioxidant and ACE inhibitory activities. In addition, a combinedantioxidant and ACE inhibition capacity makes cocoa autolysates arich source of bioactive compounds that improve cardiovascularhealth or control related diseases. Further research is needed toidentify effective peptide from cocoa autolysate and to investigate itsaforementioned activities in vivo.

References

Abbe, M. J., Amin, I., Chong, P. P., Muhajir, H., & Hasbullah, S. K. (2008). Effects of cocoaextract on glucometabolism, oxidative stress, and antioxidant enzymes in obese-diabetic (ob-db) rats. Journal of Agriculure and Food Chemistry, 56, 7877−7884.

Amin, I., Jinap, S., Jamilah, B., Harikrisna, K., & Biehl, B. (2002). Oligopeptide patternsproduced from Theobroma cacao L of various genetic origins. Journal of the Science ofFood and Agriculture, 82, 733−737.

AOAC (2000). Association of Official Analytical Chemists,Washington, DC.Benzie, I. F. F., & Strain, J. J. (1996). The ferric reducing ability of plasma (FRAP) as a

measure of “antioxidant power”: The FRAP assay. Analytical Biochemistry, 239,70−76.

Bougatef, A., Nedjar-Arroume, N., Ravallec-Plé, R., Leroy, Y., Guillochon, D., & Barkia, A.(2008). Angiotensin I-converting enzyme (ACE) inhibitory activities of sardinelle(Sardinella aurita) by-products protein hydrolysates obtained by treatment withmicrobial and visceral fish serine proteases. Food Chemistry, 111, 350−356.

Byun, H. G., & Kim, S. K. (2002). Structure and activity of angiotensin I convertingenzyme inhibitory peptides derived from Alaskan pollack skin. Journal ofBiochemistry and Molecular Biology, 35, 239−243.

Chen, H. M., Muramoto, K., Yamauchi, F., Fujimoto, K., & Nokihara, K. (1998).Antioxidative properties of histidine-containing peptides designed from peptidefragments found in the digests of a soybean protein. Journal of Agriculture and FoodChemistry, 46, 49−53.

Cheung, H. S., Wang, F. L., Ondetti, M. A., Sabo, E. F., & Cushman, D. W. (1980). Binding ofpeptide substrate and inhibition of angiotensin-converting enzyme: importance of theCOOH-terminal dipeptides sequence. The Journal of Biological Chemistry, 255, 401−407.

Cushman, D.W., & Cheung, H. S. (1971). Spectrophotometric assay and properties of theangiotensin-converting enzyme of rabbit lung. Biochemical Pharmacology, 20,1637−1648.

de Cavanagh, E. M., Inserra, F., Ferder, L., & Fraga, C. G. (2000). Enalapril and captoprilenhance glutathione-dependent antioxidant defences in mouse tissues. AmericanJournal of Physiology – Regulatory, Integrative and Comparative Physiology, 278,R572−R577.

Erdmann, K., Grosser, N., Schipporeit, K., & Schroder, H. (2006). The ACE inhibitorydipeptide Met–Tyr diminishes free radical formation in human endothelial cells viainduction of heme oxygenase-1 and ferritin. Journal of Nutrtion, 136, 2148−2152.

Halliwell, B. (1994). Free radicals, antioxidants, and human disease: Curiosity, cause, orconsequence. Lancet, 344, 721−724.

Hancock, J. T., Desikan, R., & Neill, S. J. (2001). Role of reactive oxygen species in cellsignaling pathways. Biochemical Society Transactions, 29, 345−350.

He, H. L., Chen, X. L., Wu, H., Sun, C. Y., Zhang, Y. Z., & Zhou, B. C. (2007). High throughputand rapid screening of marine protein hydrolysates enriched in peptides withangiotensin-I-converting enzyme inhibitory activity by capillary electrophoresis.Bioresource Technology, 98, 3499−3505.

He, Z., & Xia, W. (2007). Analysis of phenolic compounds in Chinese olive (Canariumalbum L.) fruit by RPHPLC–DAD–ESI–MS. Food Chemistry, 105, 1307−1311.

Jamdar, S. N., Rajalakshmi, V., Pednekar, M. D., Juan, F., Yardi, V., & Sharma, A. (2010).Influence of degree of hydrolysis on functional properties, antioxidant activity andACE inhibitory activity of peanut protein hydrolysates. Food Chemistry, 121,178−184.

Joo, S., Kies, C., & Schnepf, M. (1995). Chocolate and chocolate-like products: impact oncopper status of humans. Journal of Applied Nutrtion, 47, 67−77.

Khan, J. K., Kuo, Y. H., Kebede, N., & Lambein, F. (1994). Determination of nonproteinamino acids and toxins in Lathyrus by high performance liquid chromatographywith precolumn phenylisothiocyanate derivatization. Journal of Chromatography A,687, 113−119.

Korhonen, H., & Pihlanto, A. (2003). Food-derived bioactive peptides—opportunities fordesigning future foods. Current Pharmaceutical Design, 9, 1297−1308.

Kuba, M., Tana, C., Tawata, S., & Yasuda, M. (2005). Production of angiotensin Iconverting enzyme inhibitory peptides from soybean protein with Monascuspurpureus acid proteinase. Process Biochemistry, 40, 2191−2196.

Li, Y., Jiang, B., Zhang, T., Mu, W., & Liu, J. (2008). Antioxidant and free radical-scavenging activities of chickpea protein hydrolysate (CPH). Food Chemistry, 106(2), 444−450.

Li, G. H., Le, G. W., Shi, Y. H., & Shrestha, S. (2004). Angiotensin I-converting enzymeinhibitory peptides derived from food proteins and their physiological andpharmacological effects. Nutrition Research, 24, 469−486.

296 B. Sarmadi et al. / Food Research International 44 (2011) 290–296

Liu, Q., Kong, B., Xiong, Y. L., & Xia, X. (2010). Antioxidant activity and functionalproperties of porcine plasma protein hydrolysate as influenced by the degree ofhydrolysis. Food Chemistry, 118(2), 403−410.

Marcuse, R. (1962). The effect of some amino acids on the oxidation of linoleic acid andits methyl ester. The journal of the American oil chemists' soceity, 39, 97−103.

Peng, X., Xiong, Y. L., & Kong, B. (2009). Antioxidant activity of peptide fractions fromwhey protein hydrolysates as measured by electron spin resonance. FoodChemistry, 113, 196−201.

Pihlanto, A., Akkanen, S., & Korhonen, H. J. (2008). ACE-inhibitory and antioxidantproperties of potato (Solanum tuberosum). Food Chemistry, 109, 104−112.

Qian, Z. J., Jung, W. K., & Kim, S. K. (2008). Free radical scavenging activity of a novelantioxidative peptide purified from hydrolysate of bullfrog skin, Rana catesbeianaShaw. Bioresource Technology, 99, 1690−1698.

Rajapakse, N., Mendis, E., Jung, W. K., Je, J. Y., & Kim, S. K. (2005). Purification of a radicalscavenging peptide from fermented mussel sauce and its antioxidant properties.Food Research International, 38, 175−182.

Saiga, A., Tanabe, S., & Nishimura, T. (2003). Antioxidant activity of peptides obtainedfrom porcine myofibrillar proteins by protease treatment. Journal of Agriculturaland Food Chemistry, 51, 3661−3667.

Sakanaka, S., & Tachibana, Y. (2006). Active oxygen scavenging activity of egg-yolkprotein hydrolysates and their effects on lipid oxidation in beef and tunahomogenates. Food Chemistry, 95, 243−249.

Sarmadi, B., & Amin, I. (2010). Antioxidative peptides from food proteins: A review.Peptides, 31, 1949−1956.

Schiffrin, E. L., & Touyz, R. M. (2004). From bedside to bench to bedside: Role of renin–angiotensin–aldosterone system in remodeling of resistance arteries in hypertension.American Journal of Physiology – Heart and Circulatory Physiology, 287, H435−H446.

Shahidi, F., & Amarowicz, R. (1996). Antioxidant activity of protein hydrolyzates, fromaquatic species. The journal of the American oil chemists' soceity, 73, 1197−1199.

Sheih, I. C., Fang, T. J., & Wu, T. K. (2009). Isolation and characterisation of a novelangiotensin I-converting enzyme (ACE) inhibitory peptide from the algae proteinwaste. Food Chemistry, 115, 279−284.

Unger, T. (2002). The role of the renin–angiotensin system in the development ofcardiovascular disease. American Journal of Cardiology, 89, 3A−9A.

Velioglu, Y. S., Mazza, G., Gao, L., & Oomah, B. D. (1998). Antioxidant activity and totalphenolics in selected fruits, vegetables and grain products. Journal of Agricultureand Food Chemistry, 46, 4113−4117.

Vercruysse, L., Smagghe, G., Beckers, T., & Van Camp, J. (2009). Antioxidative and ACEinhibitory activities in enzymatic hydrolysates of the cotton leafworm, Spodopteralittoralis. Food Chemistry, 114, 38−43.

Voigt, J., Biehl, B., Heinrichs, H., Kamaruddin, S., Marsoner, G. G., & Hugi, A. (1994). In-vitro formation of cocoa-specific aroma precursors: aroma-related peptidesgenerated from cocoa-seed protein by co-operation of an aspartic endoproteaseand a carboxypeptidase. Food Chemistry, 49, 173−180.

Wang, W. Y., & De Mejia, E. G. (2005). A new frontier in soy bioactive peptides that mayprevent age-related chronic diseases. Comprehensive Reviews in Food Science andFood Safety, 4, 63−78.

Wang, X. S., Tang, C. H., Chen, L., & Yang, X. Q. (2009). Characterization and antioxidantproperties of hemp protein hydrolysates obtained with Neutrase®. Food Technol.Biotechnol, 47(4), 428−434.

Wu, C. H., Chen, H. M., & Shiau, C. Y. (2003). Free amino acids and peptides as related toantioxidant properties in protein hydrolysates of mackerel (Scomber austriasicus).Food Research International, 36, 949−957.

Zak, D. L., & Keeney, P. G. (1976a). Extraction and fractionation of cocoa proteins asapplied to several varieties of cocoa beans. Food Chemistry, 24, 479−483.

Zak, D. L., & Keeney, P. G. (1976b). Changes in cocoa proteins during ripening of fruit,fermentation, and further processing of cocoa beans. Journal of Agricultural andFood Chemistry, 24, 483−486.