Food Research International 44 (2011) 290296

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Food Research International

j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / f o o d r e s

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 b c

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

a r t i c l e

i n f o

a b s t r a c t
The present study investigated antioxidant and angiotensin converting enzyme (ACE) inhibitory activities of cocoa autolysates. After removal of cocoa fat, alkaloids and polyphenols, the remaining proteinous powder was autolyzed at pH 3.5 and 5.2. At similar concentrations, autolysates produced at pH 3.5 indicated the highest reducing power and ACE inhibition activity. However, those generated at pH 5.2 showed the highest antioxidant activity based on -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 the results from the assays were not due to the polyphenols of cocoa autolysates. Based on the results no polyphenols could be detected from cocoa autolysates. It can be indicated that among other useful substances of cocoa, its peptides and amino acids could contribute to its health-promoting properties. Furthermore, these bioactive substances can be exploited into functional foods or used as a source of nutraceuticals. 2010 Elsevier Ltd. All rights reserved.

Article history: Received 14 July 2010 Accepted 8 October 2010 Keywords: Cocoa (Theobroma cacao L.) Autolysate Antioxidant activity ACE inhibition

1. Introduction Free radicals are generated through normal reactions within the body during respiration in aerobic organisms. They can exert diverse functions like signaling roles and providing defense against infections (Hancock, Desikan, & Neill, 2001). However, any excessive amount of reactive radicals can cause cellular damage which, in turn, initiates several diseases like atherosclerosis, arthritis, diabetes and cancer (Halliwell, 1994). Therefore, in certain circumstances that endogenous defense system fails to protect the body against reactive radicals on its own, external supply of antioxidant is needed. Several peptides from protein ingredients have been found to possess antioxidant capacity. Bioactive peptides are regarded as specic protein fragments which are inactive in the parent protein sequence. They can exert several physiological functions after they are released by enzymatic hydrolysis (Korhonen & Pihlanto, 2003; Sarmadi & Amin, 2010). The amino acid composition and sequences affect the bioactive peptide activity (Chen, Muramoto, Yamauchi, Fujimoto, & Nokihara, 1998). Moreover, several peptides have been found to possess multifunctional properties such as antioxidant and ACE inhibitory capacity (Pihlanto, Akkanen, & Korhonen, 2008).

Corresponding author. Department of Nutrition and Dietetics, Faculty of Medicine and Health Sciences, Universiti Putra Malaysia, 43400 Serdang, Selangor, Malaysia. Tel.: +60 3 89472435; fax: +60 3 89472459. E-mail address: amin@medic.upm.edu.my (A. Ismail). 0963-9969/$ see front matter 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.foodres.2010.10.017

Antihypertensive peptides derived from food proteins are the most comprehensively studied bioactive peptides. Angiotensin-converting enzyme (ACE) is a dipeptidyl carboxypeptidase (EC 3.4.15.1) and is widely distributed in mammalian tissues (Li, Le, Shi, & Shrestha, 2004). It converts angiotensin I to angiotensin II, a vasoconstrictor. It also inactivates bradykinin which is a vasodilator peptide. Therefore, this enzyme plays an important role in the regulation of blood pressure through these two mechanisms (Unger, 2002). ACE inhibitors have been shown to be effective antihypertensive agents. Further, in the conditions of hypertension, angiotensin II amplies the oxidative stress as it intervenes many of its cellular functions through stimulating the formation of intracellular reactive radical species (ROS) (Schiffrin & Touyz, 2004). Therefore, in addition to blood pressure control, ACE inhibitors have been shown to intensify the antioxidant defense system in animals and humans by inhibition of angiotensin II formation (de Cavanagh, Inserra, Ferder, & Fraga, 2000). Antioxidant and ACE inhibitory activities have been reported for peptides and hydrolysates from plant and animal sources including peanut protein hydrolysates (Jamdar et al., 2010), sardine by-product protein hydrolysates (Bougatef et al., 2008), whey protein hydrolysates (Peng, Xiong, & Kong, 2009), peptide from the algae protein waste (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 its unprocessed beans are referred to as cacao. Cocoa polyphenols have

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long been reported to have health benets. As an example, supplementation of cocoa extracts to obese-diabetic rats decreased plasma oxidative stress biomarker and increased superoxide dismutase activity, attributable to its polyphenols and methylxanthines (Abbe, Amin, Chong, Muhajir, & Hasbullah, 2008). Cacao bean contains four proteins including: albumin, globulin, prolamin and glutelin (Zak & Keeney, 1976a,b). According to previous studies cocoa protein could be cleaved to hydrophilic and hydrophobic peptides and amino acids through autolysis under different pHs (Amin, Jinap, Jamilah, Harikrisna, & Biehl, 2002; Voigt et al., 1994). Among other components of cocoa, these valuable compounds stand unexplored. This assumption leads us to hypothesize that besides cocoa polyphenols, its peptides and amino acids can contribute to its health effects. To our knowledge, this is the rst study reported on antioxidant and ACE inhibitory activities of cocoa autolysate. Its ndings can provide fundamental information for further work in this area. Besides, the bioactive substances of cocoa autolysates can be used as ingredients of functional foods and dietary supplements as well as a pharmaceuticals and nutraceuticals. 2. Materials and methods 2.1. Materials -carotene, linoleic acid, Tween 20, butylated hydroxytoluene (BHT), thioglycollic acid, ascorbic acid, 2,4,6-tripyridyl-s-triazine (TPTZ), ferric chloride (FeCl36H2O), ferrous sulphate (FeSO47H2O), ethyl acetate, sodium phosphate dibasic, sodium phosphate monobasic, sodium acetate trihydrate, and angiotensin converting enzyme (ACE) from rabbit lung and hipouril histidine leucine (HHL) were purchased 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 from Malaysian Cocoa Board, Jengka, Pahang, Malaysia. Unfermented and fresh beans were taken from the pods immediately after arrival and shock-frozen in liquid nitrogen. After removal of testae and radiculae, they were freeze-dried. The freeze-dried cotyledons were crushed with mortar and pestle. They were defatted using a Soxhlet apparatus with petroleum ether (bp 4060 C) for 8 h, twice. Purine alkaloids were partially extracted with chloroform for 8 h in a Soxhlet apparatus. In order to remove the polyphenols a method described by Amin et 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 an airtight glass container before use. The efciency of polyphenol extraction was checked by qualitative and quantitative tests. For qualitative test, a small portion of AcDP was heated with 2 ml of 5 M HC1 for a few seconds (appearance of red color indicates the presence of residual polyphenols). Quantitatively, RPHPLC analysis for detection of polyphenols in cocoa autolysates was carried out following a method described by He and Xia (2007). The phenolic compounds in cocoa autolysate were identied by comparing their retention time with an authentic standard. Detection of phenolic compounds was carried out in a spectrum from = 230540 nm. 2.3. Preparation of cocoa autolysate Cocoa cotyledons contain endogenous endopeptidase (aspartic endoprotease, optimum pH 3.5) and exopeptidase (carboxypeptidase, optimum pH 5.8) both of which can be activated and cleave the protein precursor under optimum pH and temperature in vitro, in laboratory condition, and during fermentation process (Voigt et al., 1994). Therefore, to obtain cocoa autolysates, a method was applied following

a method described by Voigt et al. (1994) in which cocoa AcDP (1 g) was suspended both in acetic acid pH 3.5 (100 ml) and in sodium acetate buffer 10 mM, pH 5.2 (100 ml). The suspensions were incubated at 50 C, in a shaking water bath for 16 h for autolysis. Autolysis was stopped by adding 70% methanol (v/v) to the suspensions. Subsequently, the suspensions were stirred at room temperature for 1 h and centrifuged at 20,000 g for 30 min. The supernatants were collected, and the methanol was removed under pressure at 40 C by means of a rotary evaporator. Finally, the aqueous solutions were freeze-dried and stored at 20 C before use. In this article, autolysate of PBC 140 at pH 3.5, PBC 140 at pH 5.2, UIT1 at pH 3.5 and UIT1 at pH 5.2 will be referred to as P3, P5, U3 and U5, respectively. 2.4. Determination of protein content The protein contents of autolysates were measured by the methods of Kjeldahl (AOAC, 2000). 2.5. Determination of amino acid composition The Pico Tag method, with modication, was used for determining the 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 for 24 h. Internal standard (-aminobutyric acid, 10 ml) was added to the mixture. Mobile phase A (0.1 M ammonium acetate, pH 6.5) and mobile phase B (0.1 M ammonium acetate containing acetonitrile and methanol, 44:46:10, v/v, pH 6.5) were utilized. Sample (100 l) was injected on a C18 reversed-phase column (250 mm 4 mm, i.d. and particle size 5 m, Merck KGaA, Darmstadt, Germany) and amino acids were detected at 254 nm. 2.6. Determination of antioxidant capacity 2.6.1. Ferric reducing/antioxidant power (FRAP) FRAP assay was determined based on the reduction of Fe3+-TPTZ to a blue colored Fe2+ TPTZ (Benzie & Strain, 1996). The FRAP reagent was prepared by mixing 300 mM acetate buffer (pH 3.6), 10 mM TPTZ and 20 mM FeCl36H2O in a ratio of 10:1:1. Forty microlitres of the sample was added to the test tubes containing 3 ml of freshly prepared FRAP reagent. Absorbance was measured at 593 nm using spectrophotometer (Shimadzu UV 1601, Japan). In the FRAP assay, the antioxidant potential of sample was determined from a standard curve using FeSO47H2O at a concentration range between 62.5 and 1000 M. 2.6.2. -carotenelinoleate bleaching assay -carotene bleaching assay was conducted using a method developed by Velioglu, Mazza, Gao, and Oomah (1998) with slight modication. -carotene (0.2 mg in 1 ml chloroform), linoleic acid (0.02 ml) and Tween 20 (0.2 ml) were transferred into a round bottomed ask. Chloroform was removed using a rotary evaporator at 50 C. Following evaporation, 50 ml of distilled water was added to the mixture and then shaken vigorously to form an emulsion. Next, 2 ml of the emulsion were pipetted into test tubes containing 0.2 ml of cocoa autolysates or standard (BHT) or control (methanol 70%) and immediately placed in a water bath at 50 C. The absorbance was read at 20 min intervals for 100 min at 470 nm. Antioxidant activity (AA) was expressed as percent of inhibition relative to the control, using the following formula: A A% = 1A0 At = A0c Atc 100 where A0 is absorbance of sample/Std at t = 0, At is absorbance of sample/Std at t = 100, A0c is absorbance of control at t = 0 and Atc is absorbance 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 in vitro was measured according to the spectrophotometric method of Cushman and Cheung (1971). Briey, 100 l of cocoa autolysate and 100 l of hippuryl-L-histidyl-L-leucine (HHL, 12.5 mM in 0.05 M sodium borate buffer containing NaCl 0.4 M, pH 8.3) were incubated at 37 C for 5 min. Then, 150 l of ACE was added and the mixture was incubated for an hour. The enzymatic reaction was stopped by adding 250 l of 0.5 N HCl. The hippuric acid formed by the action of the ACE on HHL was extracted from the acidied solution into 1.5 ml ethyl acetate by vortexing for 15 s. The mixture was centrifuged at 3290 g for 10 min at 4 C, and a 0.5 ml aliquot of each ethyl acetate layer was transferred to clean tubes and evaporated by heating at 120 C for 20 min on a heating plate. The hippuric acid was redissolved in 3 ml of 1 M NaCl, and the amount formed was determined by its absorbance at 228 nm. Assay mixture without cocoa autolysate was referred as control. The IC50 value was dened as the concentration of hydrolysate 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 single experiment was replicated 3 times. The results were expressed as mean SD. Signicant differences among means of samples were evaluated by one-way analysis of variance (ANOVA) at P b 0.05, using SPSS 15. Pearson's correlation was applied to nd any signicant (P b 0.05) correlation between means. 3. Results and discussion 3.1. Cocoa autolysate Two clones of cocoa, namely PBC 140 and UIT 1, were used for this study. These clones are commonly grown by Malaysian farmers.
Table 1 Amino acids composition of cocoa autolysate based on their chemical properties. Amino acid (%) pH 3.5 Ser Lys Gly His Arg Thr Aspa1 Glub1 Val2,4 Ile2,4 Leu2,4 Ala4 Pro4 Phe3,4 Met4,5 Cys5 Tyr3 1 Acid/basic amino acid 2 Branched chain amino acid 3 Aromatic amino acid 4 Hydrophobic amino acid 5 S-containing amino acid 6.7 0.01 7.7 0.1 5.4 0.03 2.2 0.009 5.2 0.01 5.9 0.03 10.4 0.06 15.9 0.03 5.8 0.03 3.7 0.12 6.9 0.04 5.8 0.08 4.9 0.05 7.3 0.03 1.4 0.02 NDc 4.4 0.01 26.3 0.1 16.5 0.2 11.8 0.02 36 0.1 1.42 0.02 PBC-140

However, the main objective of this study was to determine contribution of cocoa peptide and amino acids in its antioxidant and ACE inhibitory capacity; thus, selection of these clones was not based on a specic rationale. In order to avoid any intervening effects of cocoa polyphenols in the results, its polyphenol contents were removed. The efciency of polyphenol removal was checked using qualitative and quantitative tests. When the retention time of the autolysate was compared with an authentic standard, no phenolic compounds could be detected. The results from both tests revealed that cocoa AcDP was free from polyphenols. AcDP of both cocoa clones were autolyzed under optimum pHs and four types of cocoa autolysates were produced. Our previous study displayed that during autolysis of AcDP at pH 3.5 mainly hydrophobic fractions were generated (Amin et al., 2002). In addition, it has been reported that mostly hydrophilic fractions and hydrophobic free amino acids 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 to hydrolyze substrates at their hydrophobic amino acid residues, resulting in formation of oligopeptides with hydrophobic amino acid residues at C-terminal. Lower proportions of free amino acids were generated compared to pH 5.2 (Amin et al., 2002; Voigt et al., 1994). Moreover, during fermentation-like incubation of cocoa AcDP at pH 5.2, carboxypeptidase of cocoa cotyledon prefers to cleave hydrophobic residues at their carboxyterminal ends (Voigt et al., 1994). Therefore, mostly hydrophilic peptides and hydrophobic amino acids were produced. Leucine, alanine, phenylalanine, and valine were the predominant free amino acids accumulated during autolysis of AcDP at pH 5.2 (Voigt et al., 1994). Hydrolysis of food proteins results in production of a broad variety of peptides and free amino acids. During hydrolysis, enzyme specicity affects the size and amino acid sequence of peptides as well as level and composition of free amino acids which subsequently could inuence 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 peptides and amino acids with different characteristics. The yields of cocoa autolysates were as follows: 9% 0.2 for P3, 10% for P5 0.3,

UIT-1 pH 5.2 6.1 0.03 8.6 0.2 4.7 0.06 2.7 0.02 7.04 0.005 4.2 0.009 11.4 0.19 20.1 0.003 4.9 0.09 3.1 0.02 5.9 0.04 4.1 0.1 4.2 0.003 6.3 0.05 1.6 0.04 ND 4.6 0.02 31.4 0.2 14 0.07 11 0.08 30.3 0.06 1.61 0.04 pH 3.5 6.3 0.01 7.3 0.01 5.3 0.008 2.06 0.005 5.6 0.21 4.2 0.17 12.2 0.27 20.8 0.15 5.08 0.03 2.9 0.06 5.9 0.06 4.6 0.17 4.4 0.03 6.9 0.004 1.3 0.001 0.4 0.01 4.6 0.005 33 0.4 14 0.16 11.5 0.01 31.3 0.05 1.7 0.02 pH 5.2 5.9 0.06 7.7 0.2 5.2 0.0 1.8 0.02 6.4 0.006 4.7 0.01 9.5 0.1 20.1 0.05 5.7 0.04 3.3 0.01 6.6 0.0002 4.5 0.08 4.7 0.02 7.3 0.06 1.5 0.004 0.3 0.02 4.8 0.009 29.7 0.06 15.5 0.05 12.2 0.07 33.5 0.003 1.77 0.3

Data correspond to the mean SD of two experiments. a Aspartic acid + Asparagine. b Glutamic acid + glutamine. c ND: not detected.

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12% 0.12 for U3 and 17% 0.14 for U5 0.37. Protein content of cocoa autolysate was high and included: 68 0.13 mg/100 mg of autolysate 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 for U5. A variation was observed among protein content of cocoa autolysates. Among them, autolysates produced from PBC 140 at pH 5.2 contained the highest amount of protein. The high protein content of autolysates can be attributed to the removal of fat from cocoa powder and elimination of insoluble undigested substances after autolysis. 3.2. Amino acid composition Analysis of the amino acid composition revealed that there was not much difference between cocoa autolysates. In all autolysates hydrophobic amino acids outweighed the other amino acids (Table 1). Many food-derived peptides with hydrophobic amino acids 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 cocoa autolysates may be related to the high amount of their hydrophobic amino acids. Other amino acids in cocoa autolysates include acidic and basic amino acids which exist in high amounts followed by aromatic amino acids. While carboxyl and amino groups in the side chains act as chelator of metal ions (Saiga, Tanabe, & Nishimura, 2003) and as hydrogen donor (Qian et al., 2008), ACE substrate or its competitive inhibitors with Cterminal dicarboxylic amino acids, like Glu, have little afnity for the enzyme (Cheung, Wang, Ondetti, Sabo, & Cushman, 1980). Aromatic residues are effective radical scavengers since they can donate protons to electron decient radicals to make them stable and meanwhile, they can keep the stability of molecule through resonance structure (Rajapakse, Mendis, Jung, Je, & Kim, 2005). It has also been reported that the peptide with comparatively low IC50 value contains a high content of branched and aromatic amino acids such as Ile, Val, Phe, and Tyr in its peptide sequence (He et al., 2007). Thus, it is very likely that aromatic amino acids along with hydrophobic amino acids contribute to antioxidant and ACE inhibition activity of cocoa autolysates. In addition, Glu and Asp may contribute in antioxidant activity of cocoa autolysates, yet they do not seem to play important roles in terms of their ACE inhibitory activity. However, not only amino acid composition of peptides but their structure and amino acid sequence are also responsible for their activity (Chen et al., 1998). Our previous results have displayed that the oligopeptide pattern of cocoa autolysates from PBC 140 deviated signicantly from other clones with regard to the peak at 3945 min retention time (Amin et al., 2002), indicating the discrepancy between peptides in the autolysates. Thus, although the amino acid composition of cocoa from both clones was similar, the discrepancy among fractions of the two clones could have affected autolysate properties. Other factors can also affect antioxidant and ACE inhibition activities of cocoa autolysates that will be discussed in the following sections. 3.3. Antioxidant capacity The exact mechanism underlying the antioxidant activity of peptides has not fully been understood, yet various studies have displayed 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 been reported that antioxidant peptides keep cells safe from damage by ROS through the induction of genes (Erdmann, Grosser, Schipporeit, & Schroder, 2006). Tyr, Trp, Met, Lys, Cys, and His are examples of amino acids that have antioxidant activity (Wang & De Mejia, 2005). In

addition to the presence of proper amino acids, their correct positioning in peptide sequence plays an important role in antioxidant activity of peptides (Rajapakse et al., 2005). Several methods have been developed to assess antioxidant capacity. However, none of them can be used as an ofcial standardized method. Hence, in the research, evaluation of antioxidant capacity is usually carried out by various methods of measurement in different oxidation conditions. The present work sought to investigate the antioxidant capacity of cocoa autolysates based on two different reaction mechanisms, i.e., -carotenelinoleate bleaching and 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 colored form Fe2+-TPTZ with maximum absorbance at 593 nm (Benzie & Strain, 1996). The reducing power results revealed that cocoa autolysates have abilities to donate electron which is involved in the antioxidant activity. The reducing power of cocoa autolysates augmented as their concentrations (1.25, 2.5, 5, 10 mg/ml) increased (Fig. 1). The effect of concentration on enhanced reducing power has been observed for chickpea protein hydrolysates (Li, Jiang, Zhang, Mu, & Liu, 2008). Antioxidant activity of U3 was signicantly (P b 0.05) higher than other autolysate following the order of P3 N P5 N U5 in similar concentrations. Whey protein hydrolysates (45 mg/ml) after 5 h hydrolysis had the FRAP value of about 1150 M (Peng et al., 2009). In our study, FRAP value of U3 at 10 mg/ml was 723 5 M which is more comparable to FRAP results of plasma protein hydrolysates with FRAP value of 713.1 8.8 M (Liu, Kong, Xiong, & Xia, 2010). High reducing power ability of hydrolysates is possibly due to amplied levels of hydrogen ions (protons and electrons) following hydrolysis (Liu et al., 2010). This could have been a possible explanation for the higher reducing power of U3 in our study. Reducing power has been reported for fraction IV from chickpea hydrolysates. This fraction had the highest amount of total hydrophobic amino acid and high hydrophobicity (Li et al., 2008). The reducing power of hemp protein hydrolysates was reported to be unrelated to the hydrophobic amino acids yet positively and signicantly (P b 0.10) correlated with their surface hydrophobicity (Wang, Tang, Chen, & Yang, 2009). Antioxidant activity of protein hydrolysates is affected by amount and composition of free amino acid and peptides (Wu et al., 2003), the type of protease, degree of hydrolysis (Liu et al., 2010) as well as size (Wu et al., 2003), structure, amino acid composition and amino acid sequences of peptides in hydrolysates (Chen et al., 1998). In this study, the differences between cocoa autolysates regarding their amino acid composition are too little to be considered affecting

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

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factors. Saiga et al. (2003) have pointed out that a disparity in activity of two similar hydrolysates (regarding their amino acid compositions) can be related to the structure and length of the peptides in the hydrolysates. Correspondingly, various factors can be important in reducing power of cocoa autolysates. These can include molecular size of peptides, their amino acid sequences, structural properties and degree of hydrolysis, any of which can lead to differences in FRAP values of autolysates. A signicant and high (r2 = 0.827) correlation was observed between FRAP results and protein content of autolysates. 3.3.2. -carotene bleaching inhibition activity -carotenelinoleic bleaching inhibition assay simulates membrane lipid oxidation and can be considered a good model for membrane based lipid peroxidation. In this oilwater emulsion-based system, linoleic acid acts as a free radical generator that produces peroxyl radicals under thermally induced oxidation. The produced free radicals attack the -carotene chromophore resulting in bleaching effect, which can be inhibited by a free-radical scavenger. Results showed that cocoa autolysates demonstrated an ability to inhibit the discoloration of -carotene by scavenging linoleatederived free radicals dose-dependently (Fig. 2). Concentration dependency trend has also demonstrated for peanut protein hydrolysates (Jamdar et al., 2010) and egg protein hydrolysates (Sakanaka & Tachibana, 2006). The most potent antioxidant autolysate was U5 followed by P5, U3 and P3, respectively. Antioxidant activity of autolysates that was produced under pH 5.2 was signicantly (P b 0.05) higher than those produced at pH 3.5. Antioxidant activity of peptides or protein in the free radical-mediated lipid peroxidation system is inuenced by molecular size, chemical properties and electron transferring ability of amino acid residues in the sequence (Qian et al., 2008). Therefore, the discrepancy in antioxidant activity of cocoa autolysate may be related to differences in the sequences of peptides and/or their molecular size. Further, the difference can be due to synergistic effect of Tween 20 with existing amino acids or peptides. It has been stated that the antioxidant activity of amino acids can be increased and their pro-oxidant activity can be lessened or reverted to antioxidant activity by addition of an emulsier or phosphate. Emulsiers can reduce the particle size of peptides and enlarge the contact surface of the phases (Marcuse, 1962). Antioxidant activity of all extracts was relatively low, ranging from 28% to 54% 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 or peptides and catalyze oxidation of linoleic acid strongly (Marcuse, 1962). In addition, it is possible that cocoa autolysates could exert higher antioxidant activity at higher concentrations. To examine

whether increased doses of cocoa autolysates can enhance their antioxidant activity, two more concentrations were added (20 and 40 mg/ml). Antioxidant activity of P5, U3 and U5 rose signicantly (P b 0.05). Under these concentrations all autolysates were signicantly lower than BHT (a synthetic antioxidant). However, at high concentrations autolysates did not differ signicantly except P3 whose antioxidant activity was signicantly lower than all. This autolysate (P3) did not demonstrate a pronounced antioxidant activity, which did not rise signicantly even at higher concentrations. This could be due to the presence of peptides with weak antioxidant activities, hindering effect of other compounds in autolysates or prooxidant effects of some amino acids (e.g., Cys, His) and peptides. Prooxidative effect was also observed for 10 mg/ml of seal protein hydrolysates (Shahidi & Amarowicz, 1996). A signicant and high (r2 = 0.762) correlation was observed between protein content and antioxidant activity of autolysates. The decrease in the absorbance of -carotene in the presence of 10 mg/ml of cocoa autolysates and 70% methanol (as a control) was recorded as a function of time (Fig. 3). Cocoa autolysate suppressed bleaching of -carotene compared with control in emulsion system indicating their antioxidant activity. The absorbance of U5 dropped slowly 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 percent of ACE inhibition by samples. Based on the results, it was revealed that all autolysates have the ability to inhibit ACE. Autolysates produced at pH 3.5 exerted more ACE inhibitory activity than autolysates produced at pH 5.2 (P b 0.05) (Fig. 4). At 2.5 mg/ml of U5 the ACE % inhibition was 44 1.6 which is comparable to 2 mg/ml of sardine hydrolysates 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 of oyster, scallop, codsh 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 (a synthetic ACE inhibitor) was 2 0.57 M. The ACE inhibitory activity results were in parallel with FRAP values where P3 and U3 displayed higher reducing power. It seems that autolysates at pH 3.5 have multifunctional roles. However, it remains to be elucidated whether the same peptide can have both functions in autolysates. Other studies

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

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

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concentrations. Cocoa autolysates produced at pH 3.5 contained more reducing agents and ACE inhibitors than those formed under pH 5.2. In addition, cocoa autolysates produced more antioxidants under pH 5.2 using the linoleic acid oxidation system. This indicates the effect of various enzymes on formation of peptides with different potential activities. It can be suggested that besides other compounds of cocoa, its peptides and amino acid compositions could contribute to its antioxidant and ACE inhibitory activities. In addition, a combined antioxidant and ACE inhibition capacity makes cocoa autolysates a rich source of bioactive compounds that improve cardiovascular health or control related diseases. Further research is needed to identify effective peptide from cocoa autolysate and to investigate its aforementioned activities in vivo.

References
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have also reported multifunctional roles for hydrolysates (Jamdar et al., 2010; Vercruysse et al., 2009). It has been reported that ACE indicates a preference to substrates or competitive inhibitors containing branched chain amino acid residues at the N-terminal position and hydrophobic amino acid residues (aromatic or branched-side chains) at the C-terminal position (Byun & Kim, 2002; Cheung et al., 1980). He et al. (2007) have suggested that high amounts of branched and aromatic amino acids (Pro, Glu, Val, Phe and Tyr) in marine protein can be responsible for relatively low IC50 values of its hydrolysates. Similarly, high contents of hydrophobic and aromatic amino acids in cocoa autolysates and high amounts of hydrophobic fractions in P3 and U3 autolysates can contribute to their higher ACE inhibitory activity. The hydrophilic amino acid residues in the peptide sequence disrupt the access of the peptide to the active site of ACE affecting the inhibitory activity (Sheih et al., 2009). Therefore, the lower activity of autolysates generated at pH 5.2 can be explained by their high content of hydrophilic fractions. However, Li et al. (2004) have proposed that the hydrophilichydrophobic partitioning in the sequence can be a crucial factor in the inhibitory activity. Therefore, for a better explanation of autolysates activities, further work is required to identify amino acid sequence of effective peptides. In addition, carboxypeptidase cleaves one or more amino acids from C-terminal positions which can result in production of peptides with low or no ACE inhibition activity. This can provide another probable reason for lower ACE inhibitory activity of P5 and U5. A signicant and moderate correlation was found between protein content and ACE inhibitory activity (r2 = 0.649). It is also possible that other compounds like alkaloids or oligosaccharides contribute to ACE inhibitory activity of cocoa autolysates. However, the ACE inhibitory activity of autolysates varied using different enzymes, suggesting that ACE inhibitor could be the product of cocoa protein hydrolysis. 4. Conclusion Adverse effects of synthetic compounds lead to a growing interest for extraction of natural compounds from food sources. In addition, there is an increasing tendency among public to choose them since these natural compounds are of no or little side effects. Accordingly, cocoa autolysates can be utilized as natural sources of peptides and amino acids with antioxidative and antihypertensive properties. An additional merit of bioactive peptides to other natural compounds is that they have nutritional and functional advantages. The present study indicated that cocoa autolysates exhibit antioxidant and ACE inhibitory activities, depending on their

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