Effect of heat and enzymatic treatment on the antihypertensive activity of whey protein hydrolysates

ARTICLE IN PRESS

International Dairy Journal 17 (2007) 632–640 www.elsevier.com/locate/idairyj

Effect of heat and enzymatic treatment on the antihypertensive activity of whey protein hydrolysates Elizabete Lourenc- o da Costaa, Jose´ Antonio da Rocha Gontijob, Flavia Maria Nettoa, a

Department of Food and Nutrition, Faculty of Food Engineering, State University of Campinas—UNICAMP, R. Monteiro Lobato, 80, CP6121, Campinas, SP, CEP:130862, Brazil b Faculty of Medical Sciences, State University of Campinas-UNICAMP, SP, Brazil Received 22 September 2005; accepted 6 August 2006

Abstract The influence of heat and enzymatic treatments on the hypotensive activity of hydrolysates derived from whey protein isolate was examined. The whey protein isolate (WPI) was previously denatured at 65 or 95 1C and hydrolyzed using the enzymes Alcalase, a-chymotrypsin or Proteomix. The hydrolysates thus obtained were characterized and studied with regard to their angiotensin converting enzyme (ACE) inhibitory activity and hypotensive activity in spontaneously hypertensive rats (SHR). The enzyme a-chymotrypsin was found to produce hydrolysates with the highest ACE inhibitory activity. The hydrolysate that most effectively reduced blood pressure in SHR was obtained from WPI previously denatured at 65 1C and treated with the enzyme Alcalase. The hydrolysate with the highest ACE inhibitory activity was able to reduce the arterial blood pressure of the animals only after intraperitoneal administration, suggesting an interference of gastrointestinal enzymes in the absorption of active peptides from this hydrolysate. r 2006 Elsevier Ltd. All rights reserved. Keywords: Whey protein isolate; Enzymatic hydrolysis; Angiotensin converting enzyme; Functional foods; Blood pressure

1. Introduction In addition to their nutritional properties, some proteins exhibit biological activity, such as mineral binding (Toba et al., 2000), opioid (Bitri, 2004) and inmunolomodulatory (Dutta, 2002) effects, amongst others. These functions are associated with the bioactive peptides present in certain protein sequences which are released by enzymatic hydrolysis in vivo or in vitro (Korhonen, Pihlanto-Lepa¨la¨, Rantama¨ki, & Tupasela, 1998). There has been growing interest in research on peptides with respect to possible anti-hypertensive activity, as evidence emerges of a relationship between increased protein concentration in the diet and reduced blood pressure. Studies in this area might substantiate a nonpharmacological alternative for the prevention and control of systemic arterial hypertension, the most important risk factor for cardiovascular disease (Martin, 2003). Bovine Corresponding author. Tel.: +55 19 3521 4080; fax: +55 19 3521 4060.

E-mail address: fl[email protected] (F.M. Netto). 0958-6946/$ - see front matter r 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.idairyj.2006.09.003

milk whey has been considered one of the main raw materials for the production of functional ingredients. Several authors reported that whey protein hydrolysates showed angiotensin converting enzyme (ACE) inhibitory activity as well as hypotensive activity in animals and humans (Fujita, Yamagami, & Ohshima, 2001; Van der Ven, Grupen, Bont, & Voragen, 2002; Vermeirssen, Van Camp, Devos, & Verstraete, 2003). There is controversy in the literature as to whether it is necessary to use native protein to obtain bioactive peptides. According to Smithers et al. (1996) and Tirelli, De Noni, and Resmini (1997), the biological activity of a whey protein depends on the preservation of their native structure. On the other hand, several authors (Fitzgerald & Meisel, 1999; Takada, Aoe, & Kumegawa, 1996; Takada et al., 1997), report obtaining bioactive peptides from denaturated protein, albeit without specifying its degree of denaturation. Heat treatment can cause protein denaturation and aggregation and therefore the profile of the peptides released during digestion or in vitro hydrolysis could be

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altered, with the formation of peptides different from those produced from native protein (Meisel, 1998). Another critical factor in bioactive peptide production is the adequate match of the enzyme and protein sources. Abubakar, Saito, Kitazawa, Kawai, and Itoh (1998) observed that whey protein hydrolysis by Thermolysin and Proteinase K resulted in products with high ACE inhibitory activity, whereas hydrolysates obtained using Actinase K had low activity. The adequate combination of prior treatment of the protein and enzyme is of great importance in obtaining high-activity hydrolysates. The aim of the present work was to evaluate the effects of heat treatment and of different enzymes on the production of whey protein hydrolysates displaying ACE inhibitory activity and antihypertensive activity in spontaneously hypertensive rats (SHR). 2. Material and methods 2.1. Materials Whey protein isolate (WPI) was supplied by Davisco Foods International Inc. (Eden Praire, MN, USA). The enzymes used were Alcalase (Novo Nordisk Biochem Inc., Kalundborg, Denmark), a-chymotrypsin (Sigma Co., St. Louis, MO, USA) and Proteomix (Biobra´s, Montes Claros, MG, Brazil). The angiotensin converting enzyme (ACE, EC3.4.15.1) and its synthetic substrate hipuril-histidilleucine (HHL) were obtained from Sigma. Reagents were analytical or chromatographic grade. 2.2. Heat treatment and preparation of hydrolysates Whey protein isolate solutions in distilled water (10 g 100 mL1) were heated in a water bath at 651 or 95 1C for 15 min to obtain products with different degrees of denaturation (WPI65 and WPI95, respectively). The untreated WPI, considered to be a native protein, was termed WPIN. After the heat treatment the samples were cooled and freeze dried. The degree of denaturation was estimated by differential scanning calorimetry TA Instruments, Model 2920 Modulated DSC, New Castle, DE, USA) according to the method described by Puppo and An˜o´n (1999). The samples were suspended in deionized water (20%, w/v) and placed in hermetically sealed aluminum capsules. Heating proceeded from 251 to 100 1C at a rate of 10 1C min1. An empty capsule was used as the reference. Denaturation temperature (Td) was determined from the thermogram peak and denaturation enthalpy (DH) was calculated based on the area of the transition peak. After the run, the sample capsule was punctured and placed in an oven at 105 1C for the determination of dry mass (Puppo & An˜o´n, 1999). The calorimetric analyses were carried out at least in triplicate. Solutions of untreated and heat treated WPI, 10% (w/v) protein in distilled water at pH 7, were hydrolyzed using

633

the enzymes Alcalase, a-chymotrypsin, and Proteomix. The conditions for hydrolysis were: 40 1C for a-chymotrypsin and Proteomix and 60 1C for Alcalase, and the enzyme-tosubstrate ratio (E/S) was 0.01 (w/w) for all enzymes and was calculated on the basis of total protein content in the enzyme preparation and in the substrate. The reaction was monitored by a pH-stat (Mettler- Toledo DL 25 titration unit, Schwerzenbach, Switzerland), and the pH maintained at a constant value by the continuous addition of 1M NH4OH. The degree of hydrolysis (DH) was calculated from the volume and molarity of NH4OH used to maintain constant pH (Adler-Nissen, 1986). When the DH reached 10%, the reaction was interrupted by heating to 90 1C for 10 min, followed by cooling, freeze-drying and storage at 18 1C for analysis. At least two hydrolysis experiments were carried out for each condition studied. This yielded the following hydrolysates: NA, 65A, 95A (WPIN, WPI65, and WPI95 hydrolyzed with Alcalase); NQ, 65Q, 95Q (WPIN, WPI65, and WPI95 hydrolyzed with a-chymotrypsin,); and NP, 65P, 95P (WPIN, WPI65, and WPI95 hydrolyzed with Proteomix). 2.3. Characterization of the hydrolysates 2.3.1. Electrophoresis Characterization by electrophoresis was carried out using a high-density polyacrylamide gel (PhastGel high density, cod. 17-0516-01, Amersham Pharmacia Biotech, Uppsala, Sweden) in the presence of sodium dodecyl sulfate (SDS-PAGE) in a PhastSystem apparatus (Amersham Pharmacia Biotech). The samples (0.5% protein) dissolved in reducing buffer (0.5 M Tris-HCl, pH 6.8, 10% SDS, 10% glycerol, 5% b-mercaptoethanol and 0.1% bromophenol blue) were heated to 95 1C for 5 min. Aliquots of 5 mL were applied to the gel. After the run, the gels were stained with 0.2% Coomassie R-250 (w/v) for 3 h, and destained in a 30% methanol and 10% acetic acid solution (v/v). Molecular masses were determined by comparison with molecular mass standards from 2.5 to 16 kDa (Amersham Pharmacia Biotech, cod. 80-1129-83). Gel densitometry was done using a Sharp JX 330 densitometer and the program Image Master (Amersham Pharmacia Biotech). 2.3.2. Reversed-phase high-performance liquid chromatography (RP-HPLC) The chromatographic profiles of the hydrolysates were determined by RP-HPLC using a C18 column (25  4.6 mm, 5 mm, AI 121570-28, Varian, Palo Alto, CA, USA) equilibrated with 70% of solvent A (0.1% trifluoroacetic acid, TFA in water) and 30% of solvent B (acetonitrile/0.1% TFA, 60:40, v/v). The samples were eluted with a linear gradient up to 80% of solvent B over 35 min. The runs were conducted at room temperature in a Varian 9012 system (Varian) with a flow of 1 mL min1 and absorbance monitored at 280 nm. Injection volume was 20 mL and sample concentration 0.25% of protein (w/v).

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Before application, the samples were previously centrifuged at 10,000  g for 10 min and filtered through 22 mm cellulose acetate membranes (Smyth & Fitzgerald, 1998). For the analysis, the chromatograms of the hydrolysates were divided into three zones: (I) low hydrophobicity: peptides that eluted in up to 5 min with a solution B gradient between 30% and 35%; (II) intermediate hydrophobicity: eluted in 5–10 min with a gradient between 35% and 40%; and (III) high hydrophobicity: eluted after more than 10 min with a gradient above 40%. The peaks were integrated. All the RP-HPLC elution profiles were duplicated. 2.4. Angiotensin converting enzyme (ACE) inhibition ACE inhibitory activity of the hydrolysates was determined by capillary electrophoresis according to the method described by Shihabi (1999) with modifications (Costa, Netto, & Nunes da Silva, 2003). A volume of 50 mL containing different concentrations of each hydrolysate was pre-incubated with ACE (4 mU, 100 mL) for 5 min at 37 1C and the substrate HHL (3.8 mM, 100 mL) added to the mixture. ACE and HHL were prepared in a 100 mM borate buffer, pH 8.3, containing 300 mM NaCl. The enzyme reaction was stopped after 30 min by adding acetonitrile (250 mL). The reaction product, hippuric acid, was separated and quantified by capillary electrophoresis system (HP-3DCE Agilent, Waldbronn, Germany). The samples were mixed and injected directly into the silica capillary column (52 cm  75 mm ID; Agilent), using the hydrodynamic mode with a pressure of 50 mBar and applied voltage of 10 kV. The hippuric acid released was detected at 228 nm using a diode array detector. The IC50 value was defined as the concentration of hydrolysate (mg mL1) required to reduce the hippuric acid peak by 50% (indicating 50% inhibition of ACE). 2.5. Antihypertensive activity To determine the antihypertensive activity of the hydrolysates, experiments were conducted on male SHR aged 14–22 weeks and weighing from 270 to 300 g, supplied by the Multidisciplinary Center for Biological Investigation (CEMIB/UNICAMP, Campinas, SP, Brazil). The animals were housed in collective cages with up to four animals per cage, in an environment with a room temperature of 2471 1C, relative humidity 6075% and automatically controlled 12-hour light/dark cycle. Commercial diet (Labina/ Purina, Paulı´ nia, SP, Brazil) and tap water were supplied ad libitum. The general guidelines established by the Brazilian College for Animal Experimentation (COBEA) were followed throughout the study. The animals were randomly divided into three groups with six animals each (n ¼ 6). To the first group (experimental group), 2 mL of the hydrolysate solution was administered by intragastric intubation (500 mg kg1 of body weight); to the second group, water was given; and

the third group received captopril (10 mg kg1 of body weight). The systolic blood pressure (BP) was measured immediately before and 2 and 4 h after administration of the solutions by the tail-cuff method using an electrosphygmomanometer (Narco Bio-Systems, Austin, TX, USA). This indirect approach permits repeated measurements with close correlation (correlation coefficient ¼ 0.975) as compared to direct intra-arterial recording (Lovenberg, 1987). The hydrolysates that exhibited the highest ACE inhibitory activity were intraperiatonially administered. For this experiment, the animals were randomly divided into three groups with eight animals each (n ¼ 8). Captopril and isotonic saline solutions were used as the controls. The blood pressure measurements were taken immediately before administration and 2, 4 and 6 h after administration of the solutions. 2.6. Statistical analyses The results of the biological experiments were recorded as the average7standard error. The statistical analysis of the data was performed using the unpaired Student t-test. A P value of 0.05 was considered to indicate significance. 3. Results 3.1. Characterization of the protein isolates and hydrolysates The whey protein transition enthalpy (DH) decreased with increasing heat treatment temperature from 2.33 J g1 of untreated protein to 1.85 J g1 after treatment at 65 1C (Table 1). When the WPI was heated to 95 1C, no peaks were observed, indicating total denaturation of the protein. The values of DH obtained for WPIN and WPI65 were similar to those observed by Erdogdu, Czuchajowska, and Pomeranz (1995) for commercial whey protein concentrate (1.82 J g1). The DH value found by Ju, Hettiarachchy, and Kilara (1999) for the WPI supplied by DaviscoTM, was 1.65 J g1. Lower enthalpy values are related to protein denaturation, although other factors can influence the results, such as protein concentration, presence of minerals and the ionic strength of the environment. Table 1 Denaturation temperatures (Td) and enthalpies (DH) of unheated whey protein isolate (WPIN) and whey protein isolates heat-treated at 65 1C (WPI65) and at 95 1C (WPI95) Product

WPIN WPI65 WPI95 a

DH (J g1 of protein)

Td (1C) a-La

b-Lg

62.0570.19 63.5270.01 NDa

72.7470.01 73.2670.09 ND

ND ¼ not detected.

2.3370.21 1.8570.37 ND

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Fig. 1 shows the chromatographic profiles (RP-HPLC) of WPIN, as well as those of the treated and untreated protein hydrolyzed with Alcalase. The WPIN chromatogram showed two peaks corresponding to a-lactalbumin (a-La) and b-lactoglobulin (b-Lg) (Fig. 1a), whereas those of the hydrolysates displayed numerous peaks, characteristic of enzyme hydrolysis (Figs. 1b–d). The elution profile of the hydrolysates can be grouped into three categories of hydrophobicity of the eluted peptides (Table 2). The profiles varied with the enzyme and the previous heat treatment of the protein isolates. The hydrolysate NA showed a higher relative concentration of high-hydrophobicity peptides (51.3%), as compared with hydrolysates of isolates that had undergone heat treatment: 43.3% and 22.9% for hydrolysates 65A and 95A respectively. Protein hydrolysis by a-chymotrypsin resulted in a higher percentage of peptides concentrated in the medium-hydrophobicity region, showing a less hydrophobic profile than hydrolysates obtained using Alcalase. No difference was observed amongst hydrolysates produced by this enzyme (NQ, 65Q and 95Q). For hydrolysates obtained using Proteomix, there was a tendency for an increase in the lowhydrophobicity peptide area, with increasing temperature of the heat treatment prior to hydrolysis (Table 2).

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A significant reduction was also observed in the area corresponding to medium-hydrophobicity peptides present in the hydrolysate 95P as compared to the hydrolysate NP. The electrophoretic profile (SDS-PAGE) of WPIN and of the hydrolysates are shown in Fig. 2. WPIN was Table 2 Hydrophobicity profiles of eluted peptides of whey protein isolate hydrolysates obtained by enzymatic hydrolysis of untreated and thermally treated isolates Hydrolysatesa

NA 65A 95A NQ 65Q 95Q NP 65P 95P

Peptide contents (%)b Low

Medium

High

18.2 23.4 38.8 22.6 21.4 26.9 26.6 27.7 35.4

30.5 33.2 38.3 49.7 44.5 36.8 40.0 37.6 28.5

51.3 43.3 22.9 27.7 34.2 36.4 37.2 34.7 36.2

a

See Material and Methods for sample names. Results calculated as the ratio of the sum of the area of the peaks within the category to total area. Measurements performed in duplicate. b

Fig. 1. RP-HPLC elution profiles of (a) unheated whey protein isolate (WPIN); and hydrolysates obtained with Alcalase; (b) NA (from WPIN); (c) 65A (from WPIN heated at 65 1C); and (d) 95A (from WPIN heated at 95 1C). The chromatograms regions I, II, III indicate the hydrophobicity of eluted peptides (low, medium and high, respectively).

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1

2

3

4

5

6

7

8

9

10

β-Lg

11

16.9 kDa 14.4 kDa

α-La

10.7 kDa 8.1 kDa

6.2 kDa 2.5 kDa Fig. 2. SDS-PAGE profiles of (1) unheated whey protein isolate (WPIN); hydrolysates with Alacalase: (2) NA (from WPIN), (3) 65A (from WPIN heated at 65 1C), and (4) 95A (from WPIN heated at 95 1C); hydrolysates with a-chymotrypsin: (5) NQ (from WPIN), (6) 65Q (from WPIN heated at 65 1C), and (7) 95Q (from WPIN heated at 95 1C); hydrolysates with Proteomix: (8) NP (from WPIN), (9) 65P (from WPIN heated at 65 1C), and (10) 95P (from WPIN heated at 65 1C); (11) molecular mass markers: Globin (16.9 kDa); Globin I+II (14.4 kDa); Globin I+III (10.7 kDa); Globin I (8.2 kDa); Globin II (6.2 kDa); and Globin III (2.5 kDa).

Fig. 3. Capillary electrophoresis separation of angiotensin converting enzyme reaction mixture (a) without inhibition, and with addition of the hydrolysate 65A at concentrations of (b) 0.25 mg mL1, (c) 0.5 mg mL1, and (d) 1.0 mg mL1 (e) Hipuril-histidil-leucine (HHL) is the substrate, and hippuric acid (HA) is the product of the reaction.

characterized by the presence of the main protein fractions a-La and b-Lg (14 and 16 kDa, respectively). The a-La and b-Lg bands were not observed in the profiles of the hydrolysates NQ, 65Q and 95Q (columns 2, 3, 4), indicating complete hydrolysis of these proteins, and the two well-defined bands with average molecular masses of 5.7 and 4.2 kDa indicated the formation of low molecular mass peptides. In hydrolysates NA, 65 and 95A (columns 2, 3, 4), the formation of low molecular mass peptides also occurred, with the prevalence of two bands with average masses around 5.6 and 7.8 kDa. The profiles of hydrolysates produced by the enzyme Proteomix (columns 8, 9, 10) showed a well-defined band with molecular mass around 10 kDa.

3.2. ACE inhibition Fig. 3 shows electrophoretic profiles of the ACE reaction mixture containing WPI hydrolysates at several concentrations. Complete separation of hippuric acid and HHL was achieved with this method. The amount of hippuric acid detected decreased as the concentration of the hydrolysate increased, indicating the inhibition of ACE by the hydrolysate. The IC50 values for ACE inhibition of all hydrolysates varied between 0.05 and 0.89 mg mL1 (Table 3). The hydrolysates with the highest inhibition were those obtained with a-chymotrypsin, and the activity was even higher when the hydrolysis product was obtained from

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Table 3 IC50 values (mg mL1) of whey protein hydrolysates obtained by enzymatic hydrolysis of untreated and thermally treated whey protein isolate

Table 4 Changes of systolic blood pressure (D mmHg) from zero time after oral administration of whey protein hydrolysates (500 mg kg1 of body weight), captopril (10 mg kg1) and watera

Hydrolysatea

IC50 (mg mL1)b

Hydrolysateb

nc

2 (h)

4 (h)

NA 65A 95A NQ 65Q 95Q NP 65P 95P

0.7370.02 0.6870.10 0.7170.06 0.4070.06 0.0570.00 0.4070.07 0.8970.05 0.6670.01 0.8470.03

NA 65A 95A NQ 65Q 95Q NP 65P 95P Captopril Water

6 6 6 6 6 6 6 6 6 9 9

16.071.3d 19.179.7d 12.975.2 0.274.1 0.374.1 13.675.8d 20.073.9 6.377.1 9.572.3 43.072.9d 4.572.7

9.374.2 14.975.3d 15.775.1 4.572.9 6.273.7 11.676.1 16.873.0 10.773.2 4.874.5 29.372.4d 4.273.9

a

See Material and methods for sample names. Values presented as average of two repetitions7standard error.

b

a

Values presented as average7standard error. See Material and methods for sample names. c n ¼ number of animals per group. d Po0.05 vs. blood pressure before hydrolysate administration. b

WPI previously treated at 65 1C (65Q), reaching a IC50 value of 0.05 mg mL1, approximately 10-fold more powerful than the products obtained using the other enzymes. When the WPI was treated at 95 1C, the hydrolysates thus obtained showed an activity similar to that from native protein (NQ). The hydrolysates obtained with Proteomix showed the lowest activity, with the hydrolysate 65P exhibiting a slight better value. Hydrolysates with Alcalase showed similar activity, independent of the previous WPI treatment. Hydrolysates obtained using Proteomix had IC50 values ranging from 0.66 to 0.89 mg mL1. Van der Ven et al. (2002), using WPI and pancreatic enzymes, obtained hydrolysates with IC50 values between 0.16 and 0.84 mg mL1. The lower activities corresponded to hydrolysates with DHo 10%, with values that were similar to those obtained in the present study. Byun and Kim (2001) obtained IC50 values between 0.629 and 0.892 mg mL1 for gelatin hydrolyzed with Alcalase, comparable with those obtained in the present study of 0.68–0.73 mg mL1. For hydrolysates obtained from blood serum albumin using Alcalase, Hyun and Shin (2000) obtained IC50 values of 0.56 and 0.89 mg mL1 after hydrolysis using trypsin. Wu and Ding (2002) obtained a soy protein hydrolysate using Alcalase with high activity (IC50 ¼ 0.06 mg mL1), similar to that of the a-chymotrypsin hydrolysate, 65Q. 3.3. Hypotensive activity of the hydrolysates The arterial BP of the SHR animals after oral administration of the hydrolysates and captopril are shown in Table 4. Only hydrolysates 95Q, NA and 65A reduced significantly (Po0.05) the BP of the animals. The highest reduction in BP occurred after administration of hydrolysate 65A: 19.1 mmHg after 2 h and 14.9 mmHg after 4 h. The effect of the hydrolysates obtained from whey proteins denatured at 65 1C (65Q, 65A and 65P) on BP was also evaluated after intraperitoneal administration (Table 5). Hydrolysate 65P was unable to reduce the BP by

Table 5 Changes of systolic blood pressure (D mmHg) from zero time after intraperitoneal administration of whey protein hydrolysates (500 mg kg1 of body weight), captopril (10 mg kg1) and isotonic saline solutiona Hydrolysateb

nc

2 (h)

4 (h)

6 (h)

65A 65Q 65P Captopril Saline

8 8 8 8 8

23.0072.26d 21.8973.78d 7.6774.78 39.3472.53d 1.4273.80

24.8473.62d 22.8973.57d 3.3474.52 37.7472.30d 3.1177.11

28.0872.74d 19.5074.60 8.3475.66 32.8072.55d 4.4976.93

a

Values presented as average7standard error. See Material and methods for sample names. c n ¼ number of animals per group. d Indicates significant difference against blood pressure before hydrolysate or captopril administration (Po0.05). b

either orally or intraperitoneal administration. The hypotensive effect of hydrolysate 65A was higher when administered intraperitoneally than when administered orally (Fig. 4). The BP after 4 h was significantly reduced from 194.2 to 169.4 mmHg (D ¼ 24.84 mmHg); after 6 h, the tendency to decrease remained (D ¼ 28.08 mmHg). Hydrolysate 65Q, which was ineffective when administered orally, exhibit a significantly hypotensive effect when intraperitoneally administered. 4. Discussion According to Smithers et al. (1998) and Meisel (1998), the biological activity of whey proteins depends on the maintenance of its native structure, a form in which increases the potential for physiological functionality. It was argued that heat treatments and mechanical damage can drastically reduce the biological activity of food proteins (Smithers et al., 1996), as they may alter the profile of peptides released during gastro-intestinal digestion, since enzymes could hydrolyze parts of the protein

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638

Δ Systolic blood pressure (mmHg)

10 0 0

1

2

3

4

5

6

7

Time (h)

-10 *

-20

* * *

-30

* * *

* * *

*

-40

* *

-50

Fig. 4. Antihypertensive effect of the hydrolysates 65A and 65Q (500 mg kg1 of body weight), and captopril (10 mg kg1) after oral (closed symbols) and intraperitoneal (open symbols) administration. *Indicates significant difference against blood pressure before hydrolysate administration (Po0.05). Symbols: 65A (K); 65Q (’); captopril (~).

that were previously inaccessible to enzymatic action (Korhonen et al., 1998). In the present study, however, the best ACE inhibition results were presented by those hydrolysates obtained from isolates previously treated at 65 1C, whereas hydrolysates obtained from untreated isolates or isolates previously treated at 95 1C had significantly lower activity. This difference in activity can be explained by the structural changes induced in the WPI by different heat treatments. Heating at 65 1C possibly induced a partially unfolded conformation in whey proteins, known as molten globule state, characterized by exposition of hydrophobic clusters (Hirose, 1993). This structure, which possibly occurred in the WPI65, could allow greater access of the enzymes to certain sites previously inaccessible to enzyme action, resulting in peptides different from those obtained from WPIN, which has a more compact structure that may have hindered enzyme access to the same sites. This fact may have special impact on the action of enzymes such as a-chymotrypsin and Alcalase that preferentially cleave peptide bonds involving hydrophobic amino acids. When the temperature of the heat treatment exceeds 90 1C, the formation of aggregates occurs, mainly due to hydrophobic interaction, resulting in insoluble aggregates of high molecular mass (La Fuente, Hemar, Tamehana, Munro, & Singh, 2002). Aggregate formation in WPI95 as well as the compact structure of the untreated isolate (WPIN), may have hampered enzyme access to specific sites of the protein, releasing peptides different from those liberated during the hydrolysis of WPI65. The different hydrolysis patterns due to different heat treatments are evidenced by the molecular mass profile of the hydrolysates and by chromatographic analysis (HPLCRP). The electrophoresis (SDS-PAGE) results showed a tendency to decrease the molecular weight with increasing heat treatment temperature, implying a different enzyme action after protein denaturation. Given that all hydrolysates have the same DH, their diverse profiles indicate a possible modification in the hydrolysis mechanism due to

protein denaturation. The chromatographic profiles of the hydrolysates showed differences in the hydrophobic characteristics of the peptides released from isolates with different denaturation degrees; an increased proportion of low hydrophilicity peptides with increasing heat treatment temperature was observed for all enzymes. These differences amongst the hydrolysates may explain the variability in the ACE inhibitory activity, which is known to depend on characteristics such as low molecular mass, with less than 12 amino acid residues (Byun & Kim, 2001; Fujita et al., 2001; Yang, Marczak, Yokoo, Usui, & Yoshikawa, 2003) and structural characteristics (Yust et al., 2003). The preferred ACE inhibitors are those peptides that contain dicarboxylic amino acids in the N-terminal position, and branched-chain amino acid residues, such as valine and isoleucine (Wu and Ding, 2002). Furthermore, it is desirable to have residues of the hydrophobic amino acids tryptophan, tyrosine, phenylalanine or proline on the C-terminal (Yust et al., 2003). Of the enzymes used in the present work, a-chymotrypsin, which preferentially cleaves the C-terminal phenylalanine, tyrosine and tryptophan residues (Adler-Nissen, 1986), produced hydrolysates with the highest ACE inhibitory activity, followed by Alcalase, which shows low specificity but preferentially cleaves C-terminal hydrophobic residues. In view of the specificity of these enzymes, it is evident that the structural changes in proteins caused by different treatments, aggregate formation by hydrophobic interactions, or the presence of a compact structure with hidden hydrophobic sites, have great impact on their activity. The hydrolysates obtained with Proteomix showed the lowest activity (highest values for IC50), which may be due to the combined action of the main enzymes present, trypsin and a-chymotrypsin, which possibly fail to release peptides with an appropriate structure for inhibiting ACE. In agreement with results of the present work, Mullally, Meisel, and Fitzgerald (1997) obtained a high ACE inhibition level with a protein concentrate hydrolysate obtained with a-chymotrypsin. According to the authors, the active peptides of this hydrolysate originate from b-Lg. In order to use protein hydrolysates as a functional ingredient, it is not sufficient for them to have a high ACE inhibitory potential; they must also show an in vivo hypotensive effect. Hydrolysate 65Q, the one displaying the highest ACE-inhibitory activity, did not reduce the SHR arterial blood pressure when orally administered, whereas hydrolysates NA and 65A, which showed lower activity, with IC50 values about 10-fold higher than 65Q, were the only ones to induce a significant arterial blood pressure reduction in the animals (Po0.05). The literature shows that there may be disagreements between peptide bioactivity as determined in vitro and in vivo since ACE inhibitory peptides may not show hypotensive effect or vice versa (Fuglsang, Nilsson, & Nyborg, 2003; Fujita, Yokoyama, & Yoshikawa, 2000; Suetsuna & Nakano, 2000). Even when a high ACE inhibitory activity is present, blood pressure reduction may not occur, either because the peptide or

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hydrolysate active fraction is not absorbed intact, or because it does not reach the target site or organ. Alterations in the activity of these peptides are attributed to gastric and pancreatic enzymes and brush border peptidases. To perform their biological activity, the peptides need to be absorbed efficiently and be resistant to degradation by serum peptidases in order to reach the target organ (Fitzgerald, Murray, & Walsh, 2004). Interference by gastrointestinal enzymes in the antihypertensive activity of the hydrolysates may have been observed for hydrolysate 65Q, which, when orally administered, caused no BP reduction in the SHR but by intraperitoneal administration (in which no gastrointestinal digestion occurred), resulted in a significant reduction in BP. On the other hand, hydrolysate 65A showed the highest in vivo activity in both oral and intraperitoneal administration. Preservation of the activity after passage through the gastrointestinal tract may be related to the lower average molecular mass of this hydrolysate as compared with 65Q, which may have allowed no further hydrolysis by gastrointestinal enzymes. Interestingly, the hydrolysate 65Q showed the highest ACE inhibitory activity and also hypotensive effect after intraperitoneal administration, whereas the hydrolysate 65A showed poor ACE inhibitory activity and high hypotensive effect after intraperitoneal administration, suggesting the importance of the blood peptidases on the activity (Vermeirssen, Van Camp, & Verstraete, 2004). 5. Conclusions Partial denaturation of the whey protein isolate obtained by the heat treatment of WPIN at 65 1C for 15 min prior to enzymatic hydrolysis resulted in peptides with the highest ACE inhibitory activity, particularly when the enzyme a-chymotrypsin was used. With regard to in vivo activity, the best results in reducing arterial blood pressure in SHR were those of hydrolysates also obtained from WPI65, but with the enzyme Alcalase. These results indicated that structural changes in the protein sources and the enzyme selection are important factors for the production of hydrolysates with specific biological activity. The choice of adequate conditions can contribute to the production of hydrolysates with higher activity, reducing the need for fractionation in order to employ these products as functional ingredients. The results obtained in vivo and in vitro did not always concur, indicating that the hydrolysates may have undergone enzymatic degradation when orally administered. Thus any assessment of the potential of protein hydrolysates with regard to their antihypertensive activity should necessarily include an in vivo evaluation. Acknowledgments The authors are grateful to FAEPEX-UNICAMP (Brazil) for partially financing this project and to CNPq for the grant to author E. L. Costa.

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