Release of angiotensin converting enzyme-inhibitory peptides by simulated gastrointestinal digestion of infant formulas

ARTICLE IN PRESS

International Dairy Journal 14 (2004) 889–898

Release of angiotensin converting enzyme-inhibitory peptides by simulated gastrointestinal digestion of infant formulas Blanca Herna! ndez-Ledesma, Lourdes Amigo, Mercedes Ramos, Isidra Recio* Instituto de Fermentaciones Industriales (CSIC), Juan de la Cierva 3, Madrid 28006, Spain Received 14 November 2003; accepted 25 February 2004

Abstract The angiotensin converting enzyme (ACE)-inhibitory activity of several infant formulas was evaluated. Most of these products showed moderate inhibitory activity, but two exceptions that corresponded to an extensively hydrolysed whey formula and an extensively hydrolysed casein formula were detected. Two products (a non-hydrolysed milk protein-based formula and an extensively hydrolysed whey formula) were subjected to a two-stage in vitro enzymatic procedure, which simulates physiological digestion, in order to study the impact of digestion on ACE-inhibitory activity. The ACE-inhibitory activity of the non-hydrolysed formula increased during simulated gastrointestinal digestion, while no significant change was observed in the activity of the hydrolysed whey formula prior to and after, digestion. The peptides generated from these two products during simulated physiological digestion were sequenced by tandem spectrometry. At the end of the digestion, most peptides found in the nonhydrolysed milk protein-based formula were formed during incubation with the pancreatic extract, but, in the hydrolysed whey formula, many peptides present in the undigested product survived simulated digestion. The potential ACE-inhibitory activity of these peptides is discussed with regard to their amino-acid sequences. r 2004 Elsevier Ltd. All rights reserved. Keywords: ACE-inhibitory peptides; Infant formula; Simulated gastrointestinal digestion; Mass spectrometry; Peptide sequencing

1. Introduction Infant formulas are designed to simulate not only the content of, but also the performance of, human milk as much as possible, in order to be an adequate replacement of human milk. The most common sources of protein in infant formulas are either cows’ milk or soy protein. In some cases, in order to diminish the risk of protein allergy, these products are formulated from different types of milk protein hydrolysates (Caffarelli et al., 2002). It is now accepted that enzymatic hydrolysis of food protein releases peptides that may exhibit different biological activities. These protein fragments, known as bioactive peptides, can be formed from the precursor inactive protein during gastrointestinal digestion and/or during food processing (Meisel, 1997). Among the different groups of bioactive peptides, angiotensin converting enzyme (ACE)-inhibitory peptides are re*Corresponding author. Tel.: +34-91-5622900; fax: +34-91-5644853. E-mail address: recio@ifi.csic.es (I. Recio). 0958-6946/$ - see front matter r 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.idairyj.2004.02.011

ceiving special attention due to their potential beneficial effects related to hypertension. The formation of ACEinhibitory peptides by enzymatic hydrolysis (PihlantoLepp.al.a, Koskinen, Piilola, Tupasela, & Korhonen, 2000; Herna! ndez-Ledesma, Recio, Ramos, & Amigo, 2002), by milk fermentation (Nakamura, Yamamoto, Sakai, & Takano, 1995; Gobbetti, Ferranti, Smacchi, Goffredi, & Addeo, 2000; Leclerc, Gauthier, Bachelard, Santure, & Roy, 2002) and during cheese ripening (Saito, Nakamura, Kitazawa, Kawai, & Itoh, 2000; ! Gomez-Ruiz, Ramos, & Recio, 2002) has extensively been reported. However, once the ACE-inhibitory peptides are released by food processing they have to be able to survive the gastrointestinal system, be absorbed and reach the cardiovascular system in an active form. Several studies have revealed the importance of gastrointestinal digestion on ACE-inhibitory peptides formation. For instance Maeno, Yamamoto, and Takano (1996) identified a potent in vivo antihypertensive peptide that exhibits low in vitro ACE-inhibitory activity. These authors found that a shorter peptide

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! B. Hernandez-Ledesma et al. / International Dairy Journal 14 (2004) 889–898

with potent ACE-inhibitory activity was liberated by pacreatic digestion from the longer form. Similarly, other studies have showed an increase in ACEinhibitory activity by the action of digestive enzymes on fermented casein solutions (Pihlanto-Lepp.al.a, Rokka, & Korhonen, 1998; Vermeirssen, Van Camp, Decroos, Van Wijmelbeke, & Verstraete, 2003). However, difficulties in peptide identification limit the knowledge available on the formation of bioactive peptides and their release from the precursor proteins. Several chromatographic steps are often necessary to purify the peptides of interest and finally they are identified by mass spectrometry (MS) alone or MS combined with N-terminal sequence analysis. The development of routine and reliable liquid chromatography-MS instruments made it possible to analyse peptides included in complex mixtures without the need to separate the individual components (Papayannopoulos, 1995). If the precursor proteins are known, the fragmentation spectra can be matched to the sequences of the selected peptides with a given mass, thereby allowing the unambiguous identification of peptides from unfractionated enzyme digests of proteins (Biemann & Scoble, 1987; McLuckey, Van Berkel, Glish, Huang, & Henion, 1991). The aim of our study was first to investigate the presence of ACE-inhibitory substances, as naturally occurring components in commercial infant formulas. Two of these products were subjected to an enzymatic hydrolysis process, which simulates physiological digestion, in order to study the formation of ACE-inhibitory peptides. The peptides generated from both products during simulated physiological digestion were sequenced by tandem MS. The potential ACE-inhibitory activity of these peptides is discussed in relation to their structure.

2. Materials and methods 2.1. Samples and simulation of gastrointestinal digestion Milk protein and soy-based infant formulas (IF-1— IF-11) were purchased on the Spanish market. Infant formulas IF-2 and IF-7 corresponded to enzymatically hydrolysed casein formula and enzymatically hydrolysed whey proteins, respectively. Infant formula IF-5 was a soy proteins-based formula and the other infant formulas analysed in the study contained mainly nonhydrolysed milk proteins. Two infant formulas (IF-1 and IF-7) were selected to simulate gastrointestinal digestion. Hydrolysates were prepared from an aqueous solution of the formulas IF-1 and IF-7 (0.7%, w/v, protein). The hydrolysis was carried out according to Alting, Meijer, and van Beresteijn (1997). The samples were first hydrolysed with pepsin (EC 3.4.4.1; 1:60,000, 3400 U mg
1) (Sigma

Chemical, St. Louis, MO, USA), which was added at a level of 20 mg g
1 protein, for 90 min at 37 C at pH 3.5 . followed by hydrolysis with Corolase PPs (Rohm, Darmstadt, Germany), which was added at a level of 40 mg g
1 protein, at pH approx. 7.5 and 37 C for 240 min. Corolase PPs is a proteolytic enzyme preparation from pig pancreas glands that contains, in addition to trypsin and chymotrypsin, numerous amino- and carboxipeptidase activities. Hydrolysis was carried out in a thermally controlled water bath under constant stirring. Aliquots were withdrawn after hydrolysis with pepsin, the pH was raised to 7.5 with 1 m NaOH and they were heated at 95 C for 10 min in a water bath. During hydrolysis with Corolase PPs, aliquots were also taken at 30, 120 and 240 min.The enzyme was inactivated by heating at 95 C for 10 min, followed by cooling to room temperature. Each sample was stored at
20 C until further analysis. After the last samples had been taken, aliquots and the remaining reaction mixtures were centrifuged at 10,000  g for 30 min and the supernatants were subjected to ultrafiltration through an hydrophilic 3000 Da cut-off membrane (Centripep, Amicon, Inc., Beverly, MA, USA). The permeates were freeze-dried and kept at
20 C until required. Water-soluble extracts (WSE) of the infant formulas were obtained by dissolving 4.5 g of the product in 30 mL of distilled water. The pH was adjusted to 4.6 with 1 m HCl and the reconstituted formula was then centrifuged at 12,000  g for 20 min at 5 C and filtered through a Whatman no. 40 filter.

2.2. Measurement of ACE-inhibitory activity ACE-inhibitory activity was measured by the spectrophotometric assay of Cushman and Cheung (1971), as . modified by Kim, Yoon, Yu, Lonnerdal, and Chung (1999). Briefly, 20 mL of each sample was added to 0.1 mL of 0.1 m potasium phosphate buffer (pH 8.3) containing 0.3 m NaCl, and 5 mm hippuryl–histydil– leucine (Sigma Chemical, St. Louis, MO, USA). ACE (5 mU) (EC 3.4.15.1, 5.1 U mg
1, Sigma) was added and the reaction mixture was incubated at 37 C for 30 min.The reaction was terminated by the addition of 0.1 mL 1 m HCl. The hippuric acid formed was extracted with ethyl acetate, heat-evaporated at 95 C for 10 min, redissolved in distilled water and measured spectrophotometrically at 228 nm.The activity of each sample was tested in triplicate. The ACE-inhibitory activity of those products with ACE-inhibitory indexes higher than 50% was also calculated as the protein concentration needed to inhibit 50% the original ACE activity (IC50), and one unit of ACE-inhibitory activity was expressed as the potency showing 50% ACE inhibition under these conditions.

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100 50

IF-3

IF-4

IF-5

IF-6

IF-7

IF-8

IF-9

IF-10

IF-11

IF-3

IF-4

IF-5

IF-6

IF-7

IF-8

IF-9

IF-10

IF-11

-1

0

IF-2

In a first experiment, the ACE-inhibitory activities of the pH 4.6-WSE of the infant formulas were screened.

150

IF-2

3.1. ACE-inhibitory activity of commercial infant formulas

200

IF-1

3. Results and discussion

250

IF-1

RP-HPLC separations of the WSE were performed on a Agilent HPLC system connected on line to an EsquireLC quadrupole ion trap instrument (Bruker Daltonik GmbH, Bremen, Germany). The HPLC system was equipped with a quaternary gradient pumping system, an in-line degasser, a variable wavelength absorbance detector set at 220 nm, and an automatic injector (all 1100 Series, Agilent Technologies, Waldbronn, Germany). The column used in these experiments was a 250 mm  4.6 mm Widepore C18 column (Bio-Rad, Richmond, CA, USA). The injection volume was 50 mL. Solvent A was a mixture of water and trifluoroacetic acid at a v/v ratio of 1000–0.37, and solvent B contained acetonitrile and trifluoroacetic acid at a v/v ratio of 1000– 0.27. Peptides were eluted with a linear gradient of solvent B in A going from 0% to 45% in 60 min at a flow rate of 0.8 mL min
1. The flow was split post detector by connecting a T-piece (Valco, Houston, TX, USA) with a 75 mm ID peek outlet tube, of an adjusted length, to give a flow of approx. 20 mL min
1 which was directed into the mass spectrometer via the electrospray interface. Nitrogen was used as nebulizing agent and drying gas and operated with an estimated helium pressure of 5  10
3 bar. The capillary was held at 4 kV. Spectra were recorded over the mass/charge (m/z) range 100–2500. About 25 spectra were averaged in the MS analyses and about 5 spectra in the MS(n) analyses. The signal threshold to perform auto MS(n) analyses was 10000 (i.e., 5% of the total signal) and the precursor ions were isolated within a range of 4.0 m/z and fragmented with a voltage ramp going from 0.35 to 1.4 V. Using Data AnalysisTM (version 3.0; Bruker Daltoniks, GmbH, Bremen, Germany), the m/z spectral data were processed and transformed to spectra representing mass values. BioTools (version 2.1; Bruker Daltoniks) was used to process the MS(n) spectra and perform peptide sequencing.

Peptide nitrogen (mg 100g )

2.3. Analysis by on-line reversed-phase high performance liquid chromatography coupled on line to tandem mass spectrometry (RP-HPLC-MS/MS)

The peptide nitrogen of the pH 4.6-WSE was also determined to establish if the measured ACE-inhibitory activity of these formulas was related to their peptide content. Fig. 1 shows the measured ACE-inhibitory indexes and the peptide content of the pH 4.6-WSE of these infant formulas. The ACE-inhibitory activity of nine of the 11 products did not reach one inhibitory unit (i.e., ACE-inhibitory indexes lower than 50%) and their peptide contents ranged from 50 to 120 mg nitrogen 100 g
1. Only two of the commercial infant preparations, IF-2 and IF-7, exhibited a potent activity with ACE-inhibitory indexes of 93% and 88%, respectively. The ACE-inhibitory activity of these two infant formulas, with ACE-inhibitory indexes >50%, was also calculated as IC50, i.e., the concentration needed to inhibit 50% the original ACE activity. IC50 values of 101.3 mg peptide nitrogen mL
1 (1609 ACE-inhibitory U mL
1) and 81.4 mg peptide nitrogen mL
1 (1978 ACE-inhibitory U mL
1) were obtained for IF-2 and IF-7, respectively. These two samples corresponded to infant formulas prepared from milk protein

100 80 IACE (%)

The total nitrogen content of the WSE was determined by the Kjeldahl method. Amino acid nitrogen was measured using the Cd-nynhidrin method according to Doi, Shibata, and Matoba (1981). The content of peptide nitrogen was calculated as the difference of total nitrogen minus amino-acid nitrogen.

891

60 40 20 0

Fig. 1. Values of the ACE-inhibitory indices (IACE), expressed as percentages, and peptide content, expressed as milligrams of peptide nitrogen 100 g
1 of product, for the pH 4.6-water-soluble extracts obtained from various reconstituted commercial infant milk formulas (IF1–IF11).

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892

hydrolysates, and consequently, the peptide content of these samples (196.3 and 226 mg nitrogen 100 g
1) was higher than that observed in the pH 4.6-WSE of the other infant formulas. Results of the RP-HPLC analyses of these pH 4.6-WSE (data not shown) strongly supported the results of the peptide nitrogen analysis. To date, there is little information available on the ACE-inhibitory activity in infant milk formulas, but Meisel, Goepfert, and Gunther . (1997) found an ACEinhibitory index of 31.4% in an hypoallergenic infant food, a value which concurs with the activity found in most samples considered in this study. 3.2. Hydrolysis under simulated gastrointestinal conditions In order to study both the survival and the formation of new ACE-inhibitory peptides, two infant formulas, IF-1 and IF-7, with different ACE-inhibitory behaviour, were subjected to a hydrolysis process, which simulates physiological digestion. IF-1 corresponded to a nonhydrolysed milk protein-based formula and IF-7 to an extensively hydrolysed whey protein formula. During simulated physiological digestion, the ACE-inhibitory index of IF-1 significantly increased after the hydrolysis with pepsin while no significant change was observed during hydrolysis with the pancreatic extract (Fig. 2). The ACE-inhibitory activity of IF-7, which was relatively high before digestion (79%), remained stable after hydrolysis with pepsin (83%) but slightly decreased after 30-min hydrolysis with Corolase PP (56%) (Fig. 2). As a result, the ACE-inhibitory activities of IF-1 and IF-7 after simulated physiological digestion were higher than (IF-1), or similar to (IF-7), those observed before hydrolysis. It can be concluded that the constituent proteins of IF-1 act as precursors of ACEinhibitory peptides by the action of gastrointestinal

enzymes. In the case of hydrolysed formula (IF-7), probably some ACE-inhibitory peptides present in the undigested product may resist simulated gastrointestinal digestion. The ACE-inhibitory activity and the peptide content of permeates, obtained following ultrafiltration of the hydrolysates, were also assayed. It has to be noted that the permeates exhibited activities similar to those of the total hydrolysates. Moreover, changes in the activity of the permeates during simulated physiological digestion of the infant formulas followed a trend similar to that observed for the total hydrolysates (Fig. 2). Therefore, it can be inferred that the peptides responsible for the ACE-inhibitory activity in the undigested and digested infant milk formulas have low molecular masses (o3000 Da). The fact that small peptides make a considerable contribution to the ACE-inhibitory activity of protein hydrolysates had previously been suggested by other authors (Mullally, Meisel, & Fitzgerald, 1997). Moreover, low molecular mass peptides have good potential to play a physiological antihypertensive role in vivo. 3.3. Identification of peptides during simulated physiological digestion In order to identify peptide sequences, the 3 kDapermeates from IF-1 and IF-7 prior to, and after, simulated physiological digestion were analyzed by RPHPLC coupled on-line with a mass spectrometer. In our case, the mass spectrometer was a quadrupole ion trap capable of multiple stages of mass analysis from a single precursor ion. Fig. 3A shows the UV-chromatograms obtained for IF-7 before and after simulated digestion. The mass spectrum of one selected peak is shown in Fig. 3B and the MS/MS spectrum of a single charged ion with m/z 775.5 and the amino acid sequence of the 100

100

IF-7

80

80

60

60

IACE (%)

IACE (%)

IF-1

40

40

20

20

0

0 Undigested

Pepsin 90 min

Corolase 30 min Corolase 120 min Corolase 240 min

Progression of simulated digestion Total hydrolysate

Fraction < 3000 Da

Undigested

Pepsin 90 min

Corolase 30 min Corolase 120 min Corolase 240 min

Progression of simulated digestion Total hydrolysate

Fraction < 3000 Da

Fig. 2. ACE-inhibitory activity (IACE) of two infant milk formulas, IF-1 and IF-7 (’), and of the corresponding permeates () obtained on ultrafiltration through a 3 kDa membrane of aliquots of the reconstituted formula withdrawn during simulated physiological digestion with pepsin and Corolase PP. See text Section 2.1 for details of digestion.

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893

1600

Absorbance 220 nm (mAU)

1400 1200 1000 800

after simulated digestion

600 400 200

before simulated digestion

0 0

(A)

10

20

30

40

50

Time (min) 775.5

4

Intensity⋅104

3 28 433.224 y4

433.3

2

1001.4

24

546.4 674.4

1

Intensity⋅103

20

610.368 b6

0 200

(B)

400

600

800

1000

m/z

16

12

8

546.257 y5

265.066 y2

511.321 b5

4

757.436 b7

0

(C)

300

400

500

600

700

800

900

1000

m/z

b ions

5

T K I y´´ ions

5

P A 4

6

V

F

2

Fig. 3. (A) UV-chromatograms of the permeate obtained on ultrafiltration (through a membrane with a molecular mass cut-off of 3 kDa) of the reconstituted infant milk formula IF-7 before and after simulated physiological digestion. (B) Mass spectrum of the selected chromatographic peak in Fig. 3A. (C) Tandem mass spectrum of ion m/z 775.5. Following sequence interpretation and database searching, the MS/MS spectrum was matched to b-lactoglobulin f(76–81). The sequence of this peptide is displayed with the fragment ions observed in the spectrum. Fragment ions are labeled according to the nomenclature proposed by Roepstorff and Fohlman (1984). For clarity, only the b and the y00 fragment ions are labelled.

identified peptide with the major fragment ions is shown in Fig. 3C. The major fragment ion was observed at m/z 433.2 that corresponded to the y00 - ion adjacent to the amino-acid proline. This amino acid is associated with very abundant y00 -type ions which are often easily identifiable because these ions are over-represented in the spectrum (Papayannopoulus, 1995). All peptides of the total ion chromatogram with a signal higher than 10,000 units were considered for peptide sequencing. However, few detected masses and the corresponding

fragmentation spectra, obtained by tandem MS, could not be matched with any peptide fragment obtained by milk protein hydrolysis. The formation of the peptides during simulated gastrointestinal digestion could be followed by the analysis of the ultrafiltration permeates (using a 3 kDa cut-off membrane) of the aliquots withdrawn during simulated digestion by HPLC-MS, and by the extraction of the characteristic ion of the peptides previously identified in the digested, or in the undigested, infant formulas.

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Table 1 Peptides identified in the permeate obtained on ultrafiltration through a 3 kDa membrane of a reconstituted infant milk formula IF-7 before and after simulated physiological digestion

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40

Obs. massa

Calc. massb

Protein fragment

Sequence

445.3 673.4 932.4 544.3 502.3 1040.4 645.4 436.3 459.3 528.3 627.5 774.5 427.3 398.3 674.3 374.2 445.3 715.4 640.3 346.2 459.3 386.3 803.3 446.3 331.2 536.3 424.3 677.3 477.2 617.4 259.1 645.3 549.4 525.3 1022.4 655.4 444.0 446.3 627.5 747.4

444.30 673.40 932.54 544.29 502.28 1040.49 645.33 436.24 459.27 528.33 627.40 774.46 427.28 398.25 673.42 374.22 445.25 715.39 640.27 346.19 460.22 386.26 803.32 446.20 331.17 536.30 424.26 677.34 477.22 618.29 259.15 645.30 549.29 525.27 1022.51 655.35 443.24 446.20 627.41 746.40

b-Lg f(1–4) b-Lg f(1–6) b-Lg f(1–8) b-Lg f(9–13) b-Lg f(11–14) b-Lg f(20–29) b-Lg f(30–35) b-Lg f(40–42) b-Lg f(72–75) b-Lg f(76–80) b-Lg f(76–81) b-Lg f(76–82) b-Lg f(77–80) b-Lg f(78–81) b-Lg f(78–83) b-Lg f(83–85) b-Lg f(83–86) b-Lg f(88–93) b-Lg f(96–100) b-Lg f(115–117) b-Lg f(120–123) b-Lg f(122–124) b-Lg f(125–131) b-Lg f(130–133) b-Lg f(131–133) b-Lg f(135–138) b-Lg f(146–148) b-Lg f(149–154) b-Lg f(151–154) b-Lg f(154–158) b-Lg f(155–156) b-Lg f(155–159) a-La f(7–10) a-La f(104–107) BSA f(69–77) BSA f(109–114) BSA f(223–226) BSA f(290–293) BSA f(454–458) BSA f(567–573)

LIVT LIVTQT LIVTQTMK GLDIQ DIQK YSLAMAASDI SLLDAQ RVY IAEK TKIPA TKIPAV TKIPAVF KIPA IPAV IPAVFK KID KIDA NENKVL DTDYK QSL QCLV LVR TPEVDDE DEAL EAL KFDK HIR LSFNPT FNPT TQLEE QL QLEEQ EVFR WLAH LFGDELCKV SPDLPK PKAE AEVE LILNR FAVEGPK

ACE-inhibitory peptidesc

IC50d (mm)

Reference

GLDIQK

580

Pihlanto-Lepp.al.a et al. (1998)

RVY

205.6

Matsufuji et al. (1994)

IPA

141

Abubakar et al. (1998)

IPA

141

Abubakar et al. (1998)

VFK

1029

Pihlanto-Lepp.al.a et al. (2000)

VLDTDYK

946

Pihlanto-Lepp.al.a et al. (2000)

LVR

14

Maruyama et al. (1989)

ALPMHIR

42.6

Mullally et al. (1997)

Peptide formatione 1, 3, 1, 1, 1, 1, 1, 1, 1, 1, 1, 1, 1, 1, 3, 1, 1, 1, 1, 3, 1, 1, 1, 1, 1, 1, 1, 1, 1, 1, 1, 1, 1, 5 5 5 1, 1, 1, 3,

2, 4, 2, 2, 2, 2, 2, 2 2, 2, 2, 2 2, 2, 4, 2, 2, 2 2, 4, 2, 2, 2, 2, 2, 2, 2 2, 2, 2, 2, 2, 2

3, 4, 5 5 3 3, 4, 5 3 3 3, 4, 5 3 3, 4, 5 3, 4, 5 3, 3, 5 3, 3, 3 5 3, 3, 3, 3, 3, 3 3, 3, 3 3, 3,

4, 5 4, 5 4, 5 4, 5

4 4, 4, 4, 4,

5 5 5 5

4, 5 4, 5 4 4, 5

2, 3, 4, 5 2, 3, 4, 5 2 4, 5

a

Observed mass (Da). Calculated monoisotopic mass (Da). c Previously described ACE-inhibitory peptides that share at least three C-terminal residues with those found in this study. d Protein concentration needed to inhibit 50% the original ACE activity. e (1) undigested product; (2) digestion with pepsin for 90 min; (3) digestion with pepsin for 90 min and Corolase PP for 30 min; (4) digestion with pepsin for 90 min and Corolase PP for 120 min; (5) digestion with pepsin for 90 min and Corolase PP for 240 min. b

A total of 40 peptide fragments were identified in the permeates of the digested and undigested IF-7 (Table 1). Most of them derived from b-lactoglobulin (b-Lg), and only 8 of the total identified peptides corresponded to alactalbumin and bovine serum albumin fragments. Of the 40 peptides, 32 sequences were already found in the permeate of the undigested product although the concentration of some of them increased markedly during digestion. Only 7 peptides could not be found in the undigested sample and these fragments were all

formed during digestion with Corolase PP (that is peptides 2, 15, 20, 34, 35, 36 and 40 in Table 1). These results demonstrate that most of the peptide fragments present in the extensively hydrolysed whey formula survived simulated gastrointestinal digestion and only some new peptides were formed during hydrolysis with the pancreatic extract. Because the UV- and the total ion current chromatograms of the undigested IF-1 permeate showed a low peptide content, peptide identification was first

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Table 2 Peptides identified in the permeate obtained on ultrafiltration through a 3 kDa membrane of a reconstituted infant milk formula IF-1 after simulated physiological digestion Obs. Massa

Calc. Massb

1 2 3

674.3 401.3 346.2

674.32 401.24 346.19

4 5 6 7 8 9 10 11

789.4 516.4 747.4 603.3 750.4 575.4 688.4 651.4

789.41 516.27 747.36 603.29 750.36 575.34 688.43 651.40

12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41

503.3 1000.5 580.4 741.5 678.4 904.4 633.4 525.3 644.4 910.4 528.3 432.2 489.3 449.3 1022.4 655.4 516.4 445.3 673.4 560.3 655.4 627.4 427.3 398.3 374.2 645.3 346.2 472.2 803.3 677.3

503.24 1000.52 580.36 741.44 678.35 904.47 633.35 525.26 644.32 910.46 528.27 431.21 488.33 450.22 1022.58 655.37 516.30 444.30 673.40 560.32 601.34 627.40 427.28 398.25 374.22 645.26 346.19 471.28 803.32 677.34

Protein fragment

Sequence

b-CN f(1–5) b-CN f(25–27) b-CN f(56–58) f(123–125) b-CN f(60–66) b-CN f(101–105) b-CN f(108–113) b-CN f(114–118) b-CN f(114–119) b-CN f(134–138) b-CN f(134–139) b-CN f(170–175)

RELEE RIN QSL YPFPGPI AMAPK EMPFPK YPVEP YPVEPF HLPLP HLPLPL VLPVPQ

b-CN f(179–182) b-CN f(193–201) b-CN f(196–201) b-CN f(203–209) aS1-CN f(8–13) aS1-CN f(24–31) aS1-CN f(104–108) aS1-CN f(125–129) aS1-CN f(133–138) aS1-CN f(163–169) aS2-CN f(106–109) aS2-CN f(125–127) aS2-CN f(150–153) k-CN f(15–17) k-CN f(24–31) k-CN f(106–111) k-CN f(111–114) b-Lg f(1–4) b-Lg f(1–6) b-Lg f(2–6) b-Lg f(11–15) b-Lg f(76–81) b-Lg f(77–80) b-Lg f(78–81) b-Lg f(83–85) b-Lg f(108–113) b-Lg f(115–117) b-Lg f(123–126) b-Lg f(125–131) b-Lg f(149–154)

PYPQ YQEPVLGPV PVLGPV GPFPIIV HQGLPQ FVAPFPEV YKVPQ EGIHA EPMIGV AWYYVPL LNPW REQ KTKL ERF KYIPIQYV MAIPPK KKNQ LIVT LIVTQT IVTQT EIVES TKIPAV KIPA IPAV KID ENSAEP QSL VRTP TPEVDDE LSFNPT

ACE-inhibitory peptidesc

IC50d (mm)

Reference

Peptide formatione 2, 3, 4, 5 3, 4, 5 3, 4, 5

YPFPGPI

500

Kayser and Meisel (1996)

MPFPKYPVQPF HLPLP HLPLP SKVLPVPQ PPQSVLSLSQSK-VLPVPQ

nr 23.6 23.6 39 25

Saito et al. (2000) Kohmura et al. (1989) Kohmura et al. (1989) Yamamoto et al. (1994)

GPV GPV LLYQQPVLGP-VRGPFPIIV RPKHPIIKKHQG-LPQ

1.2 1.2 21 nrf

Kim et al. (2001) Kim et al. (2001) Yamamoto et al. (1994) Saito et al. (2000)

KKYKVPQ

716.9

! Gomez-Ruiz et al. (2002)

GAWYYVPL

>1000

Yamamoto et al. (1994)

IPA

141

Abubakar et al. (1998)

5 3, 3, 3, 3, 3, 3, 3,

4, 4, 4, 4, 4, 4, 4,

5 5 5 5 5 5 5

3, 3, 3, 3, 3, 3, 3, 3, 3, 3, 3, 4, 3, 2, 3, 3, 3, 3, 3, 3, 3, 3, 3, 3, 3, 3, 3, 3, 3, 3,

4, 4, 4, 4, 4, 4, 4, 4, 4, 4, 4, 5 4, 3, 4, 4, 4, 4, 4, 4, 4, 4, 4, 4, 4, 4, 4, 4, 4, 4,

5 5 5 5 5 5 5 5 5 5 5 5 4, 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5

a

Observed mass (Da). Calculated monoisotopic mass (Da). c Previously described ACE-inhibitory peptides that share at least three C-terminal residues with those found in this study. d Protein concentration needed to inhibit 50% the original ACE activity e (1) undigested product (2) digestion with pepsin for 90 min; (3) digestion with pepsin for 90 min and Corolase PP for 30 min; (4) digestion with pepsin for 90 min and Corolase PP for 120 min;(5) digestion with pepsin for 90 min and Corolase PP for 240 min. f Not reported. b

performed in the permeate of the digested sample. From the 41 identified sequences, 15 corresponded to b-casein fragments, 9 to as1- and as2-casein fragments, 4 to kcasein fragments, and 13 to b-Lg fragments (Table 2). Ion extraction of the ions corresponding to these peptides in the permeate of the sample prior to hydrolysis revealed that none of these fragments existed

in the undigested sample. Most of the peptides appeared after 30 min hydrolysis with Corolase PP and only two peptides (peptides 1 and 25 in Table 2), which were formed by peptic hydrolysis, survived further hydrolysis with the pancreatic extract. Among the identified peptides, we found four peptides that had previously been described as ACE-inhibitors.

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The b-Lg peptides RVY and LVR (peptides 8 and 22 in Table 1) had demonstrated strong ACE-inhibitory activity with IC50 values as low as 205.6 and 14 mm, respectively (Matsufuji et al., 1994; Maruyama, Miyoshi, & Tanaka, 1989). Similary, b-casein peptides, YPFPGPI and HLPLP (peptide 4 and 9, in Table 2), have been found to exhibit ACE-inhibitory activities with IC50 values of 500 and 23.6 mm, respectively (Kayser & Meisel, 1996; Kohmura, Nio, Kubo, Minoshima, & Munekata, 1989). Moreover, some of the peptides identified in this study share some of structural features with ACE-inhibitory peptides previously described (Pihlanto-Lepp.al.a et al., 2000; Yamamoto, Akino, & Takano, 1994). ACE appears to prefer substrates or competitive inhibitors containing hydrophobic amino acids at the three C-terminal positions. Among the most favorable C-terminal amino acids are aromatic amino acids, as well as the imino acid proline. ACE binds only weakly to peptides that have terminal dicarboxylic amino acids (Cheung, Wang, Ondetti, Sabo, & Cushman, 1980). Some peptides included in Tables 1 and 2 ended with proline or aromatic residues (for example, peptides 7, 9 and 22 in Table 2). In addition, some peptides share some C-terminal residues with different ACE-inhibitory peptides previously described in the literature (Tables 1 and 2). For instance, peptides 10 and 13 (Table 1) and peptide 34 (Table 2) share the three C-terminal residues with peptide IPA. This ACE-inhibitory tri-peptide (IC50 value of 141 mm) had previously been found in a hydrolysate of whey proteins with proteinase K (Abubakar, Saito, Kitazawa, Kawai, & Itoh, 1998). Structure–activity correlations of many ACE-inhibitory peptides indicate that their C-terminal tripeptide residues play a predominant role in competitive binding to the active site of ACE. Although the activity of di- or tri-peptides with ACEinhibitory activity can not always be strictly extrapolated to larger peptides (Cushman, Cheung, Sabo, & Ondetti, 1977), the structural similarity of the Cterminal region may allow the prediction similar activity. Similarly, peptides 13 and 14 in Table 2 share the C-terminal tri-peptide with GPV, an active peptide isolated from bovine skin-gelatin hydrolysate (Kim, Byun, Park, & Shahidi, 2001). The presence of these sequences in the hydrolysate can explain the ACEinhibitory activities found in the digested IF-1, and in the ultrafiltration permeates corresponding to the undigested and digested IF-7 formula. The structure of some of the peptide sequences found in the digested permeates may suggest the presence of other biological activities in this hydrolysate. For instance, b-Lg f(78–83) (peptide 15 in Table 1) has been found to exert antimicrobial activity (Pellegrini, Dettling, Thomas, & Hunziker, 2001) and peptide b-Lg f(146–148) (peptide 27 in Table 1) is very similar to b-Lg f(146–149) which possesses opioid activity (Pihlanto-

Lepp.al.a, Paakkari, Rinta-Koski, & Antila, 1997). Similarly, b-casein f(60–66) (peptide 4 in Table 2) has immunomodulatory and opioid activities (Kayser & Meisel, 1996; Meisel, Frister, & Schlimme, 1989; Migliore-Samour, Floc’h, & Jolle" s, 1989). The peptide b-casein f(170-175) (peptide 11 in Table 2) may possess antioxidant activity, as occurs with b-casein f(170-176) (Rival, Boeriu, & Wichers, 2001). The peptides b-casein f(193-201) and b-casein f(106-111) (peptide 13 and 27) have the same sequence, with the exception of the C-terminal residue, as b-casein f(193–202) and b-casein f(106–112) which exhibit immunomodulatory and antithrombotic activity, respectively (Meisel & Schlimme, 1994; Bouhallab, Molle! , & Le! onil, 1992).

4. Conclusion The present results demonstrated the presence of low in vitro ACE-inhibitory activity in most of the commercial infant milk formulas considered. However, two formulas, which corresponded to extensively hydrolysed milk protein formulas, showed potent ACE-inhibitory activity. The ACE-inhibitory activity of a non-hydrolysed milk protein-based formula (IF-1) increased after simulated gastrointestinal digestion, but the activity of a hydrolysed-whey formula (IF-7) did not change after hydrolysis with gastrointestinal enzymes. The use of tandem MS and extraction of the ion of interest allowed us to identify the peptides formed at the end of the simulated digestion, and to follow their formation during simulated physiological digestion. Most of peptides found at the end of the simulated gastrointestinal digestion of the milk protein-based formula (IF-1) were formed during incubation with the pancreatic extract. However, the peptides identified in the ultrafiltration permeates of both the undigested, and digested hydrolysed-whey formula (IF-7) were similar, thereby demonstrating that most of the peptides in the hydrolysed-whey formula survive physiological digestion. Some of the peptides identified in the hydrolysates correspond to previously described ACEinhibitors, and to peptides which had been previously reported as antimicrobials, immunomodulators and opioid fragments.

Acknowledgements This work has received financial support from the Projects AGL 2000-1480 and CAL01-046-C2. The . authors acknowledge Rohm Enzyme Gmbh for providing the enzymatic preparation (Corolase PP). B.H.-L. was the recipient of a fellowship from the Ministerio de ! y Cultura, Spain. Educacion

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