Identification and Characterization of Gastrointestinal-Resistant

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Cite This: J. Agric. Food Chem. 2019, 67, 7147−7156

Identification and Characterization of Gastrointestinal-Resistant Angiotensin-Converting Enzyme Inhibitory Peptides from Egg White Proteins Hongbing Fan, Jiapei Wang, Wang Liao, Xu Jiang, and Jianping Wu* Department of Agricultural Food and Nutritional Science, University of Alberta, 4-10 Ag/For Building, Edmonton, Alberta T6G 2P5, Canada Downloaded via UNIV OF SOUTHERN INDIANA on July 28, 2019 at 02:48:24 (UTC). See https://pubs.acs.org/sharingguidelines for options on how to legitimately share published articles.

S Supporting Information *

ABSTRACT: Egg proteins are recognized as excellent sources of bioactive peptides, such as angiotensin-converting enzyme inhibitory (ACEi) peptides. Oral administration of a thermolysin-digested egg white hydrolysate (T-EWH) caused a significant blood pressure reduction in spontaneously hypertensive rats; a further ACEi assay implied that its ACEi activity was enhanced after in vitro gastrointestinal (GI) digestion. These results indicated that T-EWH contained ACEi peptides resisting GI digestion and/or being further released during GI digestion. Therefore, the objective of this study was to identify these responsible ACEi peptides from T-EWH. The conventionally activity-guided fractionation was applied, coupled with a synchronized GI digestion throughout, during which both peptide yield and ACEi activity before and after the GI digestion were measured. Finally, six ACEi peptides (LAPYK, LKISQ, LKYAT, INKVVR, LFLIKH, and LGHWVY) with good GI resistance were identified with IC50 values 97%; Piscataway, NJ, USA). Preparation of T-EWH. A 900 g amount of manually separated liquid EW (∼11% protein) was dissolved in 2 L of deionized distilled (dd) H2O. After being heated at 90 °C for 10 min, the EW slurry was hydrolyzed by thermolysin (0.1% enzyme/substrate, E/S, w/w protein) for 1.5 h at 65 °C, pH 8.0 in a jacket beaker, connected with a circulating water bath (Brinkman, Mississauga, ON, Canada) for temperature control and Titrando (Metrohm, Herisan, Switzerland) for pH control. Then, the temperature was increased to 95 °C, kept for 10 min, to terminate the reaction, followed by centrifugation at 10,000g for 15 min at 4 °C. The supernatant was freeze-dried and kept at −20 °C for further analysis. Ethics Statement, Animal Model, and Telemetry Recording. Rat experimental procedures were approved by the University of Alberta Animal Welfare Committee (No. AUP 00001571) in accordance with the guidelines issued by the Canadian Council on Animal Care and also in adherance with the Guide for the Care and Use of Laboratory Animals published by the United States National Institutes of Health. Twelve- to 14-week-old SHRs (290 ± 10 g, n = 3) were obtained from the Charles River (Senneville, QC, Canada). Upon arrival, they were acclimatized for 1 week in the University of Alberta animal core facility, exposed to a 12:12 h of light:dark cycle with controlled temperature and humidity. Rats were fed with standard rat chow and water ad libitum. Then, they were surgically implanted with telemetry transmitters (HD-S10, Data Sciences International, St. Paul, MN, USA) as previously described;14 a 7 day period was allowed for the postsurgery recovery. T-EWH was administrated orally to rats (1,000 mg/kg of body weight) once per day for 18 days by dissolving it in 20 mL of 10% (in ddH2O, v/v) Ensure (Abbott Nutrition, QC, Canada); the dose and timing of blood pressure recording were chosen based on our previous study.15

Blood pressure and heart rate were recorded by the telemetry system and were calculated as described previously.14 Ultrafiltration. A solution of the T-EWH was ultrafiltered using 10- and 3-kDa molecular weight cutoff (MWCO) membranes (Amicon Division, W. R. Grace & Co., Beverly, MA, USA) described by You and Wu.16 Then, three fractions of T-EWH with 10 kDa (III) were collected and freeze-dried for further uses. Purification of ACEi Peptides from T-EWH. Purification of ACEi peptides followed procedures of Majumder and Wu.9 with some modifications. T-EWH was first fractionated using a cation exchange High-Prep 16/10 SP FF column (16 mm × 100 mm, GE Healthcare Sweden) coupled with an AKTA explorer 10XT system. Samples (1.5 mL, 10 mg/mL) were linearly eluted (3 mL/min) using ammonium acetate (10 mM, pH 4.0, solvent A) and ammonium carbonate (0.5 M, pH 8.8, solvent B): 0%−0%−15%−100%−100%−0% B (0−1.5− 6.5−7.5−9.5−10 column volume). Fractions were collected and desalted (described in the Supporting Information) prior to ACEi activity assay. Two potent fractions (F3 and F4) were further purified through reverse-phase high-performance liquid chromatography (RPHPLC, Waters), with a guard column (40 mm × 10 mm, 5 μm, Waters, Milford, MA, USA), an X-bridge preparative C18 column (10 mm × 150 mm, 5 μm, Waters, Milford, MA, USA), and a 2998 photodiode array (PDA) detector, on a Waters 600 system. Samples (150 μL, 20 mg/mL) were eluted linearly (3 mL/min) using chromatographic grade H2O (0.1% TFA, solvent A) and ACN (0.1% TFA, solvent B): 5%−40% B (0−45 min). The most active fractions (50 μL, 5 mg/mL) were further subjected to the second HPLC purification on the same system using an X-bridge analytical C18 column (3.0 mm × 250 mm, 5 μm, Waters) coupled with a guard cartridge column (3.0 mm × 20 mm, 5 μm, Waters). Peptide concentration was determined by the Lowry method described in Protein Yield and Peptide Content Determination. Peaks were detected at 220 nm and samples were all dissolved in 100% solvent A. Gradients of the second HPLC fractionation are presented in Figure 4. Finally, the most active fractions in ACE inhibition were subjected to liquid chromatography−mass spectrometry/mass spectrometry (LC-MS/MS) analysis. 7148

DOI: 10.1021/acs.jafc.9b01071 J. Agric. Food Chem. 2019, 67, 7147−7156

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Journal of Agricultural and Food Chemistry

Figure 2. Preparation, simulated GI digestion, and fractionation as well as changes in protein yield and ACE inhibition of T-EWH. Indicated percentages (%, w/w) of each portion are from the original EW proteins. For thermolysin digestion, 0.1% E/S, 60 °C, and pH 8.0; for simulated GI digestion, 1% E/S, 37 °C at pH 2.0 (pepsin) and 7.5 (pancreatin). The most active fractions (F) with high yield were continuously fractionated until analyzed by LC-MS/MS (in dashed boxes). Peptide Sequence Analysis by LC-MS/MS. Peptide sequences were analyzed by a nanoAcquity reverse-phase ultraperformance liquid chromatography (UPLC) system, coupled with an Atlantis dC18 UPLC column (75 μm × 150 mm, 3 μm, Waters) and a Micromass quadrupole time-of-flight (Q-TOF) premier mass spectrometer.17 Solvents were LC/MS grade H2O (0.1% formic acid, solvent A) and ACN (0.1% formic acid, solvent B). After desalting, samples (5 μL) were injected and linearly eluted (0.3 mL/ min): 1%−60%−95% B (0−2−40−55 min). Ionization was performed using electrospray ionization technique (ESI) in a positive ion mode (capillary voltage, 3.4 kV; source temperature, 100 °C). Peptide mass was detected using a Q-TOF analyzer; acquisition ranges were m/z 200−1200 (MS mode) and 50−1000 (MS/MS mode), respectively. Data were interpreted by MassLynx software version 4.1 (Waters) by de novo sequencing. Simulated GI Digestion. Simulated GI digestion followed procedures described by Lacroix et al.18 with slight modifications. Hydrolysate (fractions) and pure peptides were dissolved in ddH2O at 5% protein (w/v) and 100 μg/mL, respectively. Then, samples were digested by pepsin (1% E/S, w/w protein) for 1.5 h at 37 °C, pH 2.0 (adjusted with 3 M HCl). After that, the digest was adjusted to pH 5.3 using 0.9 M NaHCO3 and then pH 7.5 with 1 M NaOH. The digest was halved with half as pepsin digest and another half further digested by pancreatin (1% E/S) for 1.5 h (at 37 °C). The reaction was then terminated by maintaining the mixture at 95 °C for 10 min. Digestion was performed in a 15-mL polypropylene tube; temperature was controlled by a reciprocal shaking water bath (Fisher Scientific). Each digest was prepared individually in triplicate. Degradation of Synthesized ACEi Peptides during Simulated GI Digestion. Degradation of synthesized peptides after pepsin and pancreatin digestion was analyzed through a Waters Acquity UPLC system (Waters), equipped with an Acquity BEH C18 column (1.7 μm, 2.1 mm × 100 mm) and a PDA eλ detector. Samples (10 μL) were eluted using a binary solvent system of chromatographic

grade H2O (0.1% TFA, solvent A) and ACN (0.1% TFA, solvent B) at a flow rate of 0.3 mL/min. The gradient was as follows: 1%−25%− 35%−99%−1% B (0−12−15−18−20 min). Absorbance was monitored at 220 nm. Peptides were identified (quantified) based on their standards (curves). Protein Yield and Peptide Content Determination. Protein yield was defined as the percentage (w/w protein) of each fraction from the original EW proteins. Peptide concentration was determined according to the Modified Lowry Protein Assay Kit provided by Thermo Fisher Scientific (Burlington, ON, Canada) using bovine serum albumin as the standard. ACEi Activity Assay. ACEi inhibitory activity assay was performed referring to Wu et al.19 with slight modifications. ACE, HHL, hydrolysate or peptide samples were dissolved in 100 mM potassium phosphate buffer containing 300 mM NaCl (pH 8.3). First, a 10 μL sample of hydrolysate (peptide) and 50 μL of HHL substrate (5 mM) were preincubated at 37 °C for 5 min in a 2 mL polypropylene centrifuge tube. Then, 20 μL of preincubated (37 °C) ACE (2 mU) was added and reacted (at 450 rpm) for another 30 min using an Eppendorf Thermomixer R (Brinkmann Instruments, NY, USA). The reaction was terminated by adding 125 μL of 1 M HCl solution and analyzed using the same UPLC system described in Degradation of Synthesized ACEi Peptides during Simulated GI Digestion, but with an Acquity BEH C18 column (1.7 μm, 2.1 mm × 50 mm). Samples (5 μL) were eluted using a gradient of chromatographic grade H2O (0.05% TFA, solvent A) and ACN (0.05% TFA, solvent B) at a flow rate of 0.245 mL/min as follows: 5%−60%−60%−5% B (0−3.5−4.2−5 min). Absorbance was monitored at 220 nm. HA was identified and quantified based on its standard curve. The IC50 value was defined as the sample concentration inhibiting 50% of the ACE activity. Statistical Analysis. All analyses were performed in triplicate. Blood pressure (means accompanied by standard errors) on other days were compared with that on day 0 by one-way analysis of 7149

DOI: 10.1021/acs.jafc.9b01071 J. Agric. Food Chem. 2019, 67, 7147−7156

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Figure 3. Cation exchange chromatogram of T-EWH ( 0.05) after pepsin digestion, whereas those of fractions I and III were significantly (P < 0.05) increased. These results implied that pepsin could further release potent ACEi peptides from T-EWH (Figure 2).21 The pellet of T-EWH was partly (7.05%) turned into supernatant after pepsin digestion, with an ACEi IC50 value of 0.49 mg/mL. However, all pepsin digests experienced a slight reduction in ACE inhibition after further digestion by pancreatin. This trend was possibly because trypsin cleaves Arg and Lys, which reduce the potency of ACEi peptides.22 After simulated GI digestion, the supernatant of pellet and fractions I, II, and III had ACEi IC50 values of 0.57, 0.10, 2.99, and 1.07 mg/mL, respectively; all of these samples increased significantly (P < 0.05) in ACE inhibition except fraction II. It was interesting to note that the supernatant of the pellet showed a higher potency than that of fraction III, which was higher than fraction II, after pepsin digestion, indicating more potent peptides were released from the pellet. EWH prepared by pepsin has been reported at a higher ACE inhibition than that by thermolysin by using a similar E/S under their respective enzymatic conditions.16,23 In addition, a non-negligible loss of sample was

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RESULTS AND DISCUSSION Preparation, Simulated GI Digestion, and Fractionation of T-EWH. Thermolysin is an effective enzyme with broad cleavage sites for the production of potent ACEi peptides from various proteins.20 Orally feeding T-EWH to SHRs at a dose of 1,000 (mg/(kg of body weight))/day (Figure 1) significantly reduced systolic, diastolic, and mean arterial blood pressure from 171.7, 139.4, and 155.5 mmHg on day 0 to 135.2, 94.0, and 113.5 mmHg on day 6 and to 112.3, 83.0, and 96.9 mmHg on day 18, respectively (P < 0.01); heart rate did not change (P > 0.05) during this study. In addition, ACEi activity of T-EWH was enhanced after simulated GI digestion (P < 0.05) (Table S1). This evidence suggested that certain ACEi peptides could resist GI digestion and/or can be further released during GI digestion. Therefore, it is interesting to identify the responsible peptides resisting GI digestion. Preparation, ultrafiltration, and simulated GI digestion of TEWH are presented in Figure 2. T-EWH had a hydrolysis yield of 94.3% (w/w), and the supernatant had an ACEi IC50 value of 1.0 mg/mL. This ACEi potency seemed to be slightly lower than most previously reported EWHs, typically with IC50 value of 0.05−0.5 mg/mL,7,16 which was possibly due to a low dose of enzyme (0.1% E/S) applied during hydrolysis. The pellet 7150

DOI: 10.1021/acs.jafc.9b01071 J. Agric. Food Chem. 2019, 67, 7147−7156

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Figure 4. HPLC chromatograms of the most active fractions of F3 and F4 from cation exchange chromatography of T-EWH ( 10 mg/mL were marked as N.D. (not detected).

Table 2. Yield and ACEi Activity before/after Simulated GI Digestion of Fractions from the Second HPLC Fractionation ACEi IC50 values (μg/mL) peptide yield (μg)

−

pepsin

pancreatin

F3-4-1 F3-4-2 F3-4-3 F3-4-4 F3-4-5 F3-4-6

514A 644A 626A 267B 199B 195B

2333Aa 76Ba 10Ca 5Da 3Da 14Ca

2388Aa 79Ba 3Db 2Db 1Db 11Ca

2568Aa 75Ba 4Db 2Db 1Db 12Ca

F3-5-1 F3-5-2 F3-5-3 F3-5-4 F3-5-5 F3-5-6

225C 493A 482A 303B 222C 133D

29Ca 43BCa 66ABa 95Aa 7Ea 14Da

19Ca 32Ba 61Aa 81Aa 1Eb 7Db

22Ca 32Ba 58ABa 80Aa 2Eb 10Dab

fraction F3-4

ACEi IC50 values (μg/mL) peptide yield (μg)

−

pepsin

pancreatin

F4-3-1 F4-3-2 F4-3-3 F4-3-4 F4-3-5 F4-3-6

202B 330A 196B 113C 33D 32D

844Cb 1450Ab 1102Ba 655Da 133Eb 89Eb

1690Aa 2056Aa 989Ba 617Ca 180Da 129Da

1104Bb 1946Aa 961Ba 585Ca 153Dab 123Da

F4-4-1 F4-4-2 F4-4-3 F4-4-4 F4-4-5 F4-4-6 F4-4-7

208A 208A 82C 251A 34D 143B 111BC

195Cb 403Bb 352BCa 426Bb 1Da 1184Aa N.D.

341Ca 889Ba 155Db 1013ABa 1Ea 1258Aa N.D.

360Ca 799Ba 226Cb 1037Aa 1Da 1048Aa N.D.

F4-5-1 F4-5-2 F4-5-3 F4-5-4 F4-5-5

173C 631B 776B 956AB 1111A

N.D. 3Ca 7Ca 48Aa 23Bb

N.D. 5Ba 7Ba 30Ab 34Aa

1431A 5Ca 7Ca 33Bb 31Ba

F4-11-1 F4-11-2 F4-11-3 F4-11-4 F4-11-5 F4-11-6

63B 308A 372A 408A 71B 47B

N.D. 10Da 8Da 19Ca 84Ba 472Ab

1404Aa 14Ca 3Db 15Ca 98Ba N.D.

1396Aa 12Da 3Eb 14Da 91Ca 1097Ba

fraction F4-3

F3-5

F4-4

F4-5

F4-11

a Data represent mean values (n = 3). Values that do not share a common letter (A−E each column, or a−c each row of ACE inhibition) differ significantly (P < 0.05). Fractions in bold were the most active ones. Fractions with IC50 values > 10 mg/mL were marked as N.D. (not detected).

yield than those of F3 (1.02%) and F4 (0.15%) (P < 0.05), F3 and F4 were finally selected for further HPLC purification of ACEi peptides. After the first HPLC purification, F3 was further fractionated into 15 subfractions (Figure 4a); their peptide amount and

ACEi activity are presented in Tables 1 and 2. Among them, F3-4 and F3-5 were the two most potent ones in ACE inhibition, both before and after GI digestion with high yield. Then, the second HPLC purification of F3-4 and F3-5 was performed (Figure 4b,c). As a result, six subfractions were 7152

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7153

EKR EQR KLP NLR PRI PYK AERF APYK INKV VIRW LAPYK LKISQ LKYAT PVLKD SQAVH QAVHA ERQEKR INKVVR KIMKGE LFLIKH LGHWVY SSHEKY VQKATY VYLPRM LTSVLMA FDKLPGFGD VNGSEKSKF

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

*

*

*

*

* *

>1000

* * *

*

*

*

*

*

>500

*

*

*

*

*

>100

1.02 (1.44 μM)Ba 1.84 (2.32 μM)Ba

6.52 (8.47 μM)Da 4.83 (6.25 μM)Ca

4.15 (5.70 μM)Ca

9.95 (16.85 μM)Ea 6.25 (10.64 μM)D 0.054 (0.09 μM)A

1000

*

*

*

>500

136.34Cb

*

*

*

*

>100

IC50 (μg/mL)

17.63 (24.89 μM)Bb

11.24 (14.61 μM)ABb 8.43 (13.41 μM)Ab

6.30 (8.65 μM)Ab

7.19 (12.18 μM)Ab

1,000 μM) and LAPYK (16.85 μM) in ACE inhibition further supported this conclusion. Indeed, the fifth and sixth positions of ACEi peptides from the C-terminus were surrounded by a cluster of hydrophobic residues of sACE.25 In addition, these peptides had a structural similarity at the tetrapeptide C-termini, consisting of hydrophobic and positively charged amino acid residues. C-terminal hydrophobicity was preferred for potent ACEi peptides,33 while the role of positive charge remains unknown. It appeared that one or two positive charges in the Cterminal tetrapeptide residue of ACEi peptide slightly affected its activity, but their positions caused a difference. A positive charge at the fourth (e.g., LKYAT, VQKATY) or second (VYLPRM) position from the C-terminus could enhance ACE inhibition. This phenomenon might be due to the presence of negatively charged Glu403 and Glu162 at the S2 and S1′ subsites of the sACE C domain, respectively.24,34,35 The preference of positive charges at these two subsites might enhance their selectivity to the sACE C domain over N domain, thus a stronger ACE inhibition in Ang II generation.34,35 The presence of a positively charged amino acid residue at the second position from the C-terminus enhances ACE inhibition of tripeptides; however, its contribution at specific positions to the activity of larger peptides awaits more fundamental studies. Besides, it was noteworthy that peptides with a C-terminal tetrapeptide composition of Lys, Tyr, Ala, and Thr showed the highest ACE inhibition, especially LKYAT (0.09 μM) which had a comparable activity with certain synthetic ACE inhibitors such as enalapril (146 nM);36 however, the underlying mechanism is yet to be explored. In conclusion, these potent ACEi pentaand hexapeptides in our study preferred both a hydrophobic N-terminus and a hydrophobic tetrapeptide C-terminus; the positive charges at the second and fourth positions from the Cterminus appeared to further enhance their activity. This conclusion may also be applicable to peptides larger than 6 amino acids with a C-terminus of the above structural features. Effect of GI Digestion on ACEi Activity of Peptides. Recent in vivo studies revealed that larger peptides could be absorbed into the circulation;37 however, peptides must resist GI digestion before being absorbed and reaching their target organs and tissues. Table 3 presents the effect of simulated GI digestion on ACEi activity of all identified peptides. Most peptides were degraded to a varying degree after 3 h GI

obtained from F3-4 and F3-5, respectively. Four fractions (F34-3/-4/-5/-6) from F3-4 and two fractions (F3-5-5/-6) from F3-5 showed increased activities after GI digestion, which indicated a possibility of newly produced and stronger ACEi peptides or a synergistic effect of smaller peptides released by GI enzymes.25 Similarly, F4 was also separated into 15 subfractions (Figure 4d), in which four fractions (F4-3/-4/-5/11) have been selected for the second HPLC purification. Six subfractions, F4-4-5, F4-5-2/-3, and F4-11-2/-3/-4, were chosen for further characterization (Figure 4e−h). Taken together, 12 subfractions were selected and analyzed by LCMS/MS, with ACEi IC50 values that ranged in 1−19 μg/mL before and after GI digestion. Identification and Structure−Activity Relationship (SAR) of ACEi Peptides from T-EWH. According to the MS/MS spectrum of the above-purified fractions, 27 individual peptides were identified after de novo sequencing. Table 3 reports their activities and parent proteins from EW (molecular weight and sources of fractions are shown in Table S2). These peptides varied in peptide lengths of 3−9 amino acid residues and had considerable differences in ACE inhibition. Longer peptides such as LTSVLMA, FDKLPGFGD, and VNGSEKSKF showed lower ACE inhibition (IC50 value > 500 μM). Tri- and tetrapeptides also showed relatively low activity (>100 μM), although smaller ACEi peptides are generally more potent in ACE inhibition.24The most potent ACEi peptides were penta- and hexapeptides. Their IC50 values were approximately or below 10 μM, comparable to those of potent EW-derived peptides, such as IRW (0.64 μM), IQW (1.59 μM), RADHPFL (6.20 μM), and YAEERYPIL (4.70 μM),9,26 but much lower than those of many others, such as IVF (33.9 μM), DILN (73.4 μM), RADHP (153 μM), TNGIIR (70 μM), and QIGLF (75 μM).26−30 SARs of ACEi di- and tripeptides have been described previously.22 A potent ACEi tripeptide requires a hydrophobic, a positively charged, and an aromatic/cyclic amino acid residue at the N-terminal, middle, and C-terminal positions, respectively.31 Moreover, SAR of tripeptides is also applicable to larger peptides under certain circumstances, reflecting their C-terminal tripeptide residues.24 For example, PYK and EKR showed ACE inhibition (>1,000 μM) comparable to those of APYK and ERQEKR. Also, other weak ACEi peptides such as AERF, INKV, SQAVH, and KIMKGE (>500 μM), as well as PVLKD, QAVHA, and SSHEKY (>100 μM), did not have potent C-terminal tripeptide residues. Interestingly, however, an additional N-terminal Val of VIRW (>100 μM) remarkably reduced the potency of IRW (0.64 μM).9 Moreover, two potent ACEi peptides, VQKATY (1.44 μM) and LKYAT (0.09 μM), did not have potent C-terminal tripeptide residues. These features suggested that the SARs of larger peptides could not always be extrapolated from those of their Cterminal tripeptide residues, implying the important roles of other positions (e.g., the fourth position from the C-terminus) in dictating their ACEi potency. Previous studies on ACEi peptides with 4−10 amino acids proposed that the fourth amino acid residue was even more important than the second and third ones from the C-terminus.32 This conclusion was favored by a comparison between INKV (>500 μg/mL) and INKVVR (4.15 μg/mL). Since VVR and NKV were both not potent C-terminal tripeptide residues, the other positions of INKV and INKVVR might lead to their different ACE inhibition. It has been proposed for several lentil-derived ACEi peptides that the hexapeptide C-termini played crucial 7154

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digestion, and overall, larger peptides were more likely to be degraded than smaller ones. However, certain smaller peptides, such as ERK, EQR, PRI, PYK, and AERF, were hydrolyzed >50%, while larger peptides, such as FDKLPGFGD, were barely degraded, indicating the importance of amino acid compositions and sequence for GI resistance.25 Therefore, cleavage of any amino acid residues might cause an either increased or decreased ACE inhibition.9,25 However, under certain circumstances, GI digestion can be an efficient way to generate new bioactive peptides from their parent peptides. 38,39 For example, GI digestion of YAEERYPIL, RADHPFL, and IWHHT liberated YPI, RADHP, IWH, and IW, respectively.39,40 However, there were barely new potent ACEi peptides generated in our study, manifested by a lack of increase in ACEi potency (compared at the same molar concentration) after GI digestion; we therefore mainly focused on the eight potent ACEi peptides. LAPYK was the only peptide whose activity was improved after GI digestion to a small degree, during which a slightly stronger ACEi peptide, possibly LAP, might be generated. As a whole, GI digestion mostly reduced ACEi activity of the other peptides. The activity of VQKATY and VYLPRM decreased most significantly (P < 0.05), approximately by 74- and 17-fold, respectively, followed by that of LGHWVY, LFLIKH, and INKVVR, which were slightly degraded. Overall, these peptides did not generate stronger ACEi peptides but exhibited good resistance during GI digestion, except VQKATY and VYLPRM. In addition, we observed that a discrepancy existed between the practical and theoretical cleavage sites (Arg and Lys) of trypsin, since certain peptides such as LKYAT, INKVVR, and LFLIKH were found not cleaved by trypsin in our study (Figure S1). In this study, eight potent ACEi penta- and hexapeptides were identified from T-EWH using a combined method of fractionation in parallel with GI digestion; six of them showed good GI-resistant stability. The GI resistance indicated their potential in vivo antihypertensive efficacy, although further investigation on their bioavailability and physiological efficacy are warranted. A hydrophobic N-terminus and a hydrophobic tetrapeptide C-terminus were important structural features for potent ACEi penta- and hexapeptides while the contribution of positive charges required further clarification. One limitation of this study is the number of peptides used, which is relatively too small to draw a sound SAR for ACEi penta- and hexapeptides; however, their characterization will shed light on the SAR of large ACEi peptides. Besides, these peptides have been reported for the first time from EWH, which will complement current research on EWH-derived bioactive peptides and further facilitate the application of EWH as functional food ingredients against hypertension.

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Article

AUTHOR INFORMATION

Corresponding Author

*Tel.: +1 780 492 6885. Fax: +1 780 492 4265. E-mail: jwu3@ ualberta.ca. ORCID

Hongbing Fan: 0000-0003-1049-6815 Wang Liao: 0000-0001-8319-2199 Jianping Wu: 0000-0003-2574-5191 Funding

This work was supported by funding from Natural Sciences and Engineering Research Council of Canada, Egg Farmers of Canada, and Burnbrae Farms Ltd. H.F. is the recipient of Doctoral Scholarships from China Scholarship Council, Alberta Innovates Technology Futures, and the Killam Trusts. Notes

The authors declare no competing financial interest.

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ABBREVIATIONS USED ACE, angiotensin-converting enzyme; ACEi, ACE inhibitory; ACN, acetonitrile; Ang I/II, angiotensin I/II; ddH2O, deionized distilled water; EW, egg white; EWH, egg white hydrolysate; GI, gastrointestinal; HA, hippuric acid; HHL, hippuryl-His-Leu; LC-MS/MS, liquid chromatography−mass spectrometry/mass spectrometry; MWCO, molecular weight cutoff; PDA, photodiode array; RAS, renin-angiotensin system; RP-HPLC, reverse-phase high-performance liquid chromatography; sACE, somatic ACE; SAR, structure−activity relationship; SHRs, spontaneously hypertensive rats; T-EWH, thermolysin-digested egg white hydrolysate; TFA, trifluoroacetic acid; UPLC, ultraperformance liquid chromatography.

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REFERENCES

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jafc.9b01071. ACEi activity of T-EWH after simulated GI digestion (Table S1); molecular weight and sources of fractions of the identified peptides (Table S2); chromatograms of peptides after pepsin and trypsin digestion (Figure S1); desalting protocol (PDF) 7155

DOI: 10.1021/acs.jafc.9b01071 J. Agric. Food Chem. 2019, 67, 7147−7156

Article

Journal of Agricultural and Food Chemistry

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DOI: 10.1021/acs.jafc.9b01071 J. Agric. Food Chem. 2019, 67, 7147−7156