Identification of novel angiotensin I-converting enzyme (ACE) inhibitory peptides from wheat gluten hydrolysate by the protease of Pseudomonas aeruginosa

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Identification of novel angiotensin I-converting enzyme (ACE) inhibitory peptides from wheat gluten hydrolysate by the protease of Pseudomonas aeruginosa Peng Zhanga, Chang Changb, Haijie Liub, Bo Lib, Qiaojuan Yana, , Zhengqiang Jiangb, ⁎

⁎

a

Key Laboratory of Food Bioengineering (China National Light Industry), College of Engineering, China Agricultural University, Beijing 100083, China Beijing Advanced Innovation Center for Food Nutrition and Human Health, College of Food Science and Nutritional Engineering, China Agricultural University, Beijing 100083, China

b

ARTICLE INFO

ABSTRACT

Keywords: Protease Pseudomonas aeruginosa Wheat gluten Antihypertensive peptides

The feasibility of a protease of Pseudomonas aeruginosa (PaproA) to prepare angiotensin I-converting enzyme (ACE) inhibitory peptides from wheat gluten was evaluated according to physiochemical and antihypertensive performances. The wheat gluten hydrolyzed by Alcalase plus PaproA (WGH_APae) to produce small fragments (< 1 kDa) was particularly preferred by the superior protein recovery rate, degree of hydrolysis, ACE inhibitory potential, and stability against gastrointestinal digestion. Among the resultant fractions from WGH_APae, the lower IC50 value of fraction 7 indicated proper concentrations of proline and negatively charged amino acids were critical to modulate ionic and hydrophobic interactions on ACE catalytic sites to inhibit ACE activity. Subsequently, fraction 7 was purified to identify two successful antihypertensive peptides containing tryptophan at the carboxyl-end: SAGGYIW and APATPSFW with IC50 values of 0.002 mg/mL and 0.036 mg/mL, respectively, suggesting both peptides had potentials in the nutraceuticals and functional foods to prevent and/or treat hypertension.

1. Introduction Hypertension has been recognized as one of the major public chronic disease worldwide relating to obesity, prediabetes, and atherosclerosis to threat health in one-quarter of the world’s adult population (Iwaniak, Minkiewicz, & Darewicz, 2014). It is diagnosed as systolic and diastolic blood pressures are ≥140 mmHg and 90 mmHg, respectively. Regarding to normal blood pressure regulation, renin-angiotensin system is a major pathway to conduct blood vessel contraction, in which renin converts angiotensinogen to angiotensin I (an inactive peptide fragment), and angiotensin I was then converted to angiotensin II by angiotensin I-converting enzyme (ACE) to bind vascular wall receptor to control blood vessel contraction. However, under abnormal metabolism, ACE catalyzes degradation and inactivation of bradykinin (a vasodilator) to trigger excessive levels of angiotensin II to cause hypertension. Thus, modulation of ACE activity to generate homeostatic angiotensin II level is the key to treat hypertension (Aluko, 2015). Hypertension is currently treated using several synthetic ACE inhibitory drugs, such as Capoten (Captopril), Prinivil (Lisinopril), Casotec

(Enalapril), and Altace (Ramipril), but they are regularly associated with adverse effects, such as dry cough, edema, diarrhea, fatigue, dizziness, headache, and erectile dysfunction (Abassi, Winaver, & Feuerstein, 2009). Therefore, researchers are making efforts on the discovery and production of natural and safe food resources (e.g., protein-derived peptides), which provide less and even none harmful side impacts, to replace the traditional drugs to lower blood pressure. Numerous food proteins, including milk, dairy, egg, meat, fish, and plant proteins, have been enzymatically hydrolyzed to prepare ACE inhibitory peptides presenting lower side effects (Chiozzi et al., 2016; Hartmann & Meisel, 2007; Iwaniak et al., 2014; Li, Le, Shi, & Shrestha, 2004). Wheat gluten, mainly consisting of gliadin (50% w/w, responsible for viscosity) and glutenin (50% w/w, responsible for elasticity), is the uppermost protein (accounting for 85% of the total protein content) in wheat (Nongonierma, Hennemann, Paolella, & FitzGerald, 2017). It has been used as an alternative to soybean-based products and meat substitutes to provide unique chewy and stringy textures (Joye & McClements, 2014). However, coeliac disease, an immune reaction to

⁎ Corresponding authors at: College of Engineering, China Agricultural University, 17 Qinghua Donglu, Beijing 100083, China (Q. Yan). College of Food Science and Nutritional Engineering, China Agricultural University, 17 Qinghua Donglu, Beijing 100083, China (Z. Jiang). E-mail addresses: [email protected] (Q. Yan), [email protected] (Z. Jiang).

https://doi.org/10.1016/j.jff.2019.103751 Received 28 September 2019; Received in revised form 13 December 2019; Accepted 21 December 2019 1756-4646/ © 2020 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/BY-NC-ND/4.0/).

Please cite this article as: Peng Zhang, et al., Journal of Functional Foods, https://doi.org/10.1016/j.jff.2019.103751

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damage small intestine by gluten consumption, strictly restricted the utilization of wheat gluten (Biesiekierski, 2017). Therefore, modification of wheat gluten to explore advanced applications is imperative to provoke economic values of this byproduct from wheat starch industry. Preparation of bioactive peptides from wheat protein by enzymatic hydrolysis and microbial fermentation is one of feasible option, in which more emphasis is given to the enzymatic hydrolysis by its advantages of economy efficiency, high yield, and safe process (Saadi, Saari, Anwar, Hamid, & Ghazali, 2015). Various bioactive peptides, such as VPL, WL, WP and IAP, have been isolated from wheat gluten hydrolysates to express different bioactivities (e.g., antihypertensive, antioxidant, and antidiabetic activities) (Cavazos & de Mejia, 2013), in which ACE inhibitory ability was most commonly proposed by the abundant presence of hydrophobic amino acids (especially proline and tryptophan) in the wheat gluten hydrolysates (Iwaniak et al., 2014). Although commercial proteases (e.g., Alcalase, pepsin, pancreatin, Neutrase, Protamex, and trypsin) have previously been used to prepare hydrolysates and bioactive peptides from wheat gluten, the isolated fractions and peptides exhibited limited ACE inhibitory activities. The untargeted cleavage of protein backbone and restricted hydrolytic ability of the proteases leading to ineffective proteolysis contributed to this result (Cavazos & de Mejia, 2013; Iwaniak et al., 2014). A novel protease (named PaproA) from Pseudomonas aeruginosa CAU342A exhibited an optimal activity at pH 8.0 and 55 °C, and has been applied to hydrolyze casein, skimmed milk, and protamine sulfate to show higher hydrolysis efficiency and broad substrate specificity (Sun, Zhang, Yan, & Jiang, 2017). Therefore, the objective of this study was to evaluate the feasibility of PaproA on the generation of ACE inhibitory peptides during enzymatic hydrolysis of wheat gluten, in order to develop more effective antihypertensive peptides.

Table 1 Various proteases for wheat gluten hydrolysis at different conditions. Hydrolysate

Protease A

Protease B

Conditions

WGH_Alc WGH_Pro WGH_Pae WGH_APro

Alcalase Protamex PaproA Alcalase

– – – Protamex

WGH_APae

Alcalase

PaproA

WGH_PP

Protamex

PaproA

60 °C at pH 8.5 55 °C at pH 8.0 45 °C at pH 7.0 60 °C at pH 8.5, followed by 55 °C at pH 8.0 60 °C at pH 8.5, followed by 45 °C at pH 7.0 55 °C at pH 8.0, followed by 45 °C at pH 7.0

Abbreviations include: wheat gluten hydrolyzed by Alcalase (WGH_Alc), wheat gluten hydrolyzed by Protamex (WGH_Pro), wheat gluten hydrolyzed by PaproA (WGH_Pae), wheat gluten hydrolyzed by Alcalase and Protamex (WGH_APro), wheat gluten hydrolyzed by Alcalase and PaproA (WGH_APae), and wheat gluten hydrolyzed by Protamex and PaproA (WGH_PP).

Overall, six wheat gluten hydrolysates (WGHs), including WGH prepared by Alcalase (WGH_Alc), WGH prepared by Protamex (WGH_Pro), WGH prepared by PaproA (WGH_Pae), WGH prepared by Alcalase followed by Protamex (WGH_APro), WGH prepared by Alcalase followed by PaproA (WGH_APae), and WGH prepared by Protamex followed by PaproA (WGH_PP), were produced in triplicate. The intact wheat gluten was applied as a control. The protein recovery rate (%) was calculated according to the ratio of protein content in the hydrolysate to the initial protein content in the wheat gluten. The protein content (%N × 6.25) was determined by micro-Kjeldahl method (AOAC, 2003). 2.3. Degree of hydrolysis (DH)

2. Materials and methods

According to the intereaction between primary amino groups and OPA, the DH of the wheat gluten hydrolysate was assessed as described by Nielsen, Petersen, and Dambmann (2001) with minor modifications. Briefly, di-Na-tetraborate decahydrate (1.905 g) and Na-dodecyl-sulfate (SDS, 40 mg) were completely dissolved in deionized water (30 mL). OPA (40 mg) was additionally dissolved in 1 mL ethanol. The OPA solution was then prepared by mixing the above-mentioned solutions. Subsequently, 44 mg dithiothreitol (DTT) was mixed with the OPA solution with the addition of deionized water (50 mL) to get OPA reagent. Afterwards, the hydrolysates solutions (100 mg/L, 50 μL) were reacted with OPA reagents (750 μL) for 5 s, followed by exact 2 min of stabilization. The absorbance of reaction solution at 340 nm was read by a spectrophotometer (TU-1800PC, Persee General Instrument Co. Ltd., Beijing, China). Serine solution (0.9516 meqv/L, 50 μL) and deionized water (50 μL) were applied as a standard and a blank, respectively. The DH of the hydrolysate was calculated using the following formula:

2.1. Materials Wheat gluten [68.9% wet basis (%N × 6.25)] at food grade was kindly donated by Longlive Biotechnology Co. Ltd. (Dezhou, China). PaproA was prepared by Pseudomonas aeruginosa CAU342A isolated from the fermentation of soy sauce koji as described by Sun et al. (2017). Alcalase (2.4 AU-A/g) and Protamex (1.5 AU-A/g) were provided by Novozymes (Copenhagen, Denmark). Pepsin (250 U/g) and trypsin (250 U/g) were purchased from Amresco (Solon, USA). ACE (EC 3.4.15.1, isolated from rabbit lung), N-Hippuryl-His-Leu hydrate (HHL), o-phthalaldehyde (OPA), and ferrozine (a sodium salt of 3-(2pyridyl)-5,6-diphenyl-1,2,4-triazolidynic acid) were obtained from Sigma-Aldrich (St. Louis, USA). All other chemicals were of analytical grades. 2.2. Enzymatic hydrolysis of wheat gluten

DH = h/htot × 100%

Wheat gluten was hydrolyzed by PaproA, Alcalase and Protamex as described in Table 1. In brief, wheat gluten (5% w/w) was initially hydrolyzed with protease A (1500 U/g) for 6 h, followed by the hydrolysis with protease B at the same concentration for another 4 h. According to the optimal conditions of each protease, the temperature and pH of hydrolysis were as follows: 60 °C and pH 8.5 for Alcalase, 55 °C and pH 8.0 for Protamex, 45 °C and pH 7.0 for PaproA. pH adjustment was performed using 1 M HCl and/or NaOH. The reaction was carried out in a temperature-controlled shaker (HZQ-X100, Peiying, Suzhou, China) for 10 h in total and terminated by heating at 95 °C for 10 min. Subsequently, the hydrolysates solutions were centrifuged (GL20B Refrigerated Centrifuge, Anting Scientific Instrument Co. Ltd., Shanghai, China) at 10,000 rpm for 10 min at 4 °C, and the supernatants were then lyophilized (HERMLE Z326K, HERMLE Labortechnik, Gosheim, Germany) at a temperature difference of 30 °C to collect the hydrolysates that were finally stored in a desiccator for further analysis.

h= (serine NH2 serine

NH2 = ODsample

)/

meqv/g protein

ODblank /ODstandard

ODblank × 0.9516 meqv/ L× 0.1 × 100/ X× P

where h is the number of hydrolyzed bonds in the hydrolysates; htot is the total number of the peptide bonds per protein equivalent (htot of wheat gluten = 8.3); α and β of wheat gluten is 1.0 and 0.4, respectively; serine-NH2 = meqv serine NH2/g protein; OD is the absorbance; X is the amount of sample (g); P is the protein content (%) of the hydrolysates. 2.4. Molecular mass distribution Molecular mass distributions of the intact and hydrolyzed wheat glutens were determined by a high-performance liquid chromatography (HPLC) system. In details, the wheat gluten or the lyophilized

2

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hydrolysates was initially resuspended in the mobile phase (containing acetonitrile, deionized water, and trichloroacetic acid at a mixing ratio of 45:55:0.1) at a concentration of 5 mg/mL, and 25 μL of the suspension was then filtered and injected into a TSKgel-G2000SWXL column (7.8 × 300 mm, Tosoh Corporation, Yamaguchi Prefecture, Japan) settled on an Agilent 1260 HPLC system (Agilent Technologies Inc., California, USA). The operating conditions were set at the flow rate of 0.5 mL/min, the injection volume of 25 μL, and the absorbance at 214 nm. The calibration curves of molecular mass were plotted using nine standards: ribonuclease A, cytochrome C, holo-transferrin, apomyoglobin, glycine-tyrosine, valine-tyrosine-valine, methionine enkephalin, leucine enkephalin and angiotensin II. The area under the chromatogram was separated into four molecular mass ranges (including < 1 kDa, 1–5 kDa, 5–10 kDa, and > 10 kDa) and expressed as the percentage of the total area.

2.7. Fractionation of selected wheat gluten hydrolysates The wheat gluten hydrolysate with the best ACE inhibitory activity (WGH_APae) was fractionated using a Fast Protein Liquid Chromatography system (FPLC, GE Healthcare, Chicago, USA) coupled with an ÄKTA purifier system (UPC-900, GE Healthcare, Piscataway, USA). WGH_APae solution (30 mg/mL, 2 mL) was subjected to a Sephadex G-15 column (100 cm × 12 mm, Whatman plc, Buckinghamshire, UK) equilibrated by 10 mM HCl, and was eluted with water at the flow rate of 0.8 mL/min under monitoring at 280 nm. In final, WGH_APae was separated into several fractions (1.5 mL of each), which was lyophilized for the measurements of ACE inhibitory activities and amino acid compositions. 2.8. Amino acid composition Amino acid compositions of the intact and hydrolyzed wheat gluten by Alcalase and PaproA and its fractions were evaluated using a Hitachi L-8900 amino acid analyzer (Hitachi Ltd., Tokyo, Japan) settling with an ion exchange column packed with Hitachi custom ion exchange resin (4.6 × 60 mm) following AOAC (2005) methodology. In details, the sample (0.2 mg) was placed in a digestion tube containing 10 mL of 6 M HCl. The oxygen in the tube was replaced by nitrogen for 10 min. The tube was then sealed and hydrolyzed for 24 h at 115 °C. After the hydrolysis, the solution was neutralized with 6 mL of 10 M NaOH and adjusted to 25 mL with deionized water. The mixture was then filtered through 0.2 μm membrane filters, followed by the injection (4 μL) into the amino acid analyzer for analysis. A ninhydrin buffer solution was applied as a mobile phase. The content of tryptophan was assessed according to Nurit, Tiessen, Pixley, and Palacios-Rojas (2009), on which the sample was treated with pancreatin to collect the supernatant. The colorimetric reagent (containing glyoxylic acid) was then added and incubated to measure the absorbance at 540 nm, in which α-methyltryptophan was applied to plot standard curve for the calculation.

2.5. ACE inhibitory activity IC50 (half maximal inhibitory concentration) is defined as the concentration of the intact, hydrolyzed wheat glutens and their isolated fractions and peptides required for 50% inhibition of ACE activity, which was determined based on the liberation of hippuric acid from NHippuryl-His-Leu hydrate catalyzed by ACE using the spectrophotometric method as described by Cushman and Cheung (1971) with slight modifications. Namely, the sample solution (2 mg/mL, 20 μL) with 10 μL ACE solution (0.1 U/mL) was pre-incubated at 37 °C for 5 min. The addition of 120 μL substrate (5 mM N-Hippuryl-His-Leu hydrate in 0.1 M sodium borate buffer containing 0.3 M NaCl at pH 8.3) was applied into the mixture, followed by the incubation for 60 min at 37 °C. The reaction was terminated by adding 150 μL of 1 M HCl and 1 mL ethyl acetate. The reactive solution was then centrifuged (GL-20B Refrigerated Centrifuge, Anting Scientific Instrument Co. Ltd., Shanghai, China) at 4000 rpm for 10 min at room temperature to collect the supernatant (750 μL), which was dried in an oven (DHG-9140, Jinghong, Shanghai, China) for 30 min at 105 °C. The liberated hippuric acid under the ACE action was finally dissolved in the deionized water (0.5 mL) to measure the absorbance at 228 nm (TU-1800PC, Persee General Instrument Co. Ltd., Beijing, China). 0.1 M of sodium borate buffers (containing 5 mM N-Hippuryl-His-Leu hydrate and 0.3 M NaCl at pH 8.3) without the samples and ACE were applied as a control and a blank, respectively. The ACE inhibitory activity (%) was calculated based on the following equation:

ACE inhibitory activity (%) = (Ab

A a)/(Ab

2.9. Identification of peptides in the selected WGH_APae fraction Peptide identification in the fraction 7 from WGH_APae was performed using an Acquity nano high performance liquid chromatography (nano HPLC) (Waters, Milford, USA) filled with a Thermo Acclaim PepMap C18, 125 A column (75 mm × 2 mm × 3 μm, Thermo Fisher Scientific, Massachusetts, USA). The nano HPLC was incorporated with a Thermo Q-Exactive high resolution mass spectrometer (Thermo Scientific, Waltham, USA) setting 70,000/17,500 scanning resolution on a 50–1600 m/z acquisition range. The sample was suspended in the mobile phase A (0.1% formic acid in water) at a concentration of 0.1 mg/mL, followed by the injection (7 μL) in the nano HPLC. Elution was then performed using a 1% to 35% gradient of mobile phase B (0.1% formic acid in acetonitrile) over a period of 65 min at a flow rate of 1 mL/min. Finally, peptides sequences were expressed with a PEAKS Studio software (version 7.5, Bioinformatics Solutions Inc., Waterloo, Canada).

A c) × 100%

where Ab is the absorbance of the control, Aa is the absorbance of the sample, Ac is the absorbance of the blank. IC50 value (mg/mL) was finally estimated by plotting the relative ACE inhibitory activity (%) against five different concentrations of the samples. 2.6. Simulated gastrointestinal digestion For the study of gastrointestinal digestion effect on the ACE inhibitory activity of the intact wheat gluten (WG) and the one hydrolyzed by the combination of Alcalase and PaproA (WGH_APae), in vitro stability was assayed according to the method of Tavares et al. (2011). In brief, the sample was digested with pepsin and trypsin as follows: pepsin solution (2.5% w/w) was prepared in a salt solution [consisting of NaCl, 36% (v/v) of HCl] at pH 2.0 and incubated (HZQ-X100 Incubator, Peiying, Suzhou, China) for 90 min at 37 °C while trypsin solution (2.5% w/w) was prepared in a 0.1 M potassium phosphate buffer at pH 8.0 and incubated for 2 h at 37 °C The reaction was terminated by boiling in a water bath for 10 min. After drying process, the digested residues [e.g., digested WG (WG_GD) and digested WGH_APae (WGH_APaeGD)] were analyzed for IC50 values as described in Section 2.5.

2.10. Synthesis of selected ACE inhibitory peptides According to the structure features of peptides, six peptides in fraction 7 from WGH_APae were synthesized to estimate their antihypertensive functions. The peptides were synthesized by Guotai Biotechnology Co. Ltd. (Hefei, China). Afterwards, the synthesized peptides were purified using a C18 reverse phase-high performance liquid chromatography (RP-HPLC) (Agilent Technologies Inc., California, USA) coupled with a C18 column (2.1 mm × 50 mm × 1.7 μm). 25 μL of the sample suspension (1 mg/mL in the 0.1% trifluoroacetic acid prepared in water) was eluted using solution A (0.1% trifluoroacetic acid in water) combined with solution B (0.1% trifluoroacetic acid in acetonitrile) at a linear gradient of 5–90% in 60 min at a flow rate of 3

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Table 2 Protein recovery rate, degree of hydrolysis (DH), molecular mass distribution, and ACE inhibitory activity (IC50) of the intact and hydrolyzed wheat glutens. Sample

WG WGH_Alc WGH_Pro WGH_Pae WGH_APro WGH_APae WGH_PP WG_GD WGH_APaeGD

Protein recovery rate (%)

– 52.35 54.78 48.60 63.38 66.50 62.69 – –

± ± ± ± ± ±

3.73a 4.81a 3.92a 4.81a 4.12a 6.77a

DH (%)

– 22.95 14.59 21.80 28.07 34.32 31.27 – –

± ± ± ± ± ±

Molecular mass distribution (%)

1.84c 3.51d 3.27c 1.43b 1.29a 3.10ab

IC50 (mg/mL)

< 1 kDa

1–5 kDa

5–10 kDa

> 10 kDa

2.16 87.97 75.68 82.58 94.22 95.11 89.68 – –

4.77 8.08 19.11 14.36 4.86 3.88 8.54 – –

5.97 0.49 1.88 1.34 0.05 0.05 0.62 – –

87.10 3.46 3.33 1.72 0.87 0.96 1.16 – –

2.79 0.38 0.42 0.32 0.32 0.21 0.23 0.44 0.25

± ± ± ± ± ± ± ± ±

0.10a 0.07bcd 0.09bc 0.02bcd 0.04bcd 0.02d 0.04d 0.06b 0.05 cd

Data represent the means ± one standard deviation (n = 3). Different letters (a–d) on the same column indicate significant (p < 0.05) differences among samples. Abbreviations include: wheat gluten (WG), wheat gluten hydrolyzed by Alcalase (WGH_Alc), wheat gluten hydrolyzed by Protamex (WGH_Pro), wheat gluten hydrolyzed by PaproA (WGH_Pae), wheat gluten hydrolyzed by Alcalase and Protamex (WGH_APro), wheat gluten hydrolyzed by Alcalase and PaproA (WGH_APae), wheat gluten hydrolyzed by Protamex and PaproA (WGH_PP), wheat gluten digested by simulated gastrointestinal fluids (WG_GD), and wheat gluten hydrolysate prepared by Alcalase and PaproA under the treatment of simulated gastrointestinal fluids (WGH_APaeGD).

1 mL/min. The eluted peak was observed at 214 nm to record the absorbance and collected to lyophilize for ACE inhibitory activity evaluation.

(~87.1%) in the wheat gluten was > 10 kDa, whereas the hydrolysates were dominated by smaller fragments (< 1 kDa). The two-step hydrolysis by Alcalase, Protamex, and/or PaproA promoted the release of smaller fractions in the hydrolysates than the one-step (by the single protease), which was also proved by Lin et al. (2011) in corn protein hydrolysates. Overall, Alcalase incorporated with PaproA developed the wheat gluten hydrolysate (WGH_APae) with a more desirable molecular mass distribution (~95.11% fractions < 1 kDa). From the view point of pharmacology, drug-like molecules should have molecular weights below 1 kDa to promote penetration through lipid bilayer membranes for circulation and absorption (Wang & Skolnik, 2009). So, the molecular mass profile of WGH_APae is promised for pharmaceutical and nutraceutical uses. To the best of knowledge, there is no report by others on the preparation of wheat gluten hydrolysates via PaproA hydrolysis. Previous studies revealed that Alcalase is a remarkable efficient protease for the production of wheat gluten hydrolysates with small fragments to present higher protein recovery rates and DH values in comparison to other proteases (Kong et al., 2007). Since WGH_Pae presented comparable physicochemical characteristics as WGH_Alc in the current study, PaproA displayed great hydrolytic efficiency as commercial Alcalase to enrich the selectivity of enzymes for hydrolysis. During the proteolysis, it is well acknowledged that the protease adheres on the active sites of proteins to create a link with certain amino acids to facilitate biochemical modifications (e.g., proton transfer reactions and electrostatic environment modulation) on protein structures (Saadi et al., 2015). Therefore, it is predicable that PaproA held a broad scope of specificity sites to amino acids to encourage protein hydrolysis. In practice, the bioactivity of hydrolysates is primarily dependent on the protein structure, specificity of proteases, DH, and conditions (e.g., temperature, time, and pH) during hydrolysis (Udenigwe & Aluko, 2012). The ACE inhibitory activity results of hydrolysates illustrated as a function of proteases are shown in Table 2. IC50 values that is inversely related to ACE inhibition potential reported a significant (p < 0.05) improvement of ACE inhibitory ability by the protease hydrolysis of wheat gluten, but the protease type was not significant (p > 0.05) to express the similar order of IC50, excepting Protamex by the poor solubility of wheat gluten to result in variation during hydrolysis. Overall, the combination of Alcalase and PaproA (IC50 of ~0.21 mg/mL) exhibited the highest specificity in the generation of ACE inhibitory hydrolysates followed by the combination of Protamex and PaproA (IC50 of ~0.23 mg/mL), the combination of Alcalase and Protamex (IC50 of ~0.32 mg/mL), PaproA (IC50 of ~0.32 mg/mL), Alcalase (IC50 of ~0.38 mg/mL), and Protamex (IC50 of ~0.42 mg/mL). This suggested the combination of proteases is helpful to generate bioactive fragments exposing specified carboxyl (C)-end and amine (N)-

2.11. Statistics All experiments were carried out on triplicate and reported as the mean ± one standard deviation. A one-way analysis of variance (ANOVA) and Holm-Sidak test were used to measure statistical differences in protein recovery rate, DH, and IC50 value in the various wheat gluten hydrolysates, WGH_APae fractions, and derived peptides. All statistics were performed using IBM SPSS Statistics 20.0 (Chicago, USA). 3. Results and discussion 3.1. Enzyme selection for antihypertensive hydrolysates production from wheat gluten Effect of various proteases on protein recovery rate, DH value, and molecular mass distribution are presented in Table 2. Although proteases type and composition were not significant (p > 0.05) on the protein recovery rate of hydrolysates, the combined proteases still gave higher (~12.28% more) protein recovery rate than the individual ones, especially the treatment of Alcalase followed by PaproA to yield WGH_APae (~66.5%), which expressed better protein recovery than commercial proteases to stimulate the industrial application (Kong, Zhou, & Qian, 2007). Regarding to DH value, an analysis of variance indicated that the type of proteases used to hydrolyze wheat gluten was significant (p < 0.05), in which wheat gluten hydrolysates prepared with the combination of two enzymes (e.g., WGH_APro, WGH_APae, and WGH_PP) showed significant higher (p < 0.05) DH values than those prepared by individual enzymes (e.g., WGH_Alc, WGH_Pro, and WGH_Pae). The WGH_APae exhibited the highest DH value (~34.32%), which was attributed to the mode of hydrolytic action involving synergistic endoprotease activities by Alcalase and PaproA. In addition, both of Alcalase and PaproA independently hydrolyzed the wheat gluten in higher degrees (~22.38%) than Protamex (~14.59%). This indicated that the hydrolysis efficiency of Alcalase and PaproA was much greater than that of Protamex possessing less cleavage sites on wheat gluten sequences, which is also demonstrated by Kong et al. (2007), who compared enzymatic hydrolysis of wheat gluten by several commercial proteases (e.g., Alcalase, pepsin, pancreatin, Neutrase, and Protamex). Moreover, proteases hydrolysis flipped over molecular mass distribution in the wheat gluten hydrolysates as the majority of molecules 4

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end with ACE inhibition features. This strategy has been particularly remarked in case of trypsin-chymotrypsin and pepsin-bromelain on whey protein and Ulva rigida protein, respectively (Paiva, Lima, Neto, & Baptista, 2016; Saint-Sauveur, Gauthier, Boutin, & Montoni, 2008). In comparison with other wheat gluten hydrolysates generated by commercial proteases (e.g., Pronase E with IC50 of ~0.39 mg/mL and Protease M with IC50 of ~0.42 mg/mL) (del Castillo et al., 2007; Motoi & Kodama, 2003), WGH_APae produced by Alcalase and PaproA (IC50 of ~0.21 mg/mL) displayed a stronger ability to prohibit ACE catalytic activity to lower blood pressure. It is well known that some bioactive substances can be fundamentally damaged under in vivo gastrointestinal digestion after oral administration to lose bioactive regulatory functions (Li et al., 2004). Because WGH_APae carried better physicochemical characteristics and ACE inhibitory activity than the other hydrolysates, it was selected to study the stability to against digestive proteases (pepsin and trypsin) in vitro as compared with the intact wheat gluten (Table 2). It was found that no hydrolysis of WGH_APae occurred after the subsequent digestion with pepsin and trypsin, since the IC50 value was not significantly changed (p > 0.05). However, the intact wheat gluten (IC50 of ~2.79 mg/mL) was drastically digested to small fractions (IC50 of ~0.44 mg/mL) with better ACE inhibition potential, which was still significantly (p < 0.05) lower than WGH_APae. Therefore, WGH_APae with a majority (~95.11%) of small fragments (< 1 kDa) had low susceptibility to digestion by the fast absorption of small molecules in the small intestine as compared with the intact wheat gluten containing ~87.1% of large fragments (> 10 kDa). Additionally, the exposed proline by proteases hydrolysis in WGH-APae could contribute to the resistance of hydrolysates degradation within the intestinal digestion and assimilation (Ohara, Matsumoto, Ito, Iwai, & Sato, 2007), which was also interpreted by Paiva et al. (2016) on the Ulva rigida protein hydrolysates and derived peptides to gastrointestinal enzymes (e.g., pepsin, trypsin, and chymotrypsin). As a consequence, Alcalase working with PaproA successfully fitted the hydrolysis of wheat gluten to generate a hydrolysate (WGH_APae) with tiny fractions (< 1 kDa) and great protein recovery rate to express an excellent capacity to bind ACE and stability to against simulated gastrointestinal fluids.

Fig. 1. Separation of the wheat gluten hydrolysate prepared with Alcalase and PaproA (WGH_APae) into seven fractions (A) and their ACE inhibitory activities (IC50) (B). Data represent the mean ± one standard deviation (n = 3). Different numbers of star on the bars indicate significant (p < 0.05) differences among fractions (F1-F7).

3.2. Fractionation of the selected wheat gluten hydrolysate (WGH_APae) The wheat gluten hydrolysate hydrolyzed with Alcalase and PaproA was fractionated through a Sephadex G-15 column to result in seven fractions with various ACE inhibitory activities (Fig. 1). Fractions 1–7 brought significant (p < 0.05) different binding abilities to ACE for blood pressure regulation, in which fraction 7 (IC50 of ~0.03 mg/mL) owned the strongest ability, followed by fraction 6 (IC50 of ~0.05 mg/ mL), fraction 2 (IC50 of ~0.15 mg/mL), fractions 5 and 4 (IC50 of ~0.20 mg/mL), and fractions 3 and 1 (IC50 of ~0.25 mg/mL). As compared to WGH_APae, fractions 2, 6, and 7 held significant lower IC50 values, properly attributing to the small molecules spreading in the fraction to be crucial for bioactivities. A similar result was also reported in the previous work by Paiva et al. (2016), who found Ulva rigida protein fraction corresponding to molecular weight < 1 kDa presented a higher ACE inhibitory activity (IC50 of ~0.095 mg/mL) than the unfractionated one (IC50 of ~0.483 mg/mL). Proteolytic hydrolysis has shown a direct beneficial influence on the exposure of hydrophobic, carboxyl, and free amino groups on amino acids to induce bioactive fragments (Saadi et al., 2015), so, the fraction 7 might retain abundant active groups on amino acids to modulate ACE activity. Since the structural properties of fractions (especially amino acid composition) in the hydrolysates are important components to determine ACE inhibitory activity, amino acid composition in each fraction was analyzed in comparison with the intact wheat gluten and WGH_APae (Table 3). The amino acid profiles were similar between the intact wheat gluten and its hydrolysate by Alcalase and PaproA containing relatively high levels of glutamic acid and proline coupled with

low levels of essential amino acids, such as methionine and lysine, which were previously informed by Zilic, Barac, Pesic, Dodig, and Ignjatovic-Micic (2011). However, both of them displayed dramatic different antihypertensive performances, thus, it is meaningful to separate WGH_APae by advanced chromatographic technique to ultimately illuminate the operative fragments really functioned on ACE activity. Resembling the intact WGH_APae, the first fraction (F1) coming out from the elution retained extraordinary higher amounts of glutamic acid (~41.89%) and proline (~21.51%) than fraction 2 (loss of glutamic acid) and fractions 6–7 (~8.19% of proline), which were more powerful to block ACE activity (Fig. 1B). In theory, both glutamic acid and proline are important features contributing to ACE inhibition by chelating with zinc on the ACE active center and hydrophobic interaction with ACE, respectively (Aluko, 2015). Hence, it was proposed that ionic interaction (between glutamic acid and zinc) and regular hydrophobic interaction (between proline and ACE) competed on the catalytic active sites of ACE to initiate constraint forces that adversely impact on the ACE inhibition by fraction 1. Regarding to fractions 3–5, although hydrophobic substances made up a huge proportion of the total amino acids (~68.05%), little presence and even absence of proline and negatively charged amino acids (aspartic acid and glutamic acid) were claimed to induce poorer ACE inhibitory potencies. This indicated that both of proline and negatively charged amino acids were more influential on the antihypertensive action, which was also evidenced by fractions 6–7 with a plentiful of negatively charged amino 5

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Table 3 Amino acid compositions (%) of the intact (WG) and hydrolyzed wheat gluten prepared by Alcalase and PaproA (WGH_APae) and its fractions (F1–F7). Amino acid

Asp Thr Ser Glu Gly Ala Cys Val Met Ile Leu Tyr Phe Lys His Arg Pro Trp Hydrophobic amino acids Aromatic amino acids

WG

3.03 2.40 4.81 39.13 3.14 2.38 1.74 3.71 1.41 3.22 6.98 3.38 5.13 1.33 2.10 3.18 11.92 1.02 26.22 9.52

WGH_APae intact

F1

F2

F3

F4

F5

F6

F7

3.26 2.48 4.89 39.70 3.28 2.44 0.96 3.80 1.26 3.02 6.70 3.14 4.99 1.37 2.29 3.06 12.03 1.36 25.35 9.48

3.06 1.44 2.27 41.89 3.37 0.93 2.42 1.79 0.48 1.39 2.96 1.96 6.28 1.79 2.66 3.60 21.51 0.19 15.80 8.43

0.00 0.48 2.25 0.00 7.66 1.25 3.23 8.91 1.75 6.81 8.45 2.58 6.63 6.63 9.40 10.24 22.55 1.19 36.37 10.40

0.00 0.00 0.00 0.00 0.96 4.01 4.67 14.20 4.59 10.68 30.53 2.43 6.60 1.09 3.59 7.84 7.65 1.16 73.04 10.19

0.00 0.30 1.16 13.45 2.42 0.63 1.01 3.09 5.23 4.26 12.69 11.75 32.51 0.32 1.15 2.10 7.28 0.65 70.16 44.91

1.63 2.42 2.09 17.94 2.36 1.15 0.76 2.14 0.22 2.99 2.35 23.21 28.88 0.43 0.41 0.89 6.96 3.16 60.95 55.25

3.02 2.25 5.70 34.46 5.25 1.95 1.00 2.73 0.60 2.86 2.87 10.91 10.88 0.64 0.33 1.54 9.28 3.75 32.79 25.54

5.68 2.27 2.17 28.27 5.91 4.69 1.35 4.10 0.54 4.06 11.85 5.62 4.41 1.92 0.35 2.24 7.10 7.47 35.28 17.50

acids (~35.72%). Moreover, tryptophan at the C-end has been abundantly proved to block the C-domain on ACE for blood pressure regulation (Iwaniak et al., 2014; Lunow, Kaiser, Ruckriemen, Pohl, & Henle, 2015), thus, a preventive effect on hypertension was promised on fraction 7 that contained the highest amount of tryptophan (~7.47%). As a result, it is critical to modulate proline and negatively changed amino acids at a moderate percentage to promote a stronger ACE inhibitory activity via hydrophobic interaction and metal chelation, while the presence of tryptophan at the C-terminal was tremendously assisted the antihypertensive activity of peptides.

Table 4 Peptides identified from fraction 7 in the wheat gluten hydrolysate prepared with Alcalase and PaproA (WGH_APae). Peptide sequence

QPQQPFPQ QPQQPFPQPQ QPQQPLPQ QQPLPQPQ IHVTET TCNMSCT AGPCAPNP QPQPFPQ TCYCEMVP EEPVLHAN TPVKKQP LAPVASTT APATPSFW AATPTSTTTT AIAPPISM TCSSCSAT LAFFNSV IAFFNSV APAPRPPNAP ALTLPVNA SAGGYIW ATTCSSSP TSFCCLCT EEAIFLW AAAAPRIS EESPHCC EGGGPGGEGSEG

3.3. Identification of antihypertensive peptides from fraction 7 in WGH_APae According to the greater ACE inhibitory potency and advanced amino acid composition, the most active fraction 7 from WGH_APae was further purified on a nano HPLC Thermo Acclaim PepMap C18 column to identify peptide sequences (Table 4). A total of 27 peptides in a range of 6–12 amino acid length was recognized in fraction 7, in which > 78% peptides performed the characteristics [e.g., proline and aromatic amino acids at the C-end, and hydrophobic amino acids (except aromatic amino acids) at the N-end] to inhibit ACE activity (Manoharan, Shuib, & Abdullah, 2017; Wu, Aluko, & Nakai, 2006). In general, most of effective ACE inhibitory peptides contains 2–13 amino acids (Li et al., 2004). Short peptides are more favorable for gastrointestinal absorption and circulation, whereas the peptides with high molecular weights are more fragile within the gastrointestinal tract and hard to be carried by specific precursors to lose bioactivities (Saadi et al., 2015). Moreover, a number of researches have been pointed out the presence of proline, tryptophan, tyrosine, or phenylalanine at the Cterminal and aliphatic amino acids (e.g., glycine, alanine, isoleucine, leucine, and valine) at the N-terminal residues of a peptide sequence potentially facilitate ACE inhibition by numerous interactions (e.g., hydrophobic, hydrogen, electrostatic, and van der Waals interactions) between peptides and ACE (Kim, Ngo, & Vo, 2012; Manoharan et al., 2017; Wu et al., 2006). Taking into all considerations, IHVTET, AGPCAPNP, APATPSFW, APAPRPPNAP, and SAGGYIW were selected to verify their ACE inhibitory potency in next. In addition, ACE is a zinccontaining peptidyl dipeptide hydrolase with active sites to bind with guanidine groups of arginine and glutamic acid (Bunning, 1983), so, EEAIFLW containing glutamic acid at the N-end was picked up to evaluate its capacity on the prevention of ACE activity. As reported in

Protein accession

M9TG60 M9TG60 M9TG60 M9TG60 A0A1D6D279 A0A1D6D279 A0A1D5XQE0 Q00M56 A0A1D5U838 A0A1D5Z5F4 W5I060 A0A1D5TPW4 A0A1D6CW75 A0A1D6D317 W5H0R1 A0A1D6AYQ9 A0A1D5U6S6 W5AAC4 A0A1D5X107 A0A1D6D060 A0A1D6A1A2 A0A1D5SET0 A0A1D6RUS1 A0A1D5Z8Q7 W5DS50 A0A1D5YJY2 A0A1D5SLJ7

Fragment number

f88-95 f124-133 f162-169 f164-171 f366-371 f133-139 f105-112 f55-62 f601-608 f420-427 f81-87 f617-624 f211-218 f168-177 f130-137 f45-52 f21-27 f283-289 f67-76 f236-243 f185-191 f32-39 f97-104 f292-298 f33-40 f6-12 f86-97

ACE inhibitory peptide features C1

C2

N

√ √ √ √ – – √ √ √ – √ – – – – – – – √ – – √ – – – – –

– – – – – – – – – – – – √ – – – – – – – √ – – √ – – –

– – – – √ – √ – – – – √ √ √ √ – √ √ √ √ – √ – – √ – –

Protein accession represents protein number of wheat gluten in UniProtKB. Abbreviations include: proline at the last three positions of C-terminus (C1), aromatic amino acids at C-terminus (C2), hydrophobic amino acids at N-terminus (N).

literature, TCNMSCT, EEPVLHAN, TCSSCSAT, TSFCCLCT, EESPHCC, and EGGGPGGEGSEG were excluded to considering by the deficiency of featured amino acids at the C- and N-terminals in the current study. As the representative peptides (including IHVTET, AGPCAPNP, APATPSFW, APAPRPPNAP, SAGGYIW, and EEAIFLW) were not encrypted in the wheat gluten sequences, the peptides were synthesized prior to exploring antihypertensive abilities (Table 5). They exhibited 6

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inhibitory peptides (SAGGYIW and APATPSFW) were successfully identified from WGH_APae. Overall, although the blood pressure reducing effects of SAGGYIW and APATPSFW were not as strong as the commercial drugs, their extreme low IC50 values (in vitro) still held promises to make them as therapeutic candidates to develop nutraceuticals and functional foods to prevent and/or treat hypertension.

Table 5 ACE inhibitory activities (IC50) and molecular weights (MW) of synthesized peptides. Peptide

MW (kDa)

IC50 (mg/mL)

IHVTET AGPCAPNP APATPSFW APAPRPPNAP SAGGYIW EEAIFLW

0.698 0.726 0.875 0.987 0.752 0.907

5.985 0.173 0.036 0.145 0.002 0.620

± ± ± ± ± ±

0.007a 0.002c 0.005e 0.004d 0.000f 0.005b

CRediT authorship contribution statement Peng Zhang: Data curation, Writing – original draft. Chang Chang: Formal analysis, Writing – review & editing. Haijie Liu: Supervision, Resources. Bo Li: Methodology. Qiaojuan Yan: Validation, Visualization. Zhengqiang Jiang: Funding acquisition, Project administration, Supervision, Validation, Writing – review & editing.

Data represent the means ± one standard deviation (n = 3). Different letters (a–f) on the same column indicate significant (p < 0.05) differences among peptides.

extreme different ACE inhibitory abilities in the following order: SAGGYIW > AGPCAPNP > APAPRPPNAP > AGPCAPNP > EEAIFLW > IHVTET. It suggested that the ACE inhibitor feature at the C-end (on SAGGYIW) was more powerful to prevent the angiotensin conversion (I to II) as compared with small molecular weights and featured N-ends (on IHVTET). It was also evidenced on VTPALR (0.655 kDa, IC50 of ~0.054 mg/mL) and KLPAGTLF (0.846 kDa, IC50 of ~0.011 mg/mL) by Li, Wan, Le, and Shi (2006). Although glutamic acid and alanine at the N-terminal with tryptophan and proline at the C-terminal independently impact ACE inhibitory activity (Manoharan et al., 2017; Wu et al., 2006), they started fighting on the active catalytic sites of ACE as they appeared together to substantially weaken the antihypertensive potency. This was responsible to the higher IC50 values of AGPCAPNP, APATPSFW, APAPRPPNAP, and EEAIFLW than SAGGYIW. According to the extremely lower IC50 values of SAGGYIW and APATPSFW, ACE inhibition properly enhanced by tryptophan at the Cterminus to block ACE active sites via electrostatic, hydrophobic, van der Waals, and hydrogen bond interactions (Suetsuna, 1998). Although both of them expressed much weaker antihypertensive activity than the captopril (IC50 of 0.163 ng/mL), their performance was comparable and even better than the other reported wheat gluten derived biopeptides (e.g., AQQLAAQLPAMCR with IC50 of 0.04 mg/mL and IPALLKR with IC50 of 0.07 mg/mL) (Asoodeh et al., 2014). Additionally, bioactive peptides are holding numerous advantages for health maintenance, but the toxicity is a major concern to develop peptide-based nutraceuticals. As reported by Gupta et al. (2013), the peptides containing cysteine, proline, aspartic acid, and histidine in higher percentages presented the potentiality of toxicity. Therefore, the toxicity of both SAGGYIW and APATPSFW was experimentally predicted by ToxinPred instrument (http://crdd.osdd.net/raghava/toxinpred/) to conclude a positive result with a low potentiality of toxicity. To promote both SAGGYIW and APATPSFW applications that were not displayed in BIOPEP and EROPMoscow database, the clarification of their inhibition patterns and kinetics on ACE was pre-requirements. Overall, to generate the inhibition, ACE preferred peptides holding aromatic amino acids at the C-terminal with small molecular weights. As the importance of stability to determine the feasibility of the selected peptides for oral administration, in silico gastrointestinal digestion with pepsin, trypsin, followed by chymotrypsin was performed on both SAGGYIW and APATPSFW. As a result, SAGGYIW and APATPSFW were degraded into SAGGY with IW and APATPSF with W, respectively, in which IW is an advanced ACE inhibitory peptide (IC50 of 4.6 μM) identified in BIOPEP database. This proposed that SAGGYIW could be potential for oral administration with a revolutionary ACE inhibitory activity.

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4. Conclusions The protease of Pseudomonas aeruginosa (PaproA) synergistically working with Alcalase was investigated to prepare the wheat gluten hydrolysate (WGH_APae) with ACE inhibitory activity. Two novel ACE 7

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