Purification and identification of angiotensin I-converting enzyme inhibitory peptides from fermented walnut residues

ABSTRACT

Defatted walnut residues (DWR) were hydrolysed using Bacillus subtilis to produce angiotensin-I-converting enzyme (ACE) inhibitory peptides. Fermented walnut residue extract (FWRE) was fractionated using ultrafiltration methods to identify the ACE-inhibitory peptides. The fraction with a molecular weight of <1 kDa (WPs-I) presented the greatest ACE-inhibitory activity. Reverse-phase high-performance liquid chromatography was used to separate WPs-I from the most active fractions, and the amino acid sequence of WPs-I was confirmed as VQTL (459.351 Da) and LGYEN (594.276 Da) by UPLC-ESI-MS/MS.

KEYWORDS:

• ACE-inhibitory activity
• ACE-inhibitory peptides
• Identification
• Fermented
• Walnut

Introduction

Hypertension or high blood pressure is increasing annually in populations worldwide. Approximately 13.5% of premature deaths worldwide are attributed to high blood pressure.^[1] Angiotensin-I-converting enzyme (ACE, EC 3.4.15.1) is a dipeptidyl carboxypeptidase associated with blood pressure regulation and cardiovascular function.^[2] Consequently, ACE-inhibitory substances are used to decrease the blood pressure of hypertensive patients.^[3] Potent synthetic ACE inhibitors, such as perindopril, captopril, enalapril, alacepril, lisinopril, and ramipril, are widely used to clinically treat hypertension in humans. However, these synthetic drugs exert several side effects, including cough, skin rashes, hypotension, hyperkalemia, headache, dizziness, and renal impairment.^[4,5] Food-derived ACE inhibitors are safer than synthetic compounds.^[6] To date, many ACE-inhibitory peptides have been isolated from food proteins, such as egg yolk protein, Agaricus bisporus, whey protein, and gluten.^[7–10] Some of these peptides have been found effective as antihypertensive agents in vivo without causing side effects in spontaneously hypertensive rats (SHRs) and hypertensive humans.^[11]

Walnut (Juglans regia L.) is the most widespread and oldest tree nut in the world.^[12] Walnut is a nutrient-dense food, mainly owing to its fat content, as well as protein, vitamin, and mineral profiles. Several biological properties of walnut such as antiatherogenic, anti-inflammatory, antimutagenic, and antioxidant activities have been reported.^[13] Studies have shown that bioactive peptides demonstrate functions better than those of other proteins, as a result of the enzymatic hydrolysis of nut proteins.^[14] However, the biological activity of fermented walnut products has not been investigated. Therefore, the current study aims to isolate and characterize potential ACE-inhibitory peptides from fermented walnut residue extract (FWRE).

Materials and methods

Materials

Defatted walnut residues (DWR), processed by Yunnan Huizhiyuan Food Company (Lincang City, China) in 2012, were ground to powder by a muller (mesh concentration 60 mm) with a protein content of 43.5%. Angiotensin converting enzyme (ACE) and hippuryl-L-histidyl-L-leucine (HHL) were purchased from Sigma-Aldrich Co. (St. Louis, MO, USA). Other chemicals (analytical grade) were obtained from Beijing Chemical Co., Ltd. (Kunming, China). B. subtilis GIM 1.135 was purchased from the Microbial Culture Collection Center of Guangdong (GIMCC), China.

Preparation of FWRE

Bacillus subtilis was stored at 4°C on agar slants and cultured for 16 h at 37°C prior to use for solid-state fermentation (SSF). After incubation for 16 h, part of the nutrient broth culture was used as an inoculum. Serial dilution by using sterile distilled water with glucose (0.2 g/100 mL) and KH₂PO₄ (0.26 g/100 mL) was performed to adjust the bacterial concentration to 108 CFU/mL. SSF was conducted in 500 mL Erlenmeyer flasks containing 100 g of DWRs, followed by autoclaving at 121°C for 20 min, cooling at room temperature, and use for inoculation. The fermentation conditions included a fermentation time of 82 h, temperature of 37°C, inoculum concentration of 10.40%, and water content of 1.50 mL/g.^[15]

FWRE was mixed with distilled water (1:25, g/mL) by shaking on a rotary shaker (180 rpm, 30 min, and 37°C) to prepare walnut peptides (WPs). The mixture was centrifuged (Allegra X-30R; Beckman Coulter, Brea, CA, Inc., USA) at 10,000 ×g for 10 min (4°C), and supernatants were lyophilized by a freeze dryer (LGJ-10F; Beijing Huaxing Technology Development Co., Ltd., Beijing, China).

ACE-inhibitory activity assay

ACE-inhibitory activity was measured by a spectrophotometric method^[16] with minor modifications. The reaction mixture contained 80 µL of 5 mmol/L HHL as a substrate, 10 µL of ACE powder (0.1 unit) in 100 mmol/L sodium borate buffer (pH 8.3), and 10 µL of the sample solution. The reaction was performed at 37°C for 30 min and terminated by adding 200 µL of 1 mol/L HCl and 1.2 mL of ethyl acetate (−20°C). After centrifugation, 1 mL of the supernatant was transferred into a test tube and evaporated at room temperature for 2 h in a vacuum. Hippuric acid was redissolved in 1 mL of distilled water, and the absorbance was measured at 228 nm. IC₅₀ was defined as the concentration of the peptide inhibiting 50% of the ACE activity. IC₅₀ was determined from the ACE-inhibitory activity and peptide contents of each sample after regression analysis.

Isolation of ACE-inhibitory peptides

Ultrafiltration

WPs were fractionated by ultrafiltration (UF) with 1000, 5000, and 10,000 molecular weight cut-off UF membranes (Amicon Co., Beverly, MA, USA). Four fractions with molecular weights of <1, 1–5, 5–10 kDa, and >10 kDa were obtained and lyophilized. The fractions were pooled, concentrated, and lyophilized for ACE-inhibitory activity assay.

Yield and content of the peptides isolated by ultrafiltration

The peptide content was determined in accordance with the national standard of China (GB/T 5009.124-2003). Walnut protein hydrolysate (1 g) was dispersed into 50 mL of trichloroacetic acid solution (15% TCA, w/w) and stirred magnetically for 30 min. The solution was allowed to stand for 5 min following filtration using a filter paper. The contents of the filtrated protein and free amino acid were determined by the Kjeldahl method and an automatic amino acid analyser (Hitachi L-8900, Tokyo, Japan), respectively.^[17] The peptide content and the yield of peptide were then calculated according to Eqs. (1) and (2).

(1)

(2)

ACE-inhibition pattern determination of the purified peptides

The fraction that exhibited the greatest ACE-inhibitory activity after ultrafiltration and purification was determined (ACE-inhibition pattern). The inhibition pattern was determined by spectrophotometry.^[16] ACE kinetics in the presence of the inhibitor were analysed by Lineweaver–Burk plots^[17] at three peptide concentrations (0, 0.2, and 0.4 mg/mL), in which the reciprocal of HHL concentration was used as an independent variable (X-axis), and the reciprocal of the HA production rate was used as a dependent variable (Y-axis). The Lineweaver–Burk plot, showing correlations between 1/V and 1/[S], was used to determine the X-axis intercepts of −1/K_m (without inhibitor) and −1/{K_m(1 + [I]/K_i)} (with inhibitor), which were used to calculate the inhibitory constant K _i.^[18] All the experiments were conducted in triplicate.

Purification and identification of ACE inhibitory peptide amino acid sequences (UPLC-ESI-MS/MS)

The fraction that exhibited the greatest ACE-inhibitory activity after ultrafiltration and purification was dissolved in distilled water and loaded into a Waters Acquity Ultra Performance Liquid Chromatography (UPLC) system with a reversed-phase column (Zorbax StableBond SB C18, 5 µm, 4.6 mm × 250 mm, Agilent, USA). The flow rate was set at 0.3 mL/min, and the column temperature was 30°C. Mobile phase B was 100% acetonitrile. For the analysis of samples, the solvent gradient used was 0.1% formic acid in water. The mobile phases were 5–70% B (0–20 min) and 70% B (20–25 min).^[19]

Mass spectrometry was performed on a Waters Xevo TQ-S (Waters, USA) with an electrospray ionization (ESI) source. Ionization was conducted in positive mode with a capillary voltage of 3500 V. Nitrogen was maintained at 40 psi for nebulization and 9 L/min at 350°C for evaporation temperature. Spectra were recorded over the mass/charge (m/z) range of 100–1000. Data acquisition and processing were performed using MassLynx 4.1. Peptide sequences were manually calculated using a 477A gas-phase automated sequencer (Applied Biosystems, Inc., Foster, CA USA).

Statistical analysis

All experiments were performed in at least three independent trials, and the results were reported as means ± standard deviations. Results were subjected to analysis of variance using SPSS, and the statistical significance of differences (p < 0.05) was evaluated.

Results and discussion

Isolation of ACE inhibitory peptides and its ACE inhibitory properties

The hydrolysate obtained by the digestion of walnut protein with B. subtilis showed higher ACE-inhibitory activity than that demonstrated by untreated walnut protein (Table 1). This finding indicated that the hydrolysis of walnut protein probably released a significant number of ACE-inhibitory peptides. The walnut protein hydrolysate was fractionated into four tiers by ultrafiltration. The peptide yield, peptide content, and ACE-inhibitory activities are listed in Table 1. WPs-I exhibited the highest ACE-inhibitory activity (IC₅₀ value of 0.098 ± 0.005 mg/mg) and peptide yield (62.20 ± 2.65%), whereas WPs-IV showed the lowest activity (IC₅₀ value of 0.982 ± 0.086 mg/mg) and peptide yield (1.20 ± 0.32%). WPs-II presented the highest peptide content, but it displayed a lower ACE-inhibitory activity (IC₅₀ value of 0.264 ± 0.027 mg/mg) than that of WPs-I. The molecular weight of walnut protein ranged from 12000 Da to 70000 Da,^[20] whereas most fractions of the WPs were smaller than 10 kDa. This result confirmed that B. subtilis digested the walnut protein into small peptide fragments. The peptide yield of WPs-I was higher than that of the three other fractions. The ACE-inhibitory activity of WPs-I was significantly higher than that of the enzymatic hydrolysis products of walnut protein (0.97–1.52 mg/mL).^[21] Pihlanto-Leppälä et al.^[22] found that the ACE-inhibitory activity of <1 kDa fraction in whey peptides was higher than that of the other tested fractions. Both results suggest the use of ultrafiltration to enrich the ACE-inhibitory peptides derived from walnut protein.

Table 1. Peptide yields, peptide contents, and IC50 values of fractions isolated by ultrafiltration.

Molecular weight (Da)	Peptide yield (%)	Peptide content (%)	IC50 values^a (mg/mL)
<1000	62.20 ± 2.65**	63.18 ± 1.64**	0.098 ± 0.005**
1000–5000	28.00 ± 1.05**	75.31 ± 2.68**	0.264 ± 0.027**
5000–10,000	8.60 ± 0.56**	67.43 ± 2.34**	0.335 ± 0.054**
>10,000	1.20 ± 0.32*	69.21 ± 2.16**	0.982 ± 0.086*
The walnut protein untreated by B. subtilis	ND	ND	1.831 ± 0.432

^aIC₅₀ value was defined as the concentration of inhibitor required to inhibit 50% of the ACE activity. All data are means and S.D. of triplicate measurements. *indicates statistical significance (p < 0.05); **indicates statistical significance (p < 0.01) compared to the control. ND, means not detected.

The inhibition pattern of the purified peptides against ACE

Lineweaver–Burk plots were constructed using three concentrations of WPs-I (0, 0.2, and 0.4 mg/mL), as shown in Fig. 1. The X-axis intercepts for the different concentrations of WPs-I are identical, indicating that WPs-I showed non-competitive inhibition. Accordingly, K_i was calculated to be 1.23 ± 0.15 mg/mL for this fraction. The Lineweaver–Burk plots indicated that WPs-I appeared non-competitive with HHL for the binding sites of ACE.

Figure 1. Lineweaver–Burk plot of WPs-I at three concentrations (0, 0.2, and 0.4 mg/mL).

Purification and identification of ACE inhibitory peptide amino acid sequences by LC-ESI-MS/MS analysis

WPs-I was subjected to RP-HPLC coupled online to an ion trap mass spectrometry to identify active peptides. Figure 2 shows the RP-HPLC-UV chromatographic profile corresponding to the analyses of Fra.I and Fra.II. The resulting chromatograms contained two major peaks (retaining time = 11.78 min, 16.64 min), which are the target peptides.

Figure 2. Purification of the peptides from the WPs-I fraction obtained from ultrafiltration using reversed-phase high-performance liquid chromatography on Zorbax SB-C18 column with a linear gradient of acetonitrile containing 0.1% formic acid (TFA) at a flow rate of 0.3 mL/min and column temperature 30°C.

Fraction WPs-I with relatively high ACE-inhibitory activity as previously indicated was subjected to individual peptide separation and sequence identification. Full-scan UPLC/MS data were mined post-acquisition for the calculated m/z values of the different multiple-charged Fra.I and Fra.II forms. These data were analysed by ESI-MS for molecular mass determination and then examined by ESI-MS/MS for the characterization of peptides. The MS spectrum of Fra.I is shown in Fig. 3A, and the MS/MS spectrum of a single charged ion with m/z at 459.351 Da is displayed in Fig. 3B. The MS spectrum of Fra.II is shown in Fig. 4A, and the MS/MS spectrum of a single charged ion with m/z at 594.276 Da is shown in Fig. 4B. Peptides were manually calculated. The results provided two deduced amino acid sequences: Val-Gln-Thr-Leu (VQTL, 459.351 Da) and Leu-Gly-Tyr-Glu-Asn (LGYEN, 594.276 Da). The results further confirmed the general finding, indicating that short peptides with 2–10 amino acids exhibit greater ACE-inhibitory activity,^[23] and bioactive peptides usually contain less than 20 amino acid residues per molecule.^[22] Moreover, peptides with lower molecular weights present a better chance of crossing the intestinal barrier and exerting biological effects.^[24] Majority of the related studies suggested that most ACE-inhibitory peptides derived from food sources are approximately 150–800 Da in terms of molecular weight.^[25] These findings agree with observations from other studies, thereby supporting the fact that the functional properties of ACE-inhibitory peptides are highly influenced by molecular mass. This report implies that active peptides can be selected to a degree by ultrafiltration, resulting in the production of a permeate stream with higher ACE inhibition than that of the original hydrolysate.^[26] Given the low molecular mass coupled with low allergenicity,^[27] the potential of the hydrolysate for use in functional foods to mitigate hypertension would seem to merit further clinical studies in humans. Synthetic drugs will always demonstrate greater activity than natural products such as hydrolysates or peptide fractions. However, hydrolysates not only exhibit biological activity but also improve the nutritional contribution.^[28] Peptide fractions are constituted by sets of peptides with similar molecular weights, which may synergistically act to increase their effects on an organism.^[29]

Figure 3. (A) The MS spectrum of Fra.I collected after RP-HPLC. (B) The MS/MS spectrum of a single charged ion (459.351 Da).

Figure 4. (A) The MS spectrum of Fra.II collected after RP-HPLC. (B) The MS/MS spectrum of a single charged ion (594.276 Da).

In addition to peptide size, different amino acid residues and peptide sequences may play an important role in determining ACE-inhibitory activities. Some studies suggested that most naturally occurring ACE-inhibitory peptides contain Pro, Lys, or aromatic amino acid residues.^[30–32] Peptides bound to ACE are strongly influenced by the C-terminal sequence, which would be more active with a hydrophobic amino acid at the C-terminal.^[33] In the current study, the C-terminal of VQTL is leucine, which is a hydrophobic amino acid. However, further research on the relationship between the structure and function of ACE-inhibitory peptides is needed because establishing the relationship between their structure and activity is a valuable research goal to further understand the underlying mechanisms.

Conclusion

In this study, fermented walnut extract (FWE) was proven to demonstrate extraordinary ACE-inhibitory activity. Two ACE-inhibitory peptides (VQTL and LGYEN) were identified by UPLC-ESI-MS/MS from the fraction (WPs-I) isolated from FWRE by ultrafiltration and RP-HPLC. Therefore, Fra.I and Fra.II are potentially suitable for extraction from related foods as natural ACE inhibitors for preventing hypertension. Moreover, the structural analysis laid a solid foundation for the artificial synthesis of ACE-inhibitory peptides. FWE or its isolated peptides can serve as a promising ACE-inhibitor source for functional food ingredients and pharmaceuticals.

Funding

The authors would like to acknowledge the financial support of the National Natural Science Foundation of China (No. 51376200).

Additional information

Funding

The authors would like to acknowledge the financial support of the National Natural Science Foundation of China (No. 51376200).