Food Chemistry 148 (2014) 396–401

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Food Chemistry

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Novel angiotensin I-converting enzyme inhibitory peptides derivedfrom edible mushroom Agaricus bisporus (J.E. Lange) Imbach identifiedby LC–MS/MS

0308-8146/$ - see front matter � 2013 Elsevier Ltd. All rights reserved.http://dx.doi.org/10.1016/j.foodchem.2013.10.053

⇑ Corresponding author. Tel.: +60 3 79674371; fax: +60 3 79674178.E-mail address: noorlidah@um.edu.my (N. Abdullah).

Ching Ching Lau a, Noorlidah Abdullah a,⇑, Adawiyah Suriza Shuib a,b, Norhaniza Aminudin a,b

a Mushroom Research Centre, Institute of Biological Sciences, Faculty of Science, University of Malaya, 50603 Kuala Lumpur, Malaysiab University of Malaya Centre for Proteomics Research (UMCPR), Medical Biotechnology Laboratory, Faculty of Medicine, University of Malaya, 50603 Kuala Lumpur, Malaysia

a r t i c l e i n f o a b s t r a c t

Article history:Received 20 March 2013Received in revised form 22 July 2013Accepted 10 October 2013Available online 24 October 2013

Keywords:Button mushroomMedicinal mushroomCompetitive ACE inhibitorNon-competitive ACE inhibitor

Angiotensin I-converting enzyme (ACE) inhibitors derived from foods are valuable auxiliaries to agentssuch as captopril. Eight highly functional ACE inhibitory peptides from the mushroom, Agaricus bisporus,were identified by LC–MS/MS. Among these peptides, the most potent ACE inhibitory activity was exhib-ited by AHEPVK, RIGLF and PSSNK with IC50 values of 63, 116 and 129 lM, respectively. These peptidesexhibited high ACE inhibitory activity after gastrointestinal digestion. Lineweaver–Burk plots suggestedthat AHEPVK and RIGLF act as competitive inhibitors against ACE, whereas PSSNK acts as a non-compet-itive inhibitor. Mushrooms can be a good component of dietary supplement due to their readily availablesource and, in addition, they rarely cause food allergy. Compared to ACE inhibitory peptides isolated fromother edible mushrooms, AHEPVK, RIGLF and PSSNK have lower IC50 values. Therefore, these peptidesmay serve as an ideal ingredient in the production of antihypertensive supplements.

� 2013 Elsevier Ltd. All rights reserved.

1. Introduction of A. bisporus increased from 2.4 million tons in 2001, to 4 million

Hypertension or high blood pressure is on the rise worldwide.Approximately 972 million adults had high blood pressure in2000; this is predicted to increase to a total of 1.56 billion in2025 (Kearney et al., 2005). About 13.5% of global prematuredeaths are attributed to high blood pressure (Lawes, Hoom, &Rodgers, 2008).

The renin–angiotensin system (RAS) plays a fundamental role inblood pressure regulation (Volpe et al., 2002). Consumption offruits, vegetables and low fat dairy products has been shown to sig-nificantly reduce blood pressure (Jauhiainen & Korpela, 2007). Pep-tides are a food component that may regulate the RAS throughinhibition of the angiotensin converting enzyme (ACE). A rangeof dietary ACE inhibitory peptides have been isolated from plants(Guang & Phillips, 2009), dairy (Gobbetti, Ferranti, Smacchi,Goffredi, & Addeo, 2000) and seafood, such as freshwater clams(Tsai, Lin, Chen, & Pan, 2006), sea cucumbers (Zhao et al., 2009)and oysters (Shiozaki et al., 2010). ACE inhibitory peptides havealso been successfully purified from edible mushrooms, such asGrifola frondosa (Choi, Cho, Yang, Ra, & Suh, 2001), Pholiota adiposa(Koo et al., 2006) and Pleurotus cornucopiae (Jang et al., 2011).

Agaricus bisporus (button mushroom) is one of the most culti-vated and consumed mushrooms worldwide. Global production

tons in 2009 (Sonnenberg et al., 2011; Van Griensven & Roestel,2004). Bioactive compounds from A. bisporus have been shown toexhibit antioxidant (Tian et al., 2012), hypoglycaemic (Mao, Mao,& Meng, 2013) and hypocholesterolaemic (Jeong et al., 2010) ef-fects. Peptides from A. bisporus have shown potentially high ACEinhibitory activity (Lau, Abdullah, Shuib, & Aminudin, 2012).Therefore, the objective of the current study was to isolate andcharacterise potential ACE inhibitory peptides from A. bisporus.

2. Materials and methods

2.1. Materials

Fruiting bodies (basidiocarps) of A. bisporus were purchasedfrom a supermarket. All solvents and chemicals used in this studywere of analytical and HPLC grade. Acetonitrile and trifluoroaceticacid (TFA) were obtained from Merck (Darmstadt, Germany). ACEfrom rabbit lung, hippuryl-L-histidyl-L-leucine (HHL) and gastroin-testinal proteases (pepsin, trypsin and a-chymotrypsin) werepurchased from Sigma–Aldrich (St. Louis, MO, USA).

2.2. Extraction and purification of potential ACE inhibitory peptides bysize exclusion chromatography (SEC)

Protein extraction from A. bisporus was done based on aprevious study (Lau et al., 2012). Briefly, fresh fruiting bodies of

C.C. Lau et al. / Food Chemistry 148 (2014) 396–401 397

A. bisporus were cleaned, sliced and blended with distilled water ata ratio of 1:2 (w/v). The mixture was filtered and centrifuged to re-move unwanted debris. Proteins were precipitated out from thewater extract using ammonium sulphate at 10–100% salt satura-tion. Precipitated proteins showing the highest ACE inhibitoryactivity were then fractionated by reverse phase high performanceliquid chromatography (RPHPLC). Based on the results reported byLau et al. (2012), the active RPHPLC fraction was E3AbF6. Thus, thiswas further purified in the current study by SEC using a BiosepSEC-S2000 column (300 � 7.8 mm, Phenomenex, Torrance, CA,USA). Analysis was performed on an HPLC system equipped withan SCL-10AVP system controller, LC-10ATVP solvent delivery unit,SPD-M10AVP UV–Vis diode array detector and DGU-12A degasser(Shimadzu, Kyoto, Japan). The mobile phase consisted of 45% ace-tonitrile containing 0.1% TFA. The flow rate was 1.0 ml/min andthe effluent monitored at 214 nm. The SEC eluent was collectedas individual fractions according to the peaks obtained. The SECfractions collected were freeze-dried and the ACE inhibitoryactivity was determined at a concentration of 1 lg/ml protein.The SEC fraction with the highest ACE inhibitory activity wasanalysed by liquid chromatography–mass spectrometry forsequence identification.

2.3. Estimation of protein content in the SEC protein fraction

Protein content of SEC fractions was estimated using the Pierce�

Bicinchoninic Acid (BCA) Protein Assay Kit (Thermo Scientific,Rockford, IL, USA) according to the protocol provided by the man-ufacturer. The absorbance values were measured with a Sunrise™ELISA microplate reader (Tecan, Grödig, Austria) at 562 nm. Theprotein content was determined by comparing the absorbance va-lue of the samples with a standard curve of bovine serum albumin(BSA).

2.4. Assay of ACE inhibitory activity

In the current study, the ACE inhibitory activity was determinedusing an ACE inhibitory assay kit (ACE kit-WST, Dojindo Laborato-ries, Kumamoto, Japan). The assay was carried out according to theprotocol provided by the manufacturer. Absorbances of thereactions were measured on a Sunrise™ ELISA microplate reader(Tecan, Grödig, Austria) at 450 nm. The ACE inhibitory activity ofthe samples was calculated using the formula given in the protocol.The concentration of the ACE inhibitor required to inhibit 50% ofACE activity under the above assay conditions was defined as theIC50.

2.5. Liquid chromatography–mass spectrometry (LC–MS/MS)

Identification of the peptide sequences present in SEC fractionsC1 and C4 was carried out by LC–MS/MS at Proteomics Interna-tional Pty Ltd, WA, Australia. Briefly, the SEC fractions weredigested with trypsin and the extracted peptides were analysedby electrospray ionisation mass spectrometry using an Ultimate3000 nano HPLC system (Dionex, Sunnyvale, CA, USA) coupled toa 4000 QTRAP mass spectrometer (Applied Biosystems, Foster City,CA, USA). Peptides were loaded onto a C18 PepMap100, 3 lm (LCPackings) column and separated with a linear gradient of water/acetonitrile/0.1% formic acid (v/v). Protein identification wascarried out using Mascot sequence matching software (MatrixScience) with the Ludwig NR database.

2.6. Peptide synthesis

The eight identified potential ACE inhibitory peptides werechemically synthesised by Peptron, Inc., Republic of Korea. The

purity of the synthesised peptides was >98% measured by RPHPLCand MS analysis.

2.7. Effect of gastrointestinal digestion on the selected peptides

The stability of peptides RIGLF, AHEPVK and PSSNK against gas-trointestinal proteases was assessed in vitro by the method of Wuand Ding (2002). The peptide solution (0.1 mg/ml, 0.5 ml) wasincubated with 0.5 ml of 0.05% pepsin solution (0.1 M HCl, pH2.0) for 2.5 h at 37 �C. In the successive pepsin–pancreatin diges-tion test, the peptide solution was adjusted to pH 8.0 after the pep-sin digestion. Then, 0.5 ml of pancreatin solution [potassiumphosphate buffer (0.1 M, pH 8.0) containing 0.025% (w/v) chymo-trypsin and 0.025% (w/v) trypsin] was added to the solution. Themixture was incubated for another 2.5 h at 37 �C. The control(without digestion) consisted of the peptide solution incubatedin buffer solutions (HCl and potassium phosphate buffer) andwas carried out alongside the experiment. After the enzymatictreatment, the pepsin solution and pepsin–pancreatin solutionwere boiled for 10 min to stop the digestion and then centrifugedat 10,000 rpm for 10 min. The supernatants were freeze-driedand used for the measurement of ACE inhibitory activity. The sta-bility of the peptides against gastrointestinal digestion was ana-lysed by SEC.

2.8. Determination of the inhibition pattern on ACE activity

The ACE inhibition pattern of peptides RIGLF, AHEPVK andPSSNK was determined spectrophotometrically using HHL as thesubstrate. Basically, 20 ll of the ACE solution (0.1 UN/ml) and50 ll of peptides were incubated with 200 ll of various HHL con-centrations (0.63, 1.25, 2.50 and 5.00 mM). The enzymatic reactionwas terminated by the addition of 250 ll of 1.0 M HCl. The liber-ated hippuric acid was extracted with ethyl acetate and evaporatedunder vacuum. The hippuric acid residue was re-dissolved in1.0 ml of distilled water and the absorbance was determined at228 nm, using a spectrophotometer (SmartSpec™ Plus Spectropho-tometer, Bio-Rad Laboratories, Hercules, USA). The kinetics of ACEinhibition in the presence and absence of each peptide (0.00, 0.05and 0.50 mg/ml) was determined by Lineweaver–Burk plots.

2.9. Statistical analysis

The analysis of ACE inhibitory activity was carried out in tripli-cate and the result was reported as mean ± standard deviation.Mean differences of ACE inhibitory activity in SEC fractions wasanalysed using one-way ANOVA in Statgraphics Plus 3.0 at p < 0.05.

3. Results and discussion

3.1. Purification of potential ACE inhibitory peptides by SEC

Bioassay-guided purification of proteins extracted from A. bisp-orus fruiting bodies was carried out by ammonium sulphate pre-cipitation followed by RPHPLC and SEC. The most active RPHPLCfraction, E3AbF6, as reported in Lau et al. (2012), was further frac-tionated by SEC. Seven fractions (C1 to C7) were obtained after SECas illustrated in Fig. 1. Other peaks were not collected as their peakarea was low and thus, considered as background impurities. Thepercentages of protein recovered in the SEC fractions were in therange of 0.7–5.1%, with the highest protein content eluted in C3(Table 1). The SEC fractions were collected and tested for theirACE inhibitory activity at a concentration of 1 lg/ml. Referring toTable 1, SEC fractions C1 and C4 exhibited the highest ACE inhibi-tory activity, where 18.7% and 16.1% of ACE activity was blocked,

Fig. 1. SEC chromatogram of E3AbF6. Following RPHPLC, the active protein E3AbF6 was further separated using a Biosep SEC-S2000 column (300 � 7.8 mm). The mobilephase consisted of 45% acetonitrile containing 0.1% TFA, eluting at a flow rate of 1.0 ml/min. Seven peaks eluted from the SEC column labelled C1 to C7, these were collectedand re-evaluated for ACE inhibitory activity.

Table 1Percentages of protein recovery yield and percentages of ACE inhibitory activity of theSEC fractions.

SEC fraction % Recovery % ACE inhibitory activity*

C1 4.5 18.7 ± 3.2e

C2 2.9 5.8 ± 2.3bc

C3 5.1 0.9 ± 0.6f

C4 4.0 16.1 ± 0.5e

C5 1.7 6.4 ± 1.5bcd

C6 0.7 6.6 ± 2.5bcd

C7 2.1 3.4 ± 1.8bf

Total 21.0 –

* ACE inhibitory activity of SEC fractions was tested at 1 lg/ml protein andexpressed as a mean ± standard deviation (n = 3). Different letters (b–f) within acolumn indicate the percentage of ACE inhibitory activity are significantly different(p < 0.05). The SEC fraction highlighted in bold was selected for further analysis.

Table 2List of peptide sequences, molecular masses and IC50 values of selected potential ACEinhibitors from SEC fractions C1 and C4.

SEC fraction Peptide sequences Molecular mass (Da) *IC50 (lM)

C1 KVAGPK 598.42 304.9 ± 7.1b

FALPC 550.20 690.9 ± 5.3c

RIGLF 605.30 115.9 ± 10.4a

EGEPR 587.30 728.2 ± 6.6c

APSAK 473.20 325.7 ± 6.5b

C4 AHEPVK 679.53 62.8 ± 5.8a

GVQGPM 589.30 736.7 ± 4.8c

PSSNK 532.30 129.3 ± 16.6a

Peptide sequences highlighted in bold were selected for further analysis.* IC50 values are expressed as a mean ± standard deviation (n = 3). Different let-

ters (a–c) indicate the IC50 values are significantly different (p < 0.05).

398 C.C. Lau et al. / Food Chemistry 148 (2014) 396–401

respectively. The remaining five SEC fractions exhibited 0.9–6.6%ACE inhibition. C1 and C4 were selected for further analysis byLC–MS/MS.

3.2. Identification of ACE inhibitory peptides by LC–MS/MS

The peptide sequences in C1 and C4 determined by LC–MS/MSwere selected as potential ACE inhibitors based on the characteris-tics of the amino acid composition, namely, fewer than six aminoacids with mostly hydrophobic amino acids (Pripp, Isaksson,Stepaniak, Sorhaug, & Aldo, 2005). In addition, the C- and N-ter-mini of ACE inhibitors usually consist of aromatic and branched ali-phatic amino acids, respectively (Wu, Aluko, & Nakai, 2006). In thecurrent study, five peptide sequences of potential ACE inhibitorswere identified in C1, while three were identified in C4. The aminoacid sequences and molecular weights of these peptides are listedin Table 2. The molecular masses of the eight peptides were in therange of 473–680 Da. The low molecular weight of these peptidesis an added advantage for an ACE inhibitor because large peptidemolecules are restricted from fitting into the active site of ACE(Natesh, Schwager, Sturrock, & Acharya, 2003).

Referring to Table 2, AHEPVK exhibited the most potent ACEinhibitory activity. It inhibited 50% of the ACE activity at a concen-tration of 63 lM. Besides AHEPVK, there were two other peptides,RIGLF and PSSNK, which exhibited a high ACE inhibitory activitywith IC50 values of 116 and 129 lM, respectively. These peptides

have lower IC50 value than ACE inhibitory peptides isolated fromother edible mushrooms, i.e. G. frondosa (130 lM), P. adiposa(254 lM) and P. cornucopiae (277 lM) (Choi et al., 2001; Janget al., 2011; Koo et al., 2006). KVAGPK and APSAK blocked 50% ofthe ACE activity at a concentration of 305 and 326 lM, respec-tively. Although the values were higher compared to RIGL, AHEPVKand PSSNK, they were lower compared to the IC50 values of peptideF2-1B purified from P. cornucopiae. This peptide showed a IC50

value of 540 lM (Jang et al., 2011). The remaining three peptidestested in the current study showed IC50 values in the range of691–737 lM. Based on the IC50 values, three peptides, i.e. RIGLF,AHEPVK and PSSNK having the most potent ACE inhibitory activitywere selected for further analysis to determine their stabilityagainst gastrointestinal digestion.

3.3. Effect of simulated gastrointestinal digestion on the selectedpeptides

The digestion of proteins and peptides is initiated by pepsin inthe stomach, followed by trypsin and chymotrypsin in the smallintestine. Hence, the oral route of administration for proteins andpeptides may lead to proteolysis in the gastrointestinal tract andthe high acidity of the stomach may destroy them before theyreach the intestine for absorption. Therefore, proteins and peptideshave to be able to maintain their biological activity in the gastroin-testinal tract before they reach their target site inside the body.

0

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RIGLF AHEPVK PSSNK

AC

E in

hibi

tory

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ivity

(%)

ACE inhibitory peptides

Control Pep Pep + T + C

Fig. 2. Effect of simulated gastrointestinal digestion on the ACE inhibitory activityof peptides RIGLF, AHEPVK and PSSNK. Control: The peptide solutions (0.1 mg/ml)were incubated in buffer solutions (HCl and potassium phosphate buffer). Pep: Thepeptide solutions were incubated with 0.05% pepsin solution for 2.5 h at 37 �C.Pep + T + C: The peptide solutions were successively digested with pepsin solutionfor 2.5 h. They were further incubated in pancreatin solution for another 2.5 h at37 �C. The ACE inhibitory activity are expressed as mean ± standard deviation(n = 3).

C.C. Lau et al. / Food Chemistry 148 (2014) 396–401 399

Preliminary experiments by gastrointestinal enzyme incubationin vitro provide an easy method to evaluate the fate of these pep-tides after oral administration.

Referring to Fig. 2, RIGLF and AHEPVK exhibited a high ACEinhibitory activity when they were incubated in buffers withoutgastrointestinal enzymes (control). Both peptides inhibited 80.3%

C

Pep

Pep+T +C

I

TTime (min)

mA

Um

AU

mA

U

mA

Um

AU

mA

U

Fig. 3. Stability of the synthetic peptides (I) RIGLF (II) AHEPVK and (III) PSSNK against gaon a Biosep SEC-S2000 column (300 � 7.8 mm); mobile phase consisted of 45% acetonitwithin the retention time illustrated in the box. C: The peptide solutions (0.1 mg/ml) w0.05% pepsin solution for 2.5 h at 37 �C. Pep + T + C: The peptide solutions were successivefor another 2.5 h at 37 �C.

of ACE activity. On the other hand, PSSNK exhibited 63.3% of ACEinhibitory activity. All three peptides had a high ACE inhibitoryactivity, i.e. they inhibited more than 90% of ACE activity after gas-trointestinal digestion. Previous studies have reported on peptideswhich were resistant to further gastrointestinal digestion andmaintained their biological activity after digestion (Wu & Ding,2002). However, some peptides undergo further hydrolysis bygastrointestinal enzymes to release true inhibitors (Li, Le, Shi, &Shrestha, 2004).

In order to verify the stability of the three peptides tested in thecurrent study, the changes without and following gastrointestinaldigestion were analysed by SEC. The chromatograms are illustratedin Fig. 3. The peaks for the buffer (HCl and potassium phosphatebuffer) eluted at approximately 9 and 11 min. They are revealedby the presence of two extra peaks in the chromatograms for con-trol group. The BIOPEP database (www.uwm.edu.pl/biochemia) isan online program that can serve as a tool to predict possible pro-teolysis products by gastrointestinal enzymes and define the pos-sible biological activity of the proteolysis fragments (Iwaniak &Dziuba, 2010). Therefore, the predicted proteolysis activity ana-lysed by the BIOPEP database was compared with the SEC chro-matograms of RIGLF, AHEPVK and PSSNK in the current study.

Based on the BIOPEP analysis, RIGLF is predicted to release a tet-rapeptide, RIGL after pepsin digestion and is further hydrolysed torelease a tripeptide, IGL, by pancreatin enzymes. Referring toFig. 3(I), the peptide RIGLF, which eluted at 8.50 min was not de-tected in the chromatograms of the peptide after gastrointestinaldigestion confirming the prediction by the BIOPEP analysis. Inter-estingly, the tripeptide fits the criteria of a potent ACE inhibitor.It has a branched aliphatic amino acid, isoleucine at the N-termi-nus and strong hydrophobic amino acid, leucine at the C-terminus

II III

Time (min)ime (min)

mA

Um

AU

mA

U

strointestinal enzymes observed by SEC chromatograms. Separation was performedrile containing 0.1% TFA; flow rate of 1.0 ml/min. The peptide was eluted as a peakere incubated in buffer solutions. Pep: The peptide solutions were incubated withly digested with pepsin for 2.5 h. They were further incubated in pancreatin solution

400 C.C. Lau et al. / Food Chemistry 148 (2014) 396–401

(Wu et al., 2006). This explains the enhanced ACE inhibitory activ-ity of RIGLF after gastrointestinal digestion as shown in Fig. 2.

According to BIOPEP, AHEPVK was not hydrolysed by the threeproteolytic enzymes. It was predicted to remain stable throughoutthe digestion process. Referring to Fig. 3(II), the peptide AHEPVK,which eluted at 7.80 min, showed a high intensity in the SEC chro-matograms of the control and after digestion. This confirmed thestability of AHEPVK against digestive enzymes. Additionally, Wanget al. (2011) have reported that the preferential parameters forhexapeptides with potent ACE inhibitory activity are hydrophobicand stereo properties. This may explain the high ACE inhibitoryactivity of AHEPVK both before (in control) and after digestion.

BIOPEP analysis predicted that PSSNK would remain stable dur-ing pepsin digestion, releasing a tetrapeptide, SSNK from its pre-cursor after being digested by pepsin–pancreatin enzymes. TheSEC chromatogram of PSSNK shown in Fig. 3(III) has detected thepeptide peak in both control and after digestion. SEC separatesthe protein or peptide based on their molecular weight. The releaseof the amino acid, proline from PSSNK after pepsin–pancreatin

I

II

III

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-0.5 0 0.5

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.D./

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) -1

1/[S

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-0.5 0 0.5

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.D./

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1/[S

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400

500

600

700

800

-0.5 0 0.5

1/V

(∆O

.D./

min

) -1

1/[S0.00 mg/ml 0.0

Fig. 4. Kinetics of the synthetic peptides (I) RIGLF, (II) AHEPVK and (III) PSSNK. Theconcentrations of the peptides (0.00, 0.05 and 0.50 mg/ml). Lineweaver–Burk plot for eachas a mean ± standard deviation (n = 3).

digestion may not have much effect on its molecular weight.Therefore, SSNK could have eluted at approximately the sameretention time as PSSNK at 8.30 min. According to Wang et al.(2011), a tetrapeptide with potential ACE inhibitory activity pos-sesses a stereo and electrical amino acid such as lysine. Besides,potential ACE inhibitory peptides with an amino acid serine atthe N-terminal have been isolated from garlic (Suetsuna, 1998).Therefore, the SSNK which contain serine and lysine at its N- andC-termini could have caused an enhanced ACE inhibitory activityof PSSNK after gastrointestinal digestion as shown in Fig. 2.

3.4. Inhibition pattern of ACE inhibitors

The inhibition pattern of RIGLF, AHEPVK and PSSNK against theACE enzyme was determined by the Lineweaver–Burk plot. The re-sults obtained are given in Fig. 4. According to Fig. 4(I) and (II), RIG-LF and AHEPVK showed a competitive inhibition pattern. Thisshows that RIGLF and AHEPVK tended to bind to the active siteof ACE to block it from binding to the substrate (Jao, Huang, &

1 1.5

] (1/M)

5 mg/ml 0.50 mg/ml

1 1.5] (1/M)

5 mg/ml 0.50 mg/ml

1 1.5

] (1/M)5 mg/ml 0.50 mg/ml

ACE inhibitory activity was determined in the absence and presence of differentpeptide was constructed using values of 1/v against 1/[S]. Each value are expressed

C.C. Lau et al. / Food Chemistry 148 (2014) 396–401 401

Hsu, 2012). Additionally, ACE has been reported to show a prefer-ence for competitive inhibitors that contain a hydrophobic aminoacid at the third position from the C-terminal (Chel-Guerrero,Domínguez-Magaña, Martínez-Ayala, Dávila-Ortiz, & Betancur-Ancona, 2012). This is in accordance with the amino acidsequences of RIGLF and AHEPVK, which might explain the compet-itive inhibition pattern exhibited by these peptides. A competitiveinhibition pattern has been previously reported for ACE inhibitorypeptides purified from edible mushrooms, such as G. frondosa, P.cornucopiae and P. adiposa (Choi et al., 2001; Jang et al., 2011;Koo et al., 2006). Likewise, competitive ACE inhibitory peptideshave been reported from food-derived peptides, such as milk andfreshwater clams (Gobbetti et al., 2000; Tsai et al., 2006). In addi-tion, the first orally administered ACE inhibitory drug, captopril,also exhibits competitive ACE inhibition (Bhuyan & Mugesh, 2011).

Referring to Fig. 4(III), PSSNK exhibited a non-competitive inhi-bition pattern against the ACE enzyme. This shows that PSSNKtended to bind together with the substrate to the enzyme at anygiven time and, thus, the enzyme-substrate-inhibitor complexcould not produce products (Jao et al., 2012). Non-competitiveACE inhibitory peptides have been purified from the edible mush-room P. cornucopiae (Jang et al., 2011). Some food-derived peptidessuch as those derived from soybean and oyster have also beenreported to exhibit a non-competitive inhibition pattern (Kuba,Tanaka, Tawata, Takeda, & Yasuda, 2003; Shiozaki et al., 2010).

4. Conclusion

In the current study, eight potential ACE inhibitory peptideswere isolated from A. bisporus. Among the peptides tested, RIGLF,AHEPVK and PSSNK exhibited potentially high ACE inhibitoryactivity even after in vitro gastrointestinal digestion. RIGLF andAHEPVK showed a competitive inhibition pattern while PSSNKexhibited a non-competitive inhibition pattern. Although captoprilhas stronger ACE inhibitory activity than the peptides investigatedin the current study, these peptides are derived from food sources.Additionally, mushrooms have an added advantage compared toother food sources due to their low probability to cause foodallergy. Therefore, these peptides could be applied as an ingredientin functional foods, dietary supplements or in pharmaceuticals asan antihypertensive agent.

Acknowledgements

The authors would like to thank the University of Malaya (GrantPPP: PS238/2008C, PS478/2010B, PV073-2011B) and the Ministryof Higher Education Malaysia (HIR-MOHE: F000002-21001) forfinancial support for this project.

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