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Egg-yolk protein by-product as a source of

ACE-inhibitory peptides obtained with usingunconventional proteinase from Asian pumpkin(Cucurbita ficifolia)

Ewelina Eckert, Aleksandra Zambrowicz⁎, Marta Pokora, Bartosz Setner1, Anna Dąbrowska,Marek Szołtysik, Zbigniew Szewczuk1, Antoni Polanowski,Tadeusz Trziszka, Józefa ChrzanowskaDepartment of Animal Products Technology and Quality Management, Wroclaw University of Environmental and Life Sciences,Chełmońskiego 37/41, 51-630 Wrocław, Poland

A R T I C L E I N F O

⁎ Corresponding author at: Department of Anand Life Sciences, Chełmońskiego 37/41, 51-6

E-mail address: aleksandra.zambrowicz@1 Faculty of Chemistry, University of Wrocl

http://dx.doi.org/10.1016/j.jprot.2014.08.0031874-3919/© 2014 Elsevier B.V. All rights rese

A B S T R A C T

Article history:Received 30 April 2014Accepted 6 August 2014Available online 17 August 2014

In the present study angiotensin I-converting enzyme (ACE) inhibitory peptides were isolatedfrom egg-yolk protein preparation (YP). Enzymatic hydrolysis conducted using unconvention-al enzyme from Cucurbita ficifolia (dose: 1000 U/mg of hydrolyzed YP (E/S (w/w) = 1:7.52)) wasemployed to obtain protein hydrolysates. The 4-h hydrolysate exhibited a significant (IC50 =482.5 μg/mL) ACE inhibitory activity. Moreover, hydrolysate showed no cytotoxic activityon human and animal cell lines which makes it a very useful multifunctional method forpeptide preparation. The compiled isolation procedure (ultrafiltration, size-exclusion chro-matography and RP-HPLC) of bioactive peptides from YP hydrolysate resulted in obtainingpeptides with the strong ACE inhibitory activity. One homogeneous and three heterogeneouspeptide fractions were identified. The peptides were composed of 9–18 amino-acid residues,including mainly arginine and leucine at the N-terminal positions. To confirm the selectedbioactive peptide sequences their analogs were chemically synthesized and tested. PeptideLAPSLPGKPKPD showed the strongest ACE inhibitory activity, with IC50 value of 1.97 μmol/L.

Biological significancePeptides with specific biological activity can be used in pharmaceutical, cosmetic or foodindustries. Because of their potential role as physiological modulators, as well as theirhighsafety profile, they can be used as natural pharmacological compounds or functional foodingredients. The development of biotechnological solutions to obtain peptides with desiredbiological activity is already in progress. Studies in this area are focused on usingunconventional highly specific enzymes and more efficient methods developed to conductfood process technologies. Natural peptides have many advantages. They are mainlytoxicologically safe, have wide spectra of therapeutic actions, exhibit less side effectscompared to synthetic drugs and are more efficiently absorbed in the intestinal tract.

Keywords:Cucurbita ficifoliaEnzymatic hydrolysisACE inhibitory activityEgg yolk protein

imal Products Technology and Quality Management, Wroclaw University of Environmental30 Wrocław, Poland.up.wroc.pl (A. Zambrowicz).aw, Joliot–Curie 14, 50-383 Wrocław, Poland.

rved.

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The complexity of operation of large scale technologies and high cost of purificationtechniques are limiting factors to the commercialization of food-derived bioactive peptides.Research on the isolation of bioactive peptides in order to reduce the processing time andcosts is continuously developing. Bioactive peptides can also be released from proteinby-products of the food industry, which reduce the substrate expense and production costas well as provide the added advantage of an efficient waste disposal. Moreover, proteins asprecursors of food-derived peptides are well-tolerated by the human body and thereforetheir application in drug development may reduce costs and duration of toxicologicalstudies during research, development and clinical trials.

© 2014 Elsevier B.V. All rights reserved.

1. Introduction

Yolk, otherwise known as deutoplasm is a spare substance inthe egg cell containing building materials and nutrients for thefuture embryo. It is also an excellent raw, natural and easybiorenewablematerial for obtaining proteins and peptides withthe possibility of subsequent use in a variety of industries,including medical, pharmaceutical, cosmetic and biotechno-logical [1].

The most important component of the egg yolk is γ-livetin,also referred to as immunoglobulin Y, which exhibits immuno-logical activity. This protein is used as a food additive to preventtraveler's diarrhea and fever caused by rotaviruses or prophy-lactically applied in baby food in order to increase theirresistance [2]. Phosvitin—another interesting egg yolk proteinappears to be a multifunctional protein. It shows valuableantioxidant and metal chelating properties [3]. Phosvitin isconsidered as a factor in preventing some diseases caused byoxidative stress, such as colon cancer or Alzheimer's disease[4,5]. Attention is also drawn on egg yolk phospholipids such aslecithin, which is more valuable than plant-derived lecithins,due to the specific chemical composition. It contains up to 80%phosphatidylcholine, which is an essential ingredient of thebrain and nervous tissues and also regulates lipid metabolism[6]. Currently, the egg yolks are mainly exploited for theextraction of valuable phospholipids because it is desirable forthe food, pharmaceutical and cosmetic industries [7,6]. Howev-er, a reasonable direction of the egg yolk active ingredient usageis also the utilization of by-products generated by the phospho-lipid extraction. Main by-products are partially denatured anddefatted egg yolk proteins that belong to the insoluble granulefraction. Despite the fact that the by-product is a source ofbioactive proteins, it has a limited value and functionality dueto denaturation during ethanol or hexane extraction of phos-pholipids. On the other hand, egg proteins are increasinglyregarded as precursors of bioactive and functional peptides [8].

Bioactive peptides are protein fragments, which are inactivein a precursor sequence and after its release, they interact withreceptors in the body and regulate the function of particularsystems such as nutrient uptake, immune defense, carriers ofmetal ions, opioid, antioxidant, antimicrobial, antiadhesive,anticancer and ACE-inhibitory [8,9].

One of the most common methods for the preparation ofbiologically active peptides from food proteins is enzymaticproteolysis [1,8,9]. Enzymatic degradation in vitro is usuallycarried out using commercial preparations of proteolyticenzymes obtained from animal (pepsin, trypsin, chymotrypsin,pancreatin), plant (papain, ficin, bromelain), or microbial origin

(Alcalase, Neutrase, Protamex™) [10,11]. However, it wasobserved that the use of non-conventional enzymes derivedfrom cheaper sources, may also result in the production ofhydrolysateswith beneficial features,while drastically reducingthe cost of hydrolysis [11]. One example of such enzymepreparation is the serine proteinase from Cucurbita ficifolia. Theenzyme constitutes about 15% of the total protein extractedfrom the pumpkin pulp and has a molecular weight of about60 kDa. It exhibits strong proteolytic properties, which weretested toward casein and corn gluten [12,13]. Serine proteinasefrom C. ficifolia is relatively stable under different temperatureconditions [13]. This protease was effectively used in theproduction of ACE-inhibitory peptides from egg white proteins[11]. However, there is limited informationon thepreparationofbioactive peptides in the egg-yolk protein proteolysis withregard to the use of this enzyme.

The objectives of this investigation were to (I) assess theACE-inhibitory activity of enzymatic hydrolysates obtainedfrom the by-product of egg-yolk phospholipid extraction, (II)peptide isolation, and (III) validation of the biological activityof the novel egg-yolk derived peptides.

2. Materials and methods

2.1. Substrate

The 40–45 week old laying hens of Lohman brown line werehoused in a bedding system. Eggswere automatically broken out,and macroscopic parts of eggs were separated on industry scale.Egg yolk protein preparation (YP), a by-product of phospholipidextraction using ethanol was used as a substrate [6].

2.2. Enzyme

Non-commercially available protease from C. ficifolia fruit pulpwas isolated following the procedure described for seeds byDryjański, Otlewski & Wilusz [14].

2.3. Determination of proteolytic activity of serine proteinasefrom C. ficifolia

Proteolytic activity was determined in reaction with 1% caseinas a substrate (BDH, Ltd., England) at pH 8.3 [15]. The substratewith the enzyme was incubated for 10 min at 37 °C. Thereaction was stopped by the addition of 5% trichloroacetic acid(TCA). The samples were then centrifuged, and the absorbanceof supernatants were measured at λ = 280 nm. One unit of

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enzymatic activity (U) was defined as the amount of enzymegivingan increase in absorbance at 280 nmof 0.1 under reactionconditions.

2.4. Determination of protein content

Total protein content (N × 6.25) in insoluble substrate wasdetermined using Kjeldahl method. Protein content in hydro-lysates and peptide fractions was determined by Lowry et al.'s[16] method.

2.5. Enzymatic hydrolysis

YP hydrolysis was carried out according to modified methodof Zambrowicz et al. [17]. 1% substrate suspension in 0.1 MTris–HCl buffer (pH 8.0) was hydrolyzed at 37 °C for 4 h usingC. ficifolia protease at enzyme:substrate ratio E/S = 1:7.52. Thereaction was terminated by heating the mixture at 100 °C for15 min. The hydrolysate was cooled, centrifuged (5500 ×g,10 min, 10 °C), then the supernatant was lyophilized andstored at 4 °C until used.

2.6. Degree of hydrolysis

Degree of hydrolysis (%) was determined as the percentageratio of protein soluble in 10% trichloroacetic acid (TCA) tototal protein [18]. TCA was added to the hydrolysates (1:1) andafter 1 h of incubation at 4 °C the samples were centrifuged(4500 ×g, 15 min, 20 °C). The concentration of the trichloro-acetic acid-soluble product in the supernatant was measuredspectrophotometrically and calculated from the followingequation:

DH %ð Þ ¼ mg soluble protein after hydrolysis=mLð‐mg soluble protein before hydrolysis=mLÞ � 100%:

2.7. The content of free amino acid groups

The content of free amino acid groups (FAG) (μmoL/g) wasdetermined using trinitrobenzene sulfonic acid (TNBS, Sigma)according to the modified Kuchroo et al. [19] method.

2.8. Determination of ACE-inhibitory activity

ACE (EC 3.4.15.1) inhibitory activity was measured using spec-trophotometric assay developed by Cushman and Cheung [20]in modification described by Miguel et al. [21]. A hydrolysatesolution (40 μL) mixed with the Hippuryl-His-Leu (HHL) sub-strate solution (5 mmol/L in 100 mmol/L potassium phosphatebuffer containing 300 mmol/L sodium chloride, pH 8.3) waspreincubated at 37 °C for 5 min and the reaction was initiatedby adding 20 μL (2 mU) of ACE solution and incubated for30 min at the same temperature. The enzymatic reaction wasterminated by the addition of 150 μL of 1 M HCl. The liberatedhippuric acid was extracted by 1 mL of ethyl acetate withvigorously shaking and after that, 750 μL of the upper layerwas transferred into a test tube and evaporated undervacuum. The hippuric acid left in the tubes was re-dissolvedin 800 μL of distilled water and the absorbance wasmeasuredat λ = 228 nm.

All samples were tested in 3 replications. The inhibitionactivity was calculated using the following equation:

Inhibition activity %ð Þ ¼ A–Bð Þ=A½ � � 100%

where A is the reaction blank, of which the mixture containedthe same volume of the buffer solution instead of the ACEinhibitor sample; and B is the reaction in the presence of bothACE and its inhibitor. The IC50 value was estimated from adose response curve of an inhibitor versus the ACE activity.

2.9. Determination of cytotoxicity/proliferation of human andmurine cells

Cytotoxicity/proliferation was performed in Polish branchAcademy of Sciences in Wroclaw (Poland). Cytotoxicity and/orcell proliferation assay was performed of human keratinocytes(HaCaT), hepatocytes (HepG2) andmurine fibroblasts (Balb 3T3).Cell viability was determined by MTT technique. MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) wasdissolved in PBS at a concentration of 5 mg/mL. Then it wasadded to the cells, previously treated with amixture of peptide,in an amount of 20 μL per well, and incubated at 37 °C for 3 h.After this time the media was removed from cells. Solution(4 mM HCl, 0.1% Nonidet P-40 in 2-propanol) at amount of100 μL was added to the mixture to dissolving formed MTT saltcrystals. After 30 min incubation at room temperature theabsorption was measured at λ = 560 nm.

2.10. Fractionation of the hydrolysates using ultrafiltration

The ultrafiltration was carried out in ultra/diafiltration systemusing the pressure of 1 bar. The selected hydrolysates werefractionated by using permeable membranes with molecularweight (MW) cut off of 30 kDa, in order to separate theenzyme, and then 5 kDa, in order to isolate the bioactivepeptide fraction. The fractions were collected, lyophilized andstored at 4 °C. The fractions having molecular weight >5 kDaand <5 kDa were assigned for further testing.

2.11. Separation of the peptide fractions using gel permeationchromatography

Gel permeation chromatographywas performed using theHPLCcolumn Zorbax GF-250 Agilent (4.6 × 250 mm). The fractionswere eluted with 0.02 M Tris–HCl buffer (pH 6.8) containing0.2 MNaCl at 30 °C and at a flow rate of 0.25 mL/min. The eluentwas monitored at two wavelengths (A230 and A280 nm).

2.12. Reversed-phase high-performance liquid chromatography

Peptide profiles of hydrolysates were monitored by reversed-phase high-performance liquid chromatography (RP-HPLC).Separation was performed using Zorbax XDB-C 18 Agilentcolumns at three different sizes (4.5 mm × 250 mm, 4.5 mm ×150 mm and 1.8 mm × 50 mm) in order to achieve betterseparation of peptide fractions. The operation conditions wereas follows: injection volume: 100 μL; mobile phase A—0.1% TFAin water; mobile phase B—0.1% TFA in acetonitrile, columntemperature: 30 °C. Analysis time, flow rate and gradient

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condition were varied (detailed information can be found ondrawings). The absorbance of eluent was monitored at λ =230 nm. RP-HPLC purifications and analysis of peptides obtain-ed by chemical synthesiswere carried out on Varian ProStarandThermo Separation Products liquid chromatographs. Separationwere performed on the YMC-Pack ODS-AQ12S05-2546WTcolumn and TSK gel ODS-120T 12TG08eh004 column, bothequipped with Guard Column, with a linear gradient elution of0–80% B. Mobile phase A (0.1% TFA in water) and B (80%acetonitrile in water + 0.1% TFA) were run at a flow rates of7 mL/min and 1 mL/min for Varian ProStar and ThermoSeparation Product, respectively.

2.13. Determination of peptide molecular weight

Molecular weight of the peptides was determined by ionizingparticles (electrospray ESI) in EsquireHCT spectrometer (Bruker,Germany) combined with ion trap detector. To the isolatedfractions of peptides, 1 mL of a mixture of water/methanol(70/30 v/v) supplemented with formic acid (0.1%) was added.Then the samples were shaken for about 2 min and injecteddirectly into the spectrometer at analyte flow of 180 mL/minand mass spectra were recorded. The following parameterswere used; flow rate: 220 mL/min; optimized sensitivity at700 m/z; range for samples 300–2200 m/z and 100–1800 m/z.The ion signals observed on the MS spectra were identifiedusing Mascot Search Results database.

2.14. Chemical synthesis of the peptides

Synthesis of specific peptides was carried out manually in asyringe reactor (BRAUN Inject, Germany). The preloaded Wangresins (0.58–0.79 mM/g) (Iris Biotech GmbH) were used for thesynthesis of fully protected peptides. Fmoc-protecting group(9-fluorenylmethyloxycarbonyl) was removed with 25% piper-idine solution (Sigma-Aldrich) in DMF (3 and 17 min) (Carl RothGmbH& Co. KG). Amino acid coupling reaction was carried outusing DMF as a solvent with the use of 3 equivalents TCTU (IrisBiotech GmbH) as the coupling reagent, 3 equivalents of HOBt(GL Biochem (Shanghai) Ltd.), and 6 equivalents of DIEA (IrisBiotech GmbH) as additives. The reaction was carried out for150 min. Peptides were cleaved from the resin simultaneouslywith the side chain deprotection using a mixture of TFA (IrisBiotech GmbH)/TIS (Alfa Aesar)/H2O (95:2.5:2.5, v/v/v). Thereaction was carried out for 120 min. Then the solution wastransferred into cold diethyl ether (Sigma-Aldrich). Cruderesidue was collected, dissolved in water, lyophilized, andpurified by reversed-phase high-performance liquid chroma-tography. All peptides were obtained as their trifluoroacetatesalts.

2.15. Analysis of chemically synthesized peptides usingHR-ESIMS

Analysis of chemically synthesized peptides was carried outby HR-ESI-MS mass spectrometer (FT-ICR Apex-Qe Ultra 7T,Bruker Daltonics, Bremen, Germany) equipped with standardelectrospray ion source.The instrument was operated inthe positive-ion mode. The instrument was calibrated usingTuneMix™mixture (Bruker Daltonics, Bremen, Germany). The

solutions used for themeasurement were CH3CN/H2O/HCOOH(50:50:0.1, v/v/v), with the m/z range of 100 to 1800.

2.16. Statistical analysis

Each type of hydrolysate was prepared in two independentbatches. The biological activity measurements of hydroly-sates as well as various fractions obtained in the purificationprocess were done in triplicate for each batch. The obtaineddata are the mean of three independent determinations ± SD.Statistical significance of the differences was determined byStudent's t-test (p < 0.05).

3. Results and discussion

3.1. Enzymatic hydrolysis

Enzymatic hydrolysis leads to value added protein by-productswith improved biological activity which can be used as proteiningredient in food, nutraceuticals and pharmacological for-mulations. The effect of proteolytic enzyme modification onACE-inhibitory activity of egg yolk protein was evaluated. Eggyolk protein preparation (YP), as the by-product of lecithinextraction was treated by a noncommercial serine proteinaseisolated from C. ficifolia.

Egg yolk is composed of a mixture of non-covalently asso-ciated lipids and proteins forming large lipoprotein complexes[21]. However, the properties of the protein preparations ob-tained after phospholipid extractionwere significantly differentfrom their native form due to the ethanol denaturation. In thepresent research, YP was analyzed by SDS-PAGE electrophore-sis (data not shown). Electrophoretic pattern revealed that theYP is mainly composed of a protein fraction with molecularmass of approximately 80 kDa estimated as the proteins ofHDL fraction (particularly apolipoproteins). Simultaneously, ahigh molecular weight band with a mass corresponding toabout 200 kDa was assigned as yolk plasma LDL proteins. Theband located at around 160 kDa was assigned to α-phosvitin. Itwas also determined its protein content, which amounted to70%.

To reduce the cost of enzymatic hydrolysis, a cheap sourceof enzymes was used in the current study. Easy extractableserine proteinase from C. ficifolia fruit pulp was selectedbecause of the simple procedure of enzyme isolation (datanot shown). Only one proteolytic enzyme was identified inenzyme preparation.

The progress of hydrolysis was monitored by determiningthe degree of hydrolysis (DH) (%), the concentration of the freeamino groups (FAG) (Table 1) and RP-HPLC peptide profilesanalysis (Fig. 1A). The proteolytic action of enzyme (1000 U/mgof intact YP (E/S (w/w) = 1: 7.52)) resulted in substrate degrada-tion reaching a DH value of higher than 46% after 4 h digestion.DH is an important factor controlling the composition andbiological properties of the obtained hydrolysates. The amountof enzyme can have a significant impact on DH, therefore thedose of applied enzymewas calculated as units of active proteinmeasured as caseinolytic activity. The analysis of the concen-tration of FAG (equal to 4525.1 μM Gly/g) of obtained hydroly-sate confirmed the above results.

Table 1 – Biological activities of the peptide fractions separated by ultrafiltration, gel permeation and reverse-phasechromatography of 4 h-egg-yolk protein hydrolysate treated by serine proteinase from C. ficifolia (1000 U/mg of hydrolyzedYP (E:S (w/w) = 1:7.52). All data were expressed as mean values (n = 3). Values sharing the same letter are not significantlydifferent at p < 0.05. For homogenous peptides with knowing sequences the IC50 was also expressed in mol/L (values inbrackets).

Isolation step Sample ACE IC50[μg/mL]

Hydrolysis 4-h hydrolysate(DH = 46.3%; FAG = 4525.1 μMGly/g)

482.5e

Ultrafiltration cut-off 5 kDa Retentate 540.0fPermeate 470.0e

HPLC-gel F 1 288.8dF 2 54.0bF 3 219.5cF4 47.8b

RP-HPLC SF 2.1 7.8aSF 2.2 (VVSGPYIVY;LLGAVASMGALLCAP)

5.0a

SF 4.1 6.8aSF 4.2 9.0aSF 4.3 (RASDPLLSV;RNDDLNYIQ)

5.5a

SF 4.4 4.5aRechromatography SF 4.4.1 (LAPSLPGKPKPD; AGTTCLFTPLALPYDYSH) 3.75a

SF 4.4.2(ITMIAPSAF)

3.5a; [3.24 μmol/L]

Chemical synthesis RASDPLLSV 5.1a; [4.70 μmol/L]RNDDLNYIQ 4.75a; [3.72 μmol/L]LAPSLPGKPKPD 2.75a; [1.97 μmol/L]AGTTCLFTPLALPYDYSH 0.72a; [8.08 μmol/L]

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Protein–peptide profiles obtained by RP-HPLC techniquedemonstrate the extent of YP hydrolysis. Most of the peptideswere eluted from the column at acetonitrile concentrationof 25% to 55%. The wide distribution of the degradationproducts indicates that hydrolysate is composed of peptideswith different hydrophobic properties.

3.2. The biological activity of YP enzymatic hydrolysate

Numerous antihypertensive peptides derived from egg whiteproteins by enzymatic hydrolysis have been reported [21,23].However, limited information is available about egg yolkproteins as a source of peptides exhibiting antihypertensiveactivity. The most common strategy for the identification ofantihypertensive food-derived peptides is based on in vitroACE inhibitory activity assay. The in vivo function of thepeptides is tested in spontaneously hypertensive rats (SHR),which constitutes an accepted model for human essentialhypertension [8]. In fact, many studies have shown that ACEinhibitory properties were performed primarily by the shortpeptides, obtained by the exhaustive hydrolysis of proteins[9]. The ACE inhibitory activity of hydrolysates obtained withthe C. ficifolia protease (IC50 = 482.5 μg/mL) was significantlyhigher than the levels of the other food protein hydrolysates.For example, various fermented soy foods possessed inhibi-tory activity (IC50) increased to 1.77 mg/mL and 17.80 mg/mLfor tempeh and soy sauce, respectively [24,25]. While, the ACEinhibitory potency of commercially available Chinese soybeanpaste (IC50 = 12.0 μg/mL) was almost 80 times higher thanpotency of YP hydrolysates [23]. Previously, C. ficifolia proteasewas used to generate ACE-inhibitory peptides from egg-white protein preparation by-product. Hydrolysate after 5 h

of hydrolysis showed the ACE inhibition potency of IC50 =9071.70 μg [11]. The results confirmed that the plant proteasefrom C. ficifolia is capable of producing ACE inhibitorypeptides.

Determination of the cytotoxic activity of hydrolysates withusing various cell cultures in vitro is necessary to determine theirproper impact and safety. Therefore, the hydrolysate was testedin terms of their cytotoxicity and/or proliferation activity inhuman and animal cell lines (Balb 3T3—murine keratinocytes,HepG2—human hepatocytes, HaCaT—human keratinocytes)(Table 2).

The MTT assay results indicated that hydrolysate used inconcentrations of 1% and 10% did not show toxicity to animaland human cell lines during 48 hof incubation. Moreover, itmay slightly affect the growth of Balb 3T3 cell lines. Thisindicates the possibility of using of YP hydrolysate as additivein functional foods, pharmacological substances or cosmetics.

3.3. The peptide isolation

The 4-h hydrolysate of YP, which showed the highest biologicalactivities, was ultrafiltered through a cellulotic membrane(MWCO 30 kDa) to remove protease and unhydrolyzed protein(Fig. 1B). Then the obtained permeate (fraction with molecularweight <30 kDa) was ultrafiltered through the 5 kDa in order toisolate and purify valuable biopeptides. The obtained fractionswere analyzed for ACE inhibitory activity. The peptide fractionswith molecular weights lower than 5 kDa exhibited a highACE inhibitory property (IC50 = 470.0 μg/mL) (Table 1). Mem-brane filtration is a widely used technique both in laboratoryand industrial-scale for fractionation, purification and con-centration of proteins and peptides [26]. According to Miguel

A)

D)

E) F)

C)

B)

Fig. 1 – Scheme of peptide isolation from 4 h-egg yolk protein hydrolysate. A—RP-HPLC of 4 h hydrolysate; B—ultrafiltration;C—gel chromatography; D—RP-HPLC of fraction 4; E—RP-HPLC of fraction 4.4; F—RP-HPLC of fraction 4.4.2.

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et al. [21], when the peptic hydrolysate of crude egg white(IC50 = 55.3 μg/mL) was filtered through a 3 kDamembrane, thepermeate presented approximately ten times higher inhibitoryactivity compared to the retentate [21]. Ultrafiltration was alsoeffectively used in fractionation of egg yolk hydrolysate, whichled to the purification of peptideswithMWof 1 kDa or lesswithACE-inhibitory activity [27]. The obtained permeate was sub-jected to gel filtration chromatography on a Zorbax GF-250column (Fig. 1C). The molecular filtration process allowed theisolation of four fractions, which were collected, concentrated

by lyophilization and then evaluated for ACE inhibitory activity(Table 1).

Among the four fractions, fraction no. 4 possessed thehighest biological activity, which was characterized byalmost 10-fold increase in ACE-inhibitory activity comparedto chromatographed material. Fraction no. 2 was also charac-terized by high ACE inhibitory activity (IC50 = 54.0 μg/mL).

The proposed procedure of biopeptide isolation, whichincluded ultrafiltration and gel filtration chromatography,was similar to the procedure described by Zhipeng et al. [28],

Table 2 – Cytotoxic/proliferation activity of egg yolk proteinhydrolysateobtainedwithusing a serine proteinase fromC.ficifolia (1000 U/mg of hydrolyzed YP (E:S (w/w) = 1:7.52).Tested cell lines: mouse fibroblasts (Balb3T3), humanhepatocytes (HepG2) and human keratinocytes (HaCaT). Alldata were expressed as mean values (mean ± SD, n = 3).

Hydrolysate(%)

MTT testA 560 nm

Balb 3T3 HepG2 HaCaT

0 0.07 ± 0.01 0.17 ± 0.04 0.05 ± 0.021 0.07 ± 0.02 0.17 ± 0.03 0.05 ± 0.01

10 0.13 ± 0.02 0.17 ± 0.02 0.05 ± 0.02

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who also fractionated hydrolysates based on differences inpeptide molecular weight. As a result of gel chromatographyon Sephadex G 25, they received the peptide fractions thatpossessed significantly higher ACE inhibitory activity com-pared to the unfractionated hydrolysates.

In our study, fractions 2 and 4, which exhibited the strongestbiological activity, were further purified by RP-HPLC. The F2wasapplied on Zorbax XDB-C18 column (4.5 × 250 mm) and twofractions were eluted up to 60% ACN and collected (datanot shown).Significantly higher activity was observed forsub-fraction 2.2 (Table 1). Only 5.0 μg/mL of the peptide sub-fractionwas required for 50% inhibition of the ACE activity.

Therefore, sub-fraction 2.2 was analyzed bymass spectrom-etry (Maldi-Tof) in order to identify the bioactive peptidesequences (Table 1). The protein origin of peptide sequenceswere identified using Mascot Search Results database (data notshown). The analysis indicated that peptide sub-fraction washeterogeneous and two peptides consisting of 9 and 15 aminoacid residues with the following sequences: VVSGPYIVY (frag-ment of breast cancermetastasis-suppressor 1-like protein) andLLGAVASMGALLCAP (fragment of lipase maturation factor 2)were identified.

Analogically, fraction 4 was further purified by RP-HPLC on aZorbax C18 column (4.5 × 250 mm) and the eluate was dividedinto four major fractions (4.1–4.4) (Fig. 2D). The analysis of allcollected fractions showed a wide diversity in the levels of ACEinhibitory activity (Table 1). Sub-fraction 4.4 demonstrated thehighest ACE inhibitory activity (IC50 = 4.5 μg/mL).

The mass spectrometry analysis indicated that fraction 4.3was a mixture of two peptides: RASDPLLSV and RNDDLNYIQcorresponding to protein fragment of 4-kinase type 2 alphaphosphatidylinositol-5-phosphate and fragment of vitellogenintype II, respectively (Fig. 2).

However, fraction 4.4 was a mixture of peptides and aspecific process protocol based on rechromatograpy on a ZorbaxXDB C18 column (4.5 × 150 mm) was required (Fig. 1E; F). Theprocedure allowed obtaining two fractions: 4.4.1 and 4.4.2,which showed strong ACE inhibitory properties (Table 1).Purification protocol led to the increase in ACE inhibitoryactivity, reached to 21.1% and 26.3% in the case of subfractions4.4.1 and 4.4.2, respectively.

Based on mass spectrometric analysis, two peptides withthe following sequences: LAPSLPGKPKPD and AGTTCLFTPLALPYDYSH were identified in subfraction 4.4.1 (data notshown). Whereas subfraction 4.4.2 proved to be homogeneousand contained one peptide: ITMIAPSAF was identified as a

fragment of transmembrane canal-like protein 3. In mostcases, the protein precursors of obtained peptides wereuntypical for main egg yolk proteins. However, besidesmajor vitellogenin cleavage products, apovitellenins, andIgY, multiple white and hen's serum proteins were identifiedin egg yolk [22]. One of the protein functions is the impact onthe body due to the presence of motives that show specificphysiological and biological activities. All proteins of livingorganism may be potential precursors for biologically activepeptides.

Among all obtained peptide fractions, peptide ITMIAPSAF(IC50 = 3.24 μmol/L) was characterized as the most active ACEinhibitor. ACE-inhibitory activity may contribute to the pres-ence of isoleucine and phenylalanine at N-and C-terminus atpeptide sequence, respectively. According to Hartman &Miesel[9] presence of Tyr, Phe, Trp and Pro at the C-terminus sequenceof peptide are critical factors in the ACE inhibitory activity.Furthermore, a common feature of almost all obtained peptidesis the presence of proline residues in their sequence [29]. Theobtained results confirmed that the long chain peptides derivedfrom egg proteins may have biological activity. Similar resultswere obtained by Miguel and others [21], who tested twolong-chain ACE inhibitory peptides obtained from egg whiteprotein hydrolysates. These fragments of ovalbumin with thefollowing sequence: FRADHPFL (ovokinin) and YAEERYPILshowed IC50 values at: 6.2 μM and 4.7 μM, respectively. Where-as, another peptide: FFGRCVSP obtained from peptic digest ofovalbumin exhibited IC50 value at 0.4 μM[30].

3.4. Peptide chemical synthesis

In order to clearly confirm which peptide is responsible forbiological activity, chemical synthesiswas carried out. As a resultof this process the pure peptides: RASDPLLSV; RNDDLNYIQ;LAPSLPGKPKPD and AGTTCLFTPLALPYDYSH were obtained.Purity of the synthesized compounds was confirmed by HighResolution Mass Spectrometer (HR-MS) and RP-HPLC analysis.The purity of synthesized peptides was in range of 92% to 99%,and their molecular masses were in accordance with theoreticalvalues (Table 3). Chemically synthesized peptides were evaluat-ed in terms of ACE inhibitory activity (Table 1).The highest ACEinhibitory activity (IC50 = 1.97 μmol/L) was exhibited by thepeptide with the following sequence: LAPSLPGKPKPD. Amongthe different classes of food-derived bioactive peptides, the ACEinhibitory peptides are the best characterized. General featureson the structure–activity relationship of ACE inhibitory peptideshave been described mainly for short chain peptides.

The ACE inhibitory activity of long chain peptides isstrongly influenced by their C-terminal tripeptide sequences.Most potent ACE inhibitors contain hydrophobic amino acidresidues at their three C-terminal positions [31].

Peptide LAPSLPGKPKPD, isolated from YP hydrolysate,contained six hydrophobic amino acid residues (L, P), and oneof them (P) was at the C-terminal tripeptide sequence.The lowest inhibitory potency (IC50 = 8.08 μmol/L) of peptideAGTTCLFTPLALPYDYSH might be explained by the presenceother than the hydrophobic amino acid residues at theC-terminal tripeptide sequence. However, the level of ACEinhibitory potency of the other two synthesized nona-peptideswasn't significantly different compared to their original

A)

B)

C)

Fig. 2 – Identification of the amino-acid sequence of purified subfraction no. 4.3. A—MS-ESI chromatogram of fraction 4.3;B—Mascot search results for fragment of phosphatidylinositol-5-phosphate 4-kinase type-2alpha: peptide RASDPLLV; C—Mascotsearch results for fragment of vitellogenin-2: peptide RNDDLNYIQ.

Table 3 – Synthetic peptide data.

Sequence Retention time [MIN] Experimental m/z [M + H]+ Theoretical m/z [M + H]+

RASDPLLSV 19.4 957.531 957.536RNDDLNYIQ 15.9 1150.542 1150.549LAPSLPGKPKPD 14.7 1219.701 1219.704AGTTCLFTPLALPYDYSH 25.9 985.484a 985.471a

a Peak corresponding to [M + 2H]2+ ion.

114 J O U R N A L O F P R O T E O M I C S 1 1 0 ( 2 0 1 4 ) 1 0 7 – 1 1 6

115J O U R N A L O F P R O T E O M I C S 1 1 0 ( 2 0 1 4 ) 1 0 7 – 1 1 6

mixture before fractionation (fraction SF 4.3 with IC50 value of5.5 mg/mL) (Table 1).

The levels of ACE-inhibitory activity of food-derivedpeptides from animal as well as plant origins are firmlydifferent. Luteolin and Apegenin from plant Fuchsiaproceumbens showed 50% inhibitory at concentration level of300 μmol/L. Whereas peptide lactokinin (β-lactoglobulinf(142–148)) had an ACE IC50 of 42.6 μmol/L [32]. The ACEinhibitory potency of peptides obtained from YP hydrolysate(IC50 = 1.97–8.08 μmol/L) was significantly higher than that ofpeptides mentioned above. However the potency ofYP-derived peptides was very similar to the inhibitorypotency of other egg protein derived peptides e.g. Pepticdigestion of ovalbumin led to peptides ERKIKVYL and LWwithstrong ACE inhibitory activity which reached to IC50 =1.2 μmol/L and IC50 = 6.8 μmol/L, respectively [30].

4. Conclusion

Protein preparation (YP) as a by-product of phospholipidisolation from egg yolk can be effectively hydrolyzed by serineprotease from C. ficifolia to obtain peptides with ACE-inhibitoryproperties. Peptide purification (ultrafiltration, gel filtrationchromatography and RP-HPLC) led to few peptide fractionswhich possessed strong ACE inhibitory activity. Based onmassspectrometric analysis, seven peptides composedof 9–18 aminoacid residues have been identified. Chemical synthesis ofselected sequences and measurement of their ACE inhibitoryactivity confirmed ACE inhibitory potential of YP derivedpeptides in vitro. Peptides LAPSLPGKPKPD (IC50 = 1.97 μmol/L)and ITMIAPSAF (IC50 = 3.24 μmol/L) were characterized as themost effective ACE inhibitors, among all obtained peptides.However, it is important to test in vivo effect of theACE-inhibitory peptides to establish their possible usefulness.

Conflict of interest

All the authors who have taken part in this study declare thatthey have nothing to disclose regarding competing interestsor funding from industry with respect to this manuscript.

Acknowledgments

The projects “Innovative technologies in the production ofbiopreparations based on new generation eggs”, and Innova-tive Economy Operational Programme Priority 1.3.1, thematicarea “Bio”, are co-financed by the European Union through theEuropean Regional Development Fund (project number:POIG.01.03.01-00-133/08) within the Innovative Economy Op-erational Programme, 2007–2013.

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