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Food Chemistry 135 (2012) 1245–1252

Food Chemistry 135 (2012) 1245–1252 Contents lists available at SciVerse ScienceDirect Food Chemistry journal homepage: www.elsevier

Contents lists available at SciVerse ScienceDirect

Food Chemistry

journal homepage: www.elsevier .com/locate/foodchem

Food Chemistry 135 (2012) 1245–1252 Contents lists available at SciVerse ScienceDirect Food Chemistry journal homepage: www.elsevier

Analytical Methods

ACE inhibitory peptides and antioxidant peptides derived from in vitro digestion hydrolysate of hen egg white lysozyme

Shengqi Rao a , Jun Sun b , Yuntao Liu b , Huawei Zeng c , Yujie Su b , Yanjun Yang b,

a School of Food Science and Engineering, Yangzhou University, Jiangsu, Yangzhou 225127, China b State Key Laboratory of Food Science and Technology, School of Food Science and Technology, Jiangnan University, Jiangsu, Wuxi 214122, China c Angel Yeast Co. Ltd., Hubei, Yichang 443003, China

article info

Article history:

Received 24 October 2011 Received in revised form 12 April 2012

Accepted 11 May 2012 Available online 19 May 2012

Keywords:

Angiotensin I-converting enzyme ACE inhibitory peptide Antioxidant peptide Lysozyme Gastrointestinal enzymes MALDI-TOF-TOF

abstract

Lysozyme from hen egg white is a well-known antimicrobial protein with high ratio of hydrophobic and positively charged amino acid residues. In order to explore functional bioactivities of enzymatic hydrol- ysates of lysozyme, the protein was subjected to a simulated gastrointestinal digestion and the resulting hydrolysate (LPH2) showed a strong competitive angiotensin I-converting enzyme (ACE) inhibitory activ- ity (IC 50 = 12.6 lg/ml) and a remarkable antioxidant activity. The LPH2 was fractionated using a 3 kDa

cut-off membrane and the obtained permeate LPH2–3 kDa was analysed by MALDI-TOF-TOF MS. Using this technology, 38 different peptides were identified and some of these peptides were well fit with struc- ture requirements of ACE inhibitory peptides and/or antioxidant peptides. The findings from this study suggest that the protein containing high proportion of hydrophobic and positively charged residues have the potential to generate multifunctional peptides, and these peptides would be beneficial ingredient to be used in functional foods.

Crown Copyright 2012 Published by Elsevier Ltd. All rights reserved.

1. Introduction

Hypertension is a major risk factor for cardiovascular and end stage renal diseases. It can affect people of all ages and endangers approximately one billion worldwide (Murray & FitzGerald, 2007). Since angiotensin I-converting enzyme (ACE) (EC 3.4.15.1) plays a critical physiological role in raising blood pressure, the inhibition of ACE activity can lead to an overall antihypertensive effect. Endogenous and exogenous reactive oxygen species and free radi- cals also have been implicated in the occurrence of hypertension and other degenerative diseases. The amount of these reactive species is controlled by endogenous antioxidants until it reaches a level when the antioxidants are overwhelmed, a state known as oxidative stress. Exogenous dietary antioxidants can decrease the contribution of exercise-induced oxidative stress and improve the animal’s physiological condition (Yu et al., 2006). Although having obvious functional effects for improving human health, synthetic ACE inhibitors and antioxidants are reported to have various undesirable side effects (Murray & FitzGerald, 2007) and potential health hazards (Barlow & Schlatter, 2010). Recently, pro- tein hydrolysates and constituent peptides with ACE inhibitory activities or antioxidant activities have received considerable

Corresponding author. Tel./fax: +86 510 85863566. E-mail addresses: raoshengqi2001@yahoo.com.cn (S. Rao), yangyj@jiangnan. edu.cn (Y. Yang).

interest from the food industry because of the consumer’s concern over the safety of the synthetic counterpart. Some enzymatic hydrolysates of food staffs or proteins, such as flaxseed protein (Udenigwe & Aluko, 2010), camel milk casein (Salami et al., 2011), protein isolate from pumpkin oil cake (Vastag, Popovic, Popovic, Krimer, & Pericin, 2011) and others, have been reported to exert both ACE inhibitory and antioxidant activities. The combination of ACE inhibitory and antioxidant activities in protein hydrolysates or peptides could be very helpful for the con- trol of cardiovascular diseases by synergies of different regulatory mechanisms. However, most of reported enzymatic hydrolysates of various food protein sources show low bioactivities. Although the active peptides purified from these hydrolysates exhibit potent inhibitory potencies, it is very difficult for their commercialisation because of low yield, high cost, and multilink of separation and purification processes. Therefore, an effective enzymatic hydroly- sate of a protein or total proteins in food stuff with potent ACE inhibitory and antioxidant activities is greatly desirable, because it can directly be used as food additive or drug against hyperten- sion while do not need for further peptide purification. Biological activities of protein hydrolysates are related to the amino acid composition, size and configuration of peptides. For in- stance, ACE prefers inhibitors or substrates containing hydropho- bic amino acid residues at each of the three C-terminal positions (Murray & FitzGerald, 2007; Rao et al., 2012). Regarding the antiox- idant activity, the presence of certain hydrophobic amino acids

0308-8146/$ - see front matter Crown Copyright 2012 Published by Elsevier Ltd. All rights reserved.

http://dx.doi.org/10.1016/j.foodchem.2012.05.059

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S. Rao et al. / Food Chemistry 135 (2012) 1245–1252

(His, Trp, Tyr, Phe, Met, Leu, Gly or Pro) and basic amino acids (Arg or Lys) has been reported to enhance the scavenging activities of peptides (Sarmadi & Ismail, 2010). Overall, proteins with high con- tent of hydrophobic and basic amino acid residues should be of interest to produce dual-functional peptides with ACE inhibitory and antioxidative activities. In this respect, several major proteins, including ovalbumin, ovotransferrin, ovomucoid, lysozyme and ovomucin in hen egg white, were analysed using on-line software (ProtScale). It was found that lysozyme possesses high proportion of hydrophobic (43.4%) and basic (13.7%) amino acid residues, and it may be the potential to prepare ACE inhibitory peptides or antioxidant peptides. Based on the above rationale, the objectives of the present re- search are: (1) examine the in vitro ACE inhibitory and antioxidant activities of the hydrolysate obtained by gastrointestinal prote- ases; (2) identify and characterise the peptides generated in the hydrolysate.

2. Materials and methods

  • 2.1. Materials

Angiotensin I-converting enzyme (EC 3.4.15.1; 5.5 U/mg pro- tein) from rabbit lung, pepsin (E.C. 3.4.23.1; P2500 U/mg protein) from hog stomach, a-chymotrypsin (E.C. 3.4.21.1; P40 U/mg pro- tein) from bovine pancreas, trypsin (E.C. 3.4.21.3; 10,000 BAEE U/ mg protein) from New Zealand-sourced pancreas and 1,1-diphe- nyl-2-pycryl-hydrazyl (DPPH) were purchased from Sigma Chemi- cal Co. (St. Louis, MO). Hippuryl–histidyl–leucine (HHL) was obtained from the peptide Institute (Osaka, Japan). Carboxymethyl chitosan (CM-CTS) was purchased from Sigma. Fresh hen eggs were bought from local market. All other reagents, unless other- wise specified, were of analytical or guaranteed reagent grade.

  • 2.2. Preparation of superparamagnetic carboxymethyl chitosan (CM-

CTS) nanoparticles

Superparamagnetic CM-CTS nanoparticles, that is Fe 3 O 4 nano- particles with CM-CTS coating, were prepared using a chemical coprecipitating method (Sun, Su, Rao, & Yang, 2011). The average particle size and morphology of the obtained functionalised nano- particles were examined using transmission electron microscope (TEM, JEOL JEM-2100 (HR)). The aqueous dispersion of the particles was drop-cast onto a carbon-coated copper grid and the grid was air-dried at room temperature before loading into the microscope.

  • 2.3. Purification of lysozyme in hen egg white by superparamagnetic

nanoparticles

Egg white was isolated from fresh eggs and diluted to 50% (V/V) with phosphate buffer solution (PBS, 20 mM, pH 9.0). The diluted egg white was kept stirring for 6 h in an ice bath, centrifuged at 10000g for 20 min at 4 C, and then collected the supernatant. 40 ml of the supernatant was mixed with 100 mg of superparamag- netic nanoparticles and kept continuous stirring for 30 min at ambient temperature (about 25 C) for absorbing of the aimed lyso- zyme. Subsequently, the superparamagnetic nanoparticles with target lysozymes were precipitated using a magnet to remove unabsorbed hybrid proteins in the supernatant. After that, the lyso- zymes from the magnetite nanoparticles was eluted with PBS (20 mM, pH 5.0) containing 0.5 M NaCl. The purified lysozyme solu- tion was dialysed against PBS (10 mM, pH 7.5) to remove salts and then lyophilised for subsequent enzymatic hydrolysis analysis. The samples during purification process were subjected to 12% sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS–PAGE) analysis.

  • 2.4. In vitro digestion of purified lysozyme

The simulated human gastrointestinal digestion process was car- ried out according to the method of Herregods et al. (2011) with some modifications. For this purpose, five hundred milligram of lysozyme was suspended in 50 ml of HCl solution (pH 2.0) to make a 1% (w/v) slurry. After heating at 80 C in a water bath for 15 min, the protein slurry was cooled to 37 C. Subsequently, the digestion in the stomach was simulated by adding pepsin in a 1/100 (w/w) en- zyme/substrate ratio and incubating for 2 h at 37 C and pH 2.0. The further small intestine phase was estimated by adding a -chymo- trypsin and typsin (each 0.5%) and incubating for 4 h at 37 C and pH 7.5. Hydrolysis was conducted in a shaking bath (thermally con- trolled incubator) under constant stirring (150 rpm). All samples were boiled for 10 min to inactivate the enzymes and centrifuged at 10,000g for 20 min at 4 C, and the resulting supernatant was fil- tered, lyophilised, and stored at 20 C for further experiments.

  • 2.5. Ultrafiltration

Ultrafiltration was performed with a 3 kDa cut-off membrane, using an amicon ultra-15 centrifugal filter device (Millipore Corpo- ration) for a volume up to 15 ml. Fifty milligram of hydrolysate sample was dissolved in 10 ml of distilled water. The filter device with sample was centrifuged at 4000g for 1 h at 4 C, and the resulting permeate was kept at -20 C until further analysis.

  • 2.6. Assay of ACE inhibitory activity

ACE inhibitory activity of the samples was measured in vitro fol- lowing the spectrophotometric assay described by Cushman and Cheung (1971) with some modifications as explained below. A 10–100 l l of sample solution was mixed with 80 ll of a 5 mM HHL borate buffer containing 100 mM borate and 300 mM NaCl (pH 8.3), and then added to 190 l l volume by using 100 mM bo- rate. The borate buffer (100 mM, pH 8.3) was used instead of an ACE solution for blank determination. The above mixture was pre- incubated at 37 C for 5 min before adding 10 ll of ACE solution (3 mU). The reaction mixture was incubated for 30 min at the same temperature and terminated by an addition of 1 N HCl (200 ll). The released hippuric acid was extracted with 1.2 ml ethyl acetate. After centrifugation (4500g, 5 min), 0.8 ml of the upper layer was transferred to a test tube and vacuum-dried at 80 C for 30 min by using a centrifugal concentrator. The hippuric acid was redis- solved in 0.8 ml of deionised water, and absorbance was measured at 228 nm using spectrophotometer. Triplicate tests were per- formed for each sample. The concentration of each peptide re- quired to inhibit 50% of ACE activity was defined as the IC 50 value. To clarify the ACE-inhibition mode, two different peptide con- centrations (13.3 and 26.5 lg/ml) of the hydrolysate LPH2 were added to each reaction mixture described as above. The enzyme activities were measured at different substrate concentrations (0.5, 1, 2, and 3.5 mM). The inhibition kinetics of ACE in the pres- ence of inhibitory peptides was determined with a Lineveaver– Burk plot.

  • 2.7. Determination of antioxidant activities

2.7.1. DPPH radical scavenging activity

The scavenging activity of 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical was measured according to the method of Shimada, Fujika- wa, Yahara, and Nakamura (1992) with a slight modification. An ali- quot of 0.5 ml of sample solution was added with 0.5 ml of 0.2 mM DPPH in 95% ethanol. The mixture was incubated for 30 min in the darkness at room temperature. The absorbance of the resulting solution was measured at 517 nm with a spectrophotometer. A

S. Rao et al. / Food Chemistry 135 (2012) 1245–1252

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standard with glutathion was also made in the similar manner for comparison. The ethanol was used as a control. The radical scaveng- ing capacity of the tested samples was measured as a decrease in the absorbance of DPPH radical and was calculated by using the follow- ing equation:

Scavenging activity ð%Þ¼ðA control A sample Þ 100=A control

All determinations were conducted in triplicate.

  • 2.7.2. Reducing power assay

The reducing power was measured according to the method of Oyaizu (1986) with several modification. An aliquot of 1 ml sample (0.2 M PBS, pH 6.6) was mixed with 1 ml of 1% potassium ferric cyanide solution. The mixture was incubated at 50 C for 30 min followed by the addition of 1 ml 10% (w/v) TCA. One ml of the incu- bation mixture was added with 1 ml of distilled water and 0.2 ml of 0.1% (w/v) ferric chloride in test tubes. After a 10 min reaction time,

the absorbance of resulting solution was read at 700 nm. Higher absorbance suggested stronger reducing power. A standard with glutathion was also made in the similar manner for comparison.

  • 2.7.3. Lipid peroxidation inhibition assay

The lipid peroxidation inhibition activity of the samples was measured in a linoleic acid emulsion system according to the methods of Osawa and Namiki (1985). Briefly, a sample of the hydrolysate was dissolved in 2.5 ml of 50 mM phosphate buffer (pH 7.0) and added to a mixture of linoleic acid (32.5 ll) and 95% ethanol (2.5 ml), and then the final volume was adjusted to 6.25 ml with distilled water. Sample was replaced with vitamin E for comparative purposes. The mixture was incubated in a sealed tube at 42 C in a dark room, and the degree of linoleic acid oxida- tion was evaluated by measuring the ferric thiocyanate method according to Mitsuda, Yasumoto, and Iwami (1996). In short, an ali- quot (0.05 ml) of reaction mixture was mixed with 75% ethanol (2.35 ml) followed by the addition of 30% ammonium thiocyanate (0.05 ml) and 20 mM ferrous chloride solution (0.05 ml) in 3.5% HCl. After 3 min, the degree of colour development, representing the linoleic acid oxidation, was measured at 500 nm.

2.8. Analysis by online RP-HPLC-MS/MS

The hydrolysate was subjected to ultraperformance liquid chro- matography (UPLC) for peptide separation in an Acquity UPLC BEH C18 column (2.1 120 mm) (Waters Corporation, Milford, MA). The elution was made in linear gradient mode from 0.1% formic acid to 40% acetonitrile containing 0.1% formic acid (20 min) at a flow rate of 0.3 ml/min. Identification of the sequence of peptides present in the fraction exerting remarkable ACE inhibitory activity was performed by matrix-assisted laser desorption/ionisation time-of-flight/time-of-flight mass spectrometry (MALDI-TOF-TOF MS). Both MS and MS/MS data were acquired with a Waters Synapt Mass Quadrupole Time-of-Flight Mass Spectrometer (Waters Cor- poration, Milford, MA). Spectra were recorded over the mass/ charge (m/z) ranges of 100–1500 in both MS and MS/MS modes. Peptide fragmentation in the MS and MS/MS mode was respec- tively achieved by collision-induced dissociation (CID) using atmo- spheric air as the collision gas, and the collision energies were set at 6 eV and 20 eV for MS and MS/MS, respectively. The signal threshold to perform auto-MS/MS in the data-dependent acquisi- tion was 20 counts/s in the total ion current, and the precursor ions were isolated within a range of m/z 3.0. Instrumental control and data analysis were performed using MassLynx software version 4.1 (Micromass UK Ltd., Wythenshawe, Manchester, UK). Both the peptide sequencing module of the software and manual calculations were used to process the MS/MS data and to perform

peptide sequencing. The peptide sequences were matched to the published sequence of the lysozyme derived from protein data in UniProtKB.

  • 2.9. Peptide synthesis

The identified peptides in part were chemically synthesised by GenScript Corporation (Nan Jin, China). The purity (all above 99%) and sequence of these peptides were verified by analytical RP- HPLC-MS/MS. ACE inhibitory activities of these chemically pep- tides were determined as described above and expressed as IC 50 .

3. Results and discussion

  • 3.1. Purification of lysozyme from hen egg white by

superparamagnetic nanoparticles

Since low content (about 3.5%) of lysozyme in egg white total protein (You, Udenigwe, Aluko, & Wu, 2010), the classical method for lysozyme purification refers to a combination of precipitation, centrifugation, dialysis, ultrafiltration and chromatography. It makes the purification process time-consuming and high-cost for large-scale production. CM-CTS have good tolerable pH and good solubility, which may be due to the abundant of –COOH and – NH 2 groups in it. Therefore, CM-CTS can act as an ion-exchange material for carboxyl groups once modified onto the magnetic Fe 3 O 4 nanoparticles. The resultant magnetic separation technology arose from Fe 3 O 4 nanoparticles with CM-CTS provides an easy and rapid way to purify the aimed lysozyme from HEW. Typical TEM micrograph of prepared Fe 3 O 4 nanoparticles with CM-CTS coating using a chemical coprecipitating method is shown in Fig. 1(A), and the nanoparticles are spherical in shape with an average size of about 15 nm. It is reported that magnetic particles less than 25 nm will exert superparamagnetism (Sun et al., 2011), and hence our prepared Fe 3 O 4 (PEG + CM-CTS) nanoparticles have superpara- magnetic properties. Taking advantage of this character, about 28 mg lysozyme with 95% purity was obtained from 1 g HEW after one-step purification by superparamagnetic nanoparticles (Fig. 1B).

  • 3.2. ACE inhibitory activities of lysozyme hydrolysates

To verify whether the active peptides were produced efficiently by gastrointestinal digestion, the high pure lysozyme was sub- jected to the two stage hydrolysis at simulated physiological con- ditions. After digestion, the hydrolysates of lysozyme showed potent ACE inhibitory activities. The peptic hydrolysate (LPH1) had an IC 50 value of 160.2 lg/ml (Table 1). Further hydrolysis with a-chymotrypsin and trypsin (LPH2) decreased significantly IC 50 values to 12.6 l g/ml (Table 1), this result implied that intestine en- zymes should be essential for releasing more active peptides responsible for ACE inhibitory potency. The IC 50 values were seen in the range from 160 and 3770 l g/ml in a variety of other proteins or foods enzymatic hydrolysates (Herregods et al., 2011; Majum- der & Wu, 2010; Segura-Campos, Chel-Guerrero, & Betancur- Ancona, 2011; Udenigwe & Aluko, 2010; Vastag et al., 2011). In contrast, the ACE inhibitory power from lysozyme hydrolysate ob- tained by gastrointestinal enzymes was very strong. As shown in Fig. 2, the inhibition mode of the LPH2 hydrolysate was estimated by a Lineweaver–Burk plot and found to be compet- itive. The Ki value of the hydrolysate was calculated to be 13.4 lg/ ml, which was smaller than that of purified peptides (RYPSYG and DERF) from bovine casein hydrolysate prepared by AS1.398 neutral protease (Jiang, Tian, Brodkorb, & Huo, 2010). The competitive inhibitors are able to enter the ACE protein molecule, interact with

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S. Rao et al. / Food Chemistry 135 (2012) 1245–1252

1248 S. Rao et al. / Food Chemistry 135 (2012) 1245–1252 Fig. 1. Purification of lysozyme

Fig. 1. Purification of lysozyme from hen egg white by superparamagnetic CM-CTS nanoparticles. (A) Size distribution of superparamagnetic CM-CTS nanoparticles; (B) 12% SDS–PAGE analysis of purification of lysozyme. Lane 1, natural HEW solution; Lanes 2 and 4, the supernatants after adsorption; Lanes 3 and 5, eluted samples.

the active sites and prevent substrate binding, thus resulting in the inhibition of ACE activity. In order to exert an antihypertensive effect in vivo, the ACE inhibitory peptides not only have to be resistant to gastrointestinal degradation but also have to be absorbed into the bloodstream in their intact form. Subsequently, small ACE inhibitory peptides (2–6 amino acids) are the most interesting ones, because they are most likely to be absorbed into the bloodstream (Verstraete, Vermeirssen, Van Camp, Decroos, & Van Wijmelbeke, 2003). To pinpoint peptides exhibiting a potential ACE inhibitory effect, the hydrolysates LPH1 and LPH2 were respectively subjected to ultra- filtration through a 3 kDa cut-off membrane. After membrane sep- aration, the filtrate of the LPH1 (LPH1–3 kDa) showed a lower IC 50 value of 75.2 lg/ml than that of the original hydrolysate (IC 50 = 160.2 lg/ml) (Table 1). This result well agrees with many reports that the inhibitory activity of small molecular-weight (MW) peptides is generally stronger than that of the larger pep- tides (Segura-Campos et al., 2011). But the filtrate LPH2–3 kDa re- vealed an increased IC 50 value (28.6 l g/ml) compared to that of the original hydrolysate LPH2 (IC 50 = 12.6 l g/ml) (Table 1). This phe- nomenon indicated the larger MW peptides in the final hydroly- sate also greatly contribute to the inhibitory potency against ACE. Similarly, the high MW peptide (21 AA) from milk proteins fermented with Lactobacillus (Lb.) helveticus NCC 2765 (Robert, Razaname, Mutter, & Juillerat, 2004) also has been found to possess strong ACE inhibitory potencies. As for high MW active peptides, the structure–activity relationship is expected to further research.

3.3. Antioxidant activity of lysozyme hydrolysates

The final hydrolysate LPH2 and its permeate LPH2–3 kDa after ultrafiltration were also evaluated for their antioxidant activities. Many in vitro determination techniques of antioxidant activity

have been developed because of the difference of antioxidant mechanisms. The response of antioxidants depends on factors such as the solvent and substrate used in the test and affinity between substrate and antioxidant. Accordingly, it is better to use different assays based on different mechanisms to assess the antioxidant potency. The use of the DPPH assay to assess the scavenging activity of substances has been widely reported in the literature. As shown in Table 1, the LPH2 (25.7%) and LPH2–3 kDa (65.3%) showed sig- nificant DPPH radical-scavenging activity at concentration of 2.0 mg/ml. The low molecular weight fraction LPH2–3 kDa had higher scavenging activity. This value was less than and close to that of the glutathion at the same concentration and comparable to that of the enzymatic hydrolysate of alfalfa leaf protein (Xie, Huang, Xu, & Jin, 2008). Obviously, the LPH2–3 kDa had the ability to quench the DPPH radical, which suggested that the LPH2–3 kDa possibly contained some substrates which were electron donors and could react with free radicals to convert them to more stable products and terminate the radical chain reaction. The reducing power of the LPH2 and LPH2–3 kDa were also gi- ven in Table 1. Similar to the result from DPPH assay, the LPH2– 3 kDa revealed more strong reducing power when compared to LPH2. When the peptide concentration of LPH2–3 kDa was at 2 mg/ml, the reducing power was 0.29. The value was lower than and close to that of glutathion, while the concentration of LPH2– 3 kDa was ten times that of glutathion. The rapeseed protein hydrolysates prepared with alcalase have been reported to exhibit notable reducing power which was 0.51 at 2.00 mg/ml (Pan, Jiang, & Pan, 2011), and this value is slightly higher than that present in this study. There has a better correlation between antioxidant activity and the reducing power. Samples with higher reducing power have better potencies to donate electron and free radicals to form stable substances, thus interrupting the free radical chain

Table 1

ACE inhibitory and antioxidative activities of hydrolysate from lysozyme by gastrointestinal proteases.

 
 

Sample or control

ACE inhibitory activity (lg/ml)

Antioxidant activity

 

DPPH radical % (mg/ml)

Reducing power A 700 nm (mg/ml)

Lipid peroxidation inhibition % (mg/ml)

LPH1

160.2 ± 2.5 a

––

 

LPH1–3 kDa

75.2 ± 2.7 b

––

0.16 ± 0.03 a (2.0)

74.6 ± 0.09 c (2.0) 0.35 ± 0.02 c (0.2)

LPH2

LPH2–3 kDa

Glutathion

12.6 ± 1.2 c 28. 6 ± 0.8 d

25.7 ± 0.22 a (2.0) 63.2 ± 0.16 b (2.0)

0.29 ± 0.07 b (2.0)

– 76.4 ± 0.21 a (0.5) 85.2 ± 0.18 b (0.5)

Vitamin E

– –

– 78.4 ± 0.29 a (0.05)

Results are the mean values of triplicate analyses ± standard deviation. ad Means within the same column without a common letter differ significantly (P < 0.05) with Tukey’s test. The value in the bracket indicates the concentration (mg/ml) of sample or control.

S. Rao et al. / Food Chemistry 135 (2012) 1245–1252

1249

10 LPH2 Control 8 13.3 µg/ml 26.5 µg/ml 6 4 2 0 -1 -0.5 0 0.5
10
LPH2
Control
8
13.3
µg/ml
26.5
µg/ml
6
4
2
0
-1
-0.5
0
0.5
1
1.5
2
2.5
3
-2
1/[S] (mM)
1/Abs 228 nm

Fig. 2. Lineweaver–Burk plot for determination of inhibitory mode of LPH2 on ACE. ACE inhibitory was determined in the presence or absence of LPH2 as described in the text using HHL as the enzyme substrate.

reactions (Juntachote & Berghofer, 2005). The assay result of reduc- ing power demonstrated that LPH2–3 kDa possessed a moderate ability to donate electron, which was involved in the antioxidant activity.

In order to further determine the antioxidant activity in another

system, the samples were characterised for their antioxidant activ-

ities by measuring their abilities to protect linoleic acid against

oxidation. At the end of the 7-day lipid peroxidation, the inhibition

rate of LPH2 and LPH2–3 kDa were 76.4% and 85.2% respectively,

which were closer to that of the positive control, vitamin E, a

well-known lipid-soluble natural antioxidant (Table 1). The result

agreed with the free radical-scavenging activity and reducing

power analysed above.

3.4. Identification and characteristics of peptides in the LPH2–3 kDa

The sequence of the peptides in the LPH2–3 kDa was investi- gated in this work by the method of UPLC-MALDI-TOF-TOF MS. The obtained peptide separation total ion chromatography (TIC) profile was shown in Fig. 3(A). To obtain precursor ions in the sam- ples, the cone voltage and collision energy in data-dependent MS experiments were set into 30 V and 6 eV, respectively, and the resultant averaged MS spectra of the LPH2–3 kDa were seen in Fig. 3(B). In subsequent MS/MS experiments, the collision energy was changed into 20 eV and the cone voltage (30 V) was kept con- stant, to get more fragments for analysis of amino acid composi- tion. Fig. 3(C) illustrated the MS/MS spectrum of the m/z 718.29

S. Rao et al. / Food Chemistry 135 (2012) 1245–1252 1249 10 LPH2 Control 8 13.3

Fig. 3. Identification of peptide sequence. (A) TIC chromatogram corresponding to the separation of the LPH2–3 kDa using a UPLC coupled to a Waters Synapt Mass Quadrupole Time-of-Flight Mass Spectrometer; (B) Averaged MS spectra of the LPH2–3 kDa corresponding to the TIC chromatogram; (C) MS/MS spectrum of the ion m/z 718.29 relevant to the peak for 5.87 min retention time in the TIC.

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Table 2

S. Rao et al. / Food Chemistry 135 (2012) 1245–1252

Identification of peptides present in the LPH2–3 kDa by MALDI-TOF-TOF.

Sequence

RT

Obsd mass b (q a )

Position

Sequence

RT

Obsd mass b (q a )

Position

NR

1.05

289.15 (1)

f(44–45) or f(113–114)

SSDITA

5.26

593.26 (1)

f(85–90)

MK

1.36

278.16 (1)

f(12–13)

ESNF

5.39

496.19 (1)

f(35–38)

MKR

1.38

434.25 (1)

f(12–14)

ITASVN

5.63

604.32 (1)

f(88–93)

AMK

1.64

349.19 (1)

f(11–13)

KVF

5.84

393.24 (1)

f(1–3)

NTQAT

2.01

534.23 (1)

f(39–43)

HGLDNY

5.87

718.29 (1)

f(15–22)

NTQATNR

2.25

804.36 (1)

f(39–45)

WIR

6.04

474.27 (1)

f(123–125)

TPGSR

2.35

517.26 (1)

f(69–73)

KIVSDGNGMNA

6.35

1106.5 (1)

f(97–107)

QINSR

3.17

617.32 (1)

f(57–61)

YSLGN

6.43

553.25 (1)

f(23–27)

LSSD

3.17

421.19 (1)

f(84–87)

LSSDITA

7.37

706.35 (1)

f(84–90)

RGY

3.21

359.20 (1)

f(21–23)

GIL

8.74

302.20 (1)

f(54–56)

GTDVQ

3.38

519.24 (1)

f(117–121)

VAW

8.80

375.19 (1)

f(109–111)

SLGN

3.55

390.18 (1)

f(24–27)

IVS

9.25

318.17 (1)

f(98–100)

CS

3.79

209.06 (1)

f(80–81)

SLGNW

9.86

576.26 (1)

f(24–28)

KIVSD

4.15

561.33 (1)

f(97–101)

CNIPCSAL

10.2

818.41 (1)

f(76–83)

AKF

4.27

365.21 (1)

f(32–34)

YGIL

10.71

465.26 (1)

f(53–56)

GSTDY

4.33

542.22 (1)

f(49–53)

WW

11.16

391.20 (1)

f(62–63)

GTDVQA

4.50

590.26 (1)

f(117–122)

YSLGNW

11.57

739.33 (1)

f(23–28)

NTGGSTDY

4.78

872.30 (1)

f(46–53)

GTDVQAWIR

11.98

1045.5 (1)

f(117–125)

HGLDNYR

4.91

873.40 (1)

f(15–21)

WVAW

12.49

561.25 (1)

f(108–111)

RT means retention time in the TIC profile. The sequence of lysozyme was as follows: KVFGRCELAAAMKRHGLDNYRGYSLGNWVCAAKFESNFNTQATNRNTDGSTDYGILQI NSRWWCNDGRTPGSRNLCNIPCSALLSSDITASVNCAKKIVSDGNGMNAWVAWRNRCKGTDVQAWIRGCRL (The sequence comes from protein data UniProtKB, and the record number is P00698. a Charge state of the precursor ion. b Molecular ion mass observed in the MALDI-TOF-TOF system calculated in Daltons (Da).

relevant to the peak for 5.87 min retention time in the TIC, and the corresponding peptide sequence was identified as HGLDNY, com- pared to the amino acid sequence of the lysozyme and character- ised by the peptide sequencing module of the MassLynx 4.1 software. Using this analytical method, 38 peptides in the LPH2– 3 kDa were identified in this study and listed in Table 2, and their observed and calculated mass matched as well as their sequence. Blast sequence similarity searches revealed above 98% homology of the identified sequences with lysozyme. Pepsin is an endopeptidase acting at stomach level which hydrolyses peptide bonds within protein sequences randomly to produce relatively large peptides. Savoie, Gauthier, Marin, and Pou- liot (2005) report that hydrolysis with pepsin often generates pep- tides containing Phe, Tyr, or Leu in N-terminal position. This is the case of different peptides possessing these amino acids in such a position, such as peptide LSSD, YSLGN, LSSDITA, YGIL, and YSLGNW in the LPH2–3 kDa (Table 2). Furthermore, pepsin also frequently cleaves at Phe, Tyr, Trp, or Leu in C-terminal region (Kageyama, 2002), and this cleavage character is likewise feasible for a-chymo- trypsin (Folk & Schirmer, 1965). In these respects, some peptides having these amino acids at the C-terminus have been identified in this work such as RGY, GIL, VAW, KVF, and others (Table 2). On the other hand, peptides having a C-terminal Arg or Lys fre- quently appear in the identified sequences and it would be charac- teristic of a trypsin action (Gray & Cooper, 1971), as in cases of AMK, TPGSR, and WIR (Table 2). Overall, these digestive proteases are suitable to generate peptides that contain aromatic (Phe, Tyr and Trp), basic (Arg and Lys) or Leu residues at C-terminus (Rao, Xu, Su, Sun, & Yang, 2011). Interestingly, these residues at C-termi- nus or within the sequences have great contribution to the ACE inhibitory or antioxidant activity of the peptides (Murray & FitzGerald, 2007; Sarmadi & Ismail, 2010). The relationships between the structure and activity level of various inhibitory peptides indicate that binding to ACE is strongly influenced by the C-terminal tripeptide sequence of the substrate. The hydrophobic residues in the C-terminal tripeptide may well interact with subsites at the active site of ACE (Ondetti & Cushman, 1982). Notably, ACE prefers to have substrate or competitive inhib- itors that contain aromatic residues such as Phe, Tyr, and Trp or ali- phatic residues Leu at the final position of C-terminus (Cheung,

Wang, Ondetti, Sabo, & Cushman, 1980). Activity data also suggests that the positive charge on the guanidine or e -amino group of C- terminal Arg or Lys side chains, respectively, contribute substan- tially to their inhibitory activity against ACE. Similarly, the pres- ence of aromatic, positively charged, and hydrophobic residues in the peptide are important in contributing to the radical-scavenging capacity of peptides through donating hydrogen to reactive oxygen species. Considering the high proportion of hydrophobic and basic residues in the lysozyme sequence, enzymatic characteristics of gastrointestinal enzymes and structure–activity relationships of bioactive peptides, the simulated gastrointestinal digestion of lyso- zyme was conducted to produce functional peptides in this study. As expected and shown in Table 2, the C-terminal residues of many peptides generated from lysozyme by gastrointestinal enzymes are well fit for the C-terminal residues requirements of ACE inhibitory peptides or antioxidant peptides according to the structure–activ- ity relationship described as above. To further verify bioactivities of generated peptides, we synthe- sized eight tripeptides among the identified peptides from the LPH2–3 kDa. As shown in Table 3, all tripeptides showed high ACE inhibitory activities, with IC 50 values of below 100 lM. These sequences all contained an aromatic, basic residue or Leu at the C- terminus, which devoted to the inhibitory potency against ACE. The sequences VAW yielded the strongest bioactivities with an IC 50 value of 2.8 l M. This peptide has a branched-chain aliphatic residue (Val) at the N-terminus and an aromatic residue (Trp) at the C-terminus. It was reported that the peptides with this struc- ture possessed potent ACE inhibitory potency (Cheung et al., 1980). Additionally, it is important to highlight the fact that the positively charged residue (Lys/Arg) at the middle position, has a positive influence for peptide-enzyme binding and therefore rein- forces the inhibitory potency of the tripeptides (Majumder & Wu, 2010). This kind of active peptides, such as AKF (IC 50 = 6.5 lM) and MKR (IC 50 = 25.7 l M), also showed potent ACE inhibitory activities. Davalos, Miguel, Bartolome, and Lopez-Fandino (2004) report that Tyr and Trp show the highest antioxidant activity among various natural amino acids. Moreover, Tyr and Trp are generally accepted as antioxidants contributing to the activities of the identified peptides (Chen, Muramoto, & Yamauchi, 1995). The peptides RGY, WIR and

S. Rao et al. / Food Chemistry 135 (2012) 1245–1252

Table 3

ACE inhibitory or/and antioxidant activities of synthetic peptides.

1251

Peptide

ACE inhibitory

Peptide or

ACE inhibitory

Antioxidant activity

Activity IC 50 (lM)

Control

Activity IC 50 (lM)

Reducing power a

Lipid peroxidation inhibition (%) b

KVF

14.0

RGY

61.9

1.08 ± 0.11 A

75.3 ± 1.43 A

MKR

25.7

WIR

88.5

1.32 ± 0.08 B

79.8 ± 0.83 B

AMK

94.2

VAW

2.8

0.76 ± 0.21 C

83.6 ± 1.57 C

 

AKF

6.5

Glutathion

0.91 ± 0.16 AC

GIL

53.8

Vitamin E

– 78.4 ± 1.71 AB

Results for ACE inhibitory activity are the mean values of triplicate analyses. Results for antioxidant activity are the mean values of triplicate analyses ± standard deviation. AC Means within the same column without a common letter differ significantly (P < 0.05) with Tukey’s test. a Absorbance at 700 nm for a peptide or a control tested at concentration of 5 mM. b The lipid peroxidation inhibition activity in a linoleic acid emulsion system for a peptide or a control at concentration of 0.5 mM or 0.1 mM, respectively.

VAW contain these two residues, and hence their antioxidant activi- ties were determined in this study. As expected, these peptides all showed strong antioxidant effects (Table 3). Notably, the peptide WIR revealed the highest reducing power with a value of 1.32 at 5 mM, and the values of RGY and VAW was close to that of glutathion at the same concentration. However, the lipid peroxidation inhibition of these peptides doesnot show significant difference. The inhibitions of the peptides were all above 75% at 0.5 mM, which were comparable to that of vitamin E (78.4%) at 0.1 mM. This result suggested that these peptides have significant protective effect on peroxidation of linoleic acid, and thus possessed potent antioxidant activities. It was worthy to highlight that hydrophobic (Gly, Ile, Ala and Val) and basic (Arg) residues within the sequences also played important roles in the anti- oxidant activities of these peptides.

4. Conclusion

In the present study, the in vitro digestion hydrolysate (LPH2) of hen egg white lysozyme was found to exert a potent dual activity as they showed both in vitro ACE inhibitory and antioxidant activ- ities. Moreover, conduction of ultrafiltration significantly improved the antioxidant activity of the LPH2, and 38 peptides in the filtra- tion LPH2–3 kDa were identified using the analytical technique MALDI-TOF-TOF. Of special interest were fragments KVF, MKR, AMK, AKF, RGY, WIR, VAW and GIL found in the LPH2–3 kDa, and they showed great ACE inhibitory activities. Furthermore, the pep- tides RGY, WIR and VAW were also found to exhibit strong antiox- idant activities. The results reported in this work suggest that physiological digestion of lysozyme may promote the generation of peptides with ACE inhibitory and antioxidant activities. Never- theless, further work using animal models will be needed to con- clude if these sequences and the intact lysozyme may have a physiological role in blood pressure regulation. Experiments using spontaneously hypertensive rats are currently in progress.

Acknowledgments

This work has received financial support from the National 863 Programs of China (2007AA10Z330), the Doctoral Research Funds of Jiangnan University (No. JUDCF11018), and the Graduate Educa- tion Innovation Project in Jiangsu Province (CXZZ11_0489).

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