IJFST

Original article

Purification and characterisation of a novel antioxidant peptide

from porcine haemoglobin hydrolysate

Qian Sun,1 Yongkang Luo,1* Huixing Shen,2 Xue Li1 & Lei Yao1

1 College of Food Science & Nutritional Engineering, China Agricultural University; Beijing Higher Institution Engineering Research Center of

Animal Product. Beijing 100083, China

2 College of Science, China Agricultural University, Beijing 100083, China

(Received 26 February 2011; Accepted in revised form 14 September 2011)

Summary Porcine haemoglobin, which is normally discarded as a by-product of meat industry, was hydrolysed using

pepsin, AS1398 neutrase, trypsin, flavorzyme, papain and alcalase respectively. The peptic hydrolysate

exhibited the highest antioxidant activities than those of other hydrolysates, which was separated using

ultrafiltration membranes, and consecutively using chromatographic methods including ion-exchange

chromatography on SP Sephadex C-25 column, gel filtration chromatography on Sephadex G-25 column

and reversed-phase high performance liquid chromatography. Finally, a novel antioxidant peptide from

porcine haemoglobin (APPH) was purified, and its sequence was identified to be ARRLGHDFNPDVQAA

(1666 Da) using mass spectrometry. APPH exhibited significant higher lipid peroxidation inhibitory ability

than that of a-tocopherol as positive control (P < 0.05), and efficiently quenched hydroxyl radical

(IC50 = 26.9 lm). APPH agrees with the 115–129 residues of the b-chain from porcine haemoglobin. These

results indicate that APPH would be a beneficial ingredient for functional food and pharmaceuticals.

Keywords Antioxidant peptide, characterisation, lipid peroxidation, porcine haemoglobin hydrolysate, purification, radicals scavenging

activity.

Introduction

Reactive oxygen species (ROS) are thought to createoxidative stress, thereby causing various degenerativediseases, such as atherosclerosis, rheumatoid arthritis,diabetes mellitus and Alzheimer’s diseases (Athukoralaet al., 2006; Manso et al., 2008; Mamelona et al., 2010).The chemical activity of hydroxyl radical is the strongestamong ROS, and it easily induces severe damage inDNA leading to carcinogenesis, mutagenesis and cyto-toxicity (Ardestani & Yazdanparast, 2007). In foods,lipid peroxidation (LPO) can lead to the development ofundesirable off-flavours and potentially toxic reactionproducts (Thiansilakul et al., 2007). To prevent foodsfrom deterioration and to provide protection againstserious diseases, it is very important to inhibit LPOoccurring in foodstuffs and the living body. Antioxi-dants can protect the human body from ROS effects andretard the progress of many chronic diseases as well asdiscoloration and deterioration in foods (Je et al., 2004).Using synthetic antioxidants such as butylated hydroxy-anisole, butylated hydroxytoluene and propyl gallate in

food products is under strict regulation because of theirpotential health hazards. Therefore, the search fornatural antioxidants as alternatives to synthetic ones isof great interest among researchers. It has been reportedthat antioxidant peptides purified from many proteinhydrolysates, such as canola (Cumby et al., 2008),smooth hound protein (Bougatef et al., 2009), marinespecies gelatin (Aleman et al., 2011) and pork hams(Park & Chin, 2011), possess antioxidant activitiesagainst free radicals and ROS.The slaughter of animals produces a considerable

amount of by-products with high biological value,among which one of the most important is bloodprotein. The haemoglobin represents 80% of the proteincontent of blood, which has excellent nutritional valuedue to its physiologically well-balanced amino acidcompositions (Piot et al., 1988). Hence, efficient recov-ery and utilisation of porcine haemoglobin are veryimportant to reduce environmental problems and tomaximise economic benefits. An interesting approachfor the effective application of haemoglobin would beenzymatic hydrolysis, which is widely used in foodindustry to convert to value-added products for upgrad-ing the functional and nutritional properties of proteins.Recently, a study on the antioxidant activity of porcine

*Correspondent: Fax: +86 10 62737385;

e-mails: [email protected] or [email protected]

International Journal of Food Science and Technology 2012, 47, 148–154148

doi:10.1111/j.1365-2621.2011.02820.x

� 2011 The Authors. International Journal of Food Science and Technology � 2011 Institute of Food Science and Technology

haemoglobin hydrolysate (PHH) which produced usingalcalase and flavourzyme has been performed (Changet al., 2007). However, the antioxidant peptides fromhaemoglobin were not further purified and identified.In this study, we investigated the antioxidant activity of

PHH prepared using six different enzymes respectively.Furthermore, we adopted ultrafiltration (UF) and severalchromatography techniques to isolate the most potentantioxidant peptide from PHH, and the amino acidsequences of the purified peptide were also determined.

Materials and methods

Materials and chemicals

Six independent batches of the porcine blood were takenfrom Shunxin agricultural Co. Ltd (Shun-Yi District,Beijing). Pepsin (pH 2.0, 37 �C, from porcine stomachmucosa) used for protein hydrolysis with declared activ-ities of 1:3000 U g)1 and a-tocopherol, were purchasedfrom Amresco (Solon, OH, USA). Alcalase (pH 8.0,55 �C) and flavorzyme (pH 6.5, 45 �C) were fromNovozymes (Novo Nordisk, Bagsvaerd, Denmark). Thecrude protease A.S.1398 (pH 7.0, 45 �C) was fromDonghua Qiangsheng Biotechnology Co., Ltd (Beijing,China) and Papain (pH 6.5, 37 �C) from Javely BiologicalProducts Co., Ltd (Nanning, China). Trypsin (pH 7.5,45 �C) was from Amresco. 2-Deoxy-D-ribose (D5899),linoleic acid (�99%) (L1376) and 1, 1-diphenyl-2-pic-rylhydrazyl radical (DPPH, D9132) were purchased fromSigma–Aldrich (St. Louis, MO, USA). The UF mem-brane reactor system was from XuBang membraneequipment Co., Ltd (Beijing, China). SP Sephadex C-25and Sephadex G-25 were purchased from Pharmacia(Uppsala, Sweden). All reagents were of analytical grade.

Preparation of PHH by pepsin

The haemoglobin was isolated from quarantine qualifiedporcine blood and transferred to the lab at 4 �Cimmediately, and was lyophilised to dry powder thenstored at 4 �C until use. Five grams of dried haemoglo-bin were dissolved in 100 mL distilled water anddigested by pepsin (enzyme to substrate ratio = 1.6%,w ⁄w) at 40.4 �C for 60 min. During hydrolysis, pH wasmaintained at 1.6 by addition of 1 m HCl. After heatingat 100 �C for 10 min to inactivate the enzyme, thehydrolysate was centrifuged at 4200 g and 4 �C for10 min to remove insoluble substrate, was lyophilised todry powder and then storage at )20 �C.

Antioxidant activity

Antioxidant activity in Linoleic acid ⁄FTC model systemThe antioxidant activity was determined according tothe method of Saha et al. (2004). The degree of

oxidation was evaluated using the ferric thiocyanate(FTC) method (Mitsuda et al., 1996). The colourdevelopment degree was measured at 500 nm (ModelUV-2600A; UNICO, Shanghai, China). a-tocopherolwas used as a positive control.

DPPH radical scavenging activityThis method was according to the procedure describedby Rajauria et al. (2010). Test samples which dissolvedin 1.5 mL of water (1.5 mg mL)1) and were mixed with1.5 mL of 1 mm DPPH ethanol solution. This mixturewas shaken and kept at 25 �C for 30 min, and then theabsorbance of the mixture was measured at 517 nmagainst a blank.

Superoxide radical scavenging activityThis assay measured the inhibition of the auto-oxidationof pyrogallol using a slightly modified method ofMarklund & Marklund (1974). A sample solution(0.3 mL, 5 mg mL)1) and 2.61 mL of 50 mm phosphatebuffer (pH 8.2) were added into freshly prepared 90 lLof 3 mm pyrogallol (dissolved in 10 mm HCl). Afterincubation for 4 min, the reaction mixture was addedwith 100 lL of 0.2 m ascorbic acid immediately. Theabsorbance was measured at 325 nm against a blank.

Hydroxyl radical scavenging activityThis assay was measured using the deoxyribose method(Siddhuraju & Becker, 2007) with some modifications.The reactions were performed in 0.1 m, phosphate buffer(pH 7.4), containing 10 mm deoxyribose, 10 mm FeS-O4Æ7H2O, 10 mm EDTA and samples. The reaction wasactivated by adding H2O2 to a concentration of 10 mm,

and the reaction mixture was incubated for 1 h at 37 �Cin a water bath. After incubation, the colour wasdeveloped by adding 1 mL of 1% thiobarbituric acidand 1 mL of 2.8% ice-cold trichloroacetic acid andheated at 100 �C for 10 min. After cooling, the absor-bance was measured at 532 nm.

Purification and characterisation of antioxidant peptide

Ultrafiltration (UF) systemPorcine haemoglobin hydrolysate was fractionatedthrough three different UF membranes having a mole-cular weight cutoff (MWCO) range of 10, 5 and 3 kDarespectively. All peptide fractions (PHH-I with MWdistribution >10 kDa, PHH-II with MW distribution of5–10 kDa, PHH-III with MW distribution of 3–5 kDaand PHH-IV with MW distribution <3 kDa) recoveredwere lyophilised in a freeze drier and stored at )20 �C.

Ion-exchange chromatography and Gel filtrationchromatographyAmong the UF fractions, PHH-IV was separated usingSP Sephadex C-25 ion-exchange column (3.5 · 40 cm).

A novel antioxidant peptide from porcine haemoglobin hydrolysate Q. Sun et al. 149

� 2011 The Authors International Journal of Food Science and Technology 2012

International Journal of Food Science and Technology � 2011 Institute of Food Science and Technology

The lyophilised PHH-IV (4 mg mL)1) was loaded ontoa the column equilibrated with 20 mm ammoniumacetate buffer (pH 3.6) and eluted with a linear gradientof ammonium formate (0–1 m) in the same buffer at aflow rate of 1 mL min)1, and 5 mL fractions werecollected. Each fraction was monitored at 280 nm andthe fraction eluted under the same elution peak werecollected, lyophilised and tested for antioxidant activity.The pooled fraction (10 mg mL)1) with the highestantioxidant activity was consecutively purified on aSephadex G-25 gel filtration column (2.6 · 100 cm)equilibrated with ammonium acetate buffer (pH 3.6).Fractions (4 mL each) were collected at a flow rate of0.8 mL min)1, and absorbance was measured at 280 nmto determine the elution profile of the sample. These twochromatographic runs were repeated 20 times. Fractionsassociated with each peak showing antioxidant activitywere pooled and freeze-dried for next step.

Reversed-phase high pressure liquid chromatography(RP-HPLC)The fraction exhibiting the highest antioxidant activitywas further purified using RP-HPLC on a Daisogel C18100A (20 · 250 mm; Daiso, Osaka, Japan) column witha linear gradient of acetonitrile (5% in 0–5 min, 5–45%in 5–40 min) containing 0.1% trifluoroacetic acid (TFA)at a flow rate of 3.0 mL min)1. Elution peaks weredetected at 220 nm, and active peak was concentratedusing a rotary evaporator. For further purification, themost potent peak was loaded onto a Kromasil-5 C18 RP-HPLC analytical column (4.6 · 250 mm; Akzo Nobel,Sweden) with a linear gradient of acetonitrile (5% in0–5 min, 5–50% in 25 min) containing 0.1% TFA at aflow rate of 1.0 mL min)1. Elution peaks were detectedat 220 nm, and the potent peaks were collected, evalu-ated for antioxidant activity and then lyophilised. Thechromatographic run was repeated 35 times. The finalpurified peptide was analysed for amino acid sequence.

Matrix-assisted laser desorption ionisation-time of flight(MALDI-TOF) mass spectrometryMolecular weight and amino acid sequence of thepurified peptide was analysed using a MALDI-TOF-TOF Bruker Ultraflex II (Bruker, Germany). Thepeptide was mixed with matrix solution (50% acetoni-trile and 0.1% TFA), and the mixture was spotted onthe MALDI target plate. The spectrum was acquired inpositive ion mode at 20 kV in linear mode.

Statistical analysis

The antioxidant activity assays were carried out intriplicates, and data were expressed as means withstandard deviations. A significant difference was con-sidered at P < 0.05 (SAS Institute Inc., Cary, NC,USA; 1999).

Results and discussion

Enzymatic hydrolysis of porcine haemoglobin

Porcine haemoglobin was separately hydrolysed with sixenzymes (Pepsin, AS1398 Neutrase, Trypsin, Flavor-zyme Papain and Alcalase) at optimal conditions. Thepeptic hydrolysate exhibited higher DPPH radical scav-enging ability (67.0 ± 1.8%) and linoleic acid autoxi-dation inhibitory ability (86.1 ± 2.1%) than otherhydrolysates. The LPO inhibitory ability of PHH couldbe due to the properties that strong affinity for oil andother nonpolar substances of porcine haemoglobin.Furthermore, pepsin preferably digests peptide bondsby cleaving after the N-terminal of aromatic aminoacids such as Phe, Trp and Tyr. The phenyl groups ofthe residues at peptide ends were likely to scavenge thefree radical to prevent DNA damages. Similar to ourstudy, Je et al. (2007) also found that peptic hydroly-sates from tuna backbone protein exerted the highestantioxidant activity among six hydrolysates prepared bydifferent enzymes.

Purification and identification of antioxidant peptide

Initially, the peptic hydrolysate was separated usingthree kinds of UF membranes (10-, 5- and 3-kDaMWCO membranes), and four kinds of permeates(PHH-I, PHH-II, PHH-III and PHH-IV) wereobtained. PHH-IV group had a higher LPO inhibitionability than the other fractions. The high inhibitoryeffect by PHH-IV was found to be 93.80 ± 0.97%(5 mg mL)1). Furthermore, PHH-IV exhibited signifi-cant higher radical scavenging effects than those of othergroups (P < 0.05) (Table 1). Therefore, PHH-IV wasemployed for the purification and identification of theantioxidant peptide.Porcine haemoglobin hydrolysate-IV was further

loaded onto a SP Sephadex C-25 column, and threeabsorbed portions were eluted with a linear gradient ofammonium formate (Fig. 1a). Fraction C possessed thehighest hydroxyl radical scavenging activity among all

Table 1 Free radical scavenging activities (%) of fractionated porcine

haemoglobin hydrolysate (PHH)a

Fractions

DPPH

radical

Hydroxyl

radical

Superoxide

radical

PHH-I 69.9 ± 1.7b 38.0 ± 4.6b 11.9 ± 1.1c

PHH-II 54.2 ± 2.0c 22.3 ± 5.7c 14.8 ± 0.8c

PHH-III 66.7 ± 3.1b 31.6 ± 2.8bc 18.1 ± 1.3b

PHH-IV 83.4 ± 3.8a 55.8 ± 2.2a 27.8 ± 1.6a

aValues are means of three determinations ± standard deviation. Means

in the same columns with different letters are significantly different

(P < 0.05).

A novel antioxidant peptide from porcine haemoglobin hydrolysate Q. Sun et al.150

International Journal of Food Science and Technology 2012 � 2011 The Authors


fractions. Following activity analysis, the lyophilisedactive fraction C was subjected on a Sephadex G-25column equilibrated with ammonium acetate buffer(20 mm, pH 3.6) (Fig. 1b). The potent fraction CII wasfurther separated using RP-HPLC on a Daisogel C18100A (20 · 250 mm) column, and more than 20 peakswere detected using this chromatography (Fig. 2).There were eight peaks exhibited potent antioxidantactivities, and the CII-4 fraction possessed the strong-est activity. To obtain a purified peptide, we rechro-matographed CII-4 fraction on a Kromasil-5 C18(4.6 · 250 mm) RP-HPLC analytical column (Fig. 3).Finally, we obtained a purified antioxidant peptide

from porcine haemoglobin (APPH). This purified pep-tide can effectively quench in hydroxyl radical (IC50 =26.9 lm). Then, the molecular mass and aminosequence of APPH was determined using MALDI-TOF mass spectroscopy. As shown in Fig. 4, APPHwas composed of 15 amino acid residues, which wasdetermined to be Ala-Arg-Arg-Leu-Gly-His-Asp-Phe-Asn-Pro-Asp-Val-Gln-Ala-Ala (MW = 1666 Da). Thisamino acid sequence agrees with the 115–129 residuesof the b-chain from porcine haemoglobin. The molec-ular mass of the APPH determined using MALDI-TOF spectroscopy was in excellent agreement withtheoretical mass calculated from the sequence. This

Figure 1 Purification profiles of antioxidant

peptides from porcine haemoglobin. (a) Ion-

exchange chromatography on SP Sephadex

C-25 chromatography. (b) Gel filtration

chromatography on Sephadex G25 column.

Upper panels represent the antioxidant

activity measured using hydroxyl radical

scavenging assay.

Figure 2 Reversed-phase high performance

liquid chromatography pattern on a Daisogel

C18 100A column of fraction CII obtained

using gel chromatography. Upper panels

represent the antioxidant activity measured

using hydroxyl radical scavenging assay.




identified peptide is a novel peptide with antioxidantactivity that had never been reported.

Antioxidant activities of purified peptide (APPH)

The antioxidant activity of APPH was further investi-gated using LPO inhibition assay. We adopteda-tocopherol, a widely used natural antioxidant, as thepositive control to evaluate the LPO inhibitory ability ofAPPH. As shown in Fig. 5, APPH effectively inhibited

LPO in linoleic acid emulsion system up to the 48 h, andthe activity was significant higher than that of a-tocoph-erol (P < 0.05).Cheng et al. (2003) reported that phenolic compounds

afforded their protective actions in LPO by scavengingthe lipid derived radicals (R·, RO· or ROO·) to stop thechain reactions in a heterogeneous lipid phase. Further-more, Tong et al. (2000) revealed that high molecularweight fraction of whey protein was able to inhibit LPOvia scavenging of free radicals. Therefore, the LPO

Figure 3 Reversed-phase high performance

liquid chromatography pattern on a Kroma-

sil-5 C18 column of subfraction CII-4 ob-

tained using Daisogel C18 100A RP-HPLC.

Elution profiles (lower panel) and antioxidant

activities of the fractions (upper panel) are

measured using hydroxyl radical scavenging

assay.

Figure 4 Identification of molecular mass and

amino acid sequence of antioxidant peptide

from porcine haemoglobin using a MALDI-

TOF-TOF mass spectrometer. Sequencing of

active peptide was acquired over the m ⁄ zrange 50–2500.




inhibitory ability of peptide or protein is dependent onmolecular size and chemical properties such as hydro-phobicity and electron transferring ability of amino acidresidues in the sequence. In the sequence of APPH,hydrophobic amino acid residue such as Ala, Leu andaromatic amino acid composed approximately 46.7% ofthe purified peptide sequence, which exert scavengingeffect of free radicals. The hydrophobic property of theAPPH may have played an important role, exerting highaffinity to linoleic acid. In addition, as hydrophobicity ofantioxidants is important for accessibility to hydropho-bic targets (Chen et al., 1996), it is presumed that thepresence of hydrophobic amino acids in purifiedpeptides may contribute to LPO inhibitory activity byincreasing solubility of peptides in lipid and therebyfacilitating better interaction with radical species. Men-dis et al. (2005) reported that hydrophobic amino acidsmight increase the affinity and reactivity to the cellmembrane in the living cells.In the APPH sequence, the Ala, Val, Leu, Pro with

non-polar aliphatic groups have high reactivity tolinoleic acid, and His, Phe with aromatic residues canmake ROS stable through direct electron transfer.Antioxidant peptides that were isolated from soybeanhydrolysate (Chen et al., 1995), egg yolk hydrolysate(Park et al., 2001) and tuna cooking juice hydrolysates(Hsu et al., 2009) have also been attributed to His due tothe proton-donation ability of its imidazole group. Ithas also been reported that His and Pro play animportant role in the antioxidant activity of peptidesfrom soybean protein hydrolysates (Chen et al., 1996).In addition, presence of Asp seems to play a vital roleirrespective of its position as observed in severalantioxidant peptide sequences (Rajapakse et al., 2005;

Uchida & Kawakishi, 1992). The hydrogen donors suchas Gly and Asp are able to quench unpaired electrons orradicals by supporting protons. The two Asp residueslocated around the central in the APPH sequence wouldbe very important to the antioxidant activity of APPH.Hsu et al. (2009) reported that the two purified

peptide from tuna cooking juice hydrolysates exhibitedstrong radical scavenging activity. The two peptides andAPPH sequence contained His-Asp sequence. There-fore, it can be expected that His-Asp sequence may beplay a vital role in the observed radical scavengingactivity of APPH. As a whole, the presence of specificamino acids and their specific positioning in thesequence could have been attributed to antioxidantactivity of the purified peptide.

Conclusions

In this study, a novel antioxidative peptide (Ala-Arg-Arg-Leu-Gly-His-Asp-Phe-Asn-Pro-Asp-Val-Gln-Ala-Ala) that could efficiently inhibited LPO and was apotent free radical scavenger was purified using UFmembranes and consecutive chromatographic methodsfrom PHH. In addition, further detailed studies onAPPH with regard to antioxidant activities in vivo andstability of this peptide when incorporated in foods, areneeded.

Acknowledgments

This work was supported financially by NationalScience Technology Ministry of China (award nr2008GA741002 and nr 2006BAD05A16) and Ministryof Agriculture of China (award nr 2011-G8).

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0

10

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25 μM 50 μM 100 μM

α-tocopherolAPPH

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ioxi

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act

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(%)

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