ARTICLE

Isolation, Purification and Characterization of Antioxidative Bioactive Elastin Peptides from Poultry Skin

Mehdi Nadalian1https://orcid.org/0000-0002-0495-0615, Nurkhuzaiah Kamaruzaman1https://orcid.org/0000-0001-8948-2154, Mohd Shakir Mohamad Yusop1,2https://orcid.org/0000-0001-7791-9363, Abdul Salam Babji1https://orcid.org/0000-0003-1585-2233, Salma Mohamad Yusop1,*https://orcid.org/0000-0002-9518-4257
Author Information & Copyright
1Department of Food Sciences, Faculty of Science and Technology, Universiti Kebangsaan Malaysia, 43600 Bangi, Selangor, Malaysia
2Institute of Systems Biology, Universiti Kebangsaan Malaysia, 43600 Bangi Selangor, Malaysia
*Corresponding author: Salma Mohamad Yusop, Department of Food Sciences, Faculty of Science and Technology, Universiti Kebangsaan Malaysia, 43600 Bangi, Selangor, Malaysia, Tel: +603-8921-5963, Fax: +603-8921-3232, E-mail: salma_my@ukm.edu.my

© Korean Society for Food Science of Animal Resources. This is an Open-Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/3.0) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

Received: Oct 25, 2019 ; Revised: Nov 20, 2019 ; Accepted: Nov 20, 2019

Published Online: Dec 31, 2019

Abstract

Muscle-based by-products are often undervalued although commonly reported having a high amount of natural bioactive peptides. In this study, elastin was isolated from the protein of broiler hen skin while its hydrolysate was prepared using Elastase. Assessment of antioxidative properties of elastin-based hydrolysate (EBH) was based on three different assays; 2,2-diphenyl-1-picryl-hydrazyl-hydrate (DPPH) radical, 2,2’-azino-bis (3-ethylbenzothiazoline-6-sulphonic acid) (ABTS) radical and metal chelating ability. The EBH was purified further using ultrafiltration, gel filtration and Reverse- Phase High-Performance Liquid Chromatography (RP-HPLC). The IC50 of ABTS radical activities for EBH were decreased as EBH further purified using ultrafiltration (EBH III; 0.66 mg/mL)>gel filtration (EB-II; 0.42 mg/mL)>RP-HPLC (EB-II4; 0.12 mg/mL). The sequential identification of the peptide was done by matrix-assisted laser desorption/ ionization time-of-flight/time-of-flight mass spectrometry (MALDI-TOF/ TOF-MS) of the potent fractions obtained from RP-HPLC (EB-II4). The presence of hydrophobic amino acids (Val and Pro) in the peptide sequences could potentially contribute to the high antioxidant activity of EBH. The sequences GAHTGPRKPFKPR, GMPGFDVR and ADASVLPK were identified as antioxidant peptides. In conclusion, the antioxidative potential from poultry skin specifically from elastin is evident and can be explored to be used in many applications such as health and pharmaceutical purposes.

Keywords: antioxidant peptides; elastin; enzymatic hydrolysis; withdrawal period; poultry skin

Introduction

Biologically active peptides are oligopeptides that can exert biological activities beyond their expected nutritional values that are proven beneficial to humans due to its health-promoting properties (Erdmann et al., 2008). These biologically active peptides–made up of sequences of amino acids encoded in the parent protein molecules – remain inactive until released via enzymatic hydrolysis by peptidases during food processing and/or during gastrointestinal digestion. An example of a parent protein molecule with high biological value is elastin (Hattori et al., 1998). Besides the apparent nutritional value of its amino acids, the protein elastin itself has been a candidate for an active source of value-added product due to its functional properties such as antihypertensive, antimicrobial, antioxidative and antiaging (Agrawal et al., 2016). Elastin is the insoluble elastic fibrous protein which forms the framework that holds the connective tissues in animals (Antonicelli et al., 2007). An elastin core is surrounded by layers of microfibrils forming elastic fibers which provides elasticity and resilience of connective tissues such as blood vessels, lungs, aorta, and skin, which are essential for human health and development (Levillain et al., 2016). Apart from the manifold components, the elastic fibers are also maintained by highly complex molecular activities firmly controlled development, multiple stages of assembly, distinct biochemistry. However, the complexity is being unraveled and understood through studies of mouse models (Kielty et al., 2002). Elastin has been investigated in fields of biomedical and tissue engineering, molecular therapy, and even cosmetics (Ganceviciene et al., 2012; Lescan et al., 2018; Yeo et al., 2015). As such, elastin is involved in multiple levels of studies and analysis, ranging from topical applications to mRNA-synthesis, thus creating a demand for elastin in various forms. Commercially, elastin is distributed in powder form and of animal origin such as bovine and porcine. The protein accumulates in the animal parts postnatally, making the extraction more intricate, hence the final product expensive (Mecham, 2008). So, finding cheaper and easier alternative with high throughput is a major concern for elastin extraction.

Generally, highly meat productive birds or poultry breeds are called broiler poultry. Broilers are young chicken (Gallus gallus domesticus) of either sex of six to eight weeks of age. There are studies suggesting the potential of functional protein extraction from by-products of poultry industry (Fauzi et al., 2016; Mohammad et al., 2014; Munasinghe et al., 2014). The study suggests that collagen – another example of parent protein molecule – could be extracted from chicken skins and bones, which then can be utilized in various fields such as pharmaceuticals, biomedicine and food. As collagen shares similar characteristics with elastin, it is worth investigating the potential of chicken skin for elastin extraction (Mecham, 2008; Munasinghe et al., 2014).

Still, there is minimal information on the extraction of elastin from chicken skin. Exploring this prospect would improve by-products management and make elastin more accessible. Also, elastin from chicken skin could be an alternative to conventional elastin as essential properties such as antihypertensive was present and have been investigated earlier (Yusop et al., 2016).

Antioxidants protect human health against ROS (reactive oxygen species) and can increase the stability of food lipids (Martinez-Maqueda et al., 2012; Nazeer and Deeptha, 2013). It is found that certain bioactive peptides do indeed have antioxidative properties and can be used as a natural substitute to synthetic antioxidants to improve health (Ryan et al., 2011). Endogenous antioxidants that are mainly enzymes – catalase, superoxide dismutase and glutathione peroxidase – must be able to control the free radicals to protect the cellular environment against oxidative stress. The biological actions of proteins can be increased by using enzymatic hydrolysis; resulting in the targeted peptides or fractions being more bioactive than the others (Babji et al., 2018). Elastase is the enzyme found to be able to break down elastin, potentially activating the peptides that possess antioxidant properties (Antonicelli et al., 2007).

The objective of this study was to isolate elastin from broiler hen skin. Furthermore, to get access to its bioavailability, its hydrolysate was produced by incorporating enzymatic hydrolysis using Elastase. Ultrafiltration and gel filtration chromatography were used preceding reversed phase high performance liquid chromatography (RP-HPLC) to further purify the antioxidative peptides. The sequential analysis of the peptides was also carried out through MALDI-TOF-TOF-MS/MS. For ease of reference, the names of the samples analyzed were listed in Table 1.

Table 1. Summary names (abbreviations) of the samples analyzed
Abbreviation Sample type
EBH Elastin-based hydrolysate
EBH III Ultrafiltrated hydrolyzed elastin with molecular weight less than 3 kDa
EB-I First fraction of gel-filtrated hydrolyzed elastin
EB-II Second fraction of gel-filtrated hydrolyzed elastin
EB-III Third fraction of gel-filtrated hydrolyzed elastin
EB-IV Fourth fraction of gel-filtrated hydrolyzed elastin
UEB Non-hydrolyzed Elastin extracted from broiler hen skin
EB-II1 First fraction from second fraction of gel-filtrated hydrolyzed elastin
EB-II2 Second fraction from second fraction of gel-filtrated hydrolyzed elastin
EB-II3 Third fraction from second fraction of gel-filtrated hydrolyzed elastin
EB-II4 Fourth fraction from second fraction of gel-filtrated hydrolyzed elastin

EBH, elastin-based hydrolysate.

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Materials and Methods

Broiler’s skin was purchased consistently from a supplier in a local market in Bangi, Malaysia. The skins were kept in the freezer at –18°C and thawed approximately 1 h before further use.

Extraction of the protein elastin

Elastin extraction from broiler was done based on the modification of Lansing method from Lansing et al. (1952) and Nadalian et al. (2015). Broiler’s skins were suspended in 1 M NaCl. The solution was put in a cold room with constant stirring for 24 h. Then, the homogenate was centrifuged (5804R, Eppendorf, Hamburg, Germany) at 13,000×g for 20 min. Afterwards, the pellet was washed with distilled water and defatted with acetone for 1 h. The treated sample then was suspended in 0.1 M NaOH and heated for 15 min in a boiling water-bath with constant shaking. After cooling and centrifugation, the residue was extracted again for 45 min in 0.1 M NaOH at 100°C. The residues of NaOH-insoluble material were then washed several times in water and lyophilized. The sample was freeze dried by using bench top freeze dryer (Labconco, Kansas City, MO, USA) at temperature −80°C and vacuum pressure of 4.5 Pa. Next, the powder obtained was immersed in oxalic acid, relative to the insoluble elastin weight, at 100°C for 40 min. The residue of insoluble elastin re-submersed for solubilizing step as water-soluble elastin.

Enzymatic hydrolysis of elastin proteins

Elastin powder was hydrolyzed by Elastase (Sigma-Aldrich, St Loius, MO, USA) at optimal conditions of combined pH (pH 8.5) and temperature (37°C). The enzyme was added to the elastin powder with the ratio of 100:1 (w/w). The freeze-dried elastin was grounded into powder, then suspended in deionized water with a ratio of 1:100 (w/v), adjusted to pH 8.5 with 0.1 M HCI and temperature of 37°C. Lastly, all the hydrolysates were heated at 95°C for 5 min for enzyme inactivation, before being centrifuged (3,000 g, 4°C, 15 min; 5804R, Eppendorf) to separate soluble hydrolysates from any non-soluble materials. The supernatants were lyophilized into powders, which then stored at −18°C.

Determination of amino acid composition

Determination of amino acid composition was done based on the method from Alaiz et al. (1992) with some modification. Broiler skin was subjected to acid hydrolysis, performic acid and alkaline hydrolysis using water AccQ.Tag Amino Acid Analyzer (Waters, Dublin, Ireland). Hydrolysis of samples began by adding 5 mL of 6 M HCl at 110°C for 24 h. Then, α-aminobutyric acid was added and filtered through 0.2 m cellulose acetate membrane (Whatman No. 1). Derivatization of amino acids was done for 10 min at 55°C. The amino acids were then run at 37°C with a flow rate of 1 mL/min using C18 AccQ-Tag Amino Acid Analysis Column (150×3.9 mm; Waters, Milford, MA, USA). For amino acid quantification, measurement of the absorbance was done at 248 nm, while the associated fluorescence detector was measured at excitation and emission of 250 nm and 395 nm respectively. Tryptophan level was quantified by alkaline hydrolysis.

2,2-Diphenyl-1-picryl-hydrazyl-hydrate (DPPH) radical-scavenging activity

Determination of free radicals scavenging activity of elastin from broiler hydrolysate (EBH) was done based on the method from Brand-Williams et al. (1995) with some modification. Elastin powder was dissolved in distilled water. 2 mL of 0.2 mM 2,2-diphenyl-1-picryl-hydrazyl-hydrate (DPPH) was added to 100 μL of sample solutions and mixed rigorously. After incubation at 0.5, 5, and 24 h, the absorbance was measured at 517 nm using a microplate reader (Biotek 259037), with distilled water as a control. DPPH scavenging activity was measured as the equation:

DPPH scavenging effect (%) = A 517 control A 517 sample A 517 control × 100

IC50 values (the concentrations of the test compounds required to reduce the produced hydroxyl radical to one-half) were calculated.

2,2’-Azino-bis (3-ethylbenzothiazoline-6-sulphonic acid) (ABTS) radical-scavenging activity

Determination of the 2,2’-azino-bis (3-ethylbenzothiazoline-6-sulphonic acid) (ABTS) radicals scavenging activity was done based on the method from Re et al. (1999). ABTS solution (7.4 mM) was prepared in 100 mM Phosphate Buffered Saline (PBS) at pH 7.4 with 0.15 M NaCl and oxidized using 2.6 mM of potassium persulfate (K2S2O8) for 12 h in the dark. The ABTS was then diluted to 734 nm absorbance with ethanol. 50 μL of the sample was mixed with 950 μL of the diluted ABTS, before being shaken rigorously for 30 s and held in a dark environment for 10 min. 50 μL of distilled water was used instead of the sample for control. Absorbance was measured at 734 nm. The percentage inhibition of ABTS.+ to ABTS was calculated using the following equation:

ABTS scavenging activity (%) = A 734 control A 734 sample A 734 control × 100

IC50 values (the concentrations of the test compounds required to reduce the produced hydroxyl radical to one-half) were calculated.

Metal chelating activity

Determination of Fe2+ chelating activity was done based on the method from Decker and Welch (1990) with some modification. 2 mL reaction composition made up of 200 μL of the sample, 50 μL FeCl2 (2 mM), and 1.75 mL deionized water was shaken and left at room temperature for 5 min to stand. 50 μL ferrozine (5 mM in methanol) was then added, mixed, left standing for another 5 min. The Fe2+ ferrozine complex was read through absorbance of 562 nm, with the positive control being EDTA. The Fe2+ chelating activity of the extract was measured as the equation:

Chelating activity (%) = A 0 A 1 A 0 × 100

Where A0 was the absorbance of the control (without sample) and A1 was the absorbance of the sample. IC50 values were calculated.

Purification of antioxidant peptides
Ultrafiltration

The hydrolysate solutions of elastin were separated into large and low molecular weight fractions by ultrafiltration at 4°C using 10 kDa Molecular Weight Cut-Off (MWCO) membrane (Vivaflow 200) (Sartorius, Göttingen, Germany), followed by a 3 kDa MWCO (Vivaflow 50) membrane. The hydrolysates were divided into 3 fractions. Fractions were referred to as the less than 10 kDa ultra-filtrated (10 kDa UF), between 3 to 10 kDa ultra-filtrated (3–10 kDa UF) and less than 3 kDa ultra-filtrated (3 kDa UF) hydrolysates, respectively. All fractions were freeze-dried and stored at –18°C until further use.

Size exclusion chromatography (SEC)/gel-filtration chromatography

After ultrafiltration, a gel filtration chromatography column, Hiprep 26/60 sephacryl S-100HR (26×600 mm) (GE Healthcare, Buckinghamshire, UK) was used to further purify fractions with the highest ABTS radicals scavenging activity. Fractions of 250 mg sample were dissolved in 2 mL sodium phosphate buffer (10 mM, pH 7.2) prior to column loading and elution with sodium phosphate buffer (10 mM, pH 7.2) at flow rate 1.5 mL/min (You et al., 2010). 5 mL of the eluted fractions were collected into several fractions based on the peaks observed by absorbance at 280 nm and freeze-dried. Each fraction was tested for ABTS radical scavenging activity assay to determine antioxidants activity.

Reversed-phased high-performance liquid chromatography (RP-HPLC)

Fractions from gel-filtration chromatography with the highest antioxidant activity were further separated using RP-HPLC (Waters, Milford, MA, USA). 5 mg of the peptide fractions were dissolved in 2 mL of sodium phosphate buffer (10 mM, pH 7.2) before being filtered with a 0.22 μm filter. 200 μL of the sample was then loaded onto XBridge BEH130 Prep C18 (10×250 mm, 5 μm) (Waters, Milford, MA, USA). The solvents involved are; solvent A, 0.1% (v/v) trifluoroacetic acid (TFA) in deionized water; solvent B, 0.1% (v/v) TFA in 100% (v/v) acetonitrile solution. The flow rate was 4.73 mg/min and the UV absorbance of the eluents was measured at 214 nm. The samples are then put into a centrifugal concentrator at 380×g for 3 h, which are then used for ABTS radical scavenging activity.

Identification of peptides by MALDI-TOF/TOF-MS

The trypsin-digestion and peptides extraction of the targeted fractions was done based on several studies (Bringans et al., 2008; Garg et al., 2013; Sharma et al., 2007). Analysis of peptides was done by MALDI TOF/TOF (matrix-assisted laser desorption/ionization time-of-flight) mass spectrometer. This method is directly derived from Garg et al. (2013), as the method is deemed the most suitable for this experiment. The full, detailed approach could be obtained from the study. This method resulted in the recognition of the peptide sequences based on the matching identity ’protein spots’ with the highest ion score.

Statistical analysis

All analyses were directed in triplicate. A one-way analysis of variance (ANOVA) was implemented, and the mean comparisons were analyzed conferring to Tukey range test at significant level 95% (p<0.05). The statistical analyses were completed by using SPSS package (SPSS 23.0 for windows, SPSS IBM, Chicago, IL, USA).

Results and Discussion

Antioxidant activities of elastin hydrolysates

The hydrolysates were measured for antioxidant activities through DPPH, ABTS and metal-chelating activity assays. It was evident that the elastin hydrolysates possess antioxidative potentials, with ABTS value reflects the radicals scavenging activity (IC50 of 1.10±0.08 mg/mL), DPPH (2.80±0.37 mg/mL) and metal chelating activity (1.21±0.09 mg/mL) as shown in Table 2. Antioxidant properties of food protein hydrolysates are found to be influenced by their amino acid composition. A high value of DPPH could be correlated to a high amount of hydrophobic peptide fraction of the peptides involved, especially the ones derived from natural protein sources (Li et al., 2008; Pownall et al., 2010). A similar point was made in a study of rapeseed peptides, where the DPPH radical scavenging activities of the peptides are found to have a direct correlation with hydrophobicity (Zhang et al., 2008). In addition, the peptides could serve as food preservatives by protecting food lipids from metal ion-dependent oxidative damage. Side chains of amino acids with carboxyl and amino groups could potentially be essential in chelating metal ions (Saiga et al., 2003).

Table 2. Antioxidant activity of elastin hydrolysates from broiler skin
Analysis IC50 (mg/mL) Antioxidative activity (%)
DPPH radical-scavenging activity 2.80±0.37 55.60±2.14
ABTS radical-scavenging activity 1.10±0.08 69.21±1.63
Metal chelating activity 1.21±0.09 65.32±1.48

All values are mean±SD of three replicates (n=3).

DPPH, 2,2-diphenyl-1-picryl-hydrazyl-hydrate; ABTS, 2,2’-azino-bis (3-ethylbenzothiazoline-6-sulphonic acid).

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Purification of antioxidant peptides
Ultrafiltration

Bioactivity of protein hydrolysates could be affected by the molecular weights of the peptides (Mohan et al., 2016). Table 3 outlines the ABTS radical scavenging activity and the IC50 value of EBH of the three fractions with different molecular weights. Significant differences in IC50 values were observed in all membrane cut-off MW sizes (p<0.05) with the lowest IC50 showed by EBH III fraction (<3 kDa) (0.66 mg/mL). It has been described that temperature treatment through hydrolysis surges liability of peptide bonds to the elastase and consequently releasing significant antioxidant peptides (Cudennec et al., 2016). The results are in line with a study confirming that the sanitized peptide fraction of fish proteins containing molecular weight less than 1,000 Da has the strongest antioxidant activity compared to other hydrolysates fractions (Centenaro et al., 2014).

Table 3. ABTS radical scavenging activity and IC50 values of ultrafiltrated EBH hydrolysates
Membrane MWCO (kDa) ABTS scavenging activity (%) IC50 value (mg/mL)
<10 UF 50.36±0.38c 0.96±0.03a
3–10 UF 52.84±0.38b 0.82±0.02b
<3 UF 55.58±0.38a 0.66±0.01c

All values are mean±SD of three replicates (n=3).

ABTS radical scavenging effects were tested at a concentration of 1 mg/mL.

a-c Means in the same column with different letters are significantly different (p<0.05).

EBH, elastin-based hydrolysate; MWCO, kDa molecular weight cut-off; ABTS, 2,2’-azino-bis (3-ethylbenzothiazoline-6-sulphonic acid).

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Gel-filtration chromatography/size exclusion chromatography (SEC)

Gel filtration is a method that separates substances based on differences in molecular dimensions. EBH III fraction (<3 kDa) from ultrafiltration which showed the highest ABTS radical scavenging activity was separated through gel filtration chromatography.Fig. 1A shows the ABTS radical scavenging activity (IC50) and gel filtration chromatography elution profile of the EBH III fraction. It is found that EBH III fraction was separated into four fractions (EBI-EBIV), where EB-II fraction exhibits the strongest ABTS radical trapping activity with the lowest IC50 values (0.42 mg/mL) compared to other fractions (p<0.05).

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Fig. 1. Data obtained to indicate the antioxidant profiles of elastin. (A) ABTS radical scavenging activity (IC50) and gel filtration chromatography elution profiles of EBH III. (B) ABTS radical scavenging activity (IC50) and the elution profile of RP-HPLC of EB-II. ABTS, 2,2’-azino-bis (3-ethylbenzothiazoline-6-sulphonic acid); EBH, elastin-based hydrolysate; RP-HPLC, high performance liquid chromatography.
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Amino acid composition of fractions from ultrafiltration and gel filtration

The amino acid composition of non-hydrolysed elastin (UEB) and fractions obtained from ultrafiltration (EBH III) and gel filtration chromatography (EB-II) are summarized in Table 4. The main amino acids of elastin (EBH III) fraction and potent fraction EB-II are Gly, Glu, Ala, and Pro where the highest was showed by Glycine content. The Glycine in EBH III, EB-II and UEB fractions are 20.64%, 19.63%, and 18.36%, respectively (p<0.05). While it is unknown what are the contents found in this stage, there are assumptions that could be done based on studies that found out that elastin extracts from broiler consist of amino acids of two main types; the basics, and the radical-scavenging. The free radical-scavenging amino acids (Glu, Cys, Met, Tyr, and Lys) are found in elastin (Cao et al., 2009; Chen et al., 1996). From the results, it is found that these amino acids could increase the antioxidant activity of the corresponding peptides; UEB=38.66%, EBH III=37.33% and EB-II=41.98%, suggesting that the hydrophobic amino acids found are indeed the radical-scavenging types.

Table 4. Amino acid composition (%) of non-hydrolysed elastin and its fractions obtained from ultrafiltration and gel filtration
AA UEB (%) EBH III (%) EB-II (%)
Asp 7.35±0.04b 9.86±0.01a 6.99±0.01c
Ser 3.01±0.12a 2.85±0.06b 2.78±0.01b
Gly 18.36±0.44c 20.64±0.01a 19.63±0.01b
Glu 11.28±0.2b 9.86±0.01c 12.31±0.01a
His 1.6±0.03b 1.58±0.01b 1.86±0.01a
Arg 7.89±0.27a 6.67±0.01c 7.07±0.01b
Thr 1.56±0.04a 1.13±0.01b 1.35±0.01b
Ala 8.13±0.08c 8.41±0.01b 8.29±0.01b
Pro 10.84±0.12a 11.38±0.07b 10.52±0.01b
Tyr 1.65±0.03b 1.7±0.01a 4.92±0.01a
Val 2.99±0.07a 2.57±0.01a 2.4±0.01a
Met 1.72±0.11a 1.73±0.01a 0.89±0.01b
Lys 4.08±0.27a 4.3±0.01a 4.15±0.01a
Ile 1.72±0.11c 1.98±0.01a 1.82±0.01b
Leu 4.24±0.04a 3.46±0.01b 3.35±0.05b
Phe 0.24±0.16c 1.51±0.01b 2.7±0.01a
Cys 0.14±0.1a 0.11±0.01a 0.2±0.01a
Trp 0.14±0.1a 0.11±0.01a 0.2±0.01a
Hyp 10.4±0.27a 7.87±0.01b 5.68±0.01c

All values are mean±SD of three replicates (n=3).

Hydrophobic amino acids (Gly, Ala, Val, Leu, Ile, Pro, Phe, Cys, Trp, and Met).

a-c Mean in the same row without a common superscript letter differ significantly (p<0.05).

AA, amino acid; UEB, non-hydrolysed elastin; EBH, elastin-based hydrolysate.

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Observation of antioxidant properties in amino acids is not necessarily new (Chen et al., 1996). Amino acids such as Met, Lys, Tyr, His, and Trp has been found to show antioxidant properties in safflower oil (Riisom et al., 1980). There are reports suggesting aromatic amino acids (Phe, His, Tyr, and Trp) also portray antioxidative property – converting radicals into stable molecules – while simultaneously maintaining own stability due to their resonance structure. In other words, aromatic amino acids have better radical-scavenging properties compared to other amino acids (Rajapakse et al., 2005; Sarmadi and Ismail, 2010).

Reversed-phased high performance liquid chromatography (RP-HPLC)

The fractions of EB-II from gel filtration with the highest radical scavenging activity were separated by RP-HPLC. The sample solution containing differences in molecular polarity form the basis of RP-HPLC, where the antioxidant peptide of EB-II fraction of EBH was separated and thus, identified. Fig. 1B portrays ABTS radical scavenging activity (IC50) and the elution profile of RP-HPLC for the EB-II fraction that generates four fractions (EB-II1-EB-II4). The EB-II4 has the elution time of 11:94 min and has been verified with the highest ABTS radical scavenging activity, with IC50 of 0.12 mg/mL (p<0.05).

The timing in the hydrophobic chromatography column is essential in determining antioxidative peptides based on hydrophobic properties. Longer elution time means the sample is more hydrophobic, which correlates with antioxidative activity. The time taken for peptides in EBH reflects the antioxidative activity in the peptide sequences, which is further supported by the ABTS assay. Another study shows a similar idea of hydrophobic chromatography column where loach peptides with a higher amount of hydrophobic amino acids have longer retention time (Rajapakse et al., 2005).

Identification of antioxidative peptides by MALDI-TOF/TOF-MS

The EB-II4 fraction which has the highest antioxidant activity was analyzed by matrix-assisted laser desorption/ionization time-of-flight/time-of-flight mass spectrometry (MALDI-TOF/TOF-MS) for identification of amino acid sequence. Fig. 2 shows the mass spectra of the antioxidant peptide from elastin hydrolysate (EBH). The amino acid sequence of the antioxidant peptides from EBH, as shown in Table 5 is characterized by the Ludwig NR databases. The following peptides were found from the results of mass spectral analysis: Gly-Ala-His-Thr-Gly-Pro-Arg-Lys-Pro-Lys-Pro-Arg (GAHTGPRKPKPR), of the parent protein trA0A021W2G6AZ78_21380 RNA helicase; peptide Gly- Pro-Gly-Phe-Asp-Val-Arg (GPGFDVR), of the parent protein H1W5B7 in parent protein, trH1W5B7CH063_00691 AMP-binding enzyme and peptide Ala-Asp-Ala-Ser-Val-Leu-Pro-Lye (ADASVLPK), of the parent protein trA0A011VSV8|RASY3_15265 Thiamine-phosphate pyrophosphorylase. It was evident that the amino acids Proline (P) and Valine (V) were present in all antioxidant peptides from the EBH. The molecular weight of antioxidant peptides of EBH is ranging from 799.38-1,447.82 Da (Table 5). The molecular weight of the fractions decreased after purification; ultrafiltration (<3 kDa)>gel filtration (0.5–3 kDa)>RP-HPLC (<1 kDa). These results confirmed that functional antioxidative peptides are highly affected by molecular structure and molecular mass, which is also the case in other studies involving other sources of peptides from marine and casein (Jeon et al., 1999; Suetsuna and Chen, 2002). The results in this study also show that antioxidative peptides of EBH have between 8-13 amino acid residues, which is supported by Pihlanto-Leppala (2000), where bioactive peptides are found to have typically between 2–20 amino acid residues.

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Fig. 2. The mass spectra (MS/MS) of the antioxidant peptides from EB-II4 fraction. (A) The peptide sequence of GAHTGPRKPFKPR with a corresponding observed the mass of 1,448.69 Da. (B) The peptide sequence of GMPGFDVR with a corresponding observed the mass of 878.42 Da. (C) The peptide sequence of ADASVLPK with a corresponding observed mass of 800.39 Da. Proline (P) and valine (V), amino acids were present in all antioxidant peptides from the EBH. EBH, elastin-based hydrolysate.
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Table 5. The amino acid sequence of antioxidant peptides obtained from RP-HPLC (EB-II4) that has the highest antioxidative activity which is characterized by the Ludwig NR database
Parent protein and accession number Sequence Observed mass (Da) Ions score Mr (calc)
trA0A021W2G6AZ78_21380 RNA helicase GAHTGPRKPFKPR 1,448.6958 36 1,447.8160
H1W5B7 in parent protein, trH1W5B7CH063_00691 AMP-binding enzyme GMPGFDVR 878.4228 18 877.4116
trA0A011VSV8|RASY3_15265 Thiamine-phosphate pyrophosphorylase ADASVLPK 800.3921 5 799.4440

RP-HPLC, high performance liquid chromatography.

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Overall, peptides of elastin extracted from EBH that have antioxidant activity contained relatively lower molecular weights and relatively hydrophobic amino acids, namely Glycine (G), Alanine (A), valine (V), and Phenylalanine (F). The results demonstrate the antioxidant capacity of elastin extracted from broiler, where the antioxidant peptides contain amino acid sequences involving aspartic acid, hydrophobic amino acids (valine and alanine), and hydrophilic amino acids (histidine and proline). Furthermore, the antioxidant property of EBH was reflected by the high ABTS radical scavenging activity thus proving the fact that free radical scavenging is the main antioxidant mechanism of EBH antioxidant peptides. The evidence that elastin hydrolysates from broiler (EBH) can be a novel source of antioxidants is also supported by a study on elastin isolated from neck ligaments (Rajapakse et al., 2005). These pepsin-solubilized and acid-solubilized elastin peptides possessed a low molecular weight and also confirmed having high antioxidant activity (Hattori et al., 1998).

Conclusion

In this study, elastin hydrolysate has been successfully extracted from poultry skin by using the enzyme elastase. Antioxidant peptides were separated using ultrafiltration, gel filtration chromatography and high-performance liquid chromatography (RP-HPLC). It was evident that the antioxidative activities were found in all the fractions. Further studies on elastin hydrolysate should include the use of a cell culture system to examine antioxidant properties of target peptides in vivo. The antioxidative protein hydrolysates obtained from the present study could potentially be a stepping stone on future animal and clinical studies.

Notes

Conflict of Interest

The authors declare no potential conflict of interest.

Acknowledgments

This experiment was a part of the research project titled ‘Isolation of Bioactive Elastin Peptides from Livestock By-Products’ funded by the Malaysian Ministry of Science, Technology and Innovation (02-01-02-SF0831) and FRGS/1/2018/ STG05/UKM/02/8, Ministry of Education of Malaysia

Notes

Author Contributions

Conceptualization: Nadalian M, Babji AS, Yusop SM. Data curation: Nadalian M, Kamaruzaman N, Babji AS, Yusop SM. Formal analysis: Nadalian M, Kamaruzaman N, Yusop SM. Methodology: Nadalian M, Kamaruzaman N, Yusop SM. Software: Nadalian M, Kamaruzaman N. Validation: Nadalian M, Kamaruzaman N, Yusop MSM. Investigation: Kamaruzaman N, Yusop MSM, Yusop SM. Writing - original draft: Nadalian M, Kamaruzaman N. Writing - review & editing: Nadalian M, Kamaruzaman N, Yusop MSM, Babji AS, Yusop SM.

Notes

Ethics Approval

This article does not require IRB/IACUC approval because there are no human and animal participants.

References

1.

Agrawal H, Joshi R, Gupta M. 2016; Isolation, purification and characterization of antioxidative peptide of pearl millet (Pennisetum glaucum) protein hydrolysate. Food Chem. 204:365-372

2.

Alaiz M, Navarro JL, Giron J, Vioque E. 1992; Amino acid analysis byhigh-performance liquid chromatography after derivatization with diethyl ethoxymethylenemalonate. J Chromatogr. 591:181-186

3.

Antonicelli F, Bellon G, Debelle L, Hornebeck W. 2007; Elastin-elastases and inflamm-aging. Curr Top Dev Biol. 79:99-155

4.

Babji AS, Etty Syarmila IK, Nur ‘Aliah D, Nurul Nadia M, Hadi Akbar D, Norrakiah AS, Ghassem M, Najafian L, Salma MY. 2018; Assessment on bioactive components of hydrolysed edible bird nest. Int Food Res J. 25:1936-1941.

5.

Brand-Williams W, Cuvelier ME, Berset C. 1995; Use of a free radical method to evaluate antioxidant activity. LWT-Food Sci Technol. 28:25-30

6.

Bringans S, Eriksen S, Kendrick T, Gopalakrishnakone P, Livk A, Lock R, Lipscombe R. 2008; Proteomic analysis of the venom of Heterometrus longimanus (Asian black scorpion). Proteomics. 8:1081-1096

7.

Cao W, Zhang C, Hong P, Ji H, Hao J, Zhang J. 2009; Autolysis of shrimp head by gradual temperature and nutritional quality of the resulting hydrolysate. LWT-Food Sci Technol. 42:244-249

8.

Centenaro GS, Salas-Mellado M, Pires C, Batista I, Nunes ML, Prentice C. 2014; Fractionation of protein hydrolysates of fish and chicken using membrane ultrafiltration: Investigation of antioxidant activity. Appl Biochem Biotechnol. 172:2877-2893

9.

Chen HM, Muramoto K, Yamauchi F, Nokihara K. 1996; Antioxidant activity of designed peptides based on the antioxidative peptide isolated from digests of a soybean protein. J Agric Food Chem. 44:2619-2623

10.

Cudennec B, Violle N, Chataigne G, Drevet P, Bisson JF, Dhulster P, Ravallec R. 2016; Evidence for an antihypertensive effect of a land snail (Helix aspersa) by-product hydrolysate: Identification of involved peptides. J Funct Foods. 22:602-611

11.

Decker EA, Welch B. 1990; Role of ferritin as a lipid oxidation catalyst in muscle food. J Agric Food Chem. 38:674-677

12.

Erdmann K, Cheung BWY, Schroder H. 2008; The possible roles of food-derived bioactive peptides in reducing the risk of cardiovascular disease. J Nutr Biochem. 19:643-654

13.

Fauzi MB, Lokanathan Y, Aminuddin BS, Ruszymah BHI, Chowdhury SR. 2016; Ovine tendon collagen: Extraction, characterisation and fabrication of thin films for tissue engineering applications. Mater Sci Eng C. 68:163-171

14.

Ganceviciene R, Liakou AI, Theodoridis A, Makrantonaki E, Zouboulis CC. 2012; Skin anti-aging strategies. Dermatoendocrinol. 4:308-319

15.

Garg H, Li H, Sivasithamparam K, Barbetti MJ. 2013; Differentially expressed proteins and associated histological and disease progression changes in cotyledon tissue of a resistant and susceptible genotype of brassica napus infected with Sclerotinia sclerotiorum. PLOS ONE. 8:e65205

16.

Hattori M, Yamaji-tsukamoto K, Kumagai H, Feng Y, Takahashi K. 1998; Antioxidative activity of soluble elastin peptides. J Agric Food Chem. 46:2167-2170

17.

Jeon YJ, Byun HG, Kim SK. 1999; Improvement of functional properties of cod frame protein hydrolysates using ultrafiltration membranes. Process Biochem. 35:471-478

18.

Kielty CM, Sherratt MJ, Shuttleworth CA. 2002; Elastic fibres. J Cell Sci. 115:2817-2828.

19.

Lansing AI, Rosenthal TB, Alex M, Dempsey EW. 1952; The structure and chemical characterization of elastic fibers as revealed by elastase and by electron microscopy. Anat Rec. 114:555-575

20.

Lescan M, Perl RM, Golombek S, Pilz M, Hann L, Yasmin M, Behring A, Keller T, Nolte A, Gruhn F, Kochba E, Levin Y, Schlensak C, Wendel HP, Avci-Adali M. 2018; De novo synthesis of elastin by exogenous delivery of synthetic modified mrna into skin and elastin-deficient cells. Mol Ther - Nucleic Acids. 11:475-484

21.

Levillain A, Orhant M, Turquier F, Hoc T. 2016; Contribution of collagen and elastin fibers to the mechanical behavior of an abdominal connective tissue. J Mech Behav Biomed Mater. 61:308-317

22.

Li Y, Jiang B, Zhang T, Mu W, Liu J. 2008; Antioxidant and free radical-scavenging activities of chickpea protein hydrolysate (CPH). Food Chem. 106:444-450

23.

Martinez-Maqueda D, Miralles B, Recio I, Hernandez-Ledesma B. 2012; Antihypertensive peptides from food proteins: A review. Food Funct. 3:350-361

24.

Mecham RP. 2008; Methods in elastic tissue biology: Elastin isolation and purification. Methods. 45:32-41

25.

Mohan A, McClements DJ, Udenigwe CC. 2016; Encapsulation of bioactive whey peptides in soy lecithin-derived nanoliposomes: Influence of peptide molecular weight. Food Chem. 213:143-148

26.

Mohammad AW, Suhimi NM, Aziz AGKA, Jahim JM. 2014; Process for production of hydrolysed collagen from agriculture resources: Potential for further development. J Appl Sci. 14:1319-1323

27.

Munasinghe KA, Schwarz JG, Nyame AK. 2014; Chicken collagen from law market value by-products as an alternate source. J Food Process. 2014:1-6

28.

Nadalian M, Yusop SM, Babji AS, Mustapha WAW, Azman MA. 2015; Effects of enzymatic hydrolysis on the antioxidant activity of water- soluble elastin extracted from broiler and spent hen skin. Int J Appl Bio Pharm Technol. 6:1-9.

29.

Nazeer RA, Deeptha R. 2013; Antioxidant activity and amino acid profiling of protein hydrolysates from the skin of Sphyraena barracuda and Lepturacanthus savala. Int J Food Prop. 16:500-511

30.

Pihlanto-Leppala A. 2000; Bioactive peptides derived from bovine whey proteins: Opioid and ace-inhibitory peptides. Trends Food Sci Technol. 11:347-356

31.

Pownall TL, Udenigwe CC, Aluko RE. 2010; Amino acid composition and antioxidant properties of pea seed (Pisum sativum L.) enzymatic protein hydrolysate fractions. J Agric Food Chem. 58:4712-4718

32.

Rajapakse N, Mendis E, Byun HG, Kim SK. 2005; Purification and in vitro antioxidative effects of giant squid muscle peptides on free radical-mediated oxidative systems. J Nutr Biochem. 16:562-569

33.

Re R, Pellegrini N, Proteggente A, Pannala A, Yang M, Rice-Evans C. 1999; Antioxidant activity applying an improved ABTS radical cation decolorization assay. Free Radic Biol Med. 26:1231-1237

34.

Riisom T, Sims RJ, Fioriti JA. 1980; Effect of amino acids on the autoxidation of safflower oil in emulsions. J Am Oil Chem Soc. 57:354-359

35.

Ryan JT, Ross RP, Bolton D, Fitzgerald GF, Stanton C. 2011; Bioactive peptides from muscle sources: Meat and fish. Nutrients. 3:350-361

36.

Saiga A, Tanabe S, Nishimura T. 2003; Antioxidant activity of peptides obtained from porcine myofibrillar proteins by protease treatment. J Agric Food Chem. 51:3661-3667

37.

Sarmadi BH, Ismail A. 2010; Antioxidative peptides from food proteins: A review. Peptides. 31:1949-1956

38.

Sharma N, Rahman MH, Strelkov S, Thiagarajah M, Bansal VK, Kav NNV. 2007; Proteome-level changes in two Brassica napus lines exhibiting differential responses to the fungal pathogen Alternaria brassicae. Plant Sci. 172:95-110

39.

Suetsuna K, Chen JR. 2002; Isolation and characterization of peptides with antioxidant activity derived from wheat gluten. Food Sci Technol Res. 8:227-230

40.

Yeo GC, Aghaei-Ghareh-Bolagh B, Brackenreg EP, Hiob MA, Lee P, Weiss AS. 2015; Fabricated elastin. Adv Healthc Mater. 4:2530-2556

41.

You L, Zhao M, Regenstein JM, Ren J. 2010; Changes in the antioxidant activity of loach (Misgurnus anguillicaudatus) protein hydrolysates during a simulated gastrointestinal digestion. Food Chem. 120:810-816

42.

Yusop SM, Nadalian M, Babji AS, Mustapha WAW, Forghani B. 2016; Production of antihypertensive elastin peptides from waste poultry skin. Int J Food Eng. 2:21-25

43.

Zhang SB, Wang Z, Xu SY. 2008; Antioxidant and antithrombotic activities of rapeseed peptides. J Am Oil Chem Soc. 85:521-527

Journal Title Change

We announce that the title of our journal and related information were changed as below from January, 2019.

 

Before (~2018.12)

After (2019.01~)

Journal Title

Korean Journal for Food Science of Animal Resources

Food Science of Animal Resources

Journal Abbreviation

Korean J. Food Sci. An.

Food Sci. Anim. Resour.

eISSN

2234-246X

2636-0780

pISSN

1225-8563

2636-0772

Journal Homepage

http://www.kosfaj.org

Same

JCR Citation Indexing

SCIE

SCIE


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