Journal of Aquatic Food Product Technology

ISSN: 1049-8850 (Print) 1547-0636 (Online) Journal homepage: http://www.tandfonline.com/loi/wafp20

Purification and Identification of Antioxidant

Peptides from Fermented Fish Sauce (Budu)

Leila Najafian & Abdul Salam Babji

To cite this article: Leila Najafian & Abdul Salam Babji (2018): Purification and Identification
of Antioxidant Peptides from Fermented Fish Sauce (Budu), Journal of Aquatic Food Product
Technology, DOI: 10.1080/10498850.2018.1559903

To link to this article: https://doi.org/10.1080/10498850.2018.1559903

Published online: 27 Dec 2018.

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JOURNAL OF AQUATIC FOOD PRODUCT TECHNOLOGY
https://doi.org/10.1080/10498850.2018.1559903

Purification and Identification of Antioxidant Peptides from

Fermented Fish Sauce (Budu)
Leila Najafiana and Abdul Salam Babjib
a
Department of Food Science and Technology, Sari Branch, Islamic Azad University, Sari, Iran; bSchool of Chemical
Sciences and Food Technology, Universiti Kebangsaan Malaysia, Bangi, Selangor, Malaysia

ABSTRACT KEYWORDS
This study aimed to isolate antioxidant peptides from Budu extract and Fermented fish; protein
identify novel antioxidant peptides using high-performance liquid chroma- hydrolysate; antioxidant
tography (HPLC) and electrospray ionization-time-of-flight mass spectro- peptide; identification
metry (ESI-TOF MS/MS). An amino acid analyzer was used to analyze the
amino acid composition of peptides. Two novel peptides, Lue-Asp-Asp-Pro-
Val-Phe-Ile-His (LDDPVFIH) and Val-Ala-Ala-Gly-Arg-Thr-Asp-Ala-Gly-Val-His
(VAAGRTDAGVH), were identified. The synthesized peptide of LDDPVFIH
showed higher antioxidant activity. The presence of hydrophobic amino
acids (Ile and Leu), acidic (Asp) and basic (His) amino acids in the peptide
sequences is believed to contribute to the high antioxidant activity of the
fermented anchovy fish (Budu) extract. Thus, the two peptides may have
potential application as functional foods and could also be used as nutra-
ceutical compounds.

Introduction
Reactive oxygen species (ROS), free radicals, and reactive nitrogen species (RNS) are produced as
normal products of cellular metabolism in aerobic organisms. A balance between free radicals
generated in the body and antioxidants is necessary. If free radicals devastate the body’s ability to
adjust them, oxidative stress is ensued. Free radicals thus adversely modify biomolecules (proteins,
DNA, and membrane lipids) and lead to a number of human diseases such as cancer, arteriosclero-
sis, heart diseases, stroke, diabetes, neurological disorders, Parkinson’s disease, and Alzheimer’s
disease (Lobo et al., 2010; Najafian and Babji, 2015). Therefore, external antioxidants can help
cope with this oxidative stress. For decades, artificial antioxidants like butylated hydroxytoluene
(BHT), butylated hydroxyanisole (BHA), propyl gallate (PG), and tert-butylhydroquinone (TBHQ)
have been commercialized (Shahidi, 2000). Although the synthetic antioxidants have a strong
antioxidant ability to inhibit or delay oxidative damage, they are also reported to have toxic effects
on human enzyme systems and DNA (Liu et al., 2016; WinatA and Lorenz, 1996). Hence, many
studies have been done to discover and increase natural antioxidant compounds and safe, antiox-
idant peptides (Sila and Bougatef, 2016).
Bioactive peptides can improve human health and prevent disease when they are used as
functional food ingredients or nutraceuticals and pharmaceuticals. These peptides usually contain
2–20 amino acid units. The sequences and amino acid composition of peptides can affect the activity
of biopeptides (Pihlanto-Leppälä et al., 1998). The importance of fish as a source of novel bioactive
substances is rapidly growing field of research (Aneiros and Garateix, 2004). Bioactive peptides
derived from fish based on their structural properties and their amino acid composition and

CONTACT Leila Najafian najafian_5828@yahoo.com Department of Food Science and Technology, Sari Branch, Islamic
Azad University, Sari, Iran.
Color versions of one or more of the figures in the article can be found online at www.tandfonline.com/wafp.
© 2018 Taylor & Francis Group, LLC
2 L. NAJAFIAN AND A. S. BABJI

sequences may be involved in various biological functions (Khora, 2013), including inhibition of
angiotensin-I-converting enzyme (ACE) (Jung et al., 2006) and antioxidant (Najafian and Babji,
2018; Samaranyaka and Li-Chan, 2008), antimicrobial (Salampessy et al., 2010) and anticoagulant (Jo
et al., 2008) activities. Antioxidant peptides derived from food sources have been considered as
perfect natural antioxidants in the medical treatment and food industries (Singh et al., 2014; Yan
et al., 2015). They can be safe and potentially exhibit a beneficial health effect due to their high
activity, easy absorption, and low toxicity (Dei Piu et al., 2014). Fish protein is thought to be a good
source from which to obtain antioxidant peptides, because fish proteins contain essential amino
acids with high availability (Majumdar and Basu, 2010). The microbial fermentation of food proteins
and lactic acid bacteria (LAB) hydrolyze proteins during fermentation of foods and can generate
bioactive peptides (Havenaar et al., 1992). Therefore, fermentation of food materials enhances the
antioxidant activity of food products. LAB accounts for about 1–24% of the microbial population of
the meat systems, and it is the stable portion of the microbial population of fish products. The LABs
not only contribute diverse flavor, aroma, and texture to fermented products but can also produce
lactic acid, antioxidant, and antimicrobial peptides (Shobharani et al., 2013).
Fish sauce is a famous food derived from fermented fish product. It is obtained through natural
hydrolysis by endogenous enzymes and microorganisms (Lopetcharat et al., 2001). Fish sauce
constitutes an important part of the diet of people in Southeast Asian countries, but it is more
important to the lower income group, providing a substantial part of the protein requirements of
these people (Mciver et al., 1982). Budu is a fish sauce that is a fermented seafood product in the east
coast region of West Malaysia, namely the states of Kelantan and Terengganu (Rosma et al., 2009).
Fish sauce is made by mixing anchovy fish with salt and fermented for 3–12 months (Velasco, 2015).
The fish product is the result of microbial proteases and hydrolysis of fish. The action of proteolytic
microorganisms surviving during fermentation process produces the flavor and aroma of Budu.
Some factories add other ingredients, including coconut sugar and tamarind. With endogenous
enzymes and fermentation of fish, peptides derived from protein hydrolysate may possess different
amino acid sequences (Wu et al., 2017). It would be beneficial to discover more new bioactive
peptides.
In this study, Budu, a fermented product, was prepared from fresh anchovy fish, and the antioxidant
activity was measured. Moreover, amino acid composition was also evaluated to elucidate its relation-
ship with antioxidant activity. Antioxidant peptides were identified after purifying by ultrafiltration,
gel filtration, and reverse phase-high performance liquid chromatography (RP-HPLC).

Materials and methods

Material
Fresh anchovies (Ilisha melastoma) were purchased from a local market in Penang, Malaysia and
sent to Fishery Research Institute, Batu Mount, Penang, Malaysia for verification of the authenticity
of the anchovy (Ilisha melastoma) species. Fish were then transported back to the laboratory in
iceboxes. Amino acid standards were purchased from Pierce (Rockford, IL, USA). 3-(2-pyridyl)-
5,6-bis(4- phenylsulphonic acid)-1,2,4-triazine(ferrozine),2,2ʹ-azino-bis(3-ethylbenzothiazoline-
6-sulphonic acid) diammonium salt (ABTS), 1,1-diphenyl-2-picrylhydrazyl (DPPH), ferric chloride,
and ammonium thiocyanate were purchased from Sigma-Aldrich (St. Louis, MO, USA). Other
chemicals of analytical grade were also purchased from Sigma-Aldrich.

Sample preparation
Fresh anchovies were washed and drained. Then, 240 g fish was mixed with 160 g salt in a 500 ml
ceramic container. The fish were submerged in liquid, and the container was then placed in a 40°C
incubator and withdrawn at 120 days of fermentation according to Fen et al. (2011). The mature
 JOURNAL OF AQUATIC FOOD PRODUCT TECHNOLOGY 3

Budu was harvested. The ground fish residue was mixed with flavor ingredients (tamarind, palm
sugar, and color) and boiled. Then, the product was cooled and filtered. The samples were
centrifuged at 710 g for 5 min (centrifuge 5810 R, Eppendorf; Hamburg, Germany) to obtain the
liquid portion.

Deproteinization of the Budu

The fermented sauce (Budu) was mixed with 0.01 N HCl (ratio of 1:4) for 8 min. The mixture was
centrifuged at 4°C at 12000 g for 20 min and filtered through glass wool. Then, three volumes of
ethanol were added, and the sample was kept for 20 min at 4°C to deproteinize the supernatant. The
sample was centrifuged again at 12000 g for 20 min at 4°C, and the supernatant was dried in a rotary
evaporator. After dissolving the dried deproteinized extract in 25 mL of 0.01 N HCl, the mixture was
filtered through a 0.45 μm membrane filter and kept at −20°C for further analysis (Budu extract).

Amino acid composition

Amino-acid compositions of Budu extract were characterized using the method of Alaiz et al.
(1992), with slight modifications. The total amount of all amino-acid residues was measured
following hydrolysis with 6-N hydrochloric acid at 110 ± 1°C for 24 h. Tryptophan is destroyed
in acid hydrolysis. Alkaline hydrolysate was performed to determine the amount of Rutherfurd
tryptophan using HPLC by the method described by Rutherfurd and GilanI (2009). The total
content of cysteine and methionine can be determined by oxidizing the protein with performic
acid (Cohen et al., 1988).

Determination of the antioxidant activity of Budu extract

DPPH radical scavenging activity
DPPH radical scavenging activity was measured using the method of Wu et al. (2003) with some
modifications. A 1.5 ml volume from each concentration of Budu extract was mixed with 1.5 ml of
0.15 mM DPPH in 95% ethanol. After vigorous mixing, the mixture was then allowed to stand for
30 min at room temperature in the dark. The absorbance was measured at 517 nm. The scavenging
effect was quantified according to the following equation:
DPPH radical scavenging activity% ¼½ðB  AÞ=B100
where A represents the absorbance of the sample, and B represents the absorbance of the control
(using distilled water instead of samples).

Reducing power
The reducing power of the Budu was estimated according to the method of Oyaizu (1988) with slight
modifications. One ml of diluted sample was dissolved in a mixture containing 2 ml of 0.2 M
phosphate buffer (pH 6.6) and 1 ml of a 1% potassium ferricyanide solution. This mixture was
incubated at 50°C for 20 min. A 1 ml aliquot of 10% trochloroacetic acid (TCA) was added to the
reaction mixture, and the reaction mixture was then centrifuged at 3000 rpm for 10 min. A 1 ml
sample from the upper layer of the solution mixture was subsequently mixed with 1 ml of distilled
water and 200 μl of a 0.1% ferric- chloride solution. After allowing this mixture to react for 10 min,
the absorbance was measured at a wavelength of 700 nm.

ABTS radical-scavenging activity assay

The ABTS radical-scavenging activities of the sample were assessed spectrophotometrically by ABTS
radical cation decolorization assay according to the method of Re et al. (1999), with slight
4 L. NAJAFIAN AND A. S. BABJI

modifications. The absorbance values were measured at 734 nm. The scavenging effect was quanti-
fied according to the following equation:
ABTS radical  scavenging activity ð%Þ¼½ðB  AÞ=B100
where A represents the absorbance of the sample, and B represents the absorbance of the control.

Purification of antioxidant peptides from the Budu extract

Hydrolyzed fish proteins exhibit different biological activities and physicochemical properties
depending on their amino acid sequence and molecular weight. Therefore, the molecular weight
of the bioactive peptide is one of the most important factors in producing bioactive peptides with
desired functional properties (Kim and Wijesekara, 2010). An ultrafiltration membrane system
can separate the peptides that have the desired molecular weights and functional properties from
fish protein hydrolysates (Je et al., 2005). The Budu extract was filtered through 10 kDa ultra-
filtration membrane followed by a 3 kDa membrane and separated into three molecular weight
peptide fractions as <10 kDa, 3–10 kDa, <3 kDa UF hydrolysates. Subsequently, for the second
purification, the samples of 250 mg containing peptides below 3 kDa were dissolved in 2 ml of
10 mM sodium phosphate buffer (pH 7.2), loaded onto a Hiprep 26/60 sephacryl S-100HR gel
filtration column, (26 × 600 mm, GE Healthcare, UK), and eluted with 10 mM sodium phosphate
buffer (pH 7.2) at a flow rate of 1 ml/min. The elution was monitored at 280 nm. Four fractions
of BI, BII, BIII, and BIV were obtained by gel filtration chromatography. The peptide fraction
with the highest antioxidant activity was further purified onto an XBridge BEH130 Prep C18
(10 × 250 mm, 5 mm, Waters, Milford, CT, USA) column. The antioxidant peptides were eluted
with linear gradient of 100% eluent 0.1% TFA in deionized water (A) for 5 min and with the
following increasing eluent B: 0–5 min, 0% eluent B; 5–10 min, 0–50% eluent B; 10–30 min,
50–65% eluent B; 30–35 min, 65–100% eluent B; 35–40 min, 100% eluent B; 40–45 min, 100–0%
eluent B at a flow rate of 4.50 mg/min. The elution peaks were monitored at 214 nm.
Fractionation resulted in two fractions, namely BIII 1 and BIII 2. The antioxidant activity was
determined by the ABTS radical scavenging activity assay.

Identification of amino acid sequences

The amino acid sequence of the purified peptides was identified by an Agilent 6520 Accurate-Mass
Q-TOF LC/MS operating in positive ion mode. The antioxidant peptides were dissolved in 0.1%
formic acid in water, and 1 μl of test sample was loaded onto C18, 160 nL enrichment column and
75 mm × 150 mm analytical column (Agilent part no: G4240- 62010). Eluent A was 0.1% formic acid
(FA) in water, and eluent B was 90% acetonitrile (ACN) in water containing 0.1% formic acid. The
spectra were recorded over the mass/charge (m/z) range of 110–3000. The Agilent ESI Q-TOF was
used for data acquisition and processing. Agilent Spectrum Mill MS Proteomics Workbench software
was used to perform a database search via SwissProt.MAR.2013.fasta.

Peptide synthesis
When a sequence has been obtained for a peptide, attention can be turned to its synthesis. The
antioxidant peptides derived from Budu extract have no utility because of lack of information on
potential functions. Thus, the identified peptides must be shown to have antioxidant properties. The
sequencing of peptides was synthesized by First BASE Laboratories Sdn Bhd (Selangar, Malaysia) for
further tests. The synthesized peptides were then purified by RP-HPLC on a Monitor C18, Column
Engineering (150 × 4.6 mm). The UV absorbance of the eluents was monitored at 214 nm. The
synthesized peptides were dissolved in distilled water to obtain different concentrations for the
antioxidant tests.
 JOURNAL OF AQUATIC FOOD PRODUCT TECHNOLOGY 5

Statistical analyses
All analyses were conducted in at least three independent trials. A one-way analysis of variance
(ANOVA) was performed, and the means were compared using Duncan’s multiple range test. The
statistical analyses were performed using SPSS (SPSS 18.0 for Windows, SPSS Inc., Chicago, IL,
USA). The statistical significance of differences (p < 0.05) was evaluated.

Results and discussion

Evaluation of the antioxidant activities of budu extract
The antioxidant activity of the Budu extract was determined using DPPH and ABTS radical
scavenging activities and reducing power assays. DPPH is a stable free radical that shows maximum
absorbance at 517 nm. When an antioxidant donates a proton or electron to the DPPH radical, the
radicals are scavenged, and the absorbance is reduced. As shown in Table 1, the IC50 value of the
Budu extract was 1.06 ± 0.03 mg/ml. DPPH radical scavenging activity of unfermented anchovy fish
mixture (UAFM) extract was too low (6.01%), and IC50 values were not calculated. Therefore, the
results suggest that Budu extract might contain peptides that scavenge the free radicals to terminate
the radical chain reaction to produce stable products. However, antioxidative peptides were inactive
within the sequences of their parent protein but released by fermentation and hydrolysis of protein.
The observed IC50 values for the ABTS radical-scavenging activity were 2.14 ± 0.43 mg/ml. The
results illustrated that the Budu extract donated hydrogen, reacted with the free radicals to produce stable
products, and terminated the radical chain reaction. ABTS radical scavenging activity of UAFM extract
was too low (5.68%), and IC50 values were not calculated. The reducing power shows the reducing
capacity of peptides. Table 1 shows that reducing power of Budu extract at a concentration of 1 mg/ml is
0.251 ± 0.007. The results illustrate that peptides and free amino acids are created by protein proteolysis
and may show antioxidant activity. The mechanisms of the antioxidant activity of Budu extract could be
attributed to the radical scavenging activity and reducing power.

Purification of antioxidative peptides from Budu extract

Most studies on the isolation and purification of antioxidant peptides from hydrolysates were carried
out using ultrafiltration or gel filtration chromatography to determine the molecular weight dis-
tribution (Wu et al., 2003) and high-performance liquid chromatography to determine the nature of
the hydrophobic peptides. An ultrafiltration membrane system can separate the peptides that have
the desired molecular weights and functional properties from fish protein hydrolysates (Je et al.,
2005; Jeon et al., 1999). Such a system can also control the molecular weight distribution of the
appropriate peptide (Kim et al., 1993). All the hydrolysates were filtered through 10 kDa ultrafiltra-
tion membrane followed by a 3 kDa membrane and separated into three molecular weight peptide
fractions as <10 kDa, 3–10 kDa, <3 kDa UF hydrolysates. Table 1 shows DPPH and ABTS radical
scavenging activities and reducing power of fermented Ilisha Melastoma (Budu) extract with

Table 1. DPPH and ABTS radical scavenging activities and reducing power of Budu extract and its ultrafiltrated peptide fractions.
IC50 (mg/ml)
Fraction DPPH radical scavenging activity ABTS radical scavenging activity Reducing power a
Budu extract 1.062 ± 0.028c 2.146 ± 0.430d 0.251 ± 0.007c
BF-I 1.011 ± 0.645 1.250 ± 0.152c 0.288 ± 0.004b
BF-II 0.983 ± 0.095b 0.841 ± 0.080b 0.298 ± 0.003b
BF-III 0.756 ± 0.185a 0.544 ± 0.310a 0.469 ± 0.006a
a
reducing power was measured at a concentration of 1 mg/ml.
- Values were reported as the mean ± SD, experiments were performed in triplicate.
- Different letters within the same parameter indicate significant differences (p < 0.05).
6 L. NAJAFIAN AND A. S. BABJI

different molecular weight fractions. The results showed that all the hydrolyzed fractions (BF-I, II,
and III) exhibited better antioxidant activities than the Budu extract. As seen in Table 1, IC50 value of
the DPPH radical inhibition of the Budu extract for BF-I, BF-II, and BF-III was 1.06 ± 0.03,
1.01 ± 0.65, 0.983 ± 0.09, and 0.756 ± 0.18 mg/ml. BF-III had the highest ABTS radical scavenging
activity (with IC50 value of 0.544 ± 0.31% mg/ml) compared to BF-II, BF-I, and Budu extract with
IC50 value of 0.841 ± 0.08, 1.25 ± 0.15, and 2.14 ± 0.43 mg/ml, respectively.
The ABTS radical-scavenging activities of samples were substantially more than the values from
DPPH radical inhibition. During centrifugation and ultrafiltration, the fractions obtained from Budu
extract consisted of water-soluble low molecular weight peptides, which can react with water-soluble
radicals (ABTS) but not with the lipid-soluble radicals (DPPH) (You et al., 2010). The result of the
reducing power assay is shown in Table 2. The hydrolysate fractions had significantly higher reducing
power values than the Budu extract (0.469 ± 0.006) (p < 0.05) at concentrations of 1 mg/ml. These results
suggested that antioxidant activity of peptides is closely related to their molecular weight.
The permeate obtained was further purified by gel filtration chromatography. The four resulting
fractions (BI, BII, BIII, and BIV) were pooled, lyophilized, and their ABTS radical scavenging activity was
assayed (Figure 1a). The BIII fraction that exhibited the strongest ABTS radical trapping activity had the
lowest IC50 values (1.09 mg/ml) compared to other fractions (p < 0.05). Currently, RP-HPLC is a key
technique in the purification of peptides. So, the BIII with the highest antioxidant activity was purified on
a HPLC column, and a total of two fractions were obtained (BIII 1 and BIII 2) and tested for ABTS radical
scavenging activity (Figure 1b). The fraction BIII 2, which showed the highest ABTS radical scavenging
activity with IC50 values (0.814 mg/ml), was then subjected to peptide characterization.

Amino acid composition of anchovy (ilisha melastoma) fish (AF), Budu extract, and BIII 2
fraction
Amino acids are the major components of peptides, and their content, type, and sequence have
a great impact on the antioxidant activity of peptides (Zhu et al., 2015). The amino acid

Table 2. Amino acid (AA) compositions of the AF, Budu extract and BIII 2 fraction (%).
AA AFa Budu ectract BIII 2
Asp 10.08 ± 0.24a 9.54 ± 0.11a 11.06 ± 0.20a
Ser 3.90 ± 0.15a 2.30 ± 0.15b 2.01 ± 0.17c
Glu 18.98 ± 0.11a 18.68 ± 0.14b 18.04 ± 0.10c
Gly 6.15 ± 0.18b 6.21 ± 0.14b 6.44 ± 0.13a
His* 3.67 ± 0.17c 4.02 ± 0.18b 4.39 ± 0.17a
Arg 1.04 ± 0.28a 0.85 ± 0.22b 0.87 ± 0.15c
Thr* 6.04 ± 0.09b 6.47 ± 0.17a 6.60 ± 0.42a
Ala 6.51 ± 0.12c 6.91 ± 0.15b 7.35 ± 0.32a
Pro 4.03 ± 0.18a 3.49 ± 0.10b 3.47 ± 0.14b
Tyr 1.62 ± 0.23a 1.04 ± 0.18b 0.72 ± 0.13c
Val* 6.02 ± 0.07c 6.24 ± 0.13b 6.49 ± 0.18a
Met* 3.08 ± 0.18c 3.42 ± 0.15b 3.51 ± 0.19a
Lys* 10.42 ± 0.11b 10.52 ± 0.21a 10.31 ± 0.09b
Ile* 5.05 ± 0.16b 5.31 ± 0.30a 5.42 ± 0.19a
Leu* 8.02 ± 0.19c 8.62 ± 0.16b 8.74 ± 0.11a
Phe* 3.80 ± 0.10b 3.85 ± 0.19b 4.09 ± 0.12a
Cys 0.66 ± 0.09a 0 .55 ± 0.08a 0.40 ± 0.11a
Trp* 0.91 ± 0.16b 1.04 ± 0.13ab 1.09 ± 0.15a
TEAAb 47.01c 49.49b 50.64a
HAAc 38.13c 38.88b 39.79a
Values are reported as the mean ± SD; experiments were performed in triplicate.
Different letters within the same parameter indicate significant differences (p < 0.05).
a
AF: Anchovy fish.
b
Total essential amino acids.
c
Hydrophobic amino acids (Ala, Val, Met, Ile, Leu, Phe, Pro and Tyr).
* Essential amino acid.
 JOURNAL OF AQUATIC FOOD PRODUCT TECHNOLOGY 7

(a) (b)

Figure 1. Separation scheme for the radical-scavenging peptide obtained from Budu extract. a: ABTS radical scavenging activity
(IC50) and gel filtration chromatography elution profiles; b: ABTS radical scavenging activity (IC50) and RP-HPLC for the BIII fraction
obtained from Budu extract.
-Values are reported as the mean ± SD, experiments were performed in triplicate.

composition of anchovy (Ilisha Melastoma) fish, Budu extract, and BIII 2 fraction obtained from
RP-HPLC is summarized in Table 2. The major amino acids of the AF, Budu extract, and BIII 2
fraction are Asp, Glu, Lys, and Leu. The BIII 2 fraction had a significant increase in essential
amino acids (50.64%) compared to AF (47.01%), whereas there was no significant difference
between the BIII 2 and Budu extract (49.49) (p < 0.05). It was found that the content of
hydrophobic amino acids (Ala, Val, Met, Ile, Leu, Phe, Pro, and Tyr) in the BIII 2 fraction
increased to 39.74% compared with smaller increases in Budu extract (38.88%) and AF (38.13%)
(p < 0.05). The presence of hydrophobic amino acids, such as His, Pro, Met, Leu, Cys, Tyr, Try,
Phe, and Val in the peptide sequences seems to improve the antioxidant activities by the
interaction with radical species and increasing peptide solubility in lipids (Kou et al., 2013;
Mendis et al., 2005). Najafian and Babji (2015) reported that hydrophobic amino acids such as
Val and Lue have antioxidant properties.
8 L. NAJAFIAN AND A. S. BABJI

Identification of antioxidant peptides by LC-MS-TOF

The BIII 2 fraction from the Budu extract with the highest antioxidant activity was collected from RP-
HPLC and subsequently subjected to LC-ESI-TOF analysis to evaluate the amino acid sequence. Figure 2
shows the mass spectra (MS/MS) of the antioxidant peptide from BIII 2 fraction. Two amino acid
sequences, Lue-Asp-Asp-Pro-Val-Phe-Ile-His (LDDPVFIH) and Val-Ala-Ala-Gly-Arg-Thr-Asp-Ala-
Gly-Val-His (VAAGRTDAGVH), were identified, which have not been reported until now. These
peptides contain 8–11 amino acid residues. The characterization of amino acid composition of the
peptide also proved important for the antioxidant properties. For antioxidant peptides from BIII 2,
histidine residue, which was located at the C-terminus, was a component of both LDDPVFIH and
VAAGRTDAGVH. Histidine is reportedly important to the antioxidative activities of peptides for its
imidazole characteristics that indicate proton-donation capability. Li et al. (2007) and Chen et al. (1996)
investigated antioxidative activities of 28 synthetic peptides designed based on an antioxidative peptide
(LLPHH) derived from a proteolytic digest of soybean protein. Results indicated that removal of the
C-terminal His residue decreased antioxidative activity, whereas removal of the N-terminal Leu had no
effect. Murase et al. (1993) and Je et al. (2005) reported that His or His-containing peptides have
chelating and lipid radical trapping ability due to the imidazole ring. Both peptides of BIII 2 have His
in C-terminal residue; according to research mentioned above, the antioxidative activity of these peptides
could be related to His. In addition, the antioxidative peptides from BIII 2 showed a high content of
hydrophobic amino acid, which included leucine (L), isoleucine (Ile), valine (V), proline (P) and
phenylalanine (F) in LDDPVFIH peptide and glycine (G), alanine (A), and valine in
VAAGRTDAGVH. These amino acid sequences were believed to contribute to the high antioxidant
activities of peptides from fermented anchovy fish (Budu) extract. Hydrophobic amino acids are
important for enhancement of the antioxidant properties of peptides, since they can increase the

(a)

(b)

Figure 2. The Mass Spectra (MS/MS) of the antioxidant peptide from Budu. a: LDDPVFIH; b: VAAGRTDAGVH.
 JOURNAL OF AQUATIC FOOD PRODUCT TECHNOLOGY 9

Table 3. Antioxidant activities of the synthesized peptides from Budu extract.

IC50 (mg/ml)
Synthesized peptides DPPH radical scavenging activity ABTS radical scavenging activity Reducing powera
VAAGRTDAGVH 1.451 ± 0.873a 0.795 ± 0.392a 0.422 ± 0.009a
LDDPVFIH 0.844 ± 0.203b 0.617 ± 0.481b 0.702 ± 0.006b
Values are reported as the mean ± SD, experiments were performed in triplicate.
In each column, different letters mean significant differences (p < 0.05).
a
reducing power was measured at a concentration of 1 mg/ml.

accessibility of the antioxidant peptides to hydrophobic polyunsaturated chain of fatty acids within
biological membranes to limit oxidative damage. This is important in biological systems because the
unsaturated fatty acids in cell membranes are very prone to oxidative damage by free radicals and oxygen
species (Aluko, 2012). Sarmadi and Ismail (2010) reported that aromatic amino acids (Phe, Tyr, and His)
could convert radicals to stable molecules by donating electrons. The high antioxidant activity of peptides
from BIII 2 is believed to these amino acid sequences. Moreover, aspartic acid (Asp) amino acid was
exhibited in both peptides. Research on antioxidant activity of fish protein hydrolysate demonstrated that
aspartic acid has electron donating ability and has also been exhibited to provide strong contributions to
the DPPH scavenging, ferric reducing, and H2O2 scavenging effects of certain food protein hydrolysates
(Aluko, 2012; Najafian and Babji, 2018)
Two identified peptides were synthesized, and their antioxidant activity was evaluated (Table 3). Both
synthesized peptides showed antioxidant activity; however, LDDPVFIH had higher DPPH and ABTS
radical scavenging activity, with IC50 values of 0.844 and 0.617 mg/ml, respectively, and reducing power
with an absorbance of 0.702 (Table 4) The presence of the N-terminal Leu and Asp residues and the
C-terminal His and Ile residues may be the reason for stronger antioxidant activity of LDDPVFIH peptide.

Conclusion
Budu was produced by hydrolysis of proteins with fermentation time of 120 days. It was deprotei-
nized to obtain antioxidative peptides from anchovy (Ilisha Melastoma) fish. Budu extract exhibited
scavenging activities for DPPH and ABTS radicals as well as reducing power and was purified using
ultrafiltration, gel filtration chromatography, and reverse-phase high- performance liquid chroma-
tography. The AF, Budu extract, and the BIII 2 fraction obtained from RP-HPLC with the highest
antioxidant activity were tested for amino acid composition. The results showed that the BIII 2
fraction had higher amounts of hydrophobic than Budu extract and AF. The BIII 2 was subjected to
LC-ESI-TOF analysis to evaluate the amino acid sequence. Two amino acid sequences, LDDPVFIH
and VAAGRTDAGVH, were identified. The presence of the hydrophobic amino acids valine,
leucine, isoleucine, phenylalanine, and proline and acidic amino acids (aspartic acid) and glycine
in the peptide sequences are believed to contribute to the high antioxidant activity in the Budu
extract. Budu may serve as source of novel peptides for natural antioxidants in a food product.

Funding
The authors acknowledge the financial support provided by Innovation Center for Confectionary Technology
(MANIS), National University Malaysia (UKM), INOVASTI-2014-006, and Sari Branch, Islamic Azad University,
Sari, Iran.

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