Food Bioscience 27 (2019) 37–45

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Food Bioscience
journal homepage: www.elsevier.com/locate/fbio

Antioxidant activity of Sind sardine hydrolysates with pistachio green hull T

(PGH) extracts
⁎ ⁎
Roghayeh Amini Sarteshnizia, Mohammad Ali Saharia, , Hassan Ahmadi Gavlighia, ,
Joe M. Regensteinb, Mehdi Nikooc
a
Department of Food Science and Technology, Faculty of Agriculture, Tarbiat Modares University, Tehran, Iran
b
Department of Food Science, Cornell University, Ithaca, NY 14853-7201, USA
c
Department of Pathobiology and Quality Control, Artemia and Aquaculture Research Institute, Urmia University, West Azerbaijan, Urmia, Iran

A R T I C LE I N FO A B S T R A C T

Keywords: High rates of oxidation during hydrolysis are one the main problems with hydrolysate production from fatty
Sind sardine fishes such as Sind sardines. The purpose of this study was to control oxidation by different pretreatments with
Pistachio green hulls pistachio green hull (PGH) extracts as an antioxidant. Different enzyme levels (1.3%, 2.5%, and 5%), pre-
Fish protein isolate treatments of fish mince (washing, defatting, fish protein isolate (FPI)), and the addition of PGH as well as
Sardinella sindensis
nitrogen gas treatment were studied on lipid oxidation during hydrolysis of Sind sardines (Sardinella sindensis).
Pistachia vera
The antioxidant properties of the hydrolysates were also studied. Results for thiobarbituric acid reactive sub-
stances indicated that the nitrogen gas and 5% (w/w) enzymatic treatment significantly decreased lipid oxi-
dation along with having a higher degree of hydrolysis and higher antioxidant activity for the hydrolysate. FPI
was more effective than washing in controlling oxidation while defatting using isopropanol was the most ef-
fective. PGH (260 µg/ml) effectively controlled the oxidation of mince and washed mince (P < 0.05) but it was
not effective for FPI as seen by the higher oxidation of heme-pigments and weaker metal chelating activity of
PGH. Interaction of hydrolysates from all treatments and PGH showed significant combined DPPH radical
scavenging and metal chelating activity (P < 0.05). Therefore, defatting using isopropanol and addition of an
antioxidant such as PGH can effectively be used for inhibition of lipid oxidation during hydrolysis and im-
provement of antioxidant activity of hydrolysates.

1. Introduction heme proteins in fish muscle, and undesirable lipid substrates

A considerable amount of by-catch and low commercial value fish
species such as sardine (Sardina pilchardus), horse mackerel (Trachurus
mediterraneus), and axillary seabream (Pagellus acarne) are caught an-
nually (García-Moreno et al., 2014). By-catch, and underutilized fish
and shellfish are generally used for the production of fishmeal (Bozzano
& Sarda, 2002; García‐Moreno et al., 2013, 2014). However, there is an
increasing need for better use of underutilized marine resources (Halim,
Yusof, & Sarbon, 2016; Harnedy & FitzGerald, 2012; Nikoo & Benjakul,
2015). These marine species contain high quality proteins for the pro-
duction of bioactive hydrolysates as potential functional food in-
gredients (Ghaly, Ramakrishnan, Brooks, Budge, & Dave, 2013; Nikoo,
Benjakul, & Rahmanifarah, 2016; Ordóñez-Del Pazo et al., 2014).
One of the main challenges during the production of fish protein
hydrolysates (FPH) is the high oxidation rate due to the presence of pro-
oxidants such as phospholipids in cell membranes, myoglobin and other
(Khantaphant, Benjakul, & Ghomi, 2011). These constituents are in-
volved in the undesirable organoleptic properties and oxidative in-
stability of hydrolysates (Benjakul, Yarnpakdee, Senphan,
Halldorsdottir, & Kristinsson, 2014). Primary products of lipid oxida-
tion break down to secondary products with a bad odor and rancid
taste. Also, oxidized unsaturated lipids can produce insoluble lipid-
protein complexes and cause quality deterioration and loss of nutri-
tional value in the resulting hydrolysates (Chaijan, 2008; Ladikos &
Lougovois, 1990). These processes may be more significant when FPH is
produced from fatty fishes such as Sardinella species as they contain
more mitochondria, myoglobin, fats, glycogen, cytochromes, and dark
muscle fibers than white fleshed-fish species (Chaijan, Benjakul,
Visessanguan, & Faustman, 2005).
Previously, FPH were produced using different fish substrate such as
fish mince (Nazeer, Kumar, & Ganesh, 2012; Zhang, Zhang, Wang,
Chen, & Luo, 2017), defatted fish mince (Chi et al., 2015; Galla,

⁎
Corresponding authors.
E-mail addresses: sahari@modares.ac.ir (M.A. Sahari), ahmadi_ha@modares.ac.ir (H.A. Gavlighi).

https://doi.org/10.1016/j.fbio.2018.11.007
Received 16 July 2018; Received in revised form 26 September 2018; Accepted 12 November 2018
Available online 15 November 2018
2212-4292/ © 2018 Elsevier Ltd. All rights reserved.
R.A. Sarteshnizi et al.
Pamidighantam, Akula, & Karakala, 2012; Klompong, Benjakul,
Kantachote, & Shahidi, 2007; Liu et al., 2014; Luo et al., 2013; Quaglia
& Orban, 1990), and fish protein isolate (FPI) (Choi, Hur, Choi, Konno,
& Park, 2009; Thuy & Lam, 2015; Zhong, Ma, Lin, & Luo, 2011).
Khantaphant et al. (2011) evaluated different pretreatment effects on
lipid oxidation and concluded that membrane separation followed by
washing resulted in the lowest myoglobin, phospholipids, and iron
content. The effects of Fucus vesiculosus (Fv) extracts on lipid oxidation
during cod bone mince hydrolysis showed the protecting effect of
natural antioxidants and gave better sensory properties to the FPH
(Halldorsdottir, Sveinsdottir, & Gudmundsdottir, 2014b).
Pistachio green hulls (PGH), the major by-product of the pistachio
industry, is a good source of phenolic compounds (Barreca et al., 2016).
The antioxidant (Goli, Barzegar, & Sahari, 2005; Rajaei, Barzegar,
Mobarez, Sahari, & Esfahani, 2010; Barreca et al., 2016), antidiabetic
(Lalegani, Gavlighi, Azizi, & Sarteshnizi, 2018), antimutagenic and
antimicrobial (Rajaei et al., 2010) properties of PGH extracts have been
reported. Because of its strong antioxidant activity, it might be an in-
teresting extract to control oxidative deterioration during production of
protein hydrolysates from fatty fishes. There is a need to learn more
about producing FPH from fatty fish and the appropriate use of anti-
oxidants during the process.
Sind sardine (Sardinella sindensis) is a small pelagic fish species
found in the southern coastal waters of Iran. It is mainly used for the
production of fishmeal. Production of bioactive peptides could be a
better way to use this fish. Previously, production of antioxidant and
antihypertensive hydrolysates from canned sardine by-products
(Sardina pilchardus) were evaluated using brewer's spent yeast and
commercial proteases (Vieira & Pinho, 2017; Vieira, Ferreira et al.,
2017). It would be interesting if lipid oxidation during hydrolysis of
fatty fish such as Sind sardine could be controlled using a local source of
antioxidants, e.g., PGH. Therefore, the purpose of this study was to
evaluate the effects of different pretreatments of protein substrates and
to test PGH extracts as natural antioxidant during hydrolysis and with
final products. In addition, the effect of different levels of hydrolysis
enzymes on lipid oxidation during production of FPH was measured.
2. Materials and methods
2.1. Materials
Sardinella (Sardinella sindensis) with a length of 7–12 cm and weight
of 15–30 g was kindly provided by local fishermen (Qeshm Island,
Iran). The fish were kept in ice during transportation and stored in
sealed plastic bags at − 80 °C until used, a maximum of 6 wk. Pistachio
green hulls (Ohadi variety) were obtained from the Rafsanjan
Agricultural Research Center of Iran. Protease from Bacillus licheni-
formis, Alcalase 2.4L (E.C. 3.4.21.62), was obtained from Novozyme®
(Bagsvaerd, Denmark). 2,2-Diphenyl-1-picrylhydrazyl (DPPH), 3-(2-
pyridyl)-5,6-diphenyl-1,2,4-triazine-4′,4"-disulfonic acid (ferrozine),
Trolox (6-hydroxy-2,5,7,8-tetramethylchromane-2-carboxylic acid),
and 1,10-phenantroline were obtained from Sigma-Aldrich
(Vallensbaek Strand, Denmark).
2.2. Preparation of pistachio green hull extract
Pistachio green hulls extracts were prepared using the method of
Lalegani et al. (2018). Briefly, hulls were oven dried at 45 °C for 12 h.
Dried hulls were then ground in a coffee grinder (Tefal Simply Invents,
Hong Kong, China), sieved (mesh size 40) and then 10 g of the PGH
powder was extracted overnight using deionized water (Millipore,
Molsheim, France) at room temperature (~ 25–30 °C). The mixture was
then filtered through Whatman No 42 filter paper (Whatman, Maid-
stone, UK). The concentrated extracts were prepared using a rotary
evaporator at 40 °C and then freeze-dried (Labconco Corp., Kansas City,
MO, USA).
38
Food Bioscience 27 (2019) 37–45
2.3. Preparation of mince, washed mince, defatted mince, and fish protein
isolate
Whole fish mince was prepared by washing, removing heads, vis-
cera, and tails, and minced using a Moulinex blender (DR5; Moulinex,
Paris, France). The mince was stored at − 80 °C until used, a maximum
of two wk. Washed mince was prepared using the procedure of
Yarnpakdee, Benjakul, Kristinsson, and Maqsood (2012). Fish mince
was homogenized with 5 volumes of cold distilled water (2–4 °C) (w/v)
using an Ultra-Turrax homogenizer (IKA®, T18 D, Staufen, Germany) at
11,000 rpm for 2 min. After stirring for 15 min at 4 °C, the homogenate
was centrifuged at 9600 ×g using 50 ml test tubes (Sigma D37520,
Rotor 19776-H, Osterode am Harz, Germany) for 10 min at 4 °C. This
process was repeated twice. The washed mince was packed and stored
at − 80 °C for a maximum of a wk. Defatted mince was prepared using
the method of Klompong et al. (2007). Fish mince was mixed with
isopropanol (1:4 (w/v), then homogenized at 11,000 rpm for 2 min and
allowed to stand at 30–35 °C for 50 min. After draining the liquid, the
residue was mixed with isopropanol again (1:4 (w/v)) and defatted at
75 °C for 90 min. The liquid was removed and the precipitate was air
dried at 30–35 °C. The alkaline process described by Hultin and
Kelleher (2000) was used for FPI production. Fish mince was homo-
genized in 9 volume of cold deionized water at 4 °C for 1 min. Then, the
pH of the homogenate was slowly increased to 11 using NaOH (2 N) for
25 min at 0–4 °C. Then, the homogenate was centrifuged at 10,000 × g
for 10 min at 4 °C and filtered through 4 layers of cheesecloth to remove
the insoluble material. The pH of the homogenate was adjusted to 5.5
for myofibrillar proteins precipitation by adding HCl (2 N) and then
centrifuged at 10,000 × g for 20 min at 4 °C.
2.4. Determination of total pigment, heme iron, and phospholipid content
Total pigment and heme iron content were determined using the
methods of Merck Index (1989) with slight modification. Freeze-dried
samples (200 mg) were mixed with 9 ml acid acetone containing 90%
acetone, 8% deionized water and 2% HCl (v/v/w). The mixtures were
kept at room temperature for 1 h and then were filtered using Whatman
No. 42. The absorbance was measured at 680 nm (Cary 60 UV–Vis
spectrophotometer, Aligent Technologies, Santa Clara, CA, USA). The
absorbance of samples were multiplied by a factor of 6800 and then
divided by the initial sample weight to obtain total pigment content as
µg/g dry sample (Hornsey, 1956). Heme iron content was calculated as
follow:
Heme iron (mg/100 g dry sample) = Total pigment (ppm) × 0.00882
The iron content in haematin is 0.0882 μg Fe/μg haematin (Clark,
Mahoney, & Carpenter, 1997).
Phospholipid content was determined using the method of
Yarnpakdee et al. (2012) with slight modification. Freeze-dried samples
(80 mg) were mixed with 20 ml NaOH (4 N) and then heated in water
bath (90–95 °C) for 30 min. After cooling at room temperature for 1 h
the samples were neutralized using HCl (4 N) and centrifuged for
10 min at 10,000 × g. Then 2 ml of supernatants were mixed with 2 ml
phosphate reagent (4.2% ammonium molybdate solution in 5 M HCl,
0.2% malachite green solution in distilled water, 1:3 v/v). The absor-
bance was measured at 620 nm after incubation at room temperature
for 30 min. A phosphate standard curve (disodium hydrogen phos-
phate) was used to obtain phosphorus content and a factor of 25 was
used to convert to phospholipid content (average molecular weight of
phosphatidyl choline/atomic weight of phosphorus) (Sigfusson &
Hultin, 2002). Phospholipid content was expressed as mg/100 g dry
sample.
2.5. Preparation of hydrolysates
Different protein powders were mixed with water to obtain a final
R.A. Sarteshnizi et al. Food Bioscience 27 (2019) 37–45

Table 1 Then, 400 µl of TCA solution was added to the homogenized samples
Hydrolysis conditions of Sind sardine with Alcalase. and centrifuged at 9400 ×g for 15 min at 4 °C using 1.5 ml micro-tubes.
Treatment Enzyme (%) (w/w Hydrolysis condition- After that, 500 µl of supernatant was mixed with an equal volume of
protein) antioxidant thiobarbituric acid (0.02 M) and heated at 95 °C for 40 min. The sam-
ples were cooled immediately and the absorbance was read against a
Mince-No Alcalase 0% N2
water blank at 532 nm. Malondialdehyde bis (dimethyl acetal) from 0
Mince-2.5% Alcalase-O2 2.5% O2
Mince-2.5% Alcalase 2.5% N2
to 4.54 µM was used for the standard curve. The TBARS values were
Mince-2.5% Alcalase-PGH 2.5% N2-PGHa expressed as µmol of MDA equivalents/kg of dry sample.
Mince-1.3% Alcalase 1.3% N2
Mince-5% Alcalase 5% N2 2.8. Determination of antioxidant activity
Washed mince 2.5% N2
Washed mince-PGH 2.5% N2-PGHa
Defatted mince 2.5% N2 2.8.1. DPPH• scavenging activity
Defatted mince-PGH 2.5% N2-PGHa DPPH• scavenging activity was estimated using the method of
FPI 2.5% N2 Shimada, Fujikawa, Yahara, and Nakamura (1992). Hydrolysates were
FPI-PGH 2.5% N2-PGHa
dissolved in distilled water and then 500 µl of the samples were mixed
PGHa: pistachio green hull (260 µg/ml); FPI: fish protein isolate.
with equal volume of 0.1 mM DPPH solution in methanol. The mixtures
were Vortexed and allowed to stand for 30 min at room temperature in
protein concentration (N × 6.25 as measured using the Kjeldahl test the dark and then the absorbance was read at 517 nm against a distilled
(AOAC, 2000)) of 3% (w/v). The pH and temperature of hydrolysis water blank. The DPPH radical scavenging activity was expressed as
were held at 8 and 50 °C, respectively (optimum condition for Alcalase) umoles of Trolox equivalents (μM TE)/g of dry sample.
(Guerard, Dufosse, De La Broise, & Binet, 2001). Different conditions of
hydrolysis were used and are shown in Table 1. After 3 h of hydrolysis, 2.8.2. Ferrous chelating activity
the enzyme was deactivated at 90–95 °C for 10 min. Finally, the mix- The ferrous chelating activity of hydrolysates was estimated using
tures were centrifuged at 1000 × g at 4 °C for 10 min and the super- the method of Decker and Welch (1990). Briefly, 1 ml of hydrolysate
natants were freeze-dried. solution, 3.7 ml distilled water and 100 µl of 2 mM FeCl2 were mixed
and allowed to stand for 3 min. Then 200 µl of 5 mM ferrozine was
added and left to stand for 20 min in the dark at room temperature. The
2.6. Determination of degree of hydrolysis (DH)
absorbance was measured at 562 nm. For the control, double distilled
water was used instead. EDTA disodium salt dihydrate (EDTA-Na2-
The degree of hydrolysis (DH), was measured using the pH-stat-
2H2O) was used for the standard curve. Ferrous ion chelating activity of
method of Adler-Nissen (1986). Hydrolysis reactions were done using a
the hydrolysates were expressed as umoles of EDTA equivalents/g of
stirring system coupled to a thermostatic water bath. The pH of 8 was
dry sample.
maintained using NaOH (4 N) and DH calculated using the following
equation:
2.8.3. Hydroxyl radical scavenging activity
DH (%) = B × Nb × 1/α × 1/MP × 1/htot × 100 Hydroxyl radical scavenging activity (HRSA) was measured using
the method of Wang et al. (2014). 1,10-Phenanthroline solution (1 ml of
Where B refers to the amount of base (ml) used to maintain the constant
1.865 mM) and 2 ml of hydrolysates samples (0.5 mg/ml) were mixed.
pH during the hydrolysis. Nb is the normality of base (eq/l), α is the
Then, 1 ml of 1.865 mM FeSO4 solution was added. The reaction was
average degree of dissociation of the α-NH2 groups expressed as:
started by addition of 1.0 ml H2O2 (0.03%, v/v). The samples were
10 pH − pK incubated at 37 °C for 60 min and the absorbance was read against the
α=
1+10 pH − pK reagent blank at 536 nm. The hydroxyl radical scavenging activity was
calculated as:
where pK is dependent on pH and temperature. At 50 °C and pH 8, pK
= 7.1 was used for calculation of the degree of amino group dissocia- HRSA (%) = [(A s −An)/(Ab −An)] × 100
tion (1/α = 1.13). The pK is the average pK value of α-amino groups where As, An, Ab refer to the absorbance of sample, negative control
liberated during hydrolysis and is calculated to adjust for temperature (the reaction mixture without any antioxidant) and blank (the reaction
according to Steinhardt and Beychok (1964): mixture without H2O2).
pKa = 7.8 + ((298 − T)/298 × T) × 2400
2.9. Analysis of peptide-phenolic complex using fluorescence spectroscopy
MP is the mass of protein of the substrate (g) and htot is the total
number of peptide bonds in the protein substrate (meq/g) (estimated Complex formation between hydrolysates of mince and poly-
from the amino acid composition of the protein by summing up the phenolic compounds of PGH was evaluated using intrinsic fluorescence
mmol of individual amino acids/g protein) from an amino acid analysis. according to the method of Guimarães Drummond e Silva et al. (2017)
For fish proteins, htot is presumed to be 8.6 meq/g of protein (Adler- with some modifications. Aqueous samples containing 2500 µg/ml hy-
Nissen, 1986). Given the assumptions made, the DH is considered to be drolysates and different concentrations of PGH (250 and 500 µg/ml)
an estimate of DH, but a comparison of the different DH of samples were prepared. The fluorescence spectra of samples was determined at
should be less influenced by these assumptions. λexc = 280 nm and λemi = from 310 to 600 nm using a Cytation 3
multi-mode reader (BioTek Instruments, Winooski, VT, USA). The effect
2.7. Determination of thiobarbituric acid reactive substances (TBARS) of complex formation between hydrolysates (2500 µg/ml) and PGH
(62.5, 250, and 500 μg/ml) on the ferrous chelating activity was
TBARS were evaluated during hydrolysis using the method of evaluated. DPPH• scavenging activity of hydrolysates (2500 µg/ml)
Lemon (1975) method with some modification. Briefly, 100 µl of containing 5 and 10 µg/ml PGH was also evaluated.
homogenate at different times of hydrolysis (0, 30, 60, 90, 120, and
180 min) was Vortexed (FAVS, Bologna, Italy) for 10 min with 600 µl of 2.10. Statistical analysis
extraction solution containing 7.5% trichloroacetic acid (TCA), 0.1%
propyl gallate, and 0.1% ethylenediaminetetraacetic acid (EDTA). Data are expressed as the mean ± standard deviation (SD) of three

39
R.A. Sarteshnizi et al. Food Bioscience 27 (2019) 37–45

Table 2
Phospholipid, total pigment, and heme iron content of mince and pretreated mince.
Sample Phospholipid content (mg/100 g dry sample) Total pigment (µg/g dry sample) Heme iron (mg/100 g dry sample)

a a
Mince 700 ± 10 2570 ± 100 23 ± 1 a
Washed mince 276 ± 3b 600 ± 80 b 5.3 ± 0.7 b
Defatted mince 690 ± 30 a 140 ± 20 c 1.2 ± 0.2 c
Fish protein isolate 420 ± 10 c 1380 ± 0.00 d
12.1 ± 0.00 d

Values are given as mean ± SD (n = 3). Means within a column with the same letter are not significantly different (P ≥ 0.05).

replications. SPSS 19 (SPSS Inc., Chicago, IL, USA) was used for data
analysis applying one-way analysis of variance (ANOVA). Significant
difference (P < 0.05) between means was done using the least sig-
nificant differences (LSD) tests.
3. Results and discussion
mince and isolate were more susceptible to protease activity, probably
due to the elimination of lipids and other water-soluble proteins,
changes of protein configuration, and a looser protein complex. De-
fatting with isopropanol decreased DH (15.3%), probably due to pro-
tein aggregation resulting from water removal from tissues in the pre-
sence of solvent (Klompong et al., 2007).

3.1. Total pigment, heme iron, and phospholipid content of different

3.3. Effect of PGH extracts and nitrogen gas on lipid oxidation during
substrates
hydrolysis
Different pretreatments showed a significant effect on total pigment
3.3.1. Fish mince
and heme iron of mince (P < 0.05) (Table 2). Defatting using solvent
Fish mince without enzyme addition (mince-No Alcalase) showed
was the most effective method and resulted in the lowest pigment and
the lowest TBARS values compared to any hydrolyzed sample (Fig. 1a).
heme iron. FPI production was less effective than washing for removing
TBARS increased during the first 30 min of reaction and the change was
pigments probably related to the precipitation of heme proteins with
not significant thereafter. Furthermore, TBARS increased significantly
decreasing pH to the isoelectric point during production of FPI
when fish mince was hydrolyzed using 2.5% enzyme with no nitrogen.
(Kristinsson & Liang, 2006). Also, FPI production and washing de-
The highest value was observed after 180 min of hydrolysis. This con-
creased the phospholipid content significantly (P < 0.05), while the
firmed that the presence of oxygen during hydrolysis affected lipid
effects of defatting using isopropanol were not significant (P ≥ 0.05).
oxidation. Thus, excluding O2 using an inert gas could decrease the rate
This could be related to the low polarity of isopropanol.
of autoxidation (Shahidi, Janitha, & Wanasundara, 1992), Lipid oxi-
Gunnlaugsdottir and Ackman (1993) confirmed that hexane/iso-
dation decreased by ~ 19% with N2. Thus, all other reactions were done
propanol were not effective for polar lipid extractions because of the
in the presence of N2.
low polarity of isopropanol and poor solubility of polar lipids in hy-
PGH at 260 µg/ml concentration reduced oxidation of mince com-
drocarbon solvents.
pared to mince-2.5% Alcalase. Additionally, enzyme concentration
significantly influenced lipid oxidation. As the enzyme level went from
3.2. Effect of pretreatments and PGH extracts on DH
1.3% to 2.5%, the TBARS increased significantly, but addition of 5%
enzyme showed a protecting effect against oxidation. The different ef-
Different levels of the enzyme (1.3%, 2.5%, and 5%) showed a
fects could be due to the released lipids and pro-oxidants as well as to
significant effect on DH (Table 3). The highest DH (22%) was obtained
the antioxidant activity of hydrolysates produced after 180 min, pos-
using 5% Alcalase. Addition of PGH extract as an antioxidant did not
sibly due to the amount and nature of the smaller peptides obtained
influence DH, showing no inhibitory effects of polyphenolic compounds
with longer hydrolysis times (Halldorsdottir et al., 2014b; Senphan &
of PGH on the proteolytic activity of Alcalase (Table 3).
Benjakul, 2015).
Washed and FPI production increased DH (19.3% and 18.8%, re-
spectively). This finding was consistent with Khantaphant et al. (2011),
who indicated that brownstripe red snapper (Lutjanus vitta) washed 3.3.2. Washed mince, defatted mince, and FPI
Washed mince, defatted mince, and FPI showed significantly lower
Table 3 TBARS than untreated fish mince (Fig. 1a–b), indicating that pretreat-
Changes in degree of hydrolysis (DH) of different substrates and hydroxyl ra- ments could remove undesirable lipid substrates or other soluble
dical scavenging activity of hydrolysates. compounds responsible for oxidation. Among pretreated muscle sam-
Treatment Degree of hydrolysis Hydroxyl radical scavenging ples, washed mince showed the highest lipid oxidation, and this value
(%) activity (%) was not significantly different from the values obtained for mince-2.5%
Alcalase-PGH and mince-5% Alcalase (P ≥ 0.05). Sarcoplasmic pro-
Mince-1.3% Alcalase 14 ± 1 a 60.7 ± 0.2 a

b a teins, fat, blood, and pigments were removed by washing

Mince-2.5% Alcalase- 17.7 ± 0.2 62.0 ± 0.4
O2 (Tongnuanchan, Benjakul, Prodpran, & Songtipya, 2011), contributing
Mince-2.5% Alcalase 17.5 ± 0.1 b 60.1 ± 0.3 a to the better oxidative stability of the resulting hydrolysates.
Mince-2.5% Alcalase- 17.6 ± 0.01 b 72.0 ± 0.2 b
The addition of PGH during hydrolysis of washed mince sig-
PGH nificantly reduced lipid oxidation. TBARS increased significantly during
Mince-5% Alcalase 22 ± 1 c 72 ± 1 b
Washed mince 19.3 ± 0.04 d 62.8 ± 0.1 a the first 30 min of hydrolysis of washed mince-PGH and the changes
Washed mince-PGH 19.0 ± 0.01 d 62 ± 1 a were not significant afterward. After 180 min, TBARS were lower for
FPI 18.8 ± 0.01 d 62 ± 1 a washed mince-PGH compared to washed mince. FPI production was
FPI-PGH 18.6 ± 0.01 d 62 ± 1 a more effective than washing in controlling the rate of oxidation after
Defatted mince-PGH 15.7 ± 0.1 e 60 ± 3 a
180 min of hydrolysis. However, addition of PGH had less benefit with
Defatted mince 15.3 ± 0.01 e 61 ± 3 a
FPI compared to washed mince (P ≥ 0.05). After 180 min, there were
PGH: pistachio green hull; FPI: fish protein isolate. no significant differences in TBARS among washed mince-PGH, FPI,
Means within a column with the same letter are not significantly different and FPI-PGH.
(P ≥ 0.05). Defatting using solvent was the most effective method to control

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R.A. Sarteshnizi et al. Food Bioscience 27 (2019) 37–45

Fig. 1. Changes in TBARS value during 180 min hydrolysis of fish mince (a); washed fish mince, defatted mince, and fish protein isolate (FPI) (b). PGH: pistachio
green hull. The data marked with different letters are significantly different (P < 0.05).

Table 4 hydrolysis. It was significantly lower than that of the mince-No Alcalase
Phenolic compounds detected in aqueous PGH extract. Data are expressed as sample (P < 0.05).
mg/g extract powder. Addition of PGH during hydrolysis of mince and washed mince ef-
Compounds Peak Rt (min) λmax Polyphenol content fectively prevented oxidation. The PGH extract contained polyphenolic
compounds of phloroglucinol, gallic acid, protocatechuic acid, 4-hy-
Phloroglucinol 1 4.44 268 65 ± 2 droxybenzoic acid, catechin, vanillic acid, eriodictyol-7-O-glucoside,
Gallic acid 2 5.33 268 5.2 ± 0.2
naringin, and cinnamic acid (Table 4) (Lalegani et al., 2018).
Protocatechuic acid 3 10.38 260 0.09 ± 0.08
4-Hydroxybenzoic acid 4 17. 3 280 0.02 ± 0.01 A previous study from this laboratory indicated that PGH had strong
Catechin 5 21.91 280 0.04 ± 0.02 DPPH• and ABTS• scavenging activity that correlated with polyphenolic
Vanillic acid 6 24.41 260 2.3 ± 0.02 compounds, especially with phloroglucinol and gallic acid (Lalegani
Eriodictyol-7-O-glucoside 7 43.10 280 0.66 ± 0.03 et al., 2018). PGH showed metal chelating activity of ferrous ions while
Naringin 8 52.40 280 0.18 ± 0.05
Cinnamic acid 9 55.48 280 n.d.
gallic acid and phloroglucinol showed no chelating activity. A weak
metal chelating activity of gallic acid was reported by Yen, Duh, and
Adapted to Lalegani et al. (2018). Tsai (2002), while Shahidi and Ambigaipalan (2015) suggested that
Values are means ± SD of three replicates. n.d. = Not detectable. flavonoids could act as metal chelators and radical scavengers.
PGH extracts contained low amounts of flavonoids and showed low
lipid oxidation with the lowest TBARS for defatted mince. TBARS did metal chelating activity, thus the decrease in lipid oxidation during
not change significantly after 30 min of hydrolysis and similar results hydrolysis of Sind sardine muscle substrates might be due to its ter-
were obtained with defatted and defatted-PGH during the entire time of mination of the free radical chain reaction. Oxidation of washed mince

41
R.A. Sarteshnizi et al.
Fig. 2. Effect of different levels of Alcalase on DPPH radical scavenging activity of hydrolysates prepared from mince (a), effect of pretreatments and pistachio green
hull (PGH) addition (b). FPI: fish protein isolate. The data marked with different letters are significantly different (P < 0.05).
is more related to the remaining lipid substrates (data not shown) and
the effect of heme pigments is less important (Table 2). According to
Kristinsson and Liang (2006), a significant removal of heme proteins
occurred with washing, while only a small amount of the lipids were
removed. Therefore, PGH with high radical scavenging capacity could
successfully inhibit oxidation. The pH shift during FPI production de-
creased the lipids and other pro-oxidants from FPI, but some lipid re-
mained in the product that led to a significant increase in fat oxidation
(Shaviklo, Thorkelsson, Arason, & Sveinsdottir, 2012). Lipid oxidation
of hydrolysates from mackerel protein isolate was correlated with the
residual phospholipids in the membranes (Yarnpakdee et al., 2012). In
addition, co-precipitation of heme proteins (hemoglobin and myo-
globin) at pH ~ 5.5 increased FPI oxidation (Kristinsson & Liang, 2006)
as hemoglobin and myoglobin convert to their oxidized form with
strong pro-oxidative activity (Baron, Skibsted, & Andersen, 2002). The
insignificant effect of PGH on controlling oxidation was probably re-
lated to its weak metal chelating activity. Among all substrates, defatted
Sind sardine mince showed the lowest oxidation during hydrolysis,
which may be related to the greatest removal of lipids from the sub-
strate using isopropanol (data not shown), and the lowest pigment and
heme iron content (Table 2).
42
Food Bioscience 27 (2019) 37–45
3.4. Effect of PGH extract on antioxidant activity of hydrolysates from Sind
sardine protein substrates
3.4.1. DPPH• scavenging activity
There was no significant difference between hydrolysates obtained
with addition of 1.3% and 5% enzyme, and hydrolysis with 2.5% en-
zyme resulted in significantly lower DPPH• scavenging activity
(Fig. 2a). This lack of correlation between enzyme dose and DPPH•
scavenging activity may be related to the interference of higher fish oil
content of mince used for hydrolysate production than other pretreated
samples in the DPPH• scavenging activity test. These results were con-
sistent with the findings of Halldorsdottir et al. (2014a) who suggested
that addition of 5% (v/v) fish oil to washed cod mince and cod protein
isolate interfered with DPPH• scavenging activity. Among all substrates
that were hydrolyzed without PGH, hydrolysates of mince showed the
lowest DPPH• scavenging activity. Defatting, washing, and FPI pro-
duction increased DPPH• scavenging activity (P < 0.05). PGH addition
showed a significant effect on DPPH• scavenging activity of hydrolysate
from mince and other pretreated substrates (P < 0.05) (Fig. 2b). Wa-
shed mince with PGH in the hydrolysates showed the highest DPPH•
scavenging activity. The combined antioxidant activity of phenol and
R.A. Sarteshnizi et al. Food Bioscience 27 (2019) 37–45

Fig. 3. Effect of different levels of Alcalase on metal chelating activity of hydrolysates prepared from mince (a), effect of pretreatments and pistachio green hull
(PGH) addition (b). FPI: fish protein isolate. The data marked with different letters are significantly different (P < 0.05).

Fig. 4. Fluorescence spectra emission of hydrolysate, pistachio green hull (PGH), and hydrolysate-PGH.

hydrolysate has been attributed to H+ transfer and electron donation
(Guimarães Drummond e Silva et al., 2017).
3.4.2. Ferrous chelating activity
Ferrous chelating activity is shown in Fig. 3. Antioxidant activity of
hydrolysate from defatted mince was significantly higher than mince,
washed mince, and FPI hydrolysates (P < 0.05) (Fig. 3b). A significant
combined effect was observed with PGH and hydrolysates with dif-
ferent substrates except for FPI (P < 0.05). Mince plus PGH showed
the highest metal chelating activity while the lowest activity was ob-
tained for washed mince and mince-2.5% Alcalase-O2. Addition of PGH
increased metal chelating activity of hydrolysates from mince, defatted
mince and washed mince. Similarly, Fv extract improved the metal
chelating properties of cod bone mince hydrolysates (Halldorsdottir

43
R.A. Sarteshnizi et al.
Fig. 5. Effect of hydrolysate and pistachio green hull (PGH) polyphenols interaction on metal chelating activity.
Fig. 6. Effect of hydrolysate and pistachio green hull (PGH) polyphenols in-
teraction on DPPH radical scavenging activity.
et al., 2014b). Highly oxidized hydrolysates produced from mince-2.5%
Alcalase-O2 showed significantly lower metal chelating activity than
mince–2.5% Alcalase-N2 hydrolysates. The negative effect of oxidation
on metal chelating activity was also reported by Halldorsdottir et al.
(2014a) who suggested that highly oxidized FPH had significantly
lower metal chelating ability than less oxidized hydrolysate.
3.4.3. Hydroxyl radical scavenging activity
There was no significant difference in antioxidant activity at 1.3%
and 2.5% enzyme (P ≥ 0.05), while the addition of 5% enzyme led to a
significant increase in hydroxyl radical scavenging activity (Table 3).
All hydrolysates of pretreated samples effectively scavenged hydroxyl
radicals at 0.5 mg/ml (Table 3). Different pretreatments and addition of
PGH did not show any significant effect on hydroxyl radical scavenging
activity of hydrolysates except for mince-PGH, where the antioxidant
activity was significantly higher (P < 0.05).
3.5. Interaction of hydrolysate-phenolic compounds and antioxidant
properties
Interactions of mince hydrolysates and PGH polyphenols are shown
in Fig. 4. Hydrolysates from mince with no phenolic compounds
showed the highest fluorescence intensity, while the lowest intensity
was obtained with PGH. Interaction of hydrolysates-PGH showed lower
fluorescence intensity than hydrolysates from mince. Fluorescence in-
tensity decreased with increasing PGH concentration. These results
suggested that complex formation between hydrolysates and PGH de-
creased the fluorescence of the aromatic amino acids.
Complex formation between hydrolysates and PGH increased the
ferrous chelating and DPPH radical scavenging activity significantly
44
Food Bioscience 27 (2019) 37–45
(P < 0.05) as shown in Figs. 5 and 6, respectively. By increasing the
PGH concentration, the antioxidant activities of hydrolysates were in-
creased. These results were consistent with the results reported by
Guimarães Drummond e Silva et al. (2017) who indicated that the
complex formation of protein/peptide-phenolics caused a synergistic
antioxidant activity.
4. Conclusions
Hydrolysis of mince in the presence of N2 combined with PGH,
especially with 5% Alcalase, significantly reduced oxidation.
Production of FPI and washed mince improved DH and decreased
oxidation. Defatting showed a negative effect on DH while effectively
reducing oxidation. Washed mince-PGH, FPI, and FPI-PGH were effec-
tive methods to reduce oxidation as seen by lower TBARS after 180 min
of hydrolysis. A combined effect of substrates and PGH on antioxidant
activity was observed. Washed mince-PGH and mince-PGH showed the
highest DPPH radical scavenging and metal chelating activity, respec-
tively. Thus, pretreatments, especially when combined with N2 treat-
ment, could successfully control lipid oxidation. Also, the use of anti-
oxidant such as PGH is a new approach for controlling lipid oxidation
and improvement of the antioxidant properties of Sind sardine protein
hydrolysates.
Acknowledgement
The authors would like to acknowledge Tarbiat Modares University
and the Iran National Science Foundation (Project no. 94013374) for
their financial support.
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