Probiotics and Antimicrobial Proteins

https://doi.org/10.1007/s12602-018-9483-y

Fractionation of Protein Hydrolysates of Fish Waste Using Membrane

Ultrafiltration: Investigation of Antibacterial and Antioxidant Activities
Samaneh Pezeshk 1 & Seyed Mahdi Ojagh 1,2 & Masoud Rezaei 2 & Bahareh Shabanpour 1

# Springer Science+Business Media, LLC, part of Springer Nature 2018

Abstract
In this study, yellowfin tuna (Thunnus albacores) viscera were hydrolyzed with protamex to obtain hydrolysate that is separated by a
membrane ultrafiltration into four molecular size fractions (< 3, 3–10, 10–30, and 30 kDa <). Antibacterial and antioxidant prop-
erties of the resulting hydrolysates and membrane fractions were characterized, and results showed that the lowermost molecular
weight fraction (< 3 kDa) had significantly the highest (P < 0.05) percentage of bacteria inhibition against Gram-positive (Listeria
and Staphylococcus) and Gram-negative (E. coli and Pseudomonas) pathogenic and fish spoilage-associated microorganisms and
scavenging activity against DPPH and ABTS radical and ferric reducing antioxidant power among the fractionated enzymatic
hydrolysates. These results suggest that the protein hydrolysate derived from yellowfin tuna by-products and its peptide fractions
could be used as an antimicrobial and antioxidant ingredient in both nutraceutical applications and functional food.

Keywords Antibacterial peptide . Antioxidant peptide . Yellowfin tuna by-products . Amino acid composition . Membrane
ultrafiltration

Introduction oxygen species (ROS) such as superoxide anions, hydroxyl

Fish processing wastes are considered to be a good pattern of
essential amino acids. Bioactive peptides are found plentifully
in fish sources protein hydrolysates [16], which usually con-
sist of natural amino acid sequences (often 2–20 residues),
encrypted in the parent or natural protein molecule, and are
inactive within the sequence of parent protein and can be
released during food processing or gastrointestinal digestion
[21]. These peptides are known to possess a wide range of
bioactivities, including immunomodulatory, antihypertensive,
anticancer, antithrombotic, antioxidant, and antimicrobial ac-
tivities [47].
Antioxidants play an important role in human nutrition and
health as they are known to protect the body against reactive
* Seyed Mahdi Ojagh
mahdi_ojagh@yahoo.com
1
Department of Fisheries, Faculty of Fisheries and the Environment,
Gorgan University of Agricultural Sciences & Natural Resources,
Gorgan, Iran
2 timicrobial peptides is necessary to their antimicrobial activity
Department of Seafood Processing, Faculty of Marin Sciences,
Tarbiat Modares University, Noor, Iran
radicals, and hydrogen peroxide [21]. Free radicals formed
during oxygen metabolism are very unstable and react rapidly
with other groups or substances in the body, leading to tissue
and cell injury [25, 32]. ROS have been implicated in the
etiology of various degenerative diseases, including cancer,
diabetes, cardiovascular disease, cataracts, neurodegenerative
disorders, and aging [36]. Enzymatic food protein-derived
peptides, in comparison to synthetic compounds such as bu-
tylated hydroxyanisole (BHA), butylated hydroxytoluene
(BHT), t-butyl hydroquinone (TBHQ), and propyl gallate
are believed to be safer natural antioxidants that can be used
as protective agents to help the human body decrease oxida-
tive damage and correlated diseases [19, 21, 45].
Antimicrobial peptides (AMPs) have been found with nu-
merous origins, such as plants, animals, and microorganisms.
Fish possess peptides that serve as a part of the innate immune
system defense against a broad spectrum of pathogens be-
cause, in addition to eliminating the invading microorganisms,
they are able to modulate inflammatory responses [20]. The
most of the antimicrobial peptides recognized are cationic
peptides with low-molecular weight and exhibit hydrophobic
properties. It is believed that the amphipathic structure of an-
[11, 12, 51]. Antimicrobial peptides have caught

investigators’ attention for their broad spectrum of potential
applications, including their therapeutic potential. In recent
years, investigators have focused on the preparation of fish
AMPs from fish organ extractions, secretes, or transgenic ex-
pression [4, 26, 29]. Reports have revealed AMPs from fish
hydrolysates [20, 43].
In the last few years, there has been an increased effort to
develop technologies capable of recognizing and quantifying
large numbers of proteins expressed within a proteome.
Different methods have been reported for separating peptide
and protein including ultracentrifugation, size-exclusion chroma-
tography, isoelectric focusing, hydrophobic interaction chroma-
tography, reversed-phase chromatography, ion-exchange chro-
matography, affinity chromatography, electrodialysis, gel electro-
phoresis, and membranes ultrafiltration (UF) [9, 10, 24, 40].
Compared to other methods, UF techniques offer advantages of
lower cost and ease to scale-up for commercial production. This
system has the main advantage that the molecular weight distri-
bution of the desired hydrolysates can be controlled by the adop-
tion of a suitable UF membrane [21]. Rapeseed protein isolate
was hydrolyzed with several proteases to get hydrolysates that
were separated by UF into four molecular size fractions (< 1, 1–
3, 3–5, and 5–10 kDa) and then were evaluated antioxidant po-
tential of protein hydrolysates and their ultrafiltration fractions
[19]. In this regards, several studies have used membrane pro-
cessing (ultrafiltration and nanofiltration) to obtain biologically
active fractions. For example, application of the multilayer mem-
brane system showed the potential to recover high ACE-
inhibitory activity from tilapia by-product protein hydrolysate
[41]. In another study, focusing on the production of protein
hydrolysate with antioxidant and antihypertensive activities re-
covered by ultrafiltration of different wastewaters from the indus-
trial processing of cuttlefish [6]. In fact, ultrafiltration at 100, 30,
and 10 kDa effectively recovers protein with bioactive properties
from cuttlefish wastewater. The antioxidant activities of tuna dark
muscle by-product hydrolysate (TPH) and its peptide fractions
were evaluated by ultrafiltration (UF) and nanofiltration (NF)
membrane processes [42]. They indicated that the effect of mem-
brane fractionation on antioxidant properties of peptides was
underlined. On the other hand, the tuna processing industry pro-
duces a large amount of solid wastes [2], so it appears as a
suitable method to achieve protein hydrolysis in order to obtain
valuable by-products from fish solid wastes. Furthermore, the
interrelationships of protein hydrolysate antioxidant and antibac-
terial activities with amino acid sequence and composition as
well as peptide molecular weight have caused increased attention
in evaluating the efficiency of proteases in releasing antioxidant
and antibacterial peptides from fish protein hydrolysate (FPH).
Hence, the objective of this study was to obtain peptide fractions
from the FPH of yellowfin tuna (Thunnus albacores) viscera
using membranes ultrafiltration, characterize the protein hydro-
lysates and fractions in relation to peptide size distribution, and
evaluate the antioxidant and antibacterial activity of fractions.
Probiotics & Antimicro. Prot.
Material and Methods
Materials
Yellowfin tuna viscera used for this study were supplied by
Darya-Khorak, a seafood processing company in Babolsar,
Iran. The viscera was minced uniformly and stored frozen at
− 20 °C until use. Protamex (from Bacillus sp) was provided
by the Iranian branch of the Danish company, Novozymes
(Novozymes, Tehran, Iran), and stored at 4 °C until used.
Bacterial strains used for the studies Escherichia coli (ATCC
10536), Pseudomonas aeruginosa (ATCC 10145),
L. monocytogenes (PTCC 1298), and Staphylococcus aureus
(ATCC 25923) were preserved strains from the National
Nutrition And Food Technology Research Institute. DPPH,
TCA, ABTS, ferrous chloride, and other antioxidant reagents
were purchased from Sigma (Sigma Chemicals, St. Louis,
MO, USA). All other chemicals used in the experiments and
ultrafiltration membranes (3, 10, and 30 kDa molecular weight
cutoff) were obtained from Fisher Scientific (Oakville, ON,
Canada). Ultrafiltration regenerated cellulose membranes
(76 mm in diameter) were used. During the filtration process,
the pressure was applied with nitrogen for each sample vol-
ume (12 mg/mL).
Preparations of Protein Hydrolysates and Membrane
Fractions
The minced viscera were homogenized with distilled wa-
ter at a ratio of 1:2 (w/v). Before the reaction, suspensions
were pre-incubated in a conical flask at optimum condi-
tions for each enzyme (41 °C and pH 6.5 for protamex)
for 15 min. The hydrolysis reaction initiated by adding
enzyme in the amount of 1.5% (w/w) with stirring at
600 rpm for 150 min. At the end of the reaction time,
the enzyme was inactivated by heating up to 90 °C for
10 min. The hydrolysates were centrifuged at 6000 g for
20 min at 4 °C and the obtained supernatant was collected
and was sequentially passed through ultrafiltration mem-
branes with molecular weight cutoff of 3, 10, and 30 kDa
in an Amicon-stirred ultrafiltration cell. The permeate
fraction from each molecular weight cutoff membrane
was collected and stored at − 20 °C until used.
Amino Acid Composition Analysis
The amino acid profiles of the samples were analyzed using an
HPLC set using C18 type column (Knauer, Germany) at the
flow rate of 1 mL/min−1 with fluorescence detector system,
after hydrolysis of protein with 6 N HCl at 110 °C for 24 h as
described by [7].
Probiotics & Antimicro. Prot.
Bacterial Strains and Culture Conditions
Several typical Gram-positive (L. monocytogenes (PTCC
1298), and Staphylococcus aureus (ATCC 25923)), Gram-
n e g a t i v e ( E s c h e r i c h i a c o l i ( AT C C 1 0 5 3 6 ) , a n d
Pseudomonas aeruginosa (ATCC 10145)) bacteria strains,
which included pathogenic bacteria as well as conditioned
pathogenic bacteria in fishes, were selected to evaluated the
antibacterial activity of the FPH and its fractions. The strains
were stored at − 20 °C in a sterile tripton soy broth (TSB) with
30% glycerol until use. Then the strains were grown in TSB
broth. Organisms were incubated at 37 °C. Subsequently, the
optical density cultures were measured at 600 nm and adjusted
to around 0.1 by addition of the TSB (OD600 = 0.1) that con-
tains approximately 108 colony-forming units per milliliter
(cfu/mL) [5].
The antibacterial properties of FPH and its fractions were
studied using the method described by [18] with some modi-
fications. Each sample was prepared by mixing TSB medium
(90 μL), the bacterial inoculum (10 μL) containing 106 cfu/
mL, and protein hydrolysate (90 μL) into each well of a 96-
well plate. Wells without peptide was considered as a control
and containing a medium, bacterial culture, and distilled wa-
ter. The plates were incubated at 37 °C for 24 h in the suitable
incubation chamber. Then, bacterial growth was evaluated by
measuring the absorbance of wells at 630 nm using ELIZA
micro-plate reader (Power Wave, X340, Bio Tek Instruments,
INC). The percentage of inhibition was calculated as [(OD
control − OD sample)/OD control)] × 100.
Minimum Inhibitory Concentration (MIC)
Determination
The minimum inhibitory concentration (MIC) of the peptide
was determined by liquid growth inhibition assay in a micro-
plate reader using sterile a 96-well microtiter plates [38]. The
MIC is the lowest peptide concentration that completely
inhibited the strain growth after 24 h of incubation at 37 °C.
Tested FPHs fraction concentrations ranged from 0/5 to
3.5 mg mL −1. Bacterial growth was measured by ELIZA at
630 nm (Table 2).
Antioxidant Activity Determination
DPPH Radical-Scavenging Capacity
DPPH radical-scavenging activity was measured using ELIZA
in accordance with the method described by [34]. A 150 μL of
each sample (or distilled water as control) was added to 150 μL
DPPH (150 μM) in ethanol solvent in the 96-well plate to a
final assay concentration of 0.5, 1.0, 1.5, 2.0, 2.5, and 3.0 mg/
mL and incubated at room temperature in the dark for 30 min.
The absorbance values of the samples (As) and control (Ac)
were measured at 517 nm. The ability to scavenge DPPH rad-
icals of the samples was calculated using the following equa-
tion: DPPH radical scavenging activity ð%Þ ¼ Ac−As
Ac *100:
ABTS Radical-Scavenging Activity Assay
ABTS radical-scavenging activity assay was determined ac-
cording to [52] with some modifications. ABTS•+ was pre-
pared by reacting 7-mM ABTS solution and 2.45-mM potas-
sium persulfate in a 10-mM phosphate buffer (pH 7.4) and
kept in the dark for 16 h. The ABTS•+ solution was diluted
with methanol to attain absorbance of 0.7 ± 0.02 at 734 nm.
Then, 250 μL of samples (0.5, 1.0, 1.5, 2.5, and 3.5 mg/mL)
was added to 750 μL of ABTS•+ solution and then, the reac-
tion mixture was kept in the dark for 20 min and absorbance
was monitored at 734 nm. The scavenging percentage was
calculated as described for the DPPH assay.
Reducing Power Assays
The ability of FPH and its fractions to reduce iron was mea-
sured using the method described by [35] with slight modifi-
cations. Briefly, a 100 μL sample aliquot was added to 250 μL
0.2 M phosphate buffer (pH 6.6) and 250 μL 1% potassium
ferricyanide solution. The mixture was incubated at 50 °C for
30 min, followed by the addition of 250 μL of 10% TCA.
Then, the mixture was centrifuged at 1650g for 10 min.
Finally, 250 μL of the supernatant solution was mixed with
250 μL of distilled water and 50 μL of 0.1% (w/v) ferric
chloride. After a 10-min reaction time, the absorbance was
measured at 700 nm.
Statistical Analyses
All experiments were conducted in triplicate. Data were
expressed as means with standard deviations. Data were ana-
lyzed using analysis of variance (ANOVA). The statistical anal-
ysis was performed using an SPSS package (SPSS 17.0 for
Windows, SPSS Inc., Chicago, IL, USA). The significant differ-
ence was measured with a 95% confidence interval (P < 0.05).
Result and Discussion
Amino Acid Composition
The peptide fractions prepared from ultrafiltration membranes
were subjected to amino acid analyses in order to illustrate the
possible effect of the amino acid profile on antioxidant and
antibacterial activity. Amino acid compositions of FPH and its
fractions are shown in Table 1. The function of any peptide is
mostly dependent on its levels and compositions of amino
 Probiotics & Antimicro. Prot.

Table 1 Amino acid composition of FPH and the resulting fractions of activity than the high-molecular-weight peptides, which is in
F1 (30 kDa <), F2 (10–30 kDa), F3 (3–10 kDa), and F4 (< 3kDa), (n = 3)
agreement with similar findings obtained for squid and tuna skin
Amino acid FPH F1 F2 F3 F4 gelatin protein hydrolysates fractions [20]. [20] showed that the
better interaction between molecular weight and bacterial mem-
Aspartic 10.54a 11.12a 11.54a 11.63a 9.75a branes could be related to the omission, presence of the amino
Glutamic 12.69c 13.23b 12.69c 13.66a 12.42d acids and their charges, and structure acquisition. In fact, peptides
Serine 5.22c 5.29c 5.66a 5.90b 5.61a have commonly been reported to interact with cell membranes as
Histidine 1.45c 1.48b 1.09d 1.46c 1.55a part of their action against bacteria. Carpet-like peptide bindings
Glycine 13.75d 14.65b 15.62a 14.52c 11.22e and particular peptide-lipid interactions have led to the forma-
Threonine 1.24e 2.67a 2.55b 1.55c 1.53d tions of the separation channel and disorganizing lipid bilayers.
Arginine 8.54a 7.96c 6.60d 7.92c 9.78b This interaction occurs between the cationic peptide and nega-
Alanine 9.14b 8.97d 8.39e 9c 9.28a tively charged components present in the bacterial cell wall, such
Tyrosine 2.78b 2.19d 2.50c 2.11d 3.54a as lipoteichoic acids present on the surface of Gram-positive
Methionine 8.91b 8.67c 8.66c 8.3d 9.29a bacteria or phosphate groups within the outer membrane of
Valine 3.04b 2.85c 2.81d 2.76e 3.39a Gram-negative bacteria [1]. This comes to phase separation,
Phenylalanine 5.36b 5.15d 4.94e 5.32c 5.65a membrane detergent-like solubilization, and even peptide pene-
Isoleucine 8.35b 8.09c 7.52e 7.88d 8.77a tration into the cell, leaving the membrane intact [29].
Leucine 2.35a 2.18b 1.34d 2.08c 2.07c Antibacterial activity of peptides is more broadly influenced by
Lysine 0.49b 0.46c 0.86a 0.4d 0.48b their sequence, molecular weight, and structural features like
peptide helicity, hydrophobicity, hydrophobic moment, and a
Different letters within the same row indicate differences between mean combination of specific amino acids such as histidine, arginine,
values (P < 0.05)
alanine, valine, and leucine [37]. This result indicated that low-
molecular-weight peptides possess better antibacter than the
acids [44]. The amino acids of Arg, Ala, Val, Leu, and Lys
high-molecular-weight (HMW) peptides, which is in agreement
were reported to appear frequently in the amino acid sequence
with similar findings obtained for quinoa protein hydrolysates
of antimicrobial peptides. As for proteins with a high content
fractions
of cationic amino acids, the most antibacterial activity is path-
Since it exhibited the highest rates of activity and intensity,
ogenic bacteria is generally observed [46]. The higher concen-
the F4 peptide was selected and maintained for further analy-
tration of cationic amino acids was observed in F4, consistent
sis. The minimal inhibitory concentration (MIC) values of the
with the result of the antibacterial assay (Table 1). The pres-
F4 was calculated against both Gram-positive (Listeria and
ence of Val, Met, Tyr, Leu, Phe, Lys, His, and Arg have been
Staphylococcus) and Gram-negative bacteria (E. coli and
shown to contribute greatly to the potency of antioxidant pep-
Pseudomonas). In this study, F4 peptide exerted effective in-
tides were detected in all of the groups. In addition, the hy-
hibition on the growth of Escherichia coli, Pseudomonas,
drophobic properties of peptides can increase their interaction
Staphylococcus aureus, Listeria monocytogenes, with MIC
with lipid targets or entrance of the peptides into target organs
values of 0.5 mg/mL (Table 2). These findings were in accord
via hydrophobic compositions, which is desirable to achieve
with the results of [29, 37], which expressed that the shortest
antioxidant effects [52]. The fraction of F4 (< 3 kDa)
peptides are most active and do not have the classical charac-
contained a higher concentration of hydrophobic amino acid
teristics of the antibacterial peptides.
when compared to FPH, fractions of F1, F2, and F3. Further,
some di- and tri-peptides containing aromatic amino acid res-
Antioxidant Activity of FPH and the Peptide Fractions
idues show strong antioxidant activity. The aromatic amino
acids could make active oxygen stable through direct electron
DPPH Radical-Scavenging Capacity
transfer and easily donate protons to electron-deficient radi-
cals, which would make antioxidant activity [17].
The DPPH radical-scavenging activity assay is a stable
nitrogen-centered free-radical compound and is widely used
Antibacterial Activity of FPH and the Peptide in the measurement of the peptide, phenolic, and food antioxi-
Fractions dant capacity [28]. The ability of FPH and peptide fractions to
scavenge DPPH radical is shown in Fig. 2. All experimented
The results presented in Fig. 1 show that, at 2 mg/mL, FPH and groups exhibited significant hydroxyl radical-scavenging activ-
all fractions displayed antibacterial activity against tested bacte- ity (P < 0.05). Our findings are similar to previous studies re-
rial species. The F4 fraction showed the highest level of activity ported by [8, 26, 30] who reported that the DPPH-scavenging
among the active fractions and FPH. This result indicated that activity increased with increasing concentrations. Among the
low-molecular-weight peptides possess better antibacterial different experimented groups, F4 exhibited the highest DPPH
Probiotics & Antimicro. Prot.

120 120

Percentage of Staphylococcus
monocytogenes inhibitation
a a
b

Percentage of Listeria
100 c 100 b
d c c
80 80

inhibitation
e

aureus
60 60 d
40 40
20 20
0 0
FPH F1 F2 F3 F4 FPH F1 F2 F3 F4
2 (mg/ml) 2 (mg/ml)

120 120
Pecentage of Escherichia coli

Pecentage of Pseudomonas
a a
100 b 100 b
c c b
c

inhibitation
inhibitation

80 80
d
60 d 60

40 40
20 20

0 0
FPH F1 F2 F3 F4 FPH F1 F2 F3 F4
2 (mg/ml) 2 (mg/ml)
Fig. 1 Antibacterial activity of fish protein hydrolysates and fractions of F1 (< 30 kDa), F2 (10–30 kDa), F3 (3–10 kDa), and F4 (< 3 kDa) against both
Gram-positive (Listeria and Staphylococcus) and Gram-negative bacteria (E. coli and Pseudomonas), (n = 3)

radical-scavenging activity at all concentrations tested (at 1, 2, pairing the single electron of the DPPH radicals [27]. In addi-
and 3 mg), while the lowest DPPH radical-scavenging activities tion, it has been reported that the high level of DPPH free
were obtained with F1. Similar findings showed that peptides radical-scavenging activity of protein hydrolysates or peptide
with low-molecular weight separated from tuna protein hydro- fractions is related to a high amount of hydrophobic and posi-
lysate [22] and salmon hydrolysate [3] have higher DPPH tively charged amino acids or peptide [15].
radical-scavenging activity. The IC50 is a parameter that is
widely used to evaluate anti-radio performance and IC50 low ABTS Radical-Scavenging Activity
exhibit high distribution activity. In this study, the F4 was the
most active (lowest IC50 value) against DPPH (at 1.8 mg/mL) ABTS radical assay, to determine antioxidative activity, especial-
(Table 3). The results are in line with those reported by [21, 48, ly where the radical is quenched to form the ABTS radical
55], who showed that peptides in the lowest molecular size
from protein hydrolysate had the highest DPPH radical- 90
a FPH
scavenging activity when compared to higher molecular pep- 80
tides present in peptide fractions. The decrease of DPPH in the F1
DPPH radical scavenging activity

presence of the FPH and peptide fractions indicates that mixed 70 F2

b
peptide amino acids were able of quenching DPPH likely by 60 a F3
F4
50 b c
cd d
Table 2 Minimum inhibitory concentration (MIC) of F4 (< 3 kDa), 40 a c
(n = 3) ab d
30 b d
Bacterial strains Percent of bacteria MIC b
inhibition (mg/mL) 20
c
Gram-negative 10
Escherichia coli 99.7 0.5 0
Pseudomonas aeruginosa 99.76 0.5 1 2 3
Gram-positive mg/ml
L. monocytogenes 99.88 0.5 Fig. 2 Free radical scavenging activities of fish protein hydrolysates and
Staphylococcus aureus 99.76 0.5 their membrane ultrafiltration peptide fractions (F1 (< 30 kDa), F2 (10–
30 kDa), F3 (3–10 kDa), and F4 (< 3 kDa)) against DPPH radicals, (n = 3)
 Probiotics & Antimicro. Prot.

0.14 a
Table 3 Antioxidant activity (IC50, mg/mL) of FPH and peptide FPH
fractions of F1 (< 30 kDa), F2 (10–30 kDa), F3 (3–10 kDa), and F4 (< 0.12 a a F1
a b
b

Reducing power
3 kDa), (n = 3) 0.1 c F2
b bb b b bb c cb cc F3
0.08 b
Sample IC50 (mg/mL) F4
0.06

DPPH radical scavenging ABTS radical scavenging 0.04

0.02
FPH 3.43 1.65 0
F1 3.68 1.54 1 2 3 4
mg/ml
F2 3.45 1.84
F3 2.2 1.52 Fig. 4 Reducing power of FPH and peptides fractions (F1 (< 30 kDa), F2
(10–30 kDa), F3 (3–10 kDa), and F4 (< 3 kDa)) at different
F4 1.8 1.4 concentrations, (n = 3)

complex is counted an excellent tool. The scavenging activity

against cationic ABTS radical shows the ability of peptide frac- Reducing Power
tions to act as hydrogen donors or electron donors in free radical
reactions [39]. Results of ABTS radical of the hydrolysate and Reducing power is generally used for the evaluation of the
fractionated hydrolysates are shown in Fig. 3. The results showed activity of protein hydrolysate and peptide fractions [54].
that the F4 fraction revealed the highest scavenging activity (66% The F4 showed the highest reducing power which was in
at 2 mg/mL). Our results are similar to previous reports that agreement with DPPH and ABTS radical-scavenging ac-
showed peptides with the low-molecular weight of fish gelatin tivity. The reducing powers of FPH and all peptide frac-
protein hydrolysate and protein hydrolysate from fish roe had the tions except F4 were a little below the absorbance 0.1. Low
strongest scavenging activity against ABTS radical [20, 23]. values of the absorbance can be due to the little concentra-
Antioxidant activity of peptides considerably depends on the tion of the peptide fractions in the blend solution (Fig. 4).
relative abundance of nonaromatic amino acids as well as to their In a similar study on pumpkin oil cake protein hydrolysate,
positioning in the peptide sequence [33]. The IC50 value of F4 the absorbance of reducing the power of all hydrolysate
(1.4 mg/mL) was lower than the other groups (Table 3). Similar samples was below 0.1 [49]. The decrease or increase in
results were reported by other researchers for tunicate (Styela reducing power for FPH or peptide fractions may be asso-
clava) hydrolysates and Pacific hake (Merluccius productus) hy- ciated with the exposure of electron-dense amino acid side
drolysates that the highest antioxidant activity was contributed by chain groups, such as charged or polar moieties during
peptides containing the high amount of low-molecular weights hydrolysis (Fig. 4). On the other hand, the indolic and
[14, 27]. The differences in ABTS scavenging activity among the phenolic groups of tyrosine have been reported to play
experimented groups might be due to the differences in amino important roles as hydrogen donors in a redox system
acid composition and sequence length [50]. It has been reported [53]. Generally, antioxidant activities of fish protein hydro-
that the high content of hydrophobic amino acids in FPH and lysates are commonly associated with the amino acid com-
peptide fractions was put forward as the main cause for its high position, sequence, and hydrophobicity [13].
lipid-peroxidation inhibition due to their high dependency with
linoleic acid [31].

Conclusion
80
ABTS radical scavenging activity

70 a FPH In conclusion, peptide fractions from hydrolysates of tuna

b
60 a a aa cbb F1 visceral presented both antioxidant and antimicrobial proper-
50 a a a a a a b F2 ties. These capacities were found to be higher mainly in the
a a b b b
40 b bb b c F3 lowermost molecular weight fractions. Moreover, the higher
b
30 c
c c F4 content of cationic and hydrophobic amino acids in peptide
20 fractions might be responsible for the enhanced antibacterial
10
and antioxidant activities. Those antibacterial and antioxidant
0
0.3 0.6 0.9 1.2 1.5 2
peptides might also open new opportunities for the develop-
mg/ml
ment of safe, efficient, and cost-effective strategies for the
prevention and alleviation of several food-borne diseases
Fig. 3 Free radical scavenging activities of fish protein hydrolysates and
their membrane ultrafiltration peptide fractions (F1 (< 30 kDa), F2 (10– and could prove useful as preservatives for storage and distri-
30 kDa), F3 (3–10 kDa), and F4 (< 3 kDa)) against ABTS radicals, (n = 3) bution of meat-based products.
Probiotics & Antimicro. Prot.
Compliance with Ethical Standards
Conflict of Interest The authors declare that they have no conflict of
interest.
Ethical Approval This article does not contain any studies with human
participants or animals performed by any of the authors
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