catalysts

Article
Characterization of Protein Hydrolysates from Eel
(Anguilla marmorata) and Their Application in
Herbal Eel Extracts
I-Chun Cheng 1 , Jin-Xian Liao 1 , Jhih-Ying Ciou 2 , Li-Tung Huang 3,4 , Yu-Wei Chen 5 and
Chih-Yao Hou 1, *
1 Department of Seafood Science, National Kaohsiung University of Science and Technology, No. 142,
Haijhuan Rd., Nanzih Dist., Kaohsiung City 81157, Taiwan; [email protected] (I-C.C.);
[email protected] (J.-X.L.)
2 Department of Food Science, Tunghai University, No. 1727, Section 4, Taiwan Boulevard, Xitun District,
Taichung City 40704, Taiwan; [email protected]
3 Institute for Translational Research in Biomedicine, College of Medicine, Kaohsiung Chang Gung Memorial
Hospital and Chang Gung University, Kaohsiung 833, Taiwan; [email protected]
4 Department of Traditional Medicine, Chang Gung University, Linkow 333, Taiwan
5 Department of Medicine, Chang Gung University, No. 259, Wenhua 1st Rd., Guishan Dist.,
Taoyuan City 33302, Taiwan; [email protected]
* Correspondence: [email protected]; Tel.: +886-985300345; Fax: +886-7-3640634




Received: 17 December 2019; Accepted: 7 February 2020; Published: 8 February 2020


Abstract: The enzymatic hydrolysis of fish proteins is the principle method for converting
under-utilized fish into valuable products for the pharmaceutical and health food industries. In this
study, three commercial enzymes (alcalase, bromelain, and papain) were tested for their ability to
create eel protein hydrolysates (EPHs) from whole eel (Anguilla marmorata). Freeze-dried EPHs had
almost more than 80% solubility (p < 0.05) in solutions ranging from pH 2–10. The amino acid profiles
of the EPHs showed a high percentage of essential amino acids, including histidine, threonine, valine,
isoleucine, and leucine. The emulsion activity index (EAI) of EPH resulted as follows: alcalase group
(36.8 ± 2.00) > bromelain group (21.3 ± 1.30) > papain group (16.2 ± 1.22), and the emulsion stability
index (ESI) of EPH was: alcalase group (4.00 ± 0.34) > bromelain group (2.62 ± 0.44) > papain group
(1.44 ± 0.09). As such, EPH has a high nutritional value and could be used as a supplement to diets
lacking protein. EPH showed excellent solubility and processed interfacial properties, which are
governed by its concentration. Among of them the alcalase group had the best antioxidant effect at
1,1-diphenyl-2-pyridinohydrazinyl (DPPH) radical method, determination of reducing power and
ABTS test compared with other groups. EPH may be useful in developing commercial products like
herbal eel extracts that are beneficial to human health.

Keywords: Anguilla marmorata; eel protein hydrolysates; functional properties; herbal eel extracts

1. Introduction
As the world’s population continues its expansion towards nearly 10 billion people by 2050,
increasing wealth in developing countries adds to the demand for protein [1], an essential component of
the human diet. This is due to changing food preferences and a growing recognition of the importance
of protein as a key dietary ingredient. Dietary protein supplements are becoming popular, especially
for people on restricted diets, like athletes and the elderly [2]. Dairy and soy are the main sources of
protein in nutritional beverages and herbal extract products, with a whey protein concentration of 80%
the most widely used. However, fish is an excellent source of protein, with proven satiating effects and

Catalysts 2020, 10, 205; doi:10.3390/catal10020205 www.mdpi.com/journal/catalysts

Catalysts 2020, 10, 205 2 of 12

higher protein content than most terrestrial animals. The proper utilization of limited aquatic resources
has been a topic of great interest for many decades. The use of proteases in fish processing leads to the
hydrolysis of proteins in the source material, which can then be separated from the muscle [3,4] and
used in beverage or herbal products.
Anguillid eels are one of the main high value species of fish used in aquaculture, with the Japanese
eel (Anguilla japonica) and European eel (Anguilla anguilla) the most popular [5,6]. World production
and consumption of Anguilla sp. eels in 1987 was ~100,000–110,000 tons and ~70–80% of it was
produced and consumed in Japan and Taiwan [7]. Eel has been an important aquatic export in these
countries for nearly 30 years. In recent years, there has been a severe shortage of Anguilla japonica glass
eels, as artificial propagation of freshwater eels had not yet been commercially successful. Most eel
seedlings come from natural fishing, which has prompted a search for other Anguilla species, such
as Anguilla marmorata. These adult eels are greyish-yellow with a white belly and brownish-black
marbling on their backs that can fade over time. Mass production of Anguilla marmorata has been
cultivated gradually and its breeding habits are still being explored, but little research exists on its
functionality [8]. Anguilla marmorata are not only economically valuable, but are also rich in nutritional
value due to their high levels of proteins, Carnosine, vitamins, and minerals [9,10]. However, research
into Anguilla marmorata is relatively rare. Recent research into fish protein hydrolysates (FPHs) and
their antioxidant activity, for instance skin gelatin hydrolysates from brownstripe red snapper or meat
protein hydrolysates from yellowstripe trevally (Selaroides leptolepis) [11,12], show that hydrolysis can
release functional peptides which may be used in numerous food products. R. Hartmann and H. Meisel
(2007) pointed out that many peptides released in vitro or in vivo from animal or plant proteins are
bioactive and have regulatory functions in humans beyond normal adequate nutrition. Different health
effects have been attributed to food-derived peptides [13]. Water-holding and fat-binding capacities
are functional properties that are closely related to texture based on the interactions between water, oil,
and other components [14]. Protein hydrolysates rich in bioactive compounds represent promising
ingredients for food and industrial applications. Some recent studies have gone beyond producing
and characterizing EPH and have tested their ability to fortify foods [15,16]. Because the protein
hydrolysates characterization and process application properties research of Anguilla marmorata is
relatively few, and protein hydrolysates are often used in nutritional supplements. Therefore, the
objectives of this study are to compare the use of three commercial enzymes (alcalase, bromelain and
papain) in the production of eel protein hydrolysates (EPHs) and to characterize these EPHs, compare
their chemical, functional properties, and sensory properties, and application to herbal eel extracts.

2. Results and Discussion

2.1. Enzyme Activity and Degree of Hydrolysis

Before hydrolysis, an enzyme activity assay was performed to estimate the quality of the respective
enzyme. Proteolytic activity for each enzyme was: alcalase 2.4 L at 2500 U/mg, papain at 10,000 U/mg,
and bromelain at 1250 U/mg. Alcalase is produced from microbes while the other two enzymes are
produced from plants. Of the three, bromelain had the lowest activity level. Degree of hydrolysis (DH)
is defined as the percentage of peptide bonds cleaved and it is the standard parameter commonly
used to monitor and compare the level of protein proteolysis, which also affects protein solubility,
emulsification, and foaming. Protein hydrolysates with high solubility can be easily mixed into liquid
and have excellent wettability. Different DH values affect the structure and amino acid composition.
Figure 1a shows the DH for alcalase, bromelain, and papain at optimal pH and temperature (pH 7.5
and 55 ◦ C, pH 4.5 and 45 ◦ C, and pH 6.0 and at 50 ◦ C, respectively) for different reaction times
(from 1 h to 16 h). Figure 1b shows DH depending on the amount of enzyme added (from 0.1 to
2.0 g/g). In Figure 1a, there are significant differences in DH between the enzymes, as alcalase shows
greater hydrolytic ability than bromelain or papain. Similar results are seen in Figure 1b, with alcalase
still showing the greatest degree of hydrolysis. The functionality of protein hydrolysates is a major
 Catalysts 2020, 10, 205 3 of 12

Catalysts 2020, 10, x FOR PEER REVIEW 3 of 13

factor in their success as functional supplements in food. The physicochemical properties of protein
95 hydrolysates
properties depend
of protein on the protein
hydrolysates substrate,
depend on thethe specificity
protein of the
substrate, enzyme
the usedoffor
specificity theproteolysis,
enzyme used and
96 the
for hydrolysisand
proteolysis, conditions [14]. conditions [14].
the hydrolysis
97

98
99 Figure1.1.(a)(a)
Figure Degree
Degree ofofhydrolysis
hydrolysis ofofeeleel(Anguilla
(Anguilla marmorata)
marmorata) atat differenttimes
different timeswith
withthree
threedifferent
different
100 commercial
commercial proteases
proteases (alcalase,
(alcalase, bromelain,
bromelain, and
and papain)
papain) atatoptimal
optimal pH pHand
andtemperature;
temperature;(b)
(b)Degree
Degree
101 of hydrolysis of eel (Anguilla marmorata) with different amounts of commercial
of hydrolysis of eel (Anguilla marmorata) with different amounts of commercial proteases (alcalase, proteases (alcalase,
102 bromelain,and
bromelain, and papain)
papain) atatoptimal
optimalpHpH andandtemperature. Means
temperature. in theinsame
Means the form
samewith various
form with characters
various
103 have significant differences (p < 0.05). Data are expressed as mean ± SD, from
characters have significant differences (p˂0.05). Data are expressed as mean±SD, from experiments experiments performed
104 in triplicate.
performed in triplicate.
2.2. Molecular Mass Distribution Profile
105 2.2. Molecular Mass Distribution Profile
Prior to enzymatic hydrolysis, the peptide molecular mass distribution of eel (Anguilla marmorata)
106 Prior to enzymatic hydrolysis, the peptide molecular mass distribution of eel (Anguilla
was analyzed via high performance liquid chromatography (HPLC). All EPHs were reduced to smaller
107 marmorata) was analyzed via high performance liquid chromatography (HPLC). All EPHs were
peptides with molecular mass between 1056.55 Da and 1554.53 Da (Table 1). Small molecule proteins
108 reduced to smaller peptides with molecular mass between 1056.55Da and 1554.53Da (Table 1). Small
and peptides are easier for the human body to digest and absorb which mainly include dipeptide or
109 molecule proteins and peptides are easier for the human body to digest and absorb which mainly
tripeptide [17].
110 include dipeptide or tripeptide [17].
Table 1. Average molecular mass of peptides in eel (Anguilla marmorata) following hydrolysis with
111 Table 1. Average
alcalase, molecular
bromelain, mass of peptides in eel (Anguilla marmorata) following hydrolysis with
and papain.
112 alcalase, bromelain, and papain
Enzyme Molecular mass (Da)
Alcalase 1056.55 ± 2.60 a
EPH Enzyme
* Bromelain Molecular
1482.24 ± 3.36 b mass (Da)
Papain 1554.53 ± 19.22 c
* Prior to enzymatic hydrolysis, eel (Anguilla marmorata) were analyzed by HPLC. a–c Different letters in the same
Alcalase
column denote significant differences between hydrolysates (p < 0.05). 1056.55±2.60a

2.3. Amino Acid Composition

Bromelain 1482.24±3.36b
EPH*
Amino acid levels in the hydrolyzed EPHs were higher than in unhydrolyzed eels. Among the
20 types of standard amino acids, there are 9 essential amino acids (EAAs) thatc adults cannot produce
Papain 1554.53±19.22
and must obtain from their diet; arginine (Arg), in particular, is essential for babies. Among the EPHs,
113 the levels
* Prior of histidine
to enzymatic (His), threonine
hydrolysis, (Thr), valine
eel (Anguilla (Val),were
marmorata) isoleucine
analyzed(Ile),by
and leucine
HPLC. (Leu) were
a-c Different higher
letters in
114 with
the samealcalase
columnthan forsignificant
denote bromelaindifferences
or papainbetween
(Table 2). In fact, the(p<0.05).
hydrolysates alcalase EPH provided the equivalent
of the World Health Organization’s recommended daily intake of EAAs needed to meet the protein
115 requirement
2.3. Amino Acidfor adults. Protein hydrolysates are mainly used in health products and energy drinks,
Composition
but hydrolysis results in protein structural changes, causing bitterness. Since bitterness was produced
116 Amino acid
by proline (Pro),levels
Leu, in the
Val, hydrolyzed
and EPHs(Phe),
phenylalanine were an
higher than
herbal eelinextract
unhydrolyzed
recipe was eels.
usedAmong the
to remove
117 20 types of standard amino acids, there are 9 essential amino acids (EAAs) that adults cannot produce
118 and must obtain from their diet; arginine (Arg), in particular, is essential for babies. Among the EPHs,
119 the levels of histidine (His), threonine (Thr), valine (Val), isoleucine (Ile), and leucine (Leu) were
120 higher with alcalase than for bromelain or papain (Table 2). In fact, the alcalase EPH provided the
Catalysts 2020, 10, 205 4 of 12

the unpleasant taste. The amino acid composition of the hydrolyzed eel protein solution could help
supply adults with EAAs and be used to develop nutritional supplements in the future.

Table 2. Total amino acids of the three EPHs (A = alcalase, B = bromelain, P = papain) and recommended
amino acid requirements of adults (WHO, 2002). Branched chain amino acids are highlighted in bold
font. WHO = World Health Organization.

Amino Acid Eel A B P WHO

His * 0.81 1.01 0.76 0.95 1.50
Thr * 0.63 2.98 2.75 0.71 2.30
Val * 0.31 1.66 3.07 2.23 3.90
Ile * 1.87 1.93 1.27 2.15 3.00
Leu * 1.74 2.85 1.48 0.77 5.90
Asp 4.51 5.14 5.33 6.44 -
Glu 7.18 7.10 6.96 6.95 -
Ser 0.80 1.48 2.31 0.92 -
Gly 0.30 6.38 5.58 3.09 -
Ala 5.83 5.03 5.02 6.37 -
Pro 1.06 1.37 0.70 1.02 -
Arg 0.41 2.04 1.95 0.55 -
Phe * 1.12 1.19 0.29 0.98 -
* required for adults. Results are expressed as the mean ± SD from triplicate determinations. Values in the same
column with different superscript letters are significantly different (p < 0.05).

2.4. Functional Properties of EPHs

The functional properties of the hydrolyzed protein solution are affected by amino acid composition
and the molecular weight of the peptides. The solubility, emulsifying properties, stability, turbidity,
color, oil binding capacity, and flavor are important in food processing. Functional properties can also
affect the sensory evaluation, especially for chewy and smooth textures.

2.4.1. Emulsifying Properties

EAI is a function of oil volume fraction, protein concentration, and the type of equipment used to
produce the emulsion [14] EAI (m2 g−1 ) and ESI (min) of EPHs at different concentrations (0.5%, 1%,
and 2% w/v) are shown in Table 3. All EPHs had reduced EAI as concentration increased. The most
significant difference among the EPHs was at 0.5% (w/v) concentration, with Alcalsae having the highest
EAI (36.8 ± 2.00 m2 g−1 ) and papain having the lowest (16.2 ± 1.22 m2 g−1 ). ESI was also significantly
impacted by concentration. Similar to EAI, differences in ESI were largest at a concentration of 0.5%
(w/v). Alcalase had the highest ESI (4.00 ± 0.34 min) at a concentration of 2% (w/v). Emulsification
occurs during homogenization when proteins are absorbed at the surface of the oil droplets as they
form, creating a membrane which prevents them from consolidating [18].

Table 3. Emulsifying activity index (EAI, m2 g−1 ) and emulsion stability index (ESI, min) at different
EPH concentrations (0.5%, 1%, and 2% w/v) and oil binding capacities (OBC, g/g).

(EAI m2 g−1 ) ESI (min) OBC (g/g)

EPH conc. 0.50% 1% 2% 0.50% 1% 2% -
Alcalase 36.8 ± 2.00 a 19.7 ± 1.00 a 11.3 ± 0.28 a 1.11 ± 0.31 a 2.70 ± 0.20 a 4.00 ± 0.34 a 1.58 ± 0.07 a
Bromelain 21.3 ± 1.30 b 10.2 ± 1.99 b 8.59 ± 0.87 b 0.92 ± 0.89 b 1.92 ± 0.29 b 2.62 ± 0.44 b 1.21 ± 0.01 b
Papain 16.2 ± 1.22 c 8.63 ± 0.58 c 3.25 ± 0.10 c 0.81 ± 0.96 c 1.37 ± 0.75 c 1.44 ± 0.09 c 1.12 ± 0.01 b
Values are given as mean ± SD from triplicate determinations (n = 3). a–c Different letters in the same column denote
significant differences between hydrolysates (p < 0.05).
Catalysts 2020, 10, 205 5 of 12

2.4.2. Oil Binding Capacity (OBC)

Oil binding capacity was determined by physical methods, the density of oil in the bulk, and
use the ratio of the mass of bound liquid oil to the solid fat content. In this study, alcalase has
the highest OBC value of the three EPHs (Table 3). The oil binding capacity of a protein affects its
functional properties and the taste of the end product. Water-holding and fat-binding capacities are
functional properties that are closely related to texture, due to the interactions between water, oil, and
other components.

2.5. Antioxidant Properties of EPHs

Antioxidants are very important in food preservation, and also play an important role in
improving immunity and delaying human aging. In Figure 2a, DPPH free radical scavenging activity
was compared for the three enzymes (alcalase, bromelain, and papain) at different times. Bromelain
had significantly higher scavenging activity than the other enzymes. Figure 2b shows the DPPH free
radical scavenging activity of EPHs at different concentration. The results for alcalase are significantly
higher. Alcalase addition ratios of 1.0 and 2.0 resulted in significantly higher scavenging ability.
Figure 2c,d used the reducing power assay to compare antioxidant activity at different times and with
different amounts of the enzymes. Of the three, the alcalase hydrolysate had the highest reducing
power. Figure 2e,f compare the impact hydrolysis time and the amount of enzyme have on ABTS
total antioxidant capacity. Of the three, bromelain has a significantly higher ABTS total antioxidant
capacity. Antioxidant tests were performed for three different mechanisms. As such, the antioxidant
capacities of the EPHs are clearly different, since they were hydrolyzed by different enzymes, with
different hydrolysis times, and with differing amounts of enzymes. However, based on all three assays,
the Catalysts
alcalase2020,
hydrolysate appears
10, x FOR PEER to have the greatest total antioxidant capacity.
REVIEW 6 of 13

Figure 2. Cont.
 Catalysts 2020, 10, 205 6 of 12

177
178 Figure 2. Antioxidant
Figure effect of
2. Antioxidant the three
effect of theEPHs (alcalase,
three EPHsbromelain,
(alcalase, and papain) using
bromelain, three antioxidant
and papain) using three
179 assays: (a) Antioxidant effect at different times based on DPPH free radical
antioxidant assays: (a) Antioxidant effect at different times based on DPPH free radical scavenging assay; (b)
scavenging
180 Antioxidant
assay; (b)effect at differenteffect
Antioxidant enzyme at concentrations
different enzyme basedconcentrations
on DPPH free radical
based scavenging
on DPPH assay; (c)
free radical
181 Antioxidant effect at different times based on reducing power assay; (d) Antioxidant
scavenging assay; (c) Antioxidant effect at different times based on reducing power assay; (d) effect at different
182 enzyme concentrations
Antioxidant effect at based on reducing
different enzyme power assay; (e) Antioxidant
concentrations based on effect at different
reducing powertimeassay;
based (e)
183 on ABTS total antioxidant capacity; (f) Antioxidant effect at different enzyme concentrations
Antioxidant effect at different time based on ABTS total antioxidant capacity; (f) Antioxidant effect based on at
184 ABTS total antioxidant capacity. Means in the same form with various characters
different enzyme concentrations based on ABTS total antioxidant capacity. Means in the same form denote significant
185 differences (p < 0.05).
with various Data are
characters expressed
denote as mean±SD
significant from
differences triplicate
(p˂0.05). determinations.
Data are expressed as mean±SD from
186 2.6. Nitrogen
triplicate determinations.
Solubility of EPH and Herbal Eel Extracts

187 Solubility
2.6. NitrogenisSolubility
often considered
of EPH andthe most
Herbalimportant physicochemical property in protein hydrolysates,
Eel Extracts
especially when considered in terms of fortifying herbal extracts. Many other functional properties,
188 such as emulsification
Solubility is often considered the most important physicochemical property in protein
Catalysts 2020, 10, x FORand
PEER foaming,
REVIEW are affected by solubility. Figure 3a shows the nitrogen solubility
189 profile
hydrolysates, especially when considered in terms of fortifying herbal extracts. Many 7other of 13
as a function of pH for EPHs produced from different enzymes. All three EPHs had high
190 solubility
functional properties, such as emulsification and foaming, are affected by solubility. Figure 3(a)
193 alkaline(more than 80%)
conditions. in different
Among them,pHEPH conditions
produced and 85%alcalase
from solubility in alkaline
performed conditions.
the best. At lowAmong
pH, the
191 them,
shows the nitrogen solubility profile as a function of pH for EPHs produced from different enzymes.
194 EPH produced
charges on the weaklyfrom alcalase
acidic performed
and basic the best. At low
side-chains pH,amino
of the the charges
acidsonbecome
the weakly
less acidic andand
soluble,
192 basic
All three EPHs had high solubility (more than 80%) in different pH conditions and 85% solubility in
195 side-chains
resulted of the amino[18].
in precipitation acidsThe
become less soluble,
reduced and
solubility ofresulted in precipitation
the alcalase hydrolysate [18].
may The
bereduced
due to the
196 solubility
greaterofproportion
the alcalase of hydrolysate
small molecule mayamino
be due to the
acids duegreater proportion
to their hydrophilicof small molecule
relationship to amino
the water
197 acids due to their
molecules priorhydrophilic
to nitrogen relationship
solubility to the water
analysis. Asmolecules
shown inprior
Figureto nitrogen
3(b), the solubility
pH of theanalysis.
herbal eel
198 As shown in Figure
extract was 7.0±1,3b,andthe pH of the
it showed herbal
good eel extract
solubility at higher 7.0 ±
waspH. The1, beverage
and it showed
matrixgood solubility
will contain other
199 at higher pH. The
compounds thatbeverage
promotematrix will contain
or hinder other
solubility. compounds
Higher solubilitythat promote
gives or hinder
food and solubility.
beverage products
200 Higher solubility appearance
an appealing gives food and andbeverage
a smoothproducts an appealing appearance and a smooth mouthfeel.
mouthfeel.
201

202
203 Figure 3. Nitrogen
Figure solubility
3. Nitrogen (%) of
solubility the
(%) ofthree EPHsEPHs
the three in (a)in
water at different
(a) water pH levels
at different pH (2, 4, 6,(2,
levels 8, 4,
and6, 10)
8, and
204 and10)
(b) herbal eel extracts. Means in the same form with various characters have significant
and (b) herbal eel extracts. Means in the same form with various characters have significant differences
205 (p <differences are˂ expressed
0.05). Data (p 0.05). Dataas mean
are ± SD from
expressed triplicate
as mean determinations.
± SD from triplicate determinations.

206 2.7. Color of EPH and Herbal Eel Extracts

207 In the food industry, food color is the deciding factor in consumers’ overall acceptance of a
208 product. The color of eel protein hydrolysates is based on the ingredients, enzymes, and hydrolysis
209 conditions. At a concentration of 15% (w/v), there were significant differences between the EPHs. As
Catalysts 2020, 10, 205 7 of 12

2.7. Color of EPH and Herbal Eel Extracts

In the food industry, food color is the deciding factor in consumers’ overall acceptance of a
product. The color of eel protein hydrolysates is based on the ingredients, enzymes, and hydrolysis
conditions. At a concentration of 15% (w/v), there were significant differences between the EPHs. As
summarized in Table 4, L* represents brightness, while a* and b* represent the color space. Both
the EPHs and the herbal eel extract were brownish-yellow in color. Specifically, all three EPHs were
yellowish in color, although alcalase (3.96 ± 0.04) was darker than both bromelain (6.37 ± 0.43) and
papain (8.10 ± 0.18). Based on a* value, alcalase (0.42±0.03) was preferable to bromelain (0.78 ± 0.02)
and papain (0.28 ± 0.01). The same trend was seen with the b* value, with alcalase (0.74 ± 0.07) again
preferable to both bromelain (2.39 ± 0.02) and papain (5.75 ± 0.10). The color differences were mainly
due to the ingredient sources and enzymes. Alcalase had the lowest brightness and therefore showed
the darkest color following hydrolysis.

Table 4. Color analysis of the three EPHs (alcalase, bromelain, and papain) at concentration of 15%
(1.5 g/10 mL).

Alcalase Bromelain Papain

L* 3.96 ± 0.04 c 6.37 ± 0.43 b 8.10 ± 0.18 a
EPH a* 0.42 ± 0.03 b 0.78 ± 0.02 a 0.28 ± 0.01 c
b* 0.74 ± 0.07 c 2.39 ± 0.02 b 5.75 ± 0.10 a
Herbal extract L* 2.17 ± 0.00 c 2.59 ± 0.04 b 4.52 ± 0.07 a
with 15% EPH a* 0.98 ± 0.00 a 0.62 ± 0.00 c 0.78 ± 0.11 b
(1.5 g/10 mL) b* 2.68 ± 0.00 c 3.75 ± 0.06 b 5.84 ± 0.05 a
Values are given as mean ± SD from triplicate determinations (n = 3). a–c Different letters in the same column denote
significant differences between hydrolysates (p < 0.05).

3. Materials and Methods

3.1. Materials
2,2-diphenyl-1-picrylhydrazyl, phthaldialdehyde, and trifluoroacetic acid were purchased from
Alfa Aesar (Haverhill, MA, USA). Glycerol and ascorbic acid were purchased from R&D Systems
(Minneapolis, MN, USA). Glacial acetic acid was purchased from ThermoFisher Scientific (Waltham,
MA, USA). Anhydrous iron (III) chloride, citric acid monohydrate, and sodium tetraborate decahydrate
were purchased from Showa Chemical Industry Company (Tokyo, Japan). Methanol, hydrochloric
acid, sodium bicarbonate, and anhydrous sodium acetate were purchased from Aencore Chemical
Co. (Melbourne, VIC, Australia). Disodium (hydrogen) phosphate was purchased from J.T. Baker
(Phillipsburg, NJ, USA). Hydrogen peroxide was purchased from SHOWA (Japan). Alcalase®2.5 L
was purchased from Strem Chemicals (Newburyport, MA, USA, CAS number 9014-01-1), and papain
(CAS number 9001-73-4) and bromelain (CAS number 9001-00-7) were purchased from ChappionBio
(Taiwan). Olive oil was purchased from Carrefour (Taiwan).

3.2. Methods

3.2.1. Anguilla Marmorata Raw Material Preparation

Anguilla marmorata were provided by the Honya eel farm (Dongshi Township, Cjiayi County,
Taiwan). The heads of the eels were excised, and the skin and bones were removed. The remaining fish
was sterilized at 121 ◦ C for 30 min before storage at −20 ◦ C for subsequent experiments and analysis.

3.2.2. Anguilla Marmorata Meat Hydrolysate Preparation

1 g eel meat was added to 0.1 M phosphate-citrate buffer solution (pH 7.5) at a 1:50 ratio then
homogenized, before the addition of different concentrations of the three enzymes, as shown in Table 5.
 Catalysts 2020, 10, 205 8 of 12

The enzyme was inactivated by heating the solution to 95 ◦ C for 20 min before it was freeze-dried to
obtain EPH. The sample was then stored at −20 ◦ C until further analysis. The scheme of the hydrolysis
process of Anguilla marmorata is shown in Figure 4.

Table 5. Parameters used to obtain the optimal hydrolysis conditions for eel, using three
different enzymes.

Enzyme
Factors Unit Symbol
Alcalase Bromelain Papain
Temperature ◦C T 55 55 45
Time h t 1.0, 2.0, 4.0, 8.0, 16.0
Enzyme
% E/S 0.125, 0.25, 0.5, 1.0, 2.0
concentration
Catalysts 2020, 10, x FOR PEER REVIEW 9 of 13

255
Figure 4. Scheme of the hydrolysis process of Anguilla marmorata.
256 Figure 4. Scheme of the hydrolysis process of Anguilla marmorata.
3.2.3. Herbal Eel Extracts
257 Table 5. Parameters used to obtain the optimal hydrolysis conditions for eel, using three different
258 The recipe of herbal eel extracts is to add 23 g of wolfberry, 20 g of red dates, 8 g of Astragalus
enzymes
propinquus, 8 g Angelica sinensis, 6 g Cinnamomum cassia, 6 g Licorice to the medicine bag, and put into
the 1000 mL water contains 49 g sugar and 6 g salt for boil 30 min together.
Enzyme After cooling down remove
Factors
the medicine bag and mix Unit Symbol
with 500 mL of 15% EPH as herbal eel extract for assay.
Alcalase Bromelain Papain
3.2.4. Determination of Degree of Hydrolysis (DH)
Temperature ℃ T 55 55 45
The degree of hydrolysis (DH), defined as the percentage ratio of the number of peptide bonds
cleaved to the total number of peptide bonds available in the substrate, was monitored throughout
Time h t 1.0, 2.0, 4.0, 8.0, 16.0
the reaction. 100 ppm serine (Seine) standard and a sample containing 0.08% protein content were
addedconcentration
Enzyme to 150 µL OPA colorant
% (1 E/S
mL 4% o-phthalaldehyde 0.125,in0.25,
ethanol added
0.5, 1.0, 2.0 to 40 mL 1250 ppm
sodium dodecyl sulfate in 0.125 M sodium tetraborate buffer solution, further dissolved in 44 mg of
dithiothreitol to 50 mL). The reaction was allowed to stand for 2 min, before absorbance was measured
259 3.2.3. Herbal Eel Extracts
by an ELISA reader at 340 nm [19].
260 The recipe of herbal eel extracts is to add 23g of wolfberry, 20g of red dates, 8g of Astragalus
261 DH (%) = [(OD(Sample)
propinquus, − OD(Blank))/(OD(Standard)
8g Angelica sinensis, − OD(Blank))
6g Cinnamomum cassia, × 0.9516
6g Licorice meqv/L
to the × 0.1/(Xbag,
medicine × P) and
− β]/α/h × 100%
puttotinto
262 the 1000mL water contains 49g sugar and 6g salt for boil 30 min together. After cooling down remove
263 thewhere OD bag
medicine (Standard)
and mix = with
standard
500mLabsorbance,
of 15% EPH ODas(Sample) = sample
herbal eel assay. OD(Blank) = blank
absorbance,
extract for
absorbance, X = g sample, and P = protein % in sample.
264 The values ofof
3.2.4. Determination the constants
Degree α, β, and h(DH)
of Hydrolysis tot (total number of peptide bonds per protein equivalent,
which is dependent on the amino acid composition of the raw material) for fish protein are estimated
265 toThe degree
be 1.00, ofand
0.40, hydrolysis (DH), defined as the percentage ratio of the number of peptide bonds
8.6, respectively.
266 cleaved to the total number of peptide bonds available in the substrate, was monitored throughout
267 the reaction. 100 ppm serine (Seine) standard and a sample containing 0.08% protein content were
268 added to 150 μL OPA colorant (1 ml 4% o-phthalaldehyde in ethanol added to 40 ml 1250 ppm
269 sodium dodecyl sulfate in 0.125 M sodium tetraborate buffer solution, further dissolved in 44 mg of
270 dithiothreitol to 50 ml). The reaction was allowed to stand for 2 min, before absorbance was measured
271 by an ELISA reader at 340 nm [19].
272
Catalysts 2020, 10, 205 9 of 12

3.2.5. Molecular Mass Identification

Each EPH was filtered and its molecular mass determined by HPLC (Hitachi, Chromaster, Japan)
and BioSep-SEC-S 2000 (600 × 7.8 mm). The mobile phase used was 45% (v/v) acetonitrile with 1%
trifluoracetic acid buffer at a rate of 1 mL/min by gradient. 25 µL of the sample was injected and
determined at 214 nm. The molecular mass was analyzed by the standard curve with standard proteins
maltose (360 Da), apoinin (6500 Da), ribonuclease (13,700 Da), and carbonic anhydrase (29,000 Da).

3.2.6. Analysis of Amino Acid Composition

The sample or standard was dissolved in 0.1 N HCl, before 50 µL was added to 250 µL of
50 mM NaHCO3 (pH 8.1) and 200 µL of DABS-Cl solution (1.3 mg/mL) was dissolved in acetonitrile.
The reaction was shaken for 5 min, then placed in a water bath at 70 ◦ C for 12 min. After cooling to
room temperature (about 5 min), 300 µL of 70% ethanol was added to stop the reaction and the sample
was filtered through a 0.45 µm PVDF filter, using methods previously described [20].
Mobile phase:
(1) Solution A: 25 mM sodium acetate solution (pH 6.5) containing 4% dimethylformamide
(2) Solution B: acetonitrile
Flow rate setting: 1 mL/min
Gradient setting: initial concentration (solution A: solution B = 85: 15)
0~5 min (solution A: solution B = 78.7: 21.3)
5~60 min (solution A: solution B = 70: 30)
60~70 min (solution A: solution B = 30: 70)
70~75 min (solution A: solution B = 30: 70)
75~76 min (solution A: solution B = 30: 70)
76~80 min (solution B: solution C = 15: 85)
80~86 min (solution A: solution B = 85: 15)
86~90 min (solution A: solution B = 85: 15)
Column: Inspire 5 µm C18, 250 × 4.6 mm
Detection wavelength: 436 nm

3.2.7. Functional Properties

Nitrogen Solubility of EPH and Herbal Eel Extracts

Nitrogen solubility was initially determined in distilled water over a range of pH values (2, 4,
6, 8, and 10), as described elsewhere [14]. 200 mg of EPH was added to 30 mL distilled water and
mixed, then centrifuged at room temperature (5000 rpm, 20 min) and filtered (ADVANTEC paper
no. 1). The nitrogen content of the resulting supernatant was determined using the Kjeldahl method
and the nitrogen solubility was calculated as follows:

N1
Nitrogen solubility(%) = × 100
N0

where N1 = supernatant nitrogen concentration and N0 = sample nitrogen concentration. Solubility

analysis was carried out in triplicate. Subsequently, nitrogen solubility of the herbal eel extracts was
determined following the method above.

Emulsifying Properties
The emulsion activity index (EAI) and emulsion stability index (ESI) of each EPH was determined
with some modifications [14]. EPHs were reconstituted in distilled water (15 mL) at concentrations of
0.5%, 1%, and 2% (w/v). Olive oil (5 mL) was homogenized with the EPH solution for 1 min at room
temperature. 50 µL aliquots of the emulsion were taken from the bottom of the conical flask directly
Catalysts 2020, 10, 205 10 of 12

after homogenization and again 10 min later, then diluted 100-fold in 0.1% (w/v) SDS solution. The new
solution was mixed for 10 s and the absorbance measured at 500 nm
2 × 2.303 × A0
ESI m2 g−1 =
0.25 × Protein weight ( g)

∆A
ESI = ×t
A0
where A = absorbance, ∆A = (A0 − A10) and t = 10 min (A0 = Absorbance at 0 min, A10 = absorbance
at 10 min).

3.2.8. Antioxidant Capacity Analysis

Analysis of Reducing Power

Ascorbic acid was used as a standard. 200 µL of the standard and sample were mixed with 200 µL
of 200 mM phosphate buffer solution (pH 6.6) and 200 µL of 1% potassium ferricyanide, then allowed
to react in a 50 ◦ C water bath for 20 min. 200 µL of 10% trichloroacetic acid solution was then added,
and 200 µL of the supernatant was combined with 200 µL of distilled water and 40 µL of a 0.1% ferric
chloride solution dissolved in 10% HCl. After standing for 10 min, the absorbance of the solution was
measured using an ELISA plate reader at a wavelength of 700 nm [21].

Analysis of DPPH Free Radical Scavenging Ability

Using ascorbic acid as a standard, 160 µL of the standard and sample were added to 40 µL of
1 mM DPPH dissolved in methanol, then protected from the light for 30 min. The absorbance was
measured using a reader at a wavelength of 517 nm [22].

ABTS Radical-Scavenging Activity Assay

The stock solution of 2,20 -azino-bis-(3-ethylbenzothiazoline-6-sulfonic acid (ABTS) radical was
produced by reacting 7mM ABTS with 2.45mM K2 S2 O8 (final concentration) and allowing the mixture
to stand in dark incubation for 12 h at room temperature condition. The ABTS radical working solution
was diluted with phosphate buffered saline (PBS) (pH7.4) at 730 nm to absorbance of 0.70 (±0.02). 1 mL
of diluted solution is added with 10 µL of different sample and reacted for a minute, then tested under
730 nm [23].

3.2.9. Color of EPH and Herbal Eel Extracts

Aliquots of EPH were diluted to 15% (w/v) and the herbal eel extracts to measured three times by a
color difference meter (Nippon Denshoku Industries Co., Ltd., SA2000, Japan). L* denoted brightness,
a* indicated red green and b* indicated yellow-blue values [24].

3.2.10. Statistical Analysis

Data are expressed as the mean±standard deviation of three independent replicates. Statistical
analysis was performed using the software SPSS statistics v24. All data were analyzed using one-way
analysis of variance (ANOVA). Post hoc analysis of group differences was performed using Duncan’s
new multiple range tests. The criterion for significance was set at p < 0.05.

4. Conclusions
This study has shown that eel (Anguilla marmorata) are a good source of highly nutritious
protein. Through hydrolysis, three commercial enzymes (alcalase, bromelain, and papain) produced
low molecular mass peptides that are easy to digest. Among the three, the EAA content in the
alcalase-hydrolyzed EPH was the most similar to the daily dose recommended by WHO. EAI was
Catalysts 2020, 10, 205 11 of 12

highest with the alcalase EPH (36.8 ± 2.00), while the bromelain and papain EPHs had EAI values of
21.3 ± 1.30 and 16.2 ± 1.22, respectively. Alcalase EPH also had a higher solubility (>80%) in a pH range
of 2 to 10, as well as demonstrating the greatest antioxidant capacity. Finally, the alcalase-hydrolyzed
EPH had a brighter color, good emulsifying properties, and showed good solubility, making it useful
in food processing. In future studies of our research will investigate the fractionation, purification,
structure identification of EPH then evaluate human nutrition efficacy by animal model assessment
potential application of EPH.

Author Contributions: Conceptualization, L.-T.H., Y.-W.C., and C.-Y.H.; Data curation, I-C.C., J.-X.L., Y.-W.C., and
J.-Y.C.; Formal analysis, I-C.C., J.-X.L., and Y.-W.C.; Funding acquisition, Y.-W.C., J.-Y.C., and C.-Y.H.; Investigation,
I-C.C. and J.-X.L.; Methodology, I-C.C., J.-X.L., and C.-Y.H.; Project administration, C.-Y.H.; Resources, J.-Y.C.;
Software, I-C.C., Y.-W.C., and C.-Y.H.; Supervision, L.-T.H. and J.-Y.C.; Validation, J.-X.L. and L.-T.H.; Visualization,
J.-X.L. and L.-T.H.; Writing—original draft, L.-T.H. and C.-Y.H.; Writing—review and editing, J.-Y.C. and C.-Y.H.
All authors have read and agreed to the published version of the manuscript.
Funding: This research was funded by the National Kaohsiung University of Science and Technology, grant
number NKUST-108D04 and The APC was funded by National Kaohsiung University of Science and Technology,
grant number NKUST-108D04.
Conflicts of Interest: The authors declare no conflict of interest. The authors alone are responsible for the content
and writing of the manuscript.

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