marine drugs

Article
Characterization and Antioxidant Activity of
Collagen, Gelatin, and the Derived Peptides from
Yellowfin Tuna (Thunnus albacares) Skin
Mala Nurilmala 1, *, Hanifah Husein Hizbullah 1 , Euis Karnia 1 , Eni Kusumaningtyas 2 and
Yoshihiro Ochiai 3
1 Department of Aquatic Product Technology, Faculty of Fisheries and Marine Science, Bogor Agricultural
University (IPB University), Bogor 16680, Indonesia; hhanifaa@gmail.com (H.H.H.);
euiskarniaa@gmail.com (E.K.)
2 Indonesian Research Centre for Veterinary Science, Bogor 16114, Indonesia; enikusuma@yahoo.com
3 Graduate School of Agricultural Science, Tohoku University, Sendai 980-8572, Japan; yochiai@tohoku.ac.jp
* Correspondence: mnurilmala@ipb.ac.id; Tel.: +62-857-7415-1345




Received: 9 January 2020; Accepted: 30 January 2020; Published: 31 January 2020


Abstract: Skin waste from tuna processing needs to be utilized, such as extraction of its collagen
and gelatin. Their functional properties can be improved by enzymatic hydrolysis for conversion to
peptides. Thus, the research objectives were to examine the characteristics and antioxidant activity of
collagen, gelatin, and the derived peptide from yellowfin tuna skin. Collagen was extracted using
0.75 M acetic acid at 4 ◦ C, while gelatin was prepared using 0.25% citric acid and extracted at 65 ◦ C.
Hydrolysis was carried out with 2% Alcalase, followed by fractionation with a molecular weight cut
off sieve for both collagen and gelatin. Collagen yield was 22.6% with pH value of 6.63 and whiteness
of 96.7%. Gelatin yield was 20.0% with pH value of 4.94 and whiteness of 51.0%. Hydrolysis for
three hours resulted in 52.7% and 45.2% degree of hydrolysis for collagen and gelatin, respectively.
The molecular weights of collagen peptides ranged from 2.94 to 11.93 kDa, while those of gelatin
peptides ranged from 3.54 to 16,620 kDa. Antioxidant activities of these peptides were higher than
those before hydrolysis. The high antioxidant activity (IC50 ) of collagen peptides were found in <3,
3–10, and 10–30 kDa fractions as well as in the gelatin peptides.

Keywords: antioxidant; collagen; fractionation; gelatin; hydrolysate; tuna skin

1. Introduction
Tuna is one of the marine biotas that has high economical value. The global total annual catch of
tuna species was around 7.5 million tons in 2018 based on The State of World Fisheries and Aquaculture
by Food and Agriculture Organization (FAO) [1]. The United States of America market imported
nearly 42,400 tons of fresh and frozen tuna during January to September 2016, at a value of US $439.2
million. Among these imports, frozen tuna fillets took a 51% share, followed by whole, dressed, and
fresh/chilled tuna (43%). Demands for frozen tuna loins in Japan have been increasing about 3% every
year. Thailand, which bought 13% more cooked loins at 26,000 tons for reprocessing, is the largest
importer of tuna raw material in Asia. China, Vietnam, and Indonesia are the main suppliers [2].
Fishermen of nearly 80 nations harvest tunas from the world oceans. Yellowfin tuna (Thunnus albacares)
is the second most important species of tuna, which accounts for about 30% of the global catch [3].
Tunas are commonly produced for canning and freezing. Frozen products are usually in the form of
whole fish or loins. The freshness and quality of tuna meat is usually evaluated based on the oxidation
extent of the muscle pigment, myoglobin [4]. The frozen tuna products could leave bones and skin
wastes. The proportion of tuna skin is about 5–8% of the total fish weight. Tuna bones and skins are

Mar. Drugs 2020, 18, 98; doi:10.3390/md18020098 www.mdpi.com/journal/marinedrugs

Mar. Drugs 2020, 18, 98 2 of 12

generally used only to feed cattle with reduced selling price. For value addition, the skin waste from
industrial tuna loins as well as fillets can be utilized as the materials for collagen and gelatin [5–7].
However, a large amount of tuna skin has remained underutilized, despite the fact that it can be an
excellent source of collagen and gelatin, provided that they are demonstrated to be of high quality. The
bottleneck for the utilization of tuna skin is the high fat content.
Commercial collagen and gelatin are generally produced from the skins and bones of pigs and
cows. However, their usage raises religious issues, because Hinduism forbids consumption of cows,
while Islam and Judaism forbid that of pigs. In addition, there are associated health concerns such as
bovine spongiform encephalopathy (BSE), transmissible spongiform encephalopathy (TSE), and foot
and mouth disease (FMD). Therefore, fishery by-products such as skin, bone, scale, and swim bladder
could be alternatives for collagen and gelatin production [8–10]. The yellowfin tuna skin waste could
be an alternative raw material for making safe and preferable collagen and gelatin.
Collagen is a connective tissue protein found in skin, teeth, and bones. Collagen can be converted
to gelatin by high temperature treatment. Both collagen and gelatin—which contain glycine, proline,
and hydroxyproline as the major amino acids—have significant potential as the initial substrates
to produce bioactive peptides [11,12]. The biological properties of the derived peptides are largely
dependent on the structure and molecular weight. Enzymatic hydrolysis is an effective method for
obtaining bioactive peptides [13]. Recently, the bioactive peptide with antioxidant properties from
enzymatically hydrolyzed proteins is a topic of great interest in the pharmaceutical and food processing
industries [14]. Previous researchers reported that Alcalase showed the most effective among five
kinds of proteases to hydrolyze gelatin from skipjack Katsuwonus pelamis scales, resulting in strong
antioxidant activities of the peptides obtained [8]. Antioxidant supplementation can maintain normal
skin cell function by preventing free radical accumulation [15]. Superior antioxidant activity of peptides
prevents oxidative stress which is the main cause of aging. Biomolecules with strong antioxidant
capacity are widely used in anti-aging research [16]. Under these backgrounds, this study aimed
to investigate the characteristics of tuna skin collagen and gelatin, and the antioxidant activity of
their hydrolysates.

2. Results and Discussion

2.1. Physicochemical Characteristics

Physicochemical characteristics of collagen and gelatin from yellowfin tuna are shown in Table 1.
The yields of collagen and gelatin clearly show the efficiency level of the extraction process. In this
study, the yield of collagen from the tuna skin was higher than those of sail fish (Istiophorus platypterus,
5.76%) [17], ocellate puffer fish (Takifugu rubripes, 10.7%) [18], bighead carp (Hypophthalmichthys nobilis,
17.50%) [19], redbelly yellowtail fusilier (Caesio cuning, 18.4%) [20], and balloonfish (Diodon holocanthus,
4%) [21]. In addition, the gelatin yield in this study (19.97%) was quite high when compared with
that of skipjack tuna (K. pelamis) skin (11.3%) [22], emperor fish (Lethrinus sp.) skin (4.8%) [23], and
K. pelamis bone (6.37%) [24]. The main structures of the skin inner layers and different harvesting ages
could lead to differences in the yield [25].

Table 1. Characteristics of tuna skin collagen and gelatin.

Parameter Collagen Gelatin

Yield (%) 22.63 ± 2.04 19.97 ± 0.79
pH 6.63 ± 0.03 4.94 ± 0.01
Color
L* (lightness) 97.57 ± 0.94 56.46 ± 0.78
a* (redness) 0.93 ± 0.75 2.56 ± 0.55
b* (yellowness) 1.21 ± 1.17 22.38 ± 0.38
Whiteness (%) 96.69 ± 0.35 50.97 ± 0.72
Mar. Drugs 2020, 18, 98 3 of 12

pH is an important factor for the quality of collagen and gelatin. The pH value of tuna skin
collagen preparation was 6.63, which was acceptable in accordance with Indonesian National Standard
for collagen pH range, which is 6.5–8 [26]. The pH value of the gelatin was also acceptable in accordance
with the Gelatin Manufacturers Institute of America standard as Type A, where the optimal pH is in the
range of 3.8–5.5 for the food standard and 4.5–5.5 for the pharmaceutical one [27]. The resulting gelatin
was also found to belong to Type A, as it was prepared by an acidic process. The pH value is influenced
by the type of material used in the pretreatment with acid or base and the neutralization step [22]. It is
known that optimal pH value can make it easier to apply the material to the product. Neutral pH
facilitates the interaction of proteins with water molecules, thereby improving the solubility [28].
Color does not affect the functional ability of the product. However, it remains an important
factor since consumers would prefer products in brighter colors [22]. The whiteness degree of tuna
collagen was 96.69%, higher than those of white snapper collagen (61.33–65.41%) [29] and skate
collagen (88.4%) [30]. In this study, it was shown that the resulting L* value (lightness) and whiteness
of the gelatin gave lower values compared with those of the original collagen. However, the L* value
was higher compared to that of the gelatin from giant catfish (Pangasius gigas) skin (20.43) [31]. The
a* value (redness) increased about two fold by converting the collagen to the gelatin, demonstrating
that the redness was enhanced in the gelatin. On the other hand, the b* value (yellowness) increased
nearly tenfold by the conversion, indicating that the yellowness was intensified in the gelatin. Usually,
the color of commercial gelatin is pale yellow to dark amber [32]. Darker gelatin is commonly due
to inorganic contaminants as well as proteins which are not removed during the extraction process.
The color of gelatin is greatly dependent on fish species or the original color of fish skin, as well as
conditions of extraction and drying [33].

2.2. Molecular Size and Hydrolysis Degree

Molecular size is an important parameter for determining the properties of collagen, gelatin,
and the derived hydrolysates. Their molecular weights were estimated by sodium dodecyl
sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) [34,35] as shown in Figure 1.

Figure 1. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) patterns of tuna

skin collagen and gelatin. A, markers; B, collagen; C, gelatin.
Mar. Drugs 2020, 18, 98 4 of 12

The tuna skin collagen and gelatin showed similar patterns in that both consisted of β, α1, and
α2 subunit bands. They are classified as type I collagen. The molecular weight of collagen was
estimated to be 277.17 kDa. Type I collagen consists of α1 (±120 kDa) and α2 (±110 kDa) chains, and β
(±200 kDa) chain [36,37]. The molecular weight of the β component in the gelatin was estimated to be
226.65 kDa, and those of the α1 and the α2 components were 153.85 kDa and 134.54 kDa, respectively.
Our previous study showed the yellowfin tuna skin gelatin extracted at 65 ◦ C consisted of β component
of 250 kDa, α1 of 129.67 kDa, and α2 of 116.36 kDa [4]. Tuna gelatin has high molecular weight bands
judging from abundant β components [13]. The results showed that the molecular weight of yellowfin
tuna skin gelatin had a similar molecular size range to that of tilapia scale gelatin Oreochromis spp.
(34–260 kDa) [38].
Collagen and gelatin were then hydrolyzed with the proteolytic enzyme Alcalase at a ratio of 2%
(v/v) in order to obtain the hydrolysates. The hydrolysis degree (HD) of these preparations is shown
in Table 2. The HD value, generally used as a parameter of proteolysis and the indicator of protein
hydrolysate ratio, is the ratio of the reduced peptide bonds during hydrolysis to the total amount of the
bonds in the original protein. The HD value may affect the functional properties of the hydrolysate [39].
Alcalase is a proteolytic enzyme of relatively high activity under moderate pH conditions compared
with whose which are active at neutral and acidic pH [40].

Table 2. Hydrolysis degree (HD) of tuna skin collagen and gelatin by 2% (v/v) Alcalase hydrolysis for
three hours.

Sample Hydrolysis Degree (%)

Collagen 52.71 ± 1.54
Gelatin 45.29 ± 0.01

The molecular weight of collagen hydrolysate as estimated by SDS-PAGE was in the range of
2.94–11.93 kDa as shown in Figure 2. Generally, bioactive peptides of high antioxidant activity have a
molecular size of less than 10 kDa [41]. The gelatin hydrolysate in this study was found to fall in the
range of 3.54–16.61 kDa. Guerard et al. reported that hydrolyzed gelatin prepared from tuna waste
produced peptides of about 5–25 kDa [42].

Figure 2. SDS-PAGE patterns of collagen and gelatin hydrolysate. A, markers; B, collagen hydrolysate;
C, gelatin hydrolysate.
Mar. Drugs 2020, 18, 98 5 of 12

2.3. Amino Acid Composition

It is known that collagen and its thermal derivative, gelatin, are rich in amino acids such as glycine
and proline. The amino acid compositions of collagen and gelatin from the yellowfin tuna skin and its
hydrolysates are shown in Table 3. The composition and sequence of amino acids in gelatin depend
on the species of materials, but the major ones are glycine and proline irrespective of the species so
far examined [43]. The length of polypeptide chains is closely correlated with the viscosity of gelatin.
The longer the polypeptide chain, the higher the gelatin viscosity value [33]. In addition, amino acid
composition affects gel strength and the melting point of gelatin [44]. In addition to physical properties,
amino acid composition also affects the functional properties of collagen, gelatin, and the hydrolysates,
especially the antioxidant activity.
The antioxidant properties of peptides are closely related to amino acid composition, structure,
and hydrophobic properties [13]. The amino acid composition of the hydrolysates was very similar to
that of the parent proteins, which were rich in glycine, alanine, proline, aspartic acid, and glutamic
acid. High glycine and proline contents make the antioxidant activity of fish skin gelatin higher than
that of meat protein [45]. Alcalase is able to hydrolyze the bonds of aliphatic or aromatic amino acid
peptides such as leucine, phenylalanine, and tyrosine [46]. Table 3 shows that the hydrolysis process
lowered the amino acid levels of collagen, by maintaining the amino acid composition. The arginine
content, which generally reflects the antioxidant activity, was retained through hydrolysis [47].

Table 3. Amino acid composition of tuna skin collagen, gelatin, and hydrolysates (g/100 g).

Amino Acid Collagen Collagen Hydrolysate Gelatin Gelatin Hydrolysate

Arginine 3.48 3.27 9.16 8.48
Lysine 2.24 1.78 4.21 4.86
Threonine 1.48 1.41 3.40 3.38
Phenylalanine 0.95 0.89 2.68 2.29
Leucine 1.13 1.04 2.68 2.63
Valine 0.96 0.96 2.22 2.27
Isoleucine 0.48 0.44 1.17 1.15
Histidine 0.31 0.29 0.85 0.72
Glycine 10.44 8.85 25.98 24.45
Proline 5.10 4.09 11.94 11.86
Alanine 5.03 3.64 10.50 11.20
Glutamate 5.05 3.34 10.31 11.61
Aspartate 2.73 1.93 5.12 5.83
Serine 1.53 1.35 3.78 3.67
Tyrosine 0.21 0.17 0.59 0.54

2.4. Antioxidant Activity

The antioxidant activity was measured by a 2,20 -azino-bis (3-ethylbenzothiazoline-6-sulphonic
acid) (ABTS) method as well as a 2,2-diphenyl-1-picrylhydrazyl (DPPH) method in order to examine
how much the hydrolysis and fractionation process influenced the antioxidant activities. The ABTS
radical is a radical with a nitrogen center that has the coloration of blue/green, when reduced by
antioxidant compounds. It will turn into a non-radical form, from colored to colorless, whereas DPPH
radicals are violet-colored free radicals that require electron transfer from antioxidants to transform into
a colorless non-radical solution. The antioxidant activities of the collagen, gelatin, and the hydrolysates
are shown in Table 4.
Antioxidants are the molecules capable of quenching reactive oxygen species as well as inhibiting
the oxidation of molecules that can produce free radicals. Antioxidants are electron-donating
compounds (electron donors) or reductants [48]. Antioxidant activity is associated with the progress of
reductions, which have been confirmed to be terminators of chain reactions caused by free radicals [36].
Free radicals are the major factors that can accelerate glycation [49]. They can also cause oxidative
stress and thus various diseases [50]. The antioxidant activities were increased through hydrolysis.
Mar. Drugs 2020, 18, 98 6 of 12

Scavenging activity of 100% represents complete scavenging of ABTS or DPPH radicals. Hydrolysis
processes could increase the scavenging activity for both ABTS and DPPH assays. As shown in Figure 3,
the scavenging activities of collagen, gelatin, hydrolysate, and the fractions against ABTS were much
higher compared with DPPH, as the results represent the scavenging activities against ABTS from
tenfold diluted solution in the concentration range of 10–40 µg/mL. The scavenging activities against
ABTS radicals of the collagen hydrolysate from milkfish (Chanos chanos) skin showed the effectiveness
at 1 mg/mL [51]. However, the scavenging activity of nearly 100% was reported for the peptide from
horse milk at 1 µg/mL [52]. On the other hand, the gelatin hydrolysate from skipjack scale showed the
scavenging activity in the concentration range of 0.1–5.0 mg/mL [8]. The lower the molecular weight,
the stronger the scavenging activity tends to become. These data demonstrate that the antioxidant
activities of our preparations were satisfactorily high.

Figure 3. Scavenging activity (in percentage) of tuna skin collagen, gelatin, and hydrolysates.
Upper figure, 2,20 -azino-bis (3-ethylbenzothiazoline-6-sulphonic acid (ABTS) method; lower figure,
2,2-diphenyl-1-picrylhydrazyl (DPPH) method. The different letters indicate significant differences
(p < 0.05).

In this study, IC50 values were classified as follows: Very strong, <0.05 mg/mL; strong,
0.05–0.10 mg/mL; average, 0.10–0.15 mg/mL; weak, 0.15–0.20 mg/mL; and very weak, >0.20 mg/mL. Lower
IC50 shows higher antioxidant activity [53]. The lower molecular weight peptides tend to show higher
antioxidant activities, although variable molecular weight distributions also provide striking differences
in the antioxidant properties of hydrolysates [54]. The average molecular weight of protein hydrolysates
is one of the most important factors determining biological activity, including the antioxidant one. High
antioxidant activity is generally observed for low molecular weight and severed oligopeptides. In addition,
the specificity of the protease not only affects the peptide size but also the number and sequence of amino
acids in the peptides, which then also affect the antioxidant activity of the hydrolysates [55].
Both ABTS and DPPH methods showed that the highest antioxidant activity of collagen hydrolysate
expressed by IC50 was obtained for the peptide fractions of 3–10 kDa, and for the gelatin peptide
fractions of 10–30 kDa. In this study, the antioxidant activities of gelatin and its hydrolysis were
higher than that of collagen. In addition, antioxidant activity of gelatin was found to be higher in
Mar. Drugs 2020, 18, 98 7 of 12

the ABTS method. This may be due to the fact that the ABTS assay tends to detect the hydrophobic
and hydrophilic compounds, while the DPPH assay is suitable for hydrophobic ones as shown in our
previous report [51,56]. In addition, the enzyme Alcalase can produce hydrolysates with higher radical
scavenging capacity and iron reducing activity compared to the other enzymes such as collagenase,
trypsin, or pepsin [13,45].

Table 4. Antioxidant activities of tuna skin collagen, gelatin, and their hydrolysates.

IC50 (µg protein/mL)

Samples
ABTS DPPH
Collagen 313.29 ± 0.15 f 560.51 ± 0.02 g
Hydrolyzed collagen 66.28 ± 0.12 e 119.10 ± 0.01 f
Peptides >30 kDa 64.47 ± 0.13 e 101.77 ± 0.01 f
Peptides 10–30 kDa 33.02 ± 0.05 cd 83.22 ± 0.01 de
Peptides 3–10 kDa 29.24 ± 0.02 c 75.94 ± 0.01 d
Peptides <3 kDa 35.39 ± 0.07 d 82.12 ± 0.02 de
Gelatin 62.21 ± 0.01 e 654.62 ± 0.03 g
Hydrolyzed gelatin 16.28 ± 0.01 b 78.87 ± 0.01 d
Peptides >30 kDa 19.17 ± 0.01 b 84.15 ± 0.01 e
Peptides 10–30 kDa 9.11 ± 0.01 a 15.12 ± 0.01 a
Peptides 3–10 kDa 17.06 ± 0.01 b 50.04 ± 0.01 c
Peptides <3 kDa 11.20 ± 0.01 ab 23.80 ± 0.01 b
Note: Different superscripts in the same column show significant differences (p < 0.05).

All the results obtained in the present study demonstrated that excellent quality of collagen,
gelatin, and their hydrolysates with high antioxidant activities can be obtained from the skin waste of
the tuna processing industry. These findings will be useful for effective utilization of tuna resources.
In addition, these products will be accepted to those communities where the products from livestock
sources are rejected due to religious reasons.

3. Methods

3.1. Preparation of Collagen

Collagen was extracted according to the method in our previous report [6]. Pretreatment of tuna
skin consisted of two steps, the removal of non-collagenous protein followed by the removal of fat.
After cleaning with distilled water, the skin was cut into pieces of approximately 1 × 1 cm. The removal
of non-collagenous protein was carried out by immersing the tuna skin 1:10 (w/v) in 0.1 M NaOH
aqueous solution at 4 ◦ C for 12 h. The NaOH solution was changed every 2 h.
The washed skin was neutralized with distilled water until pH value reached 7, then was immersed
in 10% 1:10 (w/v) butyl alcohol for 24 h for fat removal, and then neutralized with distilled water.
Collagen extraction was carried out with 0.75 M acetic acid at a ratio of 1:10 (w/v) at 4 ◦ C for 72 h.
Filtration through a filter paper was carried out to collect collagen. The filtered residue was extracted
with the same concentration of acetic acid for another 72 h. Subsequently, collagen was precipitated by
adding solid NaCl to a final concentration of 1.8 M and subsequent addition of 0.05 M Tris-HCl (pH
7.5) at a ratio of 1:1 (v/v) and was allowed to stand for 24 h. The mixture was centrifuged at 3500× g
at 4 ◦ C for 1 h. The pellet obtained by the centrifugation was dialyzed against 0.1 M acetic acid, and
further against distilled water for 24 h. The obtained collagen was stored frozen at −20 ◦ C.

3.2. Preparation of Gelatin

Gelatin was prepared according to our previously reported method with a slight modification [7].
The extraction was carried out by soaking the cut skin (approximately 1 × 1 cm) with 0.25% citric acid
for 12 h at a skin and citric acid ratio of 1:4 (w/v). Then, the skin was washed with distilled water
and extracted under vigorous agitation at 65 ◦ C for 7 h at a skin and distilled water ratio of 1:1 (w/v),
Mar. Drugs 2020, 18, 98 8 of 12

followed by filtration through a calico and cotton cloth. The filtrate was dried up with a vacuum
evaporator at 60 ◦ C for 50 min.

3.3. Preparation of Hydrolysates

The skin collagen and gelatin were hydrolyzed according to Chalamaiah et al. [57] with a slight
modification. The collagen and gelatin solution (6.67%, w/v) was adjusted to pH 8 with NaOH, and
hydrolyzed with Alcalase (2%, v/v) (Sigma–Aldrich, St. Louis, MO, USA) at 55 ◦ C for 3 h. The mixture
was then left at −20 ◦ C for 5 min for enzyme inactivation. The solution was then centrifuged at
10,000× g at 4 ◦ C for 15 min, and the supernatant was obtained as the hydrolysate.

3.4. Fractination
Peptides were fractionated based on their molecular sizes using a molecular weight cut-off (MWCO)
membrane 30, 10, and 3 kDa (Millipore Co. Ltd., Waltham, MA, USA) according to Kusumaningtyas
et al. [54] with a slight modification. Namely, the peptide solutions were poured into 1.5 mL Eppendorf
tubes equipped with a membrane, which were subsequently centrifuged at 5000× g at 22 ◦ C for 10, 15,
or 30 min, respectively. The resulting fractions of >30 kDa, 10–30 kDa, 3–10 kDa, and <3 kDa were
designated as F1, F2, F3, and F4, respectively.

3.5. Molecular Weight Estimation

The protein profile was analyzed based on molecular weight according to Laemmli (1970) by a
sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) method [58]. For SDS-PAGE
analysis, 3% stacking gel and 15% separating gel for collagen and gelatin were used, as well as 17.5%
for the hydrolysates. Samples of 2 mg were dissolved in 1 mL of 5% SDS, heated at 85 ◦ C for 1 h,
centrifuged at 12,400× g for 5 min. After mixing 20 µL each of the sample and 2× Laemmli buffer,
the mixture was heated at 85 ◦ C for 10 min before 15 µL of which was loaded onto the gel well.
Electrophoresis was carried out at 13 mA and 100 V for 3 h. Electrophoresis was stopped when the front
dye reached about 0.5 cm from the bottom of the gel. Gel staining was carried out using Coomassie
brilliant blue, followed by destaining in 25% methanol and 10% acetic acid. The gel was scanned using
Photocapt software.

3.6. Hydrolysis Degree Analysis

The hydrolysis degree was determined according to Baharrudin [39]. To 20 mL of hydrolysate
was added 20 mL of 20% trichloroacetic acid (w/v). The mixture was allowed to stand for 30 min, and
the precipitate was removed by centrifugation at 6000× g for 30 min. The resulting supernatant was
analyzed for nitrogen content by a Kjeldahl method (AOAC 2005). The degree of hydrolysis (DH) was
calculated using the following formula:

Nitrogen content in the supernatant

DH (%) = × 100
Nitrogen in the sample

3.7. Amino Acid Analysis

Amino acid analysis was carried out according to Nollet [59]. Samples of collagen and gelatin
(0.1 g) were dissolved in 5 mL of 6 M HCl, vortexed, and then hydrolyzed at 110 ◦ C for 22 h. The
hydrolyzed sample was transferred to a 50 mL measuring flask and set up to the boundary mark. The
sample was filtered through a 0.45 µm nitrocellulose filter (ThermoFischer Scientific, Waltham, MA,
USA), and 500 µL of the filtrate was mixed with 40 µL of α-aminobutyric acid (AABA) and 460 µL of
Aquabidest. To the mixture of 10 µL was added 70 µL of AccQ-Fluor Borate, and it was then vortexed.
To the homogenate was added 20 µL of fluorine reagent and it was then incubated at 55 ◦ C for 10 min.
The prepared samples were analyzed with the Ultra Performance Liquid Chromatography (UPLC)
system (Shimadzu, Tokyo, Japan).
Mar. Drugs 2020, 18, 98 9 of 12

The standard solution analysis was carried out by mixing 40 µL of amino acid standards with an
equal volume of the internal standard (AABA) and 920 µL of distilled water, and then homogenized.
The standard of 10 µL was pipetted and mixed with 70 µL AccQ-Fluor Borate, and then vortexed. To
the homogenate was added 20 µL of fluorine reagent and it was incubated at 55 ◦ C for 10 min. The
prepared samples were analyzed as described above.

3.8. Antioxidant Quantification

The antioxidant activity was assayed according to ABTS and DPPH methods [51]. For the ABTS
assay, a total of 100 µL sample solution was mixed with 200 µL ABTS solution in the microplate wells
and placed at room temperature for 10 min. The absorbance was measured at 405 nm. For the DPPH
assay, 0.2 mM DPPH radical was added to 96% ethanol. DPPH solution was then measured at 540 nm
until an absorbance of 1.1 ± 0.05 was obtained. To a total of 100 µL sample solution was added 200 µL
of DPPH, which was then allowed to stand for 30 min. The absorbance of the mixture was measured
at 540 nm. The scavenging activity of peptide fractions to ABTS and DPPH radicals was expressed
using equation:
(A0 − A1)
Scavenging activity (%) = × 100
A0
where
A0 = absorbance of ABTS/DPPH, and
A1 = final absorbance.
Inhibition concentration of 50% free radical activity (IC50 ) values were calculated using the linear
regression equation. IC50 values were obtained by entering y = 50 and the known values a and b. The
value of x as IC50 can be calculated by the following equation:

y = bx + a

where
y = antioxidant activity,
x = sample concentration,
a = slope,
b = intercept.
The concentration of the sample and antioxidant activity were plotted on the x and y axes,
respectively, in the linear regression equation. The linear regression equation obtained in the form
of the equation y = bx + a was used to find the value of IC50 of each sample by stating a value of y
(50) and the value of x to be obtained as IC50 . The IC50 value states the concentration of the sample
solution needed to reduce free radicals by 50%.

3.9. Statistical Analysis

The quantitative data of the test results were processed using Excel 2013 (Microsoft, Redmond,
WA, USA), Minitab 162013 (Minitab, Sate College, PA, USA), and SPSS Statistics 222013 (IBM, Armonk,
NY, USA). Data were analyzed descriptively, while the experiments were designed using completely
randomized design data for antioxidant activities. Data analysis was performed with one-way analysis
of variance (ANOVA) at a 95% confidence interval (α = 0.05). The level that could affect the response
was further checked using a Tukey test.

4. Conclusions
Collagen and gelatin could be successfully prepared from tuna skin waste, which is abundantly
available from tuna processing. Both the collagen and the gelatin were found to be of high quality
based on the standards for commercial use. The derived peptides from both the collagen and gelatin
showed strong antioxidant activities, mainly in the ABTS assay as demonstrated by the scavenging
Mar. Drugs 2020, 18, 98 10 of 12

activity and IC50 values. The highest antioxidant activity (lowest IC50 ) of collagen hydrolysate was
obtained for the peptide fractions of 3–10 kDa and for the gelatin peptide fractions of 10–30 kDa.
Those of the hydrolysates and the derived peptides of gelatin were categorized as very strong. Tuna
skin waste is thus considered to be an excellent source of antioxidant peptides, making it possible to
effectively utilize the waste and add value to tuna resources. Further study is now ongoing to identify
the strong antioxidant components obtained by high performance liquid chromatography.

Author Contributions: M.N. conceived, designed the experiments, supervised, and wrote the manuscript. H.H.H.
and E.K. (Euis Karnia) performed the experiments and analyzed the data. E.K. (Eni Kusumaningtyas) supervised
and contributed to the manipulation of analytical tools. Y.O. contributed the reagents, did the final editing and
proofread the manuscript. All authors have read and agreed to the published version of the manuscript.
Funding: The present study was supported by the Ministry of Research, Technology, and Higher Education
through competitive funding in joint research and publication for MN grant number 1586/IT3.11/PN/2018.
Acknowledgments: The authors are indebted to Sonja Kleinertz (DAAD Long Term Lecturer in IPB University,
Indonesia) for grammar correction.
Conflicts of Interest: All the authors declare no conflict of interest.

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