2018 SCIENCELINE  
Journal of Life Science and Biomedicine  
J Life Sci Biomed, 8(1): 10-18, 2018  
ISSN 2251-9939  
Antioxidant Activity of Protein Fractions Derived  
from Acrochaetium sp. (Rhodophyta) Enzymatic  
Hydrolysates  
Seto WINDARTO1,2, Happy NURSYAM1, Jue-Liang HSU2,3, Meng-Chou LEE4  
1Faculty of Fisheries and Marine Science, University of Brawijaya, Indonesia  
2Department of Biological Science and Technology, National Pingtung University of Science and Technology, Taiwan  
3Research Center for Tropic Agriculture, National Pingtung University of Science and Technology, Taiwan  
4Department of Aquaculture, National Taiwan Ocean University, Taiwan  
ABSTRACT  
Original Article  
Natural antioxidants are helpful in the prevention of human diseases. The objective  
PII: S225199391800003-8  
of this study is to isolate the potential protein fractions from Acrochaetium sp. as an  
antioxidant. Fractions were obtained by proteolytic digestion using α-chymotrypsin,  
Rec. 10 Nov. 2017  
pepsin, trypsin, thermolysin individually and in combination of two enzymes, then  
centrifuged using 3 kDa molecular weight cut-off (MWCO) ultrafiltration membrane  
and fractionated by reversed-phase high performance liquid chromatography (RP-  
HPLC). The 2,2-Diphenyl-1-picrylhydrazyl free radical (DPPH) assay was used to  
measure the antioxidant activity. Result showed that thermolysin hydrolysate and  
the combination of trypsin-thermolysin hydrolysates had the highest antioxidant  
activity compared to the other hydrolysates with IC50 value of 1.48±0.92 mg/mL and  
1.37±0.84 mg/mL after fractionated using 3 kDa MWCO ultrafiltration membrane.  
Fractionation using RP-HPLC resulted fraction 7 obtained from thermolysin  
hydrolysates showed the highest antioxidant activity with IC50 value 0.58±0.56  
mg/mL and fraction I obtained from trypsin-thermolysin hydrolysates showed the  
highest antioxidant activity with IC50 value 0.38±0.33 mg/mL. The protein fractions  
from Acrochaetium sp. hydrolysates as antioxidant still has not been reported  
previously, therefore it can indicated as a potential therapeutic source for reducing  
oxidative stress.  
Acc. 25 Dec. 2017  
Pub. 25 Jan. 2018  
Keywords  
Acrochaetium sp.,  
Antioxidant,  
DPPH,  
Enzymatic  
hydrolysates,  
Fractions,  
RP-HPLC  
INTRODUCTION  
The key cause of the pathogenic disorders and various chronic diseases is oxidation. The oxidative reaction  
is not only deteriorates the quality of food products, but also lead to various chronic diseases such as  
hypertension, cancer and Parkinson’s disease. Cellular damage is caused by the high level of oxidative stress due  
to significant imbalance between the antioxidant defense system and free radicals [1, 2]. Free radicals attacks on  
protein, lipids and nucleic acids which lead to weakening of the antioxidant enzymes and lipid peroxidation [3].  
The easiest way to prevent these diseases from human body is consume vegetables, seed, and fruits to increase  
the antioxidant capacity in human body. An antioxidant is a substance which inhibits oxidation of the substrate  
at low concentration compared to that of an oxidizable substrate [4]. Antioxidants are widely applied to  
medicine, chemical industries, and important food additive which are mainly used to prevent the oxidation of  
Windarto S, Nursyam H, Hsu J-L, Lee M-Ch. 2018. Antioxidant Activity of Protein Fractions Derived from Acrochaetium sp. (Rhodophyta)  
Enzymatic Hydrolysates. J. Life Sci. Biomed. 8(1): 10-18; www.jlsb.science-line.com  
fats and also avoid nutrition of food damaging, browning and fading by capture and neutralize the free radicals  
[5].  
Currently, synthetic antioxidants such as butyl hydroxyanisole (BHA), butylated hydroxytoluene (BHT),  
tertiary butyl hydroquinone (TBHQ) and propyl gallate (PG) are added to food products to retard lipid oxidation,  
thus inhibit the generation of reactive oxygen species (ROS). The synthetic antioxidants must be used under  
strict regulation due to their potential health hazards and when compared to natural antioxidants, natural  
antioxidants are more favored in the present life because of their pure nature, high security, non-toxicity and  
have strong antioxidant capacity [6, 7]. Therefore, there is an interest in developing natural antioxidants.  
Recently, more studies have been carried out to find antioxidant in various natural products, such as seed  
of pea [8], chickpea [9], peanuts kernels [10] and corn [11]. Several studies about marine organism as antioxidant  
also have been carried out, such as yellow stripe trevally [12], muscle of ornate threadfin bream [13], pacific hake  
[14], aquatic species [15], capelin [16], muscle proteins of harp seal [17] and rhodophyta [18].  
Marine algae are sustainable resources in marine ecosystems and mostly used as a source of food and  
medicine. Algae biomass has been used for centuries as food and medicine. Major compounds in algae are  
polysaccharides, phenolic and phlorotannins, protein, peptides and essential amino acids, lipids, terpenoids and  
steroids, vitamins and minerals [19, 20]. Algal biomass and algae-derived compounds have a very wide range of  
potential applications for nutrition and health products. Some algae are considered as rich sources of natural  
antioxidants. Macroalgae have received muchattention as potential natural antioxidants and there has been  
very limited information on antioxidant activity of macroalgae [21]. Among macroalgae, the antioxidant activity  
of Acrochaetium sp. used in this study is rarely reported. Acrochaetium sp. is a rhodophyta which distribute in  
Taiwan, South America, Atlantic Islands, Indonesia and Africa [22].  
Currently, there is attention to the function and bioactivities of protein and its hydrolysates from food  
sources that may be used as an alternative source in the prevention of some diseases. Besides, food proteins  
have been known as bio-molecule that plays an important role in human improvement with their well-known  
nutritional values [23]. Peptides derived from food proteins can be a great source of antioxidants due to its  
aromatic rings, excessive donor electrons and appropriate hydrophobic character [24]. Enzymatic hydrolysis is  
the most reliable and an effective method to produce peptides with functional properties [25].  
In this study, Acrochaetium sp. protein isolate was hydrolyzed using single (α-chymotrypsin, pepsin,  
trypsin, thermolysin) and in combination enzymatic processes. The aims of this study were to generate  
Acrochaetium sp. protein hydrolysates, fractionate the hydrolysates using RP-HPLC and evaluate the potential  
antioxidant activity of these samples using DPPH assay.  
MATERIAL AND METHODS  
Sample Preparation  
Salt, sediment, and organic debris from Acrochaetium sp. were removed using fresh water. Algae were  
o
carefully rinsed with freshwater and dried at 40 C for 2 h and ground to obtain a powder with a particle size  
lower than 1 mm and finally stored at 4 °C in plastic bags for further analysis.  
Protein extraction, digestion, and ultrafiltration  
The dried powder of Acrochaetium sp. was dissolved in 20% of trichloroacetic acid (TCA) for 12 h at 4 °C.  
The TCA was removed using acetone and the pellet was lyophilized. The dried protein then was hydrolyzed by  
α-chymotrypsin (37 °C), pepsin (37 °C), thermolysin (60 °C) and trypsin (37 °C) for 16 h. Acrochaetium sp. was also  
digested by various combinations of enzymes, for each enzyme was incubated for 3 h. The reaction was stopped  
by heating the mixture and then fractionated into < 3 kDa MWCO. The filtrate was collected and lyophilized for  
further analysis.  
Fractionation of Acrochaetium sp. Protein Hydrolysate by RP-HPLC  
Acrochaetium sp. protein hydrolysate was eluted by 5% acetonitrile (ACN) and 0.2% FA in deionized water  
and fractionated by reverse-phase high performance liquid chromatography (RP-HPLC, Hitachi Chromaster,  
Tokyo, Japan). The mobile phase of buffer A (5% ACN and 0.1% TFA in deionized water) and buffer B (95% ACN  
and 0.1% TFA in deionized water). Twenty microliters of < 3 kDa hydrolysates was loaded at a flow rate of 1  
mL/min. Absorbance of the fractions was monitored at 214 nm.  
Windarto S, Nursyam H, Hsu J-L, Lee M-Ch. 2018. Antioxidant Activity of Protein Fractions Derived from Acrochaetium sp. (Rhodophyta)  
Enzymatic Hydrolysates. J. Life Sci. Biomed. 8(1): 10-18; www.jlsb.science-line.com  
DPPH Radical Scavenging Assay  
DPPH radical scavenging assay was measured according to Yu et al. [26]. Fresh DPPH solutions (0.1 mM  
DPPH in purified ethanol) were prepared daily. The samples, which comprised 100 μl samples with 100 μl of  
DPPH solution in a 96-well plate, was mixed and incubated for 30 min in the dark at room temperature. The  
absorbance was measured by using ELISA at 517 nm (AS). Ethanol was used as the blank (Ab), and distilled water  
was used as the control (AC). The DPPH radical scavenging activity was calculated according to the following  
equation:  
where Ab is the absorbance of the blank, AS is the absorbance of the sample solution, and AC is the  
absorbance of the control.  
Statistical Analysis  
Data was expressed as the mean ± standard deviation (mean ± SD). The analysis was done by using one way  
ANOVA in SPSS 16.0 (Chicago, SPSS Inc.) followed by post-hoc Duncan’s test and accepted at the P<0.05 level to  
identify the significant differences among treatments.  
RESULTS AND DISCUSSION  
Generation of free radicals and lipid peroxidation often occur in biological and food systems. In biological  
systems, antioxidants as part of the defense mechanism can prevent oxidative damage [27] and free radical  
generation by pro-oxidative from environment such as air pollutant, ultraviolet radiation, and cigarette smoke  
[28]. Recently, there is increased interest in naturally bioactive compounds as alternatives to synthetic  
substances, even these naturally compounds often show lower activity than the synthetic substances, but they  
are nontoxic and do not leave any residues [20]. As reported by Margaret et al. [29], bioactive peptides can be  
released by enzymatic proteolysis of food proteins, therefore pancreatic enzymes; chymotrypsin and trypsin  
have been used for derivation of bioactive peptides.  
Enzymatic hydrolysis is the most effective method to produce peptides with functional properties; in this  
study we used several proteases individually and in combination. As shown in Figure 1, thermolytic hydrolysate  
of Acrochaetium sp. possessed the highest scavenging of DPPH radicals than other proteases (57.40%). These  
results are consistent with the previous studies suggested that thermolysin is specifically catalyzes peptide  
bond containing hydrophobic and aromatic amino acid, which potential as antioxidant peptide [30]. In this  
study, we also used the combination of two enzymes and resulted the combination of thermolysin-trypsin had  
the highest scavenging of DPPH radicals compared with other combination of different enzymes, as shown in  
the Figure 2. Besides thermolysin catalyzes peptide bond containing hydrophobic and aromatic amino acid, the  
using of trypsin also contribute the releasing of amino acids (2-20 residues) which formed antioxidant peptides  
and immobile in parent protein [24].  
Bioactivity of protein hydrolysates is mainly affected by the molecular weight of the peptides. The  
molecular weight of hydrolyzed protein is one of an important factor in producing protein hydrolysates [31].  
The thermolysin hydrolysate and thermolysin-trypsin hydrolysate was fractionated by ultrafiltration with  
molecular weight cut-off (MWCO) membranes of < 3 kDa. The IC50 values of the thermolysin hydrolysate were  
1.83±0.95 mg/mL (> 3 kDa) and 1.48±0.92 mg/mL (< 3 kDa) (Figure 3). The thermolysin-trypsin hydrolysate  
showed the IC50 values of 1.70±1.03 mg/mL (> 3 kDa) and 1.37±0.84 mg/mL (< 3 kDa) (Figure 4). Ultrafiltration  
membrane system was used to separate the hydrolysates into defined molecular weight ranges. It holds well in  
purification of simple peptides from various crude protein hydrolysates [32, 33]. The isolated peptide fractions  
showed higher antioxidant activity than the hydrolysate [34]. This indicated that the peptide generation plays  
an important part in antioxidant potential of proteins. Purification step will affect the IC50 value, it indicated  
that the lower and more purified molecule has higher inhibition rate, more purified the molecule, and the IC50  
will be decreased.  
RP-HPLC involves the separation of molecules on the basis of hydrophobicity. The separation depends on  
the hydrophobic binding of the solute molecule from the mobile phase to the immobilized hydrophobic ligands  
attached to the stationary phase. RP-HPLC (detected at 214 nm under an UV-vis detector) was further used to  
fractionate the antioxidant peptides and the Acrochaetium sp. was separated into 12 fractions (fraction 1-12 for  
the thermolysin hydrolysate and fraction A-L for the thermolysin-trypsin hydrolysate) as shown in the Figure 5  
Windarto S, Nursyam H, Hsu J-L, Lee M-Ch. 2018. Antioxidant Activity of Protein Fractions Derived from Acrochaetium sp. (Rhodophyta)  
Enzymatic Hydrolysates. J. Life Sci. Biomed. 8(1): 10-18; www.jlsb.science-line.com  
and 7. Each fraction was collected; freeze dried, and determined its antioxidant activity. As shown in Figure 6,  
fraction 7 exhibited the highest DPPH free radical scavenging activity with the inhibition (57.29%) and among 12  
fractions for thermolysin-trypsin hydrolysate of Acrochaetium sp., fraction I showed the highest DPPH free  
radical scavenging activity (64.54%) (Figure 8). Furthermore, the IC50 value was tested for fraction 7 and fraction  
I. Fraction 7 from Acrochaetium sp. hydrolysate using thermolysin had IC50 value of 0.58±0.56 mg/mL and in the  
other hand; fraction I from Acrochaetium sp. hydrolysate using thermolysin-trypsin had IC50 value of 0.38±0.33  
mg/mL (Figure 9). These results showed higher antioxidant activity compared by the other marine organisms,  
such as Theragra chalcogramma (1.3 mg/mL) [35], Thunnus tonggol (5 mg/mL) [36], Gadus morhua (2.5 mg/mL) [37],  
and Navodon septentrionalis (10 mg/mL) [38].  
100  
80  
60  
40  
20  
0
α-chymotrypsin  
Pepsin  
Enzyme  
Thermolysin  
Trypsin  
Figure 1. DPPH radical scavenging activity (%) of Acrochaetium sp. enzymatic hydrolysate using different single  
enzyme.  
100  
80  
60  
40  
20  
0
Chy-The  
Chy-Try  
Chy-Pep  
Pep-The  
Pep-Try  
The-Try  
Enzymes  
Figure 2. DPPH radical scavenging activity (%) of Acrochaetium sp. enzymatic hydrolysate using different  
combination of two enzymes (Chy: α-chymotrypsin; Pep: Pepsin; The: Thermolysin; Try: Trypsin).  
Windarto S, Nursyam H, Hsu J-L, Lee M-Ch. 2018. Antioxidant Activity of Protein Fractions Derived from Acrochaetium sp. (Rhodophyta)  
Enzymatic Hydrolysates. J. Life Sci. Biomed. 8(1): 10-18; www.jlsb.science-line.com  
 
1 0 0  
8 0  
6 0  
4 0  
2 0  
0
1 0 0  
8 0  
6 0  
4 0  
2 0  
0
A
B
IC 5 0 = 1 .48 0 .9 2 m g /m L  
IC 5 0 = 1 .83 0 .9 5 m g /m L  
-3 .2  
-3 .0  
-2 .8  
-2 .6  
-2 .4  
-3 .2  
-3 .0  
-2 .8  
-2 .6  
-2 .4  
L o g C o n c en tratio n  
L o g C o n c en tratio n  
Figure 3. (A) IC50 value of Acrochaetium sp. hydrolysate using thermolysin (> 3 kDa) and (B) (<3 kDa).  
1 0 0  
8 0  
6 0  
4 0  
2 0  
0
1 0 0  
8 0  
6 0  
4 0  
2 0  
0
B
A
IC 5 0 = 1 .37 0 .8 4 m g /m L  
IC 5 0 = 1 .70 1 .0 3 m g /m L  
-3 .2  
-3 .0  
-2 .8  
-2 .6  
-2 .4  
-3 .2  
-3 .0  
-2 .8  
-2 .6  
-2 .4  
L o g C o n c en tratio n  
L o g C o n c en tratio n  
Figure 4. (A) IC50 value of Acrochaetium sp. hydrolysate using thermolysin-trypsin (> 3 kDa) and (B) (<3 kDa).  
Figure 5. RP chromatogram of thermolysin hydrolysate of Acrochaetium sp.  
Windarto S, Nursyam H, Hsu J-L, Lee M-Ch. 2018. Antioxidant Activity of Protein Fractions Derived from Acrochaetium sp. (Rhodophyta)  
Enzymatic Hydrolysates. J. Life Sci. Biomed. 8(1): 10-18; www.jlsb.science-line.com  
 
100  
80  
60  
40  
20  
0
1
2
3
4
5
6
7
8
9
10  
11  
12  
RP-HPLC Fractions of Thermolysin Hydrolysate  
Figure 6. DPPH radical scavenging activity (%) of Acrochaetium sp. fractions using thermolysin.  
Figure 7. RP chromatogram of thermolysin-trypsin hydrolysate of Acrochaetium sp.  
100  
80  
60  
40  
20  
0
A
B
C
D
E
F
G
H
I
J
K
L
RP-HPLC Fractions of Thermolysin-Trypsin Hydrolysates  
Figure 8. DPPH radical scavenging activity (%) of Acrochaetium sp. fractions using thermolysin-trypsin.  
Windarto S, Nursyam H, Hsu J-L, Lee M-Ch. 2018. Antioxidant Activity of Protein Fractions Derived from Acrochaetium sp. (Rhodophyta)  
Enzymatic Hydrolysates. J. Life Sci. Biomed. 8(1): 10-18; www.jlsb.science-line.com  
 
1 0 0  
8 0  
6 0  
4 0  
2 0  
0
1 0 0  
8 0  
6 0  
4 0  
2 0  
0
A
B
IC 5 0 = 0 .58 0 .5 6 m g /m L  
IC 5 0 = 0 .38 0 .3 3 m g /m L  
-4 .0  
-3 .5  
-3 .0  
-2 .5  
-4 .0  
-3 .5  
-3 .0  
-2 .5  
L o g C o n c en tratio n  
L o g C o n c en tratio n  
Figure 9. (A) IC50 value of fraction 7 from thermolysin hydrolysate and (B) IC50 value of fraction I from  
thermolysin trypsin hydrolysate.  
CONCLUSION  
The bioactivities of Acrochaetium sp. as antioxidant used in this study is rarely reported. Peptide fractions  
showing highly antioxidant, and it obtained from the enzymatic hydrolysates using thermolysin and  
thermolysin-trypsin, respectively. Fraction obtained by ultrafiltration showed an antioxidant higher than the  
whole hydrolysate. Fractionation using RP-HPLC resulted fraction 7 from Acrochaetium sp. hydrolysate using  
thermolysin had IC50 value of 0.58±0.56 mg/mL and fraction I from Acrochaetium sp. hydrolysate using the  
combination of thermolysin-trypsin had IC50 value of 0.38±0.33 mg/mL. Due to increasing concerns about the  
safety antioxidants, Acrochaetium sp. protein hydrolysates represent a novel source of natural antioxidant  
hydrolysates and antioxidant peptides. Further works such as the identification of the peptide from the fraction  
using LC-MS/MS, simulated gastrointestinal simulation and antioxidant activity are also suggested.  
DECLARATIONS  
Authors’ Contributions  
All authors contributed equally to this work.  
Competing interests  
The authors declare that they have no competing interests that might have influenced the performance or  
presentation of the work described in this manuscript.  
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