Journal of Biotechnology 241 (2017) 175–183

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Journal of Biotechnology
journal homepage: www.elsevier.com/locate/jbiotec

Antioxidant capacities of fucoxanthin-producing algae as influenced

by their carotenoid and phenolic contents
Su Chern Foo a , Fatimah Md. Yusoff a,b,∗ , Maznah Ismail a , Mahiran Basri a,c ,
Sook Kun Yau a , Nicholas M.H. Khong a , Kim Wei Chan a , Mahdi Ebrahimi d
a
Institute of Bioscience, Universiti Putra Malaysia, 43400 UPM Serdang, Selangor Darul Ehsan, Malaysia
b
Department of Aquaculture, Faculty of Agriculture, Universiti Putra Malaysia, 43400 UPM Serdang, Selangor Darul Ehsan, Malaysia
c
Department of Chemistry, Faculty of Science, Universiti Putra Malaysia, 43400 UPM Serdang, Selangor Darul Ehsan, Malaysia
d
Department of Veterinary Preclinical Sciences, Universiti Putra Malaysia, 43400 UPM Serdang, Selangor Darul Ehsan, Malaysia

a r t i c l e i n f o a b s t r a c t

Article history: Natural antioxidants from sustainable sources are favoured to accommodate worldwide antioxidant
Received 20 July 2016 demand. In addition to bioprospecting for natural and sustainable antioxidant sources, this study aimed
Received in revised form to investigate the relationship between the bioactives (i.e. carotenoid and phenolic acids) and the antiox-
25 November 2016
idant capacities in fucoxanthin-producing algae. Total carotenoid, phenolic acid, fucoxanthin contents
Accepted 25 November 2016
and fatty acid profile of six species of algae (five microalgae and one macroalga) were quantified followed
Available online 30 November 2016
by bioactivity evaluation using four antioxidant assays. Chaetoceros calcitrans and Isochrysis galbana dis-
played the highest antioxidant activity, followed by Odontella sinensis and Skeletonema costatum which
Keywords:
Microalgae mass culture
showed moderate bioactivities. Phaeodactylum tricornutum and Saccharina japonica exhibited the least
Antioxidant activity antioxidant activities amongst the algae species examined. Pearson correlation and multiple linear regres-
Carotenoid sion showed that both carotenoids and phenolic acids were significantly correlated (p < 0.05) with the
Fucoxanthin antioxidant activities, indicating the influence of these bioactives on the algal antioxidant capacities.
Phenolic acids © 2016 Elsevier B.V. All rights reserved.
PUFA profiles

1. Introduction et al., 2014); especially for their bioactive compounds such as

carotenoids, phenolic acids and lipids. Increasing numbers of
While the market for antioxidant products is predicted to microalgae-derived products can be found in the form of supple-
approach $86 billion in 2016, a steady supply of natural bioactive ments as microalgae species like Chlorella and Spirulina are being
remains the limiting factor for biotechnology, food and nutraceu- granted GRAS (generally recognized as safe) status. Production of
tical applications (Murray et al., 2013). This problem is further standardized and quality antioxidants is the key for transition from
escalated by climate changes; leading to plant diseases and poor laboratory proof-of-concept to continuous commercial produc-
yield in terrestrial antioxidant sources (Ruiz-Vera et al., 2015; Van tions. Prior to that, the composition and characteristic of bioactive
de Perre et al., 2015). An aquatic biomass source that can sustain- responsible for a claimed bioactivity must be known.
ably supply natural biomolecules without increasing the use of Microalgae contain bioactive compounds mainly carotenoids
terrestrial resources beyond globally sustainable levels (Elkington, and phenolic acids that are responsible for antioxidant activi-
1997; Lozano, 2008) is urgently needed. ties. In order to establish the relationship between bioactive and
Microalgae are increasingly recognized as the next gener- their influence on antioxidant activities, Pearson correlation study
ation sustainable feedstock (Murray et al., 2013; Pérez-López is the most common statistical tool to do so (Goh et al., 2010;
Hajimahmoodi et al., 2010; Jiménez-Escrig et al., 2001; Li et al.,
2007). However, correlation is simply a way to describe how two
variables are related to each other. Past microalgae correlation
Abbreviations: ABTS, 2,2 azino-bis(3-ethylbenzthiazoline-6-sulphonic acid);
BCB, beta carotene bleaching; IC, iron chelating; FRAP, ferric reducing antioxidant studies have reported carotenoids as the major contributors of
properties; TPC, total phenolic content; PUFA, polyunsaturated fatty acids; FAME, antioxidant capacities (Goh et al., 2010; Li et al., 2007) whilst other
fatty acid methyl esters; TAC, total antioxidant capacity; TEAC, trolox equivalent studies claimed phenolic acids were responsible (Hajimahmoodi
antioxidant capacity; GRAS, generally recognized as safe. et al., 2010; Jiménez-Escrig et al., 2001). This discrepancy needs
∗ Corresponding author at: Institute of Bioscience, Universiti Putra Malaysia,
further inferences on the data hence; multiple linear regression
43400 UPM Serdang, Selangor Darul Ehsan, Malaysia.
E-mail addresses: [email protected], [email protected] (F.Md. analysis may be added to observe the weight of contribution (i.e.
Yusoff).

http://dx.doi.org/10.1016/j.jbiotec.2016.11.026
0168-1656/© 2016 Elsevier B.V. All rights reserved.
176 S.C. Foo et al. / Journal of Biotechnology 241 (2017) 175–183

beta coefficients) of the independent (X) and dependent (Y) vari- and biomass collection were described in our previous study (Foo
ables (Yan, 2009). et al., 2015a). At least 5.0 g of biomass was collected for each species
Despite the fact that fucoxanthin is the major carotenoid of and stored at −80 ◦ C prior to analysis. The commercial macroalga
microalgae in the class Bacillariophyceae and Prymnesiophyceae, biomass, S. japonica Aresch was procured from Changsha NutraMax
scientific investigations on fucoxanthin from microscopic sources Inc., China for comparison purposes in terms of bioactive content
and their involvement in antioxidant systems are limited (Goh et al., and bioactivity.
2010; Li et al., 2007); as compared to their larger commercialized
macroalgae counterparts (Airanthi et al., 2011; Martins et al., 2013;
2.3. Preparation of algae methanolic extracts
Sachindra et al., 2010). In addition, the exclusive marine carotenoid,
fucoxanthin is produced in a higher quantity in the microalgae
The common solvents used to extract algal antioxidants is
(e.g. Chaetoceros calcitrans: 2.33 ± 0.44 mg. g−1 DW; (Goiris et al.,
methanol (Goh et al., 2010; Kim et al., 2012; Natrah et al., 2007).
2012)) as compared to the macroalgae (e.g. Laminaria digitata:
Solvent comparison study also found that methanol has the polar-
0.468 mg. g−1 DW; (Holdt and Kraan, 2011)). In this regard, the
ity most suitable in extracting antioxidants; especially from brown
commercialized brown macroalga, Saccharina japonica (Kanazawa
microalgae. This was reflected by the good radical scavenging
et al., 2008) was included in our investigation for comparison pur-
and chelating activities (Foo et al., 2015a). As such, approxi-
poses. The five microalgae species were selected because they have
mately 1.0 g of lyophilised biomass from each species was added
gained widespread uses in aquaculture due to their rapid and sta- ®
with 250 ml methanol and homogenized (WiseTis HG–15 s dig-
ble growth rates in hatchery systems, good nutrient composition
ital homogenizer, Daihan Scientific, Korea) at 14125.93 × g for
and absence of toxins that may be transferred up the food chain
15mins. Filtrates from three extractions were pooled and methanol
(Brown, 2002).
was removed under reduced pressure using a rotary evaporator
In this study, carotenoid and phenolic acid of methanolic
(RotaVapor R210, Buchi, Poshfach, Flawil, Switzerland). Extracts
extracts were quantified followed by antioxidant evaluations. The
were then placed in a 40 ◦ C oven followed by a desiccator at
aim of this study was to identify lead compounds from each bioac-
room temperature to completely remove residual solvents. Dry
tive type and to examine statistical relationship between bioactives
weight was taken and this procedure was repeated until a con-
and antioxidant activities in the investigated species. The fatty acid
stant weight of the extract was obtained. The percentage extraction
and PUFA profile of each species was also reported in this study.
yield (%) was calculated according to the formula;yield (%) =
weight of extract (g)
weight of dry biomass (g)
× 100%.
2. Materials and methods All extracts were stored in −80 ◦ C freezer at minimal light expo-
sure.
2.1. Chemicals
2.4. Antioxidant activities
All chemicals and reagents were of analytical or HPLC
grade. Methanol and chloroform were purchased from Merck
The total amount of antioxidant of algal biomass was deter-
KGaA (Darmstadt, Germany). Fucoxanthin, boron trifluoride
mined using total antioxidant capacity (TAC) assay; which included
(BF3 ), potassium hydroxide (KOH), henicosanoic acid (C21:0),
the trolox equivalent antioxidant capacity (TEAC) and ferric reduc-
vanillin (4-hydroxy-3-methoxybenzaldehyde), tripalmitin, con-
ing antioxidant properties (FRAP) assays (Rubio et al., 2016). TEAC
centrated sulphuric acid, 3,4,5-trihydroxybenzoic acid (gallic acid),
assay is also known as ABTS radical cation scavenging assay
2,5- dihydroxybenzoic acid, cinnamic acid, protocatechuic acid,
(Aminjafari et al., 2016).
sinapic acid, caffeic acid, vanilic acid, chlorogenic acid, fer-
The ABTS·+ scavenging activity of algae extracts was determined
ulic acid, syringic acid, 4-hydroxybenzoic acid, rosmarinic acid,
according to Foo et al. (2015b). Extracts from each species were pre-
sodium hydrogen carbonate (NaHCO3 ), potassium persulfate
pared in triplicates by fully dissolving 10 mg of dried extracts each
(K2 S2 O8 ), iron (II) chloride (FeCl2 ), 3-(2-pyridyl)-5,6-diphenyl-
in 1000 ␮l methanol. ABTS·+ solution was produced by adding 50 ml
1,2-4-triazine-4 4 disulphonic acid monosodium salt (ferrozine),
of 7 mM ABTS stock solution with 50 ml of 2.45 mM potassium
2,4,6-tripyridyl-s-triazine (TPTZ), 0.1 M acetate buffer, linoleic acid,
persulfate in the dark at room temperature. After 24 h of incuba-
beta carotene (type II, synthetic), Tween 20, 6-hydroxy-2,5,7,8-
tion, ABTS·+ solution was diluted to an absorbance of 0.70 ± 0.02
tetramethylchroman-2-carboxylic acid (Trolox), Folin-Ciocalteu
at 734 nm. Then, 200 ␮l of the adjusted solution was added to
phenol reagent, sodium nitrate (NaNO3 ), silica (Na2 SiO3 ·9H2 O),
20 ␮l sample in the dark and left to react for 10mins in a 96-well
ethylenediaminetetraacetic acid disodium (Na-EDTA), boric acid
microtitre plate. The absorbance of the radicals-sample mixture
(H3 BO3 ), sodium hydrogen phosphate (NaH2 PO4 ·2H2 O), zinc
was measured at 734 nm (MultiskanTM GO UV/Vis microplate spec-
chloride (ZnCl2 ), cobalt chloride (CoCl2 ·6H2 O), copper sulphate
trophotometer, Thermo Fischer Scientific, USA). Trolox was used as
(CuSO4 ·5H2 O), iron (III) chloride (FeCl3 ·6H2 O), ammonium molyb-
the standard (3.125–100 ␮g. ml−1 ; 2 fold dilution) and antioxidant
date ((NH4 )6 Mo7 O24 ·4H2 O), manganese chloride (MnCl2 ·4H2 O),
activity was expressed in milligram trolox equivalent per gram dry
cobalamin (vitamin B12 ) and ammonium formate (NH4 HCO2 ) were
weight biomass (mg TE. g−1 DW).
purchased from Sigma-Aldrich Co. (St. Louis, MO, USA).
The iron chelating (IC) properties of microalgae extracts were
determined according to Decker and Welch (1990). To 100 ␮l of
2.2. Algae biomass collection sample extract, 135 ␮l distilled water and 5 ␮l of 2 mM FeCl2 were
added. After addition of 10 ␮l of 5 mM ferrozine, mixtures were
Tropical microalgae species, C. calcitrans (UPMC-A0010), left for 10 min and measured at 562 nm (MultiskanTM GO UV/Vis
Isochrysis galbana (UPMC-A0009), Skeletonema costatum (UPMC- microplate spectrophotometer, Thermo Fischer Scientific, USA).
A0019) and Odontella sinensis (UPMC-A0050) were isolated from Na-EDTA was used as standard (3.125–100 ␮g. ml−1 ; 2 fold dilu-
local waters. The diatom, Phaeodactylum tricornutum was a gift from tion) and methanolic extract iron chelation activity was expressed
Dr. Graziella Chini Zittelli from Italy. All microalgae species were in milligram EDTA equivalent per gram dry weight biomass (mg
maintained in the Microalgae Culture Collection Unit, Laboratory Na-EDTA .g−1 DW).
of Marine Biotechnology, Institute of Bioscience, Universiti Putra FRAP of microalgae methanolic extracts was determined accord-
Malaysia, Serdang, Selangor, Malaysia. Detailed culture conditions ing to Nilsson et al. (2005). To 50 ␮l extract, 150 ␮l of FRAP reagent
 S.C. Foo et al. / Journal of Biotechnology 241 (2017) 175–183 177

and 100 ␮l ddH2 O were added. The FRAP reagent was prepared rosmarinic acid) were chosen. Results were expressed in milligrams
from a mixture of 0.1 M acetate buffer (pH 3.6), 10 mM TPTZ, standard per gram biomass dry weight (mg. g−1 biomass DW).
and 20 mM ferric chloride (10:1:1 v:v:v). Absorbance readings at
593 nm (MultiskanTM GO UV/Vis microplate spectrophotometer, 2.7. Fatty acid methyl esters (FAME) analysis
Thermo Fischer Scientific, USA) were taken after addition of the
reagent. FRAP activity of algae methanolic extracts were calculated This analysis followed the method described by Ebrahimi et al.
and expressed in milligram trolox equivalent per gram dry weight (2012) with slight method modifications. To 20 ml of chloroform:
biomass (mg TE. g−1 DW). methanol (2:1 v/v) solvents, 0.5 g lyophilized cells were added and
The beta carotene bleaching (BCB) inhibitory activity of the mixture was sonicated and shaken at ambient temperature
algae methanolic extracts was evaluated according to procedure overnight. The next day, 10 ml of 0.9% saline was added, shaken
described by Foo et al. (2015b). Six ml of ␤ carotene solution (1 mg and centrifuged at 626.03 × g for 15 min to obtain two phases. The
␤ carotene/10 ml chloroform) was added to 120 mg of linoleic acid lower phase was collected and spiked with 100 ␮l of heneicosanoic
and 1200 mg of Tween20 followed by drying under a stream of acid (C21:0) internal standard. Solvents were removed from the
nitrogen gas. Then, 100 ml of distilled water was added to obtain extracts under a stream of nitrogen gas and extracted fatty acids
a clear ␤ carotene-linoleic acid emulsion. Next, 1.5 ml aliquots of were trans-methylated to methyl esters following AOAC (1990).
emulsion were added into test tubes containing 20 ␮l of samples Transmethylation was done by adding 2 ml of 0.66 N KOH to the
followed by incubation in 50 ◦ C water bath for 60mins. After that, dried extracts and incubated for 10mins at 70 ◦ C. After cooling to
the absorbance of standards and samples were measured at 470 nm room temperature, 2 ml 14% methanolic BF3 was added and the
(Pharmaspec UV 1601, Shimadzu, Kyoto, Japan). Antioxidant activ- mixture was incubated in a water bath for another 20mins. Finally,
ity of algae methanolic extracts was expressed in milligram trolox 4 ml of distilled deionized water (ddH2 O) and 4 ml of petroleum
equivalent per gram dry weight biomass (mg TE. g−1 DW). ether were added, mixed and centrifuged to yield another two dis-
tinctively phased solution. The upper layer was collected and stored
2.5. Total carotenoid content and fucoxanthin content in 2 ml vial prior to fatty acid profiling. The FAME were separated
by gas liquid chromatography on an Agilent 7890A GC system (Agi-
The total carotenoid content of extracts in triplicates was deter- lent, Palo Alto, CA, USA) via a 100 m × 0.25 mm ID (0.2 ␮m) Supelco
mined according to the method described by Lichtenthaler and SP-2560 capillary column (Supelco, Inc., Bellefonte, PA, USA). One
Buschmann (2001). Further fucoxanthin quantification of algae microliter of FAME was injected by an autosampler into the chro-
extracts employed the use of high performance liquid chromatog- matograph equipped with a flame ionization detector (FID). The
raphy (Agilent 1300 HPLC series, Agilent Technologies Inc., GA, USA) carrier gas was helium and split ratio was 10:1 after injection of
following Foo et al. (2015a). The mobile phase gradient selected was FAME. The injector temperature was programmed at 250 ◦ C and
100% water (A) and 100% methanol (B): starting from 0% to 100% detector temperature at 300 ◦ C. The column temperature program
A in 2 mi, 100% to 50% A in 3 min, 50% to 25% A in 4 min, 25% to initiated was run at 120 ◦ C for 5mins, increased to 170 ◦ C for 15mins
10% A in 6 min, 10% to 5% A in 8 min, and 0% to 100% B in 15 min at 2 ◦ C min−1 and then increased again to a final temperature of
at a flow rate of 1 ml. min−1 and injection volume of 20 ␮l. The 235 ◦ C for 10mins. The fatty acid, FA concentrations was expressed
absorbance of 445 nm for each run was recorded and the standard as% total identified FA of algae. A reference standard (mix C4-C-24
curve and retention times were calibrated using pure fucoxanthin methyl esters; Sigma-Aldrich, Inc., St. Louis, MO, USA) was used to
standard at six concentrations. Results were expressed as milligram determine recoveries and correction factors for the determination
fucoxanthin per gram dry weight biomass (mg FX. g−1 DW). of individual FA composition.

2.8. Statistical analysis

2.6. Total phenolic content and phenolic acid profiling

Test of normality (p > 0.05) followed by one-way analysis of

Total phenolic content (TPC) of methanolic extracts was deter-
variance (ANOVA) and Tukey HSD post hoc test were carried out
mined using the Folin-Ciocalteu reagent assay (Kabir et al., 2015).
to analyse significant differences (p < 0.05) between extracts and
Five hundred microlitres of samples from each algal species were
antioxidant activities using the statistical program, SPSS Version
reacted with 2.5 ml of 10% (v/v) Folin-Ciocalteu reagent and 2.0 ml
21.0 (SPSS Inc., Chicago, USA). Pearson correlation and multi-
of 7.5% (w/v) sodium bicarbonate solution in 15 ml clean centrifuge
ple linear regression analyses were carried out to investigate the
tubes. After 60mins incubation at 40 ◦ C, the absorbance of each
relationship of carotenoid (fucoxanthin) and phenolic contents
reaction mixture was recorded at 765 nm spectrophotometrically
(gallic acid) in each antioxidant assay. Values were given as the
(Pharmaspec UV 1601, Shimadzu, Kyoto, Japan). Gallic acid was
means ± standard deviation (SD) of three replications (n = 3). Values
used as the positive control and results expressed as milligram gal-
were considered to be statistically significant when p < 0.05.
lic acid equivalent per gram of dry weight biomass (mg GAE. g−1
DW).
Phenolic acid separations were performed on an Agilent ZOR- 3. Results and discussion
BAX SB C-18 column (150 × 4.6 mm, 5 ␮m) following the method
described by Foo et al. (2015a). Briefly, samples (20 ␮l) were 3.1. Extraction yield
injected with a flow rate of 1 ml. min−1 into an Agilent 1200 series
HPLC series (Agilent Technologies Inc., Alpharetta, GA, USA). Gradi- The extraction yield in this study was comparable to our pre-
ent elution used a mobile phase of water-acetic acid (95:5, v/v) (A) vious study on efficient extraction protocol to concentrate algal
and methanol-acetonitrile-acetic acid (95:5:1, v/v/v) (B): starting antioxidants (Foo et al., 2015a). The highest methanolic extract
from 0% to 5% B in 2 min, 5%–24% B in 8 min, 25%–40% B in 10 min, yield was found in C. calcitrans (48.47 ± 0.74%), followed by S. costa-
40%–50% B in 10 min, 50–100% B in 10 min, 100% B in 5 min, and tum (47.00 ± 0.03%). Both were significantly higher (p < 0.05) than
100%–5% B in 5 min. Wavelengths (280 nm and 320 nm) were used O. sinensis (37.63 ± 1.66%), I. galbana (31.02 ± 1.24%) and P. tricor-
and pure reference standards (i.e. 2,5 dihydroxybenzoic acid, cin- nutum (29.03 ± 0.58%). S. japonica had the least (p < 0.05) extraction
namic acid, sinapic acid, caffeic acid, vanilic acid, chlorogenic acid, yield (24.41 ± 0.39%) compared to other investigated microalgae
gallic acid, ferulic acid, syringic acid, 4-hydroxybenzoic acid and species.
178 S.C. Foo et al. / Journal of Biotechnology 241 (2017) 175–183

Fig. 1. Antioxidant activities of algae methanolic extracts evaluated using different antioxidant assays (a) ABTS·+ scavenging assay; (b) Iron chelating assay; (c) FRAP assay;
(d) ␤ carotene bleaching assay. Different letters within the same assay indicates significant difference (p < 0.05). Error bar denotes standard error of the means (n = 3).

3.2. Antioxidant assays (Fig. 1). Firstly, I. galbana (21.55 ± 1.58 mg TE. g−1 DW) and C. cal-
citrans (16.92 ± 0.87 mg TE. g−1 DW) exerted significantly higher
Overall, I. galbana and C. calcitrans displayed significantly higher (p < 0.05) ABTS·+ radical scavenging activity as compared to the
(p < 0.05) antioxidant activities in the four antioxidant assays other species. In addition, C. calcitrans (13.39 ± 1.17 mg EDTA. g−1
compared to the rest of the investigated microalgae species DW), I. galbana (8.49 ± 0.30 mg EDTA. g−1 DW) and O. sinensis
 S.C. Foo et al. / Journal of Biotechnology 241 (2017) 175–183 179

Fig. 2. Correlation between antioxidant capacity with total carotenoid and total phenolic content in (a) ABTS·+ scavenging assay; (b) Iron chelating assay; (c) FRAP assay; (d)
␤ carotene bleaching assay.

Fig. 3. Pearson correlation between (a) total carotenoid content vs. fucoxanthin content; (b) total phenolic content vs. gallic acid, showing significant positive relationship.
180 S.C. Foo et al. / Journal of Biotechnology 241 (2017) 175–183

Table 1
Comparison of extraction yields and bioactive contents in algae species. Microalgae had higher contents of carotenoid, fucoxanthin and phenolics as compared to macroalgae.

Type Species Extraction yield (%) Total carotenoid Fucoxanthin content Total phenolic content References
content (mg. g−1 DW) (mg FX. g−1 DW) (mg GAE. g−1 DW)

Microalgae Chaetoceros 48.47 ± 0.74a 6.13 ± 0.25a 5.13 ± 0.19a 3.68 ± 0.39b This study
calcitrans
Microalgae Isochrysis galbana 31.02 ± 1.24c 4.33 ± 0.04b 2.19 ± 0.02b 12.24 ± 1.61a This study
Microalgae Skeletonema 47.00 ± 0.03a 0.97 ± 0.24d 0.36 ± 0.00d 0.18 ± 0.00c This study
costatum
Microalgae Odontella sinensis 37.63 ± 1.66b 2.66 ± 0.10c 1.18 ± 0.00c 3.71 ± 0.34b This study
Microalgae Phaeodactylum 29.03 ± 0.58c 0.18 ± 0.00d 0.07 ± 0.00e 0.02 ± 0.00c This study
tricornutum
Macroalgae Saccharina japonica 24.41 ± 0.39d 0.05 ± 0.02d 0.03 ± 0.00e 0.09 ± 0.00c This study
Microalgae Isochrysis galbana – – – 3.18 Custódio et al.
T-ISO (2014)
Microalgae Nitzchia laevis – – – 2.37 ± 0.77 Li et al. (2007)
Microalgae Phaeodactylum – – 15.71 – Kim et al. (2012)
tricornutum
Microalgae Chaetoceros – 2.33 ± 0.14 – 1.84 ± 0.11 Goiris et al. (2012)
calcitrans
Microalgae Odontella aurita – – 14–15 – Xia et al. (2013)
Macroalgae Sargassum – – 0.01 – Xiao et al. (2012)
fusiforme
Macroalgae Undaria pinnatifida – – 0.876−1.277 – Kim et al. (2004)
Macroalgae Eisenia bicyclis 9.72 – 0.109 0.202 Airanthi et al.
(2011)
Macroalgae Kjellmaniella 12.03 – 0.152 0.377 Airanthi et al.
crassifolia (2011)
Macroalgae Sargassum horneri 7.25 – 0.020 0.072 Airanthi et al.
(2011)
Macroalgae Cystoseira 14.61 – 0.0041 0.086 Airanthi et al.
hakodatensis (2011)
a–f
Results presented as mean ± standard deviation (n = 3). Different letters within the same column indicate significant difference (p < 0.05).

Table 2
Identification and quantification of different phenolic acids (mg. g−1 DW) in algae species.

Sample Chaetoceros Isochyrysis Skeletonema Odontella sinensis Phaeodactylum Saccharina

calcitrans galbana costatum tricornutum japonica

2,5 dihydroxy-benzoic acid nd 0.563 ± 0.002 0.144 ± 0.000 0.516 ± 0.001 0.064 ± 0.001 0.050 ± 0.005
Cinnamic acid 1.694 ± 0.000 0.142 ± 0.004 0.241 ± 0.091 nd 0.083 ± 0.000 nd
Sinapic acid – – 0.066 ± 0.001 – 0.016 ± 0.000 –
Caffeic acid 0.277 ± 0.007 nd 0.157 ± 0.001 0.918 ± 0.010 0.090 ± 0.002 0.028 ± 0.000
Vanilic acid 0.405 ± 0.008 0.295 ± 0.009 0.066 ± 0.003 0.200 ± 0.005 0.095 ± 0.002 0.053 ± 0.002
Chlorogenic acid – 1.988 ± 0.022 0.390 ± 0.011 1.443 ± 0.017 – 0.061 ± 0.000
Gallic acid 7.568 ± 0.265 13.652 ± 0.194 4.296 ± 0.051 9.489 ± 0.117 1.612 ± 0.047 1.309 ± 0.000
Ferulic acid – 0.555 ± 0.001 0.015 ± 0.000 – 0.041 ± 0.001 0.019 ± 0.001
Syringic acid – 1.877 ± 0.052 0.939 ± 0.009 2.238 ± 0.060 0.940 ± 0.020 –
4-hydroxybenzoic acid 0.351 ± 0.020 0.425 ± 0.006 0.069 ± 0.001 1.442 ± 0.019 0.065 ± 0.001 0.037 ± 0.001
Rosmarinic acid 0.275 ± 0.000 7.252 ± 0.024 nd nd 0.285 ± 0.001 0.092 ± 0.011

(8.43 ± 0.45 mg EDTA. g−1 DW) had significantly higher (p < 0.05) studies may be due to the amount of bioactives produced as the cul-
chelating ability than P. tricornutum (1.45 ± 0.11 mg EDTA. g−1 DW) ture system and settings that differed in every experiment (Goiris
and S. japonica (3.66 ± 0.59 mg EDTA. g−1 DW). Thirdly, C. calci- et al., 2012). It can be observed from this study that microalgae
trans (1.65 ± 0.11 mg TE.g−1 DW) and I. galbana (1.96 ± 0.05 mg has a competitive edge to macroalgae as alternative fucoxanthin
TE.g−1 DW) demonstrated significantly higher reducing proper- sources. Table 1 presents a lower fucoxanthin quantity in macroal-
ties (p < 0.05) in FRAP assay compared to the others. This trend gae (Airanthi et al., 2011; Xiao et al., 2012) than microalgae. At
was also observed in BCB activity where protection against beta- present, commercial fucoxanthin is mainly sourced from macroal-
carotene bleaching by extracts of C. calcitrans (1.43 ± 0.01 mg gae (i.e. Laminaria japonica, Sargassum sp., and Undaria pinnatifida).
TE. g−1 DW) and I. galbana (1.23 ± 0.03 mg TE. g−1 DW) was sig- However, increasing number of studies are showing microalgae and
nificantly (p < 0.05) higher as compared to the rest. its versatility for industrial scale commercialization (Murray et al.,
It was found that the I. galbana methanolic extracts in this study 2013) for bioactives. In addition, microalgae can be consistently
were three times better in scavenging ABTS·+ (21.55 ± 1.58 mg cultured all year round in controlled facilities; free of diurnal and
TE.g−1 DW) in comparison to the value (5.63 mg TE. g−1 DW) as seasonal changes (Honya et al., 1994) hence allowing for stable and
reported by Goiris et al. (2012). Also, C. calcitrans (16.92 ± 0.87 mg consistent supply of bioactives.
TE. g−1 DW) in this study was a better ABTS·+ scavenger than
commercialized microalga Chlorella vulgaris (5.76 mg TE. g−1 DW) 3.3. Carotenoid and fucoxanthin contents
(Cha et al., 2010) or macroalga, Sargassum polycystum (1.89 mg
TE. g−1 DW) (Matanjun et al., 2009). Besides that, C. calcitrans In carotenoid content studies, C. calcitrans showed the
(13.39 mg EDTA. g−1 DW) chelated three times more iron than highest amount of carotenoids (6.13 ± 0.25 mg. g−1 DW)
4.88 mg EDTA. g−1 DW in another study (Goh et al., 2010). A reason and this was significantly higher (p < 0.05) than I. galbana
accounting for the differences in antioxidant capacities in different (4.33 ± 0.04 mg. g−1 DW), O. sinensis (2.66 ± 0.10 mg. g−1
 S.C. Foo et al. / Journal of Biotechnology 241 (2017) 175–183 181

Table 3
FAME profile of 7 marine algae methanolic extracts (% total FAME).

Saturates (%) Chaetoceros Isochrysis galbana Skeletonema Odontella sinensis Phaeodactylum Saccharina
calcitrans costatum tricornutum japonica

C12:0 0.57 ± 0.09b 0.29 ± 0.06b 0.84 ± 0.01b 0.68 ± 0.11b 0.25 ± 0.04b 2.17 ± 0.77a
C14:0 17.49 ± 1.84ab 14.60 ± 1.31bc 12.28 ± 1.81c 13.68 ± 0.09c 18.96 ± 0.25a 7.33 ± 0.10d
C15:0 0.85 ± 0.09c 1.31 ± 0.29bc 1.31 ± 0.29a 2.46 ± 0.11a 1.02 ± 0.12bc 1.54 ± 0.23b
C16:0 20.33 ± 1.48b 15.1 ± 2.32cd 18.21 ± 1.48bc 13.12 ± 0.08d 12.45 ± 0.56d 24.81 ± 0.79a
C17:0 3.75 ± 0.38a 0.27 ± 0.07c 0.64 ± 0.07bc 1.57 ± 0.05bc 1.76 ± 0.07b 4.41 ± 1.36a
C18:0 5.54 ± 1.14a 0.97 ± 0.09b 5.75 ± 0.45a 4.75 ± 2.71a 7.40 ± 0.05a 5.96 ± 0.75a
C14:1 1.15 ± 0.24c 0.64 ± 0.07cde 7.38 ± 0.59a 2.13 ± 0.13b 0.46 ± 0.18de 0.00 ± 0.00e
C15:1 0.00 ± 0.00e 5.78 ± 0.36b 7.39 ± 0.22a 7.42 ± 0.64a 3.00 ± 0.30d 4.17 ± 0.39c
C16:1 0.00 ± 0.00c 4.27 ± 0.36b 0.00 ± 0.00c 14.28 ± 1.23a 0.00 ± 0.00c 0.00 ± 0.00c
C16:1(n-7) 25.42 ± 3.18a 5.47 ± 1.11e 19.99 ± 2.50bc 16.31 ± 0.91cd 24.37 ± 0.60ab 0.00 ± 0.00f
C17:1 0.00 ± 0.00d 0.93 ± 0.07c 2.26 ± 0.43ab 2.42 ± 0.76ab 1.76 ± 0.07bc 1.02 ± 0.03c
C18:1(n-7) 3.53 ± 0.65a 0.16 ± 0.03c 0.34 ± 0.02c 0.90 ± 0.05bc 0.61 ± 0.01bc 1.34 ± 0.25b
C18:1(n-9) 1.28 ± 0.54c 24.22 ± 1.20a 12.52 ± 2.55b 6.65 ± 0.14c 1.39 ± 0.19c 16.04 ± 0.67b
C16:2(n-4) 0.00 ± 0.00b 0.00 ± 0.00b 0.00 ± 0.00b 0.00 ± 0.00b 0.00 ± 0.00b 4.40 ± 0.17a
C18:2(n-6) 1.13 ± 0.08ef 3.42 ± 0.24b 1.43 ± 0.09de 1.83 ± 0.23cd 0.80 ± 0.07f 9.45 ± 0.11a
C18:3(n-3) 0.10 ± 0.01d 7.00 ± 0.08a 0.38 ± 0.09cd 0.00 ± 0.00d 1.01 ± 0.24c 3.31 ± 0.40b
C18:3(n-6) 0.71 ± 0.07b 0.69 ± 0.04bc 0.88 ± 0.14b 0.28 ± 0.02d 0.75 ± 0.11b 4.26 ± 0.18a
C20:4(n-6) 2.18 ± 0.43b 0.34 ± 0.07d 1.25 ± 0.07c 0.26 ± 0.01d 2.42 ± 0.13b 9.79 ± 0.30a
C20:5(n-3) 10.94 ± 2.53b 0.77 ± 0.13d 4.19 ± 0.39c 10.46 ± 0.55b 19.12 ± 0.19a 0.00 ± 0.00d
C22:5(n-3) 0.00 ± 0.00c 1.99 ± 0.29a 0.74 ± 0.24b 0.00 ± 0.00c 0.68 ± 0.01b 0.00 ± 0.00c

C22:6(n-3) 1.20 ± 0.28b 11.76 ± 2.91a 0.76 ± 0.05b 0.80 ± 0.04b 1.78 ± 0.38b 0.00 ± 0.00b
 SFA 49.88 ± 2.28a 32,55 ± 3.99e 40.45 ± 0.87cd 36.27 ± 2.79de 41.83 ± 0.62bc 46.23 ± 0.61ab

 MUFA 16.87 ± 3.61c 25.97 ± 3.19ab 9.65 ± 0.30d 13.26 ± 0.84cd 26.54 ± 0.47ab 31.20 ± 0.46d

 PUFA 31.37 ± 1.78c 41.48 ± 1.90b 49.89 ± 0.57a 50.11 ± 1.96a 31.63 ± 0.22c 22.57 ± 0.65d

 omega-6 3.96 ± 0.77 4.45 ± 0.19 3.59 ± 0.32 2.37 ± 0.24 3.96 ± 0.07 23.49 ± 0.42a
b b b c b

omega-3 12.91 ± 2.87b 21.52 ± 3.38a 6.06 ± 0.58c 11.25 ± 0.59b 20.58 ± 0.44a 3.31 ± 0.40c
␻6/␻3 ratio 3.25 ± 0.24b 4.87 ± 0.95a 1.70 ± 0.31c 4.77 ± 0.23a 5.70 ± 0.13a 0.14 ± 0.02d
a–f
Results presented as mean ± standard deviation of three determinations. Different letters within the same row indicates significant difference (p < 0.05). SFA is saturated
fatty acids; MUFA is monounsaturated fatty acid; PUFA is polyunsaturated fatty acid and ␻ is omega.

Table 4
Multiple linear regression analysis of X-variable versus Y-variable.

X-variable Y-variable Coefficients Standard error r t Stat P-value

Total carotenoid content ABTS·+ Scavenging 0.250 0.962 0.981 0.866 0.409
Fucoxanthin 0.129 0.874 0.588 0.571
Total phenolic content 0.443 0.293 2.737 0.023
Gallic acid 0.279 0.329 1.325 0.218
Total carotenoid content Iron Chelating Activity −0.331 0.767 0.966 −0.860 0.412
Fucoxanthin 0.998 0.697 3.410 0.008
Total phenolic content −0.320 0.234 −1.481 0.173
Gallic acid 0.722 0.262 2.570 0.030
Total carotenoid content FRAP Activity 0.514 0.135 0.966 1.339 0.214
Fucoxanthin 0.115 0.123 0.396 0.701
Total phenolic content 1.006 0.041 4.674 0.001
Gallic acid −0.563 0.046 −2.010 0.075
Total carotenoid content ␤ Carotene Bleaching 0.091 0.045 0.992 0.493 0.634
Fucoxanthin −0.028 0.014 3.764 0.004
Total phenolic content −0.028 0.014 −0.267 0.795
Gallic acid 0.545 0.015 4.047 0.003

DW), S. costatum (0.97 ± 0.24 mg. g−1 DW), P. tricornutum 3.4. TPC and phenolic acid profile
(0.18 ± 0.00 mg. g−1 DW) and S. japonica (0.05 ± 0.02 mg. g−1
DW) (Table 1). Fucoxanthin is the major and unique carotenoid TPC evaluation revealed that I. galbana (12.24 ± 1.61 mg
that distinguishes Bacillariophyceae, Prymnesiophyceae and GAE. g−1 DW) had significantly higher (p < 0.05) phenolic acids
Phaeophyceae (Pennington et al., 1988) from other microalgae than the rest of the algae species. This was followed by O.
classes that do not produce the pigment. Similarly, in HPLC studies, sinensis (3.71 ± 0.34 mg GAE. g−1 DW), C. calcitrans (3.68 ± 0.39 mg
C. calcitrans showed the highest fucoxanthin (5.13 ± 0.19 mg GAE. g−1 DW), S. costatum (0.18 ± 0.00 mg GAE. g−1 DW), S. japon-
FX. g−1 DW) content. This value was significantly higher (p < 0.05) ica (0.09 ± 0.01 mg GAE.g−1 DW) and P. tricornutum (0.02 ± 0.00 mg
than those observed in I. galbana (2.19 ± 0.02 mg FX. g−1 DW), O. GAE.g−1 DW) (Table 2). Many previous microalgae studies only
sinensis (1.18 ± 0.00 mg FX. g−1 DW), S. costatum (0.36 ± 0.00 mg investigated the TPC (Goh et al., 2010; Goiris et al., 2012) and did not
FX. g−1 DW) or P. tricornutum (0.07 ± 0.00 mg FX. g−1 DW). Both the proceed for further microalga phenolic profiling. This study identi-
carotenoid and fucoxanthin contents showed significantly positive fied gallic acid as the major phenolic acid among the eleven types of
Pearson correlation of r = 0.927 (p < 0.05) (Fig. 3). In addition, it was phenolic acids investigated using HPLC. In this study, Pearson corre-
observed that significantly higher amount (p < 0.05) of carotenoid lation analysis showed a significant and high correlation (r = 0.915;
and fucoxanthin contents were found in all investigated microal- p < 0.05) between TPC and gallic acid (Fig. 3).
gae as compared to the seaweed, S. japonica (0.03 ± 0.00 mg FX. g−1
DW).
182 S.C. Foo et al. / Journal of Biotechnology 241 (2017) 175–183

3.5. FAMEs analysis and PUFA profiling cantly (p< 0.05). In addition, gallic acid being the major phenolic
acid detected in this study, demonstrated significant ABTS·+ scav-
Overall, results showed that microalgae strains are significant enging and metal chelating activities (p> 0.05). This was achieved
producers of PUFA (Table 3). Investigated strains had high PUFA by donation of hydrogen atom (H·) or electrons by the 3 hydroxyl
content ranging from 22.57 to 50.11% of total FAMEs. O. sinensis groups at the para-position to the carboxylic group in gallic acids
(50.11%) and S. costatum (59.89%) had significantly higher (p < 0.05) (Li et al., 2007; Lu et al., 2006). However, this was not so in the
PUFAs than I. galbana (41.48%), P. tricornutum (31.63%), C. calcitrans case of ABTS·+ scavenging and FRAP assays. Only TPC had a sig-
(31.37%) and S. japonica (22.57%). Besides that, fatty acid eicosapen- nificant correlation (p < 0.05) compared to the rest of the multiple
taenoic acid (C20:5n3; EPA) was found to be significantly higher linear regression analysis (Table 4). The overlapping in mechanism
(4.18-20.77%) in microalgae compared to investigated macroalga. of action might be a possible reason to account for the significant
In particular, C. calcitrans had the highest EPA content (20.77% of correlation (p < 0.05); for example, TPC works on a similar electron
total fatty acids) which was higher than previously reported EPA transfer mechanism as ABTS·+ scavenging assay (Prior et al., 2005).
values of 2.9–14.0% of total fatty acids in C. calcitrans (Shamsudin, In the meantime, both mechanisms of action in TPC and FRAP assays
1992). The PUFA content of I. galbana in our studies (41.48%) was involved reduction through the donation of an electron or hydrogen
about two times higher than a previous report (29.21%) (Custódio atom (Müller et al., 2011). Nevertheless, this study demonstrated
et al., 2014) which could be due to the differences in culture condi- that the selection of an appropriate antioxidant assay can have a
tions. Temperature, medium type and carbon dioxide levels (Wang significant impact on the results and that proper selection is of vital
et al., 2014) can have a significant effect on the fatty acid profile in importance to ensure reliable deduction from the analyses.
microalgae strains. The fatty acid profile of microalgae can be espe-
cially useful when mercury contamination poses as a threat to wild 4. Conclusion
fish stocks. For example, the fatty acid profile of C. calcitrans from
this study was rich in C14:0 (17.5%), C16:0 (20.33%), C16:1 (25.42%), This study showed that the two microalgae species (C. calci-
C20:5 (10.94%), which was similar to C. muelleri (Mendiola et al., trans and I. galbana) had significantly higher (p < 0.05) fucoxanthin
2007) and Chaetoceros sp. (Maadane et al., 2015). and gallic acid contents as well as significantly higher (p < 0.05)
antioxidant activities than the other microalgae species. This was
3.6. Influence of carotenoid and phenolic contents on antioxidant followed by O. sinensis and S. costatum with moderate antioxidant
capacities activity whereas the species with least antioxidant activity were P.
tricornutum and S. japonica.
Both carotenoid and phenolic compounds were found to be Correlation analyses (Pearson correlation and multiple regres-
responsible for the antioxidant activities in the investigated algae sion analysis) illustrated that both carotenoid and phenolic
and this was supported by Pearson correlation analysis (p < 0.05) contents significantly contributed to the antioxidant activities of
(Fig. 2). Total carotenoid content was found to positively cor- the algal species (p < 0.05). In fact, a stronger correlation was
relate to ABTS·+ scavenging assay (r = 0.8414; p < 0.05), IC assay observed when major component of carotenoid (fucoxanthin) and
(r = 0.9252; p < 0.05), FRAP assay (r = 0.8313; p < 0.05) and BCB assay phenolics (gallic acids) were used in establishing a statistical rela-
(r = 0.9545; p < 0.05) respectively. Besides that, TPC correlated to tionship (multiple linear regression) between the bioactives and
ABTS·+ scavenging assay (r = 0.8809; p < 0.05), IC assay (r = 0.4919; antioxidant capacities. It was important to note that each antioxi-
p < 0.05), FRAP assay (r = 0.8473; p < 0.05) and BCB assay (r = 0.532; dant assay has its characteristic mechanism of action, thus selection
p < 0.05). These findings were parallel to a past study (Goiris of appropriate antioxidant assay is crucial to obtain meaningful
et al., 2012) which reported a significant contribution of phenolic information.
and carotenoid contents to antioxidant capacities (FRAP, r = 0.741;
ABTS·+ scavenging, r = 0.714; p < 0.05). Authors’ contributions
In addition, multiple regression analysis showed the influence
of the investigated bioactives in explaining antioxidant activi- SCF carried out the study and drafted the manuscript. SCF and
ties (Table 4). It was found that further identification of major SKY collected, analysed and interpreted the data under the super-
carotenoid and phenolic compounds (i.e. fucoxanthin and gallic vision of FMY, MB, and MI. NMHK and KWC contributed to the
acid) resulted in significantly higher (p < 0.05) beta coefficients design and conception of the study. FMY, NMHK, MB and MI crit-
compared to the mixed compounds (i.e. total carotenoid content ically revised the manuscript. All authors have read and approved
and TPC). The standardized beta coefficient in the multiple lin- this manuscript for publication. All authors take responsibility for
ear regression is an index used to show the weight/importance the integrity of the work as a whole, from inception to the finished
of the X-variable (bioactives) in explaining the Y-variable (antioxi- article.
dant activity). In the IC assay, fucoxanthin showed a beta coefficient
of 0.998 (p< 0.05) whilst gallic acid’s beta coefficient was 0.722 Acknowledgements
(p< 0.05). Also in the BCB assay, fucoxanthin had a beta coefficient
of 0.528 (p< 0.05) compared to the total carotenoid content (0.091; The authors acknowledge the financial support of the Malaysian
p> 0.05). Similarly, gallic acid had a significantly higher (0.545; Ministry of Higher Education for Fundamental Research Grant
p< 0.05) coefficient than the TPC (−0.028; p> 0.05). From these Scheme (FRGS: 02-01-13-1224FR) and Higher Institution Centre
results, it seemed that both fucoxanthin and gallic acid contributed of Excellence (HICoE) Research Fund. The first author is a recipient
almost equally (approximately 50% each) to the antioxidant capac- of the Ministry of Higher Education (MyBrain15) scholarship and
ity in the algae. In fact, linear regression provides information on Asian Fisheries Society Kanazawa Research Fellowship 2013.
the extent of the contribution of each component which would be
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