Bioprocess and Biosystems Engineering (2018) 41:1611–1620

https://doi.org/10.1007/s00449-018-1987-z

RESEARCH PAPER

Potent biomedical applications of isolated polysaccharides

from marine microalgae Tetraselmis species
Shaheen Amna Kashif1 · You Jin Hwang2 · Jae Kweon Park1

Received: 20 October 2017 / Accepted: 15 July 2018 / Published online: 22 August 2018
© Springer-Verlag GmbH Germany, part of Springer Nature 2018

Abstract
Microalgae Tetraselmis species were used to evaluate the biological characteristics of water-soluble polysaccharides (WSPs)
as one of the significant bioactive substances (BAS) from these photosynthetic microalgae species. Compositional analysis
of these BAS shows that they are mainly composed of WSPs along with negligible amount of proteins and lipids. WSPs were
partially purified and characterized for their compositional, structural and biological properties such as antioxidant, tyrosi-
nase inhibitory activity and antifungal activies. These WSPs showed the significant antioxidant, antifungal and tyrosinase
inhibitory activities, respectively. The outcomes of this study demonstrated that WSPs can be the potent source of biological
moieties for further investigations along with specific potent biological activities.

Keywords  Water-soluble polysaccharide · Biological activities · Photosynthetic microalgae · Bioactive substances

Abbreviations are less competitive for agricultural land, produce useful

WSP Water-soluble polysaccharides by-products and are environmental friendly as compared
BAS Bioactive substances to other conventional plants [1], microalgae have already
T1 KCTC 12236BP been used commercially in various fields such as food, ani-
T2 KCTC 12432BP mal feed, cosmetics, pollution abatement, and therapeutics
DPPH 1,1-Diphenyl-2-picrylhydrazyl industries [2, 3]. The biochemical composition of microal-
FRAP Ferric reducing antioxidant power gae depends on its cultivation conditions and external factors
ABTS 2,2′-Azino-bis(3-ethylbenzothiazoline- such as temperature, light, and nutrient composition of the
6-sulfonic acid) di-ammonium salt TPTZ culture medium [4]. Microalgae are small unicellular organ-
2,4,6-tri(2-pyridyl)-s-triazine isms, covered with a relatively thick cell wall and its prod-
TLC Thin-layer chromatography ucts are usually located in globules or bound to cell mem-
branes, which cause the extraction of intracellular products
challenging. To date, various methods such as mechanical,
Introduction enzymatic, chemical, ultrasonication and microwave tech-
niques have been used for cell disintegration and product
Most of the marine microalgae are recognized as great extraction. Obviously, the efficacy of the product depends
sources of raw materials in the field of bio-refinery due to on the method of extraction. In this study, various extraction
their composition and fast growth, rich lipid content, and methods have been established to ensure low experimental
cost-effective photosynthetic mechanisms. Since microalgae cost, high product recovery and high quality of the extracted
products, called water-soluble polysaccharide WSPs.
Besides being a great source of proteins, some species of
* Jae Kweon Park microalgae produce some other valuable compounds, such
jkpark@gachon.ac.kr
as carotenoids, polysaccharides, vitamins, lipids and fats.
1
Department of Life Sciences, College of Bionano, Among various microalgal species, both Tetraselmis spe-
Gachon University, Seongnamdaero 1342, Seongnam‑si, cies KCTC 12236BP (T1) and KCTC 12432BP (T2) were
Gyeonggi‑do 461‑701, Republic of Korea isolated and used as one of the best biomasses and promising
2
Department of Biomedical Engineering, College producers of BAS that are associated with biological activi-
of Health Science, Gachon University, Incheon 21936, ties and potential health benefits. Among other species, these
Republic of Korea

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Vol.:(0123456789)

1612
two strains were selected because of their rapid growth,
ease of availability, cost-effectiveness and ease of handling
(unpublished data). In addition, most of the microalgae are
able to accumulate a large amount of lipids, which can be
extracted and used for biodiesel production. However, the
waste materials generated by this process have not been stud-
ied well, but these defatted residues or whole biomasses can
be tested for their potential use [5]. Among BAS, polysac-
charides that are homo- or hetero-monosaccharaides which
are linked together by glycosidic bonds represent a class
of highly valuable components with various applications in
food, cosmetics, fabrics, stabilizers, emulsifiers and medi-
cine [6]. Polysaccharides usually serve as antimicrobial
agents, health foods and antioxidants; they also possess
higher anti-inflammatory properties and have a role in skin
whitening; hence, they act as potent tyrosinase inhibitors in
the melanin synthesis pathway [7, 8]. Subsequently, water-
soluble polysaccharides (WSP) from microalgae are the
compounds of most interest in the present study.
Rhododendrol has been recently reported to be a potent
tyrosinase inhibitor in the melanin synthesis pathway and
is widely used these days in skin whitening cosmetics [9].
Some other chemical compounds have also been isolated/
synthesized to address tyrosinase inhibitory activity to play
pivotal role in the synthesis of melanin and to overcome skin
darkness [10]. Therefore, discovering a tyrosinase inhibitor
is important for controlling the production of melanin as
the rate-limiting enzyme in the melanin synthesis pathway.
However, since most of the polysaccharides are embedded
inside the cell wall, to break down the complex cell wall and
isolate the WSPs, various mechanical, chemical or enzymati-
cal pretreatments are required. Keeping all these in mind,
we can summarize that the main purpose of this study is to
optimize and purify WSPs from de-fatted microalgal bio-
masses of Tetraselmis species KCTC 12236BP (T1) and
KCTC 12432BP (T2), respectively, for the assessment of
their biochemical properties for their possible biological and
therapeutic applications.
Materials and methods
Chemicals
Sulfuric acid ­(H 2SO 4), 1,1-diphenyl-2-picrylhydrazyl
(DPPH), 2,2′-azino-bis (3-ethylbenzothiazoline-6-sulfonic
acid) di-ammonium salt (ABTS), 2,4,6-tri(2-pyridyl)-s-tri-
azine (TPTZ), ferric chloride, potassium persulfate, sodium
hydroxide and l-ascorbic acid were purchased from Sigma
Chemical Co. (St. Louis, MO, USA). Sodium hydroxide
(NaOH) and 4-hydroxy-benzohydrazide (PHABAH) were
used for quantification of reducing sugar and were obtained
from Junsei Chemical Co. (Tokyo, Japan). Decanoic acid,
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Bioprocess and Biosystems Engineering (2018) 41:1611–1620
gallic acid and bovine serum albumin were used for the
quantification of polyphenol; lipid and protein were obtained
from Sigma Chemical Co. (St. Louis, MO, USA). All other
reagents were purchased and used of the highest grade
available.
Biomass pretreatment and isolation of WSP
Both de-fatted microalga Tetraselmis species (sp.) KCTC
12236 BP and KCTC 12432 BP were obtained from the
laboratory of Professor Choul-Gyun Lee, INHA Univer-
sity (InCheon, Korea), and were designated as T1 and T2,
respectively. To isolate BAS, T1 and T2 were suspended,
respectively, in 1 N ­H2SO4, 1 M NaOH and distilled water
to be produce a final concentration of 10% (w/v), then vigor-
ously vortexed and allowed to react for various periods of
time, i.e., 0, 6 and 24 h. After centrifugation at 13,000 rpm
for 5 min, the supernatant from each sample was collected
and continuously subjected to ethanol precipitation (2.5-
fold of the original volume) for 1 h at − 20 °C to isolate
and purify WSP. The resultant solution was centrifuged at
13,000 rpm for 5 min; afterward, the pallet was isolated and
rinsed with ethanol twice and the purified WSPs were re-
suspended in water to isolate water-soluble substances. After
removal of water-insoluble materials, all samples were lyo-
philized and analyzed for their biochemical and biological
activities after re-suspension of these lyophilized samples in
water (up to the initial volume).
Phenol–sulfuric acid assay
Compositional elucidation of extracted WSP is critically
important to quantify the ingredients of these WSPs. The
contents of total carbohydrates in the sample were deter-
mined based on phenol–sulfuric acid assay [11]. To quantify
the total carbohydrate (TC) content of WSPs, 200 µL of the
sample was mixed with phenol–sulfuric acid solution. The
sample’s absorbance was measured at 490 nm using a UV
spectrophotometer (Infinite M200 Pro Nano-quant, TECAN,
Austria), and the TC content was quantified based on the
absorbance of the control (glucose).
Reducing sugar contents
Evaluation of reducing sugar content in WSPs is an impor-
tant process to characterize the nature of these polysaccha-
rides [12]. Briefly, 16 mM PHABAH (4-hydroxybenzohy-
drazide) solution was used to quantify the concentration of
reducing sugars in the testing samples. The prepared solu-
tion was mixed with samples in an appropriate ratio and
the resulting mixture was then heated to 100 °C for 5 min.
The sample mixture was then centrifuged at 13,000 rpm for
5 min to remove the insoluble matters. Afterward, samples
Bioprocess and Biosystems Engineering (2018) 41:1611–1620 1613
were read at 405 nm by UV-spectrophotometer (Infinite
M200 Pro Nano-quant, TECAN, Austria). Reducing sugar
concentration in the sample was determined after normal-
izing with a wide range of glucose, used as control.
Fourier‑transform infrared spectroscopy
Fourier-transform infrared (FT-IR) spectroscopy was used to
identify the functional groups of WSP extracted from both
microalga Tetraselmis sp. 12236BP (T1) and 12432BP (T2),
respectively. This analysis was performed through FT-IR
spectra (400–4000 cm−1) with a Nicolet 6700 FT-IR spec-
trophotometer (Nicolet Analytical Instruments, USA) using
Zn/Se windows. To detect the high resolution, infrared spec-
trum was selected as 8/cm and 64 scans of accumulation, as
described in the previous study [13].
TLC analysis
To find the differences in chemical composition, a thin-
layer chromatography (TLC) using silica gel (60/Kieselguhr
­F254 thin-layer plate, Darmstadt, Germany) was performed
according to a previous study [14]. A chemical mixture con-
sisting of N-propanol: ­NH4OH: ­H2O = 7:1.5:1.5 (v/v) was
prepared and used as running buffer. Separation of WSPs on
TLC plate was visualized under UV light at a wavelength of
254 and 365 nm, respectively. Following the UV light obser-
vation, the TLC plate was then developed using a solution
mixture consisting of 3% phosphoric acid and 8% copper
sulfate (v/v), which is reliable to observe sugar-containing
materials. The TLC plate was then baked at 200 °C to iden-
tify each resultant band.
DPPH radicals scavenging activity
The DPPH radical scavenging assay is widely performed to
analyze the antioxidant activities of biologically active com-
pounds derived from various sources [15]. Briefly, 1 mM
DPPH solution was mixed with the test samples in a 3:1
(v/v) ratio and allowed to react in a dark at room tempera-
ture for 20 min. To determine the best mixing ratio of the
sample with the DPPH solution, various ratios were tested.
Subsequently, the absorbance was measured at 517 nm using
a UV-spectrophotometer (Infinite M200 Pro Nano-quant,
TECAN, Austria). The scavenging relative activity (%) was
calculated as: RA (%) = [1 − (As)/Ac] × 100, where As is
the absorbance of the sample and Ac is the absorbance of
the control. Specific activity was measured on the basis of
total carbohydrates. µmol g−1 min−1. µmol is derived from
the ascorbic acid concentration, used for standard curve,
“g” is derived from the TC quantification of each sample
and “min” is the reaction time for the completion of each
experiment.
ABTS‑radical scavenging assay
The ABTS (2,2′-azino-bis (3-ethylbenzothiazoline-6-sul-
fonic acid)) radical cation scavenging assay was used to
evaluate the antioxidant activity of WSP [15]. Briefly, 7 mM
of ABTS and potassium persulfate, 7.35 mM, were dissolved
in distilled water, and the resulting solution was kept for
16 h at room temperature in dark. Then ABTS solution was
mixed with samples in a ratio of 10:1 and allowed to react at
room temperature for 6 min. After that the absorbance was
taken at 734 nm using a spectrophotometer (Infinite M200
Pro Nanoquant, TECAN, Austria). The ABTS radical scav-
enging relative activity was calculated: RA (%) = [1 − (As)/
Ac] × 100, where, As is the absorbance of the sample and Ac
is the absorbance of the control. Specific activity was meas-
ured on the basis of total carbohydrates, µmol g−1 min−1.
µmol is derived from the ascorbic acid concentration, used
for standard curve, “g” comes from TC quantification of
each sample and “min” is the reaction time for the comple-
tion of each experiment.
Ferric reducing antioxidant power (FRAP) assay
The FRAP reagent was prepared [16] by mixing TPTZ
[40 mM 2,4,6-tri (2-pyridyl)-s-triazine], 20 mM ferric chlo-
ride and 0.3 M acetate solution. The solution was mixed
with the testing sample solution and water in a 33:1:14 ratio
and the reaction allowed to continue at 37 °C for 4 min. The
absorbance of the substances was read at 595 nm using a UV
spectrophotometer (Infinite M200 Pro Nanoquant, TECAN,
Austria) to calculate the reducing power percentage of WSP:
RA (%) = [1 − (Ac/As)] × 100, where, Ac is the absorbance
of the control and As is the absorbance of the sample. Spe-
cific activity was measured on the basis of total carbohy-
drates, µmol g−1 min−1. µmole is derived from the ascorbic
acid concentration used for standard curve, “g” comes from
TC quantification of each sample and “min” is the reaction
time for the completion of each experiment.
The extracts were further analyzed for protein, lipid and
polyphenol contents according to already established proto-
cols, respectively [17–19].
Tyrosinase inhibitory activity
The method used for the detection of tyrosinase inhibitory
activity [9, 20] is briefly described as follows. 1 mM l-tyros-
ine was added to 25 mM phosphate buffer (pH 6.8) and then
mixed with 24 µL of enzyme solution consisting of 100 µg/
mL tyrosinase from mushroom in the presence or absence
of testing samples (30 µL). The mixed solution was then
incubated at room temperature for 30 min, and the absorb-
ance was monitored at 405 nm using a UV-spectrophotom-
eter (Infinite M200 Pro Nano-quant, TECAN, Austria). To
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1614
compare the effectiveness of tyrosinase inhibitory activity,
we were unable to find suitable positive control; however
lag-time delaying of tyrosinase activity in the presence of
tyrosine used as a substrate was measured by the dependence
of concentration of BAS. Zero concentration of tyrosine is
termed as negative control.
Antimicrobial assays
The microorganisms used in this study were Candida albi-
cans and Penicillium italicum, respectively, which were
subjected to antimicrobial assays according to already
established protocol [21]. Briefly, twofold dilutions of
the antimicrobial agents were prepared in a liquid growth
medium, dispensed in 96-well micro-titration plate (micro-
dilution). Then, each well was inoculated with a microbial
inoculum. Mixed thoroughly and the inoculated 96-well
micro-titration plates were incubated in a UV-spectropho-
tometer (Infinite M200 Pro Nano-quant, TECAN, Austria)
with agitation under suitable time and temperature condi-
tions depending on the microorganisms tested. Relative
growth-inhibitory activity was calculated based on the result
from the control test. For the determination of antifungal
activities, fluconazole and imidazole were used as commer-
cially available drugs to compare the antifungal activities of
partially purified polysaccharides.
Protein quantification protocol
The protein quantification was done according to a previ-
ous study [17]. Briefly, a fourfold dilution of a 2 mg/mL
Coomassie blue solution was prepared. The samples were
mixed with Coomassie blue solution in a ratio of 10:100 and
centrifuged at 13, 5000 RPM for 5 min to remove insolu-
ble matter. The absorbance of each sample was measured
at 595 nm using a UV–visible spectrophotometer (Infinite
M200 Pro Nano-quant, TECAN, and Austria). The absorb-
ance of each BSA standard was plotted as a function of its
theoretical concentration.
Lipid quantification analysis
The lipid quantification was performed according to a pre-
vious study with some modifications [18]. Briefly, 500 µL
of sample was mixed with 200 µL of neutralizing agent
(100 mM Tris–HCL). Then this mixture was combined with
200 µL of copper reagent (9:1:10 = 1 M triethanolamine:1 N
acetic acid:6.5 % of ­CuSO4) and 250 µL of chloroform. This
mixture was centrifuged at 13,000 RPM for 5 min to remove
insoluble matter. 50 µL of this reaction mixture was mixed
with 50 µL of color developer (1% diethyldithiocarbamate
in butanol). The absorbance of the samples as measured at
440 nm compared with gallic acid control absorance.
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Bioprocess and Biosystems Engineering (2018) 41:1611–1620
Total phenol content (TPC)
The TPC was determined using the Folin–Ciocalteu colori-
metric method [19, 22] with slight modifications. Briefly,
200 µL of the diluted solution was mixed with 200 µL of
Folin–Ciocalteu reagent and 500 µL of 2% ­Na2CO3 solu-
tion was added to the following mixture. After 30 min of
incubation at room temperature, the solution was then cen-
trifuged at 13,000 RPM and the absorbance was measured
at 725 nm with a UV-spectrophotometer (Infinite M200 Pro
Nano-quant, TECAN, Austria). The gallic acid equivalent
was determined based on a calibration curve generated
using gallic acid standard solution to calculate the phenolic
content.
Statistical analysis
Each sample contained at least three replicates and all exper-
iments performed in this study were repeated at least three
times. Data on the effect of the antioxidant activities were
compared using Student’s test and presented as the mean
value standard deviation (SD) with P < 0.05 indicating sta-
tistical significance.
Result and discussion
Characterization of bioactive substances
from microalga Tetraselmis species
To characterize the biochemical properties of microalgal
biomasses, we primarily isolated bioactive substances (BAS)
and purified the water-soluble polysaccharide (WSP) from
Tetraselmis sp. 12236BP (T1) and 12432BP (T2), respec-
tively. According to a previous study [13], microalgal spe-
cies have different structural components of glycans in their
cell walls. To isolate crude BAS, various chemical pre-
treatments have been done using T1 and T2, i.e., acid (1 N
­H2SO4), base (1 M NaOH) and water treatments. Afterward,
these crude extracts were subjected to ethanol treatment to
purify WSPs. Furthermore, the experimental conditions of
these isolated and partially purified WSPs by acid, base and
water pretreatment were observed for various periods of
time at different temperatures to optimize suitable working
conditions (Fig. 1). According to the results, we optimized
the best working conditions in terms of total carbohydrate
for both T1 and T2 to be 37 °C for 24 h, respectively. Based
on these primary findings, we selected 37 °C and 24 h as
the best experimental conditions for further experiments in
this study.
WSPs are one of the most promising BAS as they are
able to diffuse in the culture medium, free from cell debris,
and are easy to collect and purify [23, 24]. However, after
Bioprocess and Biosystems Engineering (2018) 41:1611–1620 1615
Fig. 1  Characterization of WSPs extracted from microalga Tet-
raselmis sp. 12236BP (T1) and 12432BP (T2) for its carbohydrate
content at various time and temperature points (optimization). a
Acid treatment, b base treatment, c water, acid 1 N H
­ 2SO4, base 1 M
NaOH
various pretreatments applied on these WSPs, the qual-
ity and recovery of WSPs comprise an important factor.
Therefore, after ethanol precipitation, the resulting WSPs
were freeze dried and ultimately the total % yield for WSPs
was calculated (Table 1). As shown in Table 1, the highest
Table 1  Quantification of % age yield of WSP extracted from microalgae Tetraselmis sp. 12236BP (T1) and 12432BP (T2) after acid and base
extraction at various temperatures and time periods
T1
Acid Base
Yield (%)a 7.0 31.5
a
 Yield = [dried biomass (g)/original biomass (g)] × 100
productivity of WSPs was observed following base treatment
and that is about 31% for T1 and 3% for T2, respectively. The
results of the present study indicate that base treatment can
lead to increase in productivity and quality of the WSP from
the de-fatted microalgal biomasses.
Biochemical profile
To evaluate the characteristics of WSPs as potent bio-
resources, the crude cellular component proportion was
determined (Table 2). As a result, the highest TC content
was observed to be 0.87 g/L after base (alkali) treatment,
whereas after acidic treatment the TC was observed to be
0.63 ± 0.07 g/L and 0.42 g/L after water extraction for T1.
In case of T2, the highest TC content was also observed
following base treatment, i.e., 0.42 g/L, whereas following
acidic and water extraction the TC content was observed
to be 0.59 and 0.35 g/L respectively. Furthermore, lipids,
polyphenols, and proteins were also quantified, since most of
the Tetraselmis species (sp.) are known to be lipid producers
that can later be transformed into biodiesel [25]. The crude
lipid levels of the microalgal species vary from 1.5 to 9.3%.
In the present study, as we used defatted microalgae and to
provide evidence, we determined lipid and protein levels and
our results showed insignificant values. The results indicated
that lipid production was reduced in case of T2 following the
same pretreatments and was observed to be 0.0019 g/L for
acid, 0.0026 g/L for base and 0.00016 g/L after water treat-
ment, respectively. On the other hand, microalgal proteins
have long been considered as an alternative protein source
in foods due to abundance of proteins and their amino acid
profiles [26]. Polyphenols in microalgae are also considered
to be biofuel producers [27]. But unfortunately, negligible
amount of proteins, lipids, and polyphenols were elucidated
from defatted microalgal biomasses (Table 2).
Chemical composition elucidation
The FT-IR spectra of WSPs extracted from microalga Tet-
raselmis sp. T1 and T2 was reconstructed by Microsoft
Excel, using the spectra of the reference substances to
determine their relative contribution to the overall constitu-
ents of WSP from T1 and T2 (Fig. 2). The band spectrum
between 4000 and 3000 cm−1 showed an OH stretch that
T2
Water Acid Base Water
10.0 4.5 34.5 9.1
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1616 Bioprocess and Biosystems Engineering (2018) 41:1611–1620

Table 2  Biochemical analysis of contents in WSP extracted from microalgae Tetraselmis sp. 12236BP (T1) and 12432BP (T2)
T1 T2
Acid Base Water Acid Base Water

TC (g L­ −1) 0.63 ± 0.07 0.87 ± 0.05 0.42 ± 0.02 0.42 ± 0.01 0.59 ± 0.05 0.35 ± 0.01

RS (g ­L−1)a 0.23 ± 0.02 0.23 ± 0.02 0.33 ± 0.04 NDb NDb NDb
Polyphenol (g ­L−1) 0.05 ± 0.03 0.05 ± 0.01 0.02 ± 0.02 ND 0.01 ± 0.02 ND
Proteins (g ­L−1) 0.0014 ± 0.01 0.00140 ± 0.01 0.00101 ± 0.01 0.00006 ± 0.06 0.00048 ± 0.07 0.000011 ± 0.02
Lipid (g ­L−1) 0.0029 ± 0.02 0.0009 ± 0.04 0.0019 ± 0.02 0.0011 ± 0.20 0.0026 ± 0.01 0.00016 ± 0.06
pH 3 9 7 3 11 7

TC total carbohydrates, RS reducing sugars

a
 Reducing sugar
b
 None detectable

Fig. 2  FT-IR analysis of WSPs

extracted from microalga
Tetraselmis sp. 12236BP (T1)
and 12432BP (T2). a Acid
treatment, b base treatment;
treatment conditions 24 h and
37 °C

was evidently higher in case of water-pretreated microalgae.
The band length at 1643–1654 cm−1 indicates –COOH along
with α helix amino acids that can be lower molecular weight
proteins, and all three pretreated groups showed a sharp peak
at 1643–1650 cm−1. Peaks absorbed at 1279–1050 cm−1 are
characteristic for absorption of C=O stretching vibration.
Absorptions around 900–800 provide α-configuration and
β-configuration sugar units. These findings also coincide
with % yield and TLC analysis, hence proposing that base
(alkali) pretreatment provides best yield along with best
extraction and composition analysis (data not shown). As
water was used as a solvent for water-soluble polysaccha-
rides, it showed almost identical peaks from O–H scissors.
Antioxidant activity
The antioxidant activity of these extracted WSPs from T1
and T2 was measured concerning their efficiency of scav-
enging free radicals generated by DPPH, ABTS, and FRAP,
respectively. For the determination of antioxidant activity

13
Bioprocess and Biosystems Engineering (2018) 41:1611–1620 1617
assays, vitamin C (ascorbic acid) was used as positive con-
trol to evaluate the potency of these partially purified poly-
saccharides. The DPPH scavenging specific activities of
WSP isolated from T1 were determined to be 7.23, 19.85,
and 6.84 (µmol/g/min) after acid, base, and water pretreat-
ments (Table 3), whereas DPPH radical scavenging specific
activity of WSP purified from T2, was observed to be 7.47,
60.33, and 8.67 (µmol/g/min) following acid, base, and water
pretreatments, respectively. These results clearly reveal that
a significant scavenging activity was observed after base
treatment for both the strains of Tetraselmis sp., T1 and T2.
Relative DPPH scavenging activity was varied between
3–15% as compared to ascorbic acid concentration, used
as control (relative to ascorbic acid 100%) for T1; however
non-significant change in relative DPPH scavenging activi-
ties for T2, i.e., 1–7% was observed. Next, FRAP antioxidant
activity was observed and the reaction was established upon
the reaction of FRAP reagent with iron(II) and SH-groups
in antioxidants [28]. The reaction starts with the reduction
of ­Fe3+ TPTZ complex (colorless) to F ­ e2+-tripyridyltriazine
(blue colored) involving the action of electron-donating
antioxidants. As a result, no significant specific activity was
observed among all treated groups of T1 and T2, indicating
that WSP may not contain sulfate or other relative functional
groups in their structure.
Next, ABTS antioxidant assay was performed and the
principle of the assay is based on the ability of a sample to
scavenge ABTS cation by addition of potassium persulfate.
This radical cation is blue in color and is highly reactive
toward most of the antioxidants. The blue ABTS radical cat-
ion is converted to its original colorless form in the presence
of an antioxidant. Table 3 shows ABTS scavenging relative
and specific activities of WSPs of T1 and T2. Data show that
the highest specific scavenging activity (65.67 µmol/g/min)
was observed following base treatment. Taken together, all
these results suggest that the total antioxidant activity can
be related to higher yields of WSPs.
Tyrosinase inhibitory activity
To explore other potent biological activities of WSPs iso-
lated from T1 and T2, we investigated tyrosinase inhibitory/
Table 3  Antioxidant activity of T1
WSP extracted from microalgae
Tetraselmis sp. 12236BP (T1) Acid
and 12432BP (T2)
Specific ­activityb (µmol g−1 ­min−1)
 DPPH 7.23 ± 0.51
 FRAP 0.64 ± 0.02
 ABTS N/D
a
 Mean values were calculated from three different experiments
b
 Specific activity was derived from calculated TC values of these WSPs
whitening activity. Tyrosinase inhibition is the most com-
mon methodology to attain skin whitening (hypopigmenta-
tion), as tyrosinase is the rate-limiting enzyme in the mela-
nin synthesis pathway [7, 8]. These facts help us find out
about tyrosinase inhibition by these WSPs extracted from
T1 and T2 (Fig. 3). All samples from T1 and T2 showed
high relative inhibitory activity (Fig. 2a) with the highest of
86% for T1 and 73% for T2, following base (alkali)-extracted
WSPs. T1 and T2 pretreated by acid showed approximately
65 and 60% relative inhibitory activity, whereas 50 and 69%
relative inhibitory activity was observed for T1 and T2 after
water extraction, respectively. On the contrary, the highest
specific activity was determined for WSP of T2, i.e., 243,499
U/min, whereas the T1 specific inhibitory activity ranged
from 8787 to 52,815 U/min (Fig. 3b). These results were
confirmed by the changes in the absorbed wavelength dur-
ing the experiment (Fig. 3c). Taken together, these results
demonstrate that WSPs from T1 and T2 are rich sources of
tyrosinase inhibitors and hence can be potently applied in
the cosmetics and therapeutic industry. 1U represents 0.1
absorbance change per gram of sample.
Antifungal activity
The study of antifungal agents has lagged far behind that
of antibacterial agents, likely because fungi were not rec-
ognized as important pathogens until several years ago [29,
30]. The associated increase in fungal infections prompted a
search for newer and safer agents to combat fungal infections
[31]. These facts prompted us to find the antifungal activity
of these WSPs of microalga Tetraselmis sp. T1 and T2. Two
different fungal strains were used in this study, i.e., C. albi-
cans and P. italicum to confirm the antifungal characteristics
of these extracted WSPs. We hypothesized that concentra-
tion-dependent inhibition of growth would be observed, and
our experiments proved that the growth of C. albicans was
inhibited up to 80% by base (alkali)-extracted WSPs from
T1, whereas the growth of C. albicans was inhibited up to
70% following the same treatment for T2 (Fig. 4). However,
WSPs extracted with water and acid treatments from T1 and
T2 were unable to inhibit C. albicans growth; similar results
were observed when WSP from T1 and T2 were applied to
T2
Base Water Acid Base Water
19.85 ± 0.91 6.84 ± 2.04 7.47 ± 1.11 60.33 ± 4.11 8.67 ± 1.51a
0.64 ± 0.03 0.61 ± 0.01 0.16 ± 0.01 N/D 0.09 ± 0.01
65.67 ± 4.12 24.15 ± 0.4 N/D 18.73 ± 3.13 N/D
13

1618
Fig. 3  Tyrosinase inhibitory activity of WSPs purified from micro-
alga Tetraselmis sp. 12236BP (T1) and 12432BP (T2). a Relative
activity, b specific activity, c modified absorbance, acid 1 N H
­ 2SO4,
base 1  M NaOH, water; treatment conditions: 24  h and 37  °C, spe-
cific activity (SA)  = U/min, 1 U  = 0.1 ΔAbs/g, relative activity
(1 − (T − C)/(A − B)) × 100. According to student’s test, the mean
value standard deviation (SD) with P < 0.05 indicating statistical sig-
nificance
P. italicum (Fig. 5). These results demonstrated that WSP
from T1 and T2 may have a broad spectrum of antimicrobial
activity.
13
Bioprocess and Biosystems Engineering (2018) 41:1611–1620
Fig. 4  Antifungal activity of WSPs extracted from microalgae Tet-
raselmis sp. 12236BP (T1) and 12432BP (T2) against Candida albi-
cans. a Tetraselmis sp. 12236BP (T1), b Tetraselmis sp. 12432BP
(T2), acid 1N ­H2SO4, base 1  N NaOH, water; treatment conditions:
24 h and 37 °C
Conclusions
Our experimental findings suggest that WSPs are one of the
most important components of BAS and can be evolved as
potent biological material in cosmeceuticals and therapeu-
tics industry. This study proposes low-cost and low-energy
consumption of the technique and a promising method in
industrial usage in terms of bio-refinery aspects, which will
enhance the properties of these BAS and WSPs from micro-
algal biomasses. Overall, these results evidently exhibit that
BAS and WSP from the microalgal biomasses have potential
biological activities.
Bioprocess and Biosystems Engineering (2018) 41:1611–1620 1619
Fig. 5  Antifungal activity of WSPs extracted from microalgae Tet-
raselmis sp. 12236BP (T1) and 12432BP (T2) against Penicillium
itallicum. a Tetraselmis sp. 12236BP (T1), b Tetraselmis sp. 12432BP
(T2), acid 1N ­H2SO4, base 1  N NaOH, water; treatment conditions:
24 h and 37 °C
Acknowledgements  This research was supported by a grant from the
Marine Biotechnology Program funded by the Ministry of Oceans and
Fisheries.
Compliance with ethical standards  of protein-dye binding. Anal Biochem 72:248–254. https​://doi.
Conflict of interest  The authors report no conflicts of interest in this
work.
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