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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1612 Bioprocess and Biosystems Engineering (2018) 41:1611–1620

two strains were selected because of their rapid growth, gallic acid and bovine serum albumin were used for the
ease of availability, cost-effectiveness and ease of handling quantification of polyphenol; lipid and protein were obtained
(unpublished data). In addition, most of the microalgae are from Sigma Chemical Co. (St. Louis, MO, USA). All other
able to accumulate a large amount of lipids, which can be reagents were purchased and used of the highest grade
extracted and used for biodiesel production. However, the available.
waste materials generated by this process have not been stud-
ied well, but these defatted residues or whole biomasses can Biomass pretreatment and isolation of WSP
be tested for their potential use [5]. Among BAS, polysac-
charides that are homo- or hetero-monosaccharaides which Both de-fatted microalga Tetraselmis species (sp.) KCTC
are linked together by glycosidic bonds represent a class 12236 BP and KCTC 12432 BP were obtained from the
of highly valuable components with various applications in laboratory of Professor Choul-Gyun Lee, INHA Univer-
food, cosmetics, fabrics, stabilizers, emulsifiers and medi- sity (InCheon, Korea), and were designated as T1 and T2,
cine [6]. Polysaccharides usually serve as antimicrobial respectively. To isolate BAS, T1 and T2 were suspended,
agents, health foods and antioxidants; they also possess respectively, in 1 N ­H2SO4, 1 M NaOH and distilled water
higher anti-inflammatory properties and have a role in skin to be produce a final concentration of 10% (w/v), then vigor-
whitening; hence, they act as potent tyrosinase inhibitors in ously vortexed and allowed to react for various periods of
the melanin synthesis pathway [7, 8]. Subsequently, water- time, i.e., 0, 6 and 24 h. After centrifugation at 13,000 rpm
soluble polysaccharides (WSP) from microalgae are the for 5 min, the supernatant from each sample was collected
compounds of most interest in the present study. and continuously subjected to ethanol precipitation (2.5-
Rhododendrol has been recently reported to be a potent fold of the original volume) for 1 h at − 20 °C to isolate
tyrosinase inhibitor in the melanin synthesis pathway and and purify WSP. The resultant solution was centrifuged at
is widely used these days in skin whitening cosmetics [9]. 13,000 rpm for 5 min; afterward, the pallet was isolated and
Some other chemical compounds have also been isolated/ rinsed with ethanol twice and the purified WSPs were re-
synthesized to address tyrosinase inhibitory activity to play suspended in water to isolate water-soluble substances. After
pivotal role in the synthesis of melanin and to overcome skin removal of water-insoluble materials, all samples were lyo-
darkness [10]. Therefore, discovering a tyrosinase inhibitor philized and analyzed for their biochemical and biological
is important for controlling the production of melanin as activities after re-suspension of these lyophilized samples in
the rate-limiting enzyme in the melanin synthesis pathway. water (up to the initial volume).
However, since most of the polysaccharides are embedded
inside the cell wall, to break down the complex cell wall and Phenol–sulfuric acid assay
isolate the WSPs, various mechanical, chemical or enzymati-
cal pretreatments are required. Keeping all these in mind, Compositional elucidation of extracted WSP is critically
we can summarize that the main purpose of this study is to important to quantify the ingredients of these WSPs. The
optimize and purify WSPs from de-fatted microalgal bio- contents of total carbohydrates in the sample were deter-
masses of Tetraselmis species KCTC 12236BP (T1) and mined based on phenol–sulfuric acid assay [11]. To quantify
KCTC 12432BP (T2), respectively, for the assessment of the total carbohydrate (TC) content of WSPs, 200 µL of the
their biochemical properties for their possible biological and sample was mixed with phenol–sulfuric acid solution. The
therapeutic applications. 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
Materials and methods absorbance of the control (glucose).

Chemicals Reducing sugar contents

Sulfuric acid ­(H 2SO 4), 1,1-diphenyl-2-picrylhydrazyl Evaluation of reducing sugar content in WSPs is an impor-
(DPPH), 2,2′-azino-bis (3-ethylbenzothiazoline-6-sulfonic tant process to characterize the nature of these polysaccha-
acid) di-ammonium salt (ABTS), 2,4,6-tri(2-pyridyl)-s-tri- rides [12]. Briefly, 16 mM PHABAH (4-hydroxybenzohy-
azine (TPTZ), ferric chloride, potassium persulfate, sodium drazide) solution was used to quantify the concentration of
hydroxide and l-ascorbic acid were purchased from Sigma reducing sugars in the testing samples. The prepared solu-
Chemical Co. (St. Louis, MO, USA). Sodium hydroxide tion was mixed with samples in an appropriate ratio and
(NaOH) and 4-hydroxy-benzohydrazide (PHABAH) were the resulting mixture was then heated to 100 °C for 5 min.
used for quantification of reducing sugar and were obtained The sample mixture was then centrifuged at 13,000 rpm for
from Junsei Chemical Co. (Tokyo, Japan). Decanoic acid, 5 min to remove the insoluble matters. Afterward, samples

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Bioprocess and Biosystems Engineering (2018) 41:1611–1620 1613

were read at 405 nm by UV-spectrophotometer (Infinite ABTS‑radical scavenging assay

M200 Pro Nano-quant, TECAN, Austria). Reducing sugar
concentration in the sample was determined after normal- The ABTS (2,2′-azino-bis (3-ethylbenzothiazoline-6-sul-
izing with a wide range of glucose, used as control. fonic acid)) radical cation scavenging assay was used to
evaluate the antioxidant activity of WSP [15]. Briefly, 7 mM
Fourier‑transform infrared spectroscopy of ABTS and potassium persulfate, 7.35 mM, were dissolved
in distilled water, and the resulting solution was kept for
Fourier-transform infrared (FT-IR) spectroscopy was used to 16 h at room temperature in dark. Then ABTS solution was
identify the functional groups of WSP extracted from both mixed with samples in a ratio of 10:1 and allowed to react at
microalga Tetraselmis sp. 12236BP (T1) and 12432BP (T2), room temperature for 6 min. After that the absorbance was
respectively. This analysis was performed through FT-IR taken at 734 nm using a spectrophotometer (Infinite M200
spectra (400–4000 cm−1) with a Nicolet 6700 FT-IR spec- Pro Nanoquant, TECAN, Austria). The ABTS radical scav-
trophotometer (Nicolet Analytical Instruments, USA) using enging relative activity was calculated: RA (%) = [1 − (As)/
Zn/Se windows. To detect the high resolution, infrared spec- Ac] × 100, where, As is the absorbance of the sample and Ac
trum was selected as 8/cm and 64 scans of accumulation, as is the absorbance of the control. Specific activity was meas-
described in the previous study [13]. ured on the basis of total carbohydrates, µmol g−1 min−1.
µmol is derived from the ascorbic acid concentration, used
TLC analysis for standard curve, “g” comes from TC quantification of
each sample and “min” is the reaction time for the comple-
To find the differences in chemical composition, a thin- tion of each experiment.
layer chromatography (TLC) using silica gel (60/Kieselguhr
­F254 thin-layer plate, Darmstadt, Germany) was performed Ferric reducing antioxidant power (FRAP) assay
according to a previous study [14]. A chemical mixture con-
sisting of N-propanol: ­NH4OH: ­H2O = 7:1.5:1.5 (v/v) was The FRAP reagent was prepared [16] by mixing TPTZ
prepared and used as running buffer. Separation of WSPs on [40 mM 2,4,6-tri (2-pyridyl)-s-triazine], 20 mM ferric chlo-
TLC plate was visualized under UV light at a wavelength of ride and 0.3 M acetate solution. The solution was mixed
254 and 365 nm, respectively. Following the UV light obser- with the testing sample solution and water in a 33:1:14 ratio
vation, the TLC plate was then developed using a solution and the reaction allowed to continue at 37 °C for 4 min. The
mixture consisting of 3% phosphoric acid and 8% copper absorbance of the substances was read at 595 nm using a UV
sulfate (v/v), which is reliable to observe sugar-containing spectrophotometer (Infinite M200 Pro Nanoquant, TECAN,
materials. The TLC plate was then baked at 200 °C to iden- Austria) to calculate the reducing power percentage of WSP:
tify each resultant band. RA (%) = [1 − (Ac/As)] × 100, where, Ac is the absorbance
of the control and As is the absorbance of the sample. Spe-
DPPH radicals scavenging activity cific activity was measured on the basis of total carbohy-
drates, µmol g−1 min−1. µmole is derived from the ascorbic
The DPPH radical scavenging assay is widely performed to acid concentration used for standard curve, “g” comes from
analyze the antioxidant activities of biologically active com- TC quantification of each sample and “min” is the reaction
pounds derived from various sources [15]. Briefly, 1 mM time for the completion of each experiment.
DPPH solution was mixed with the test samples in a 3:1 The extracts were further analyzed for protein, lipid and
(v/v) ratio and allowed to react in a dark at room tempera- polyphenol contents according to already established proto-
ture for 20 min. To determine the best mixing ratio of the cols, respectively [17–19].
sample with the DPPH solution, various ratios were tested.
Subsequently, the absorbance was measured at 517 nm using Tyrosinase inhibitory activity
a UV-spectrophotometer (Infinite M200 Pro Nano-quant,
TECAN, Austria). The scavenging relative activity (%) was The method used for the detection of tyrosinase inhibitory
calculated as: RA (%) = [1 − (As)/Ac] × 100, where As is activity [9, 20] is briefly described as follows. 1 mM l-tyros-
the absorbance of the sample and Ac is the absorbance of ine was added to 25 mM phosphate buffer (pH 6.8) and then
the control. Specific activity was measured on the basis of mixed with 24 µL of enzyme solution consisting of 100 µg/
total carbohydrates. µmol g−1 min−1. µmol is derived from mL tyrosinase from mushroom in the presence or absence
the ascorbic acid concentration, used for standard curve, of testing samples (30 µL). The mixed solution was then
“g” is derived from the TC quantification of each sample incubated at room temperature for 30 min, and the absorb-
and “min” is the reaction time for the completion of each ance was monitored at 405 nm using a UV-spectrophotom-
experiment. eter (Infinite M200 Pro Nano-quant, TECAN, Austria). To

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1614 Bioprocess and Biosystems Engineering (2018) 41:1611–1620

compare the effectiveness of tyrosinase inhibitory activity, Total phenol content (TPC)
we were unable to find suitable positive control; however
lag-time delaying of tyrosinase activity in the presence of The TPC was determined using the Folin–Ciocalteu colori-
tyrosine used as a substrate was measured by the dependence metric method [19, 22] with slight modifications. Briefly,
of concentration of BAS. Zero concentration of tyrosine is 200 µL of the diluted solution was mixed with 200 µL of
termed as negative control. Folin–Ciocalteu reagent and 500 µL of 2% ­Na2CO3 solu-
tion was added to the following mixture. After 30 min of
Antimicrobial assays incubation at room temperature, the solution was then cen-
trifuged at 13,000 RPM and the absorbance was measured
The microorganisms used in this study were Candida albi- at 725 nm with a UV-spectrophotometer (Infinite M200 Pro
cans and Penicillium italicum, respectively, which were Nano-quant, TECAN, Austria). The gallic acid equivalent
subjected to antimicrobial assays according to already was determined based on a calibration curve generated
established protocol [21]. Briefly, twofold dilutions of using gallic acid standard solution to calculate the phenolic
the antimicrobial agents were prepared in a liquid growth content.
medium, dispensed in 96-well micro-titration plate (micro-
dilution). Then, each well was inoculated with a microbial Statistical analysis
inoculum. Mixed thoroughly and the inoculated 96-well
micro-titration plates were incubated in a UV-spectropho- Each sample contained at least three replicates and all exper-
tometer (Infinite M200 Pro Nano-quant, TECAN, Austria) iments performed in this study were repeated at least three
with agitation under suitable time and temperature condi- times. Data on the effect of the antioxidant activities were
tions depending on the microorganisms tested. Relative compared using Student’s test and presented as the mean
growth-inhibitory activity was calculated based on the result value standard deviation (SD) with P < 0.05 indicating sta-
from the control test. For the determination of antifungal tistical significance.
activities, fluconazole and imidazole were used as commer-
cially available drugs to compare the antifungal activities of
partially purified polysaccharides. Result and discussion

Protein quantification protocol Characterization of bioactive substances

from microalga Tetraselmis species
The protein quantification was done according to a previ-
ous study [17]. Briefly, a fourfold dilution of a 2 mg/mL To characterize the biochemical properties of microalgal
Coomassie blue solution was prepared. The samples were biomasses, we primarily isolated bioactive substances (BAS)
mixed with Coomassie blue solution in a ratio of 10:100 and and purified the water-soluble polysaccharide (WSP) from
centrifuged at 13, 5000 RPM for 5 min to remove insolu- Tetraselmis sp. 12236BP (T1) and 12432BP (T2), respec-
ble matter. The absorbance of each sample was measured tively. According to a previous study [13], microalgal spe-
at 595 nm using a UV–visible spectrophotometer (Infinite cies have different structural components of glycans in their
M200 Pro Nano-quant, TECAN, and Austria). The absorb- cell walls. To isolate crude BAS, various chemical pre-
ance of each BSA standard was plotted as a function of its treatments have been done using T1 and T2, i.e., acid (1 N
theoretical concentration. ­H2SO4), base (1 M NaOH) and water treatments. Afterward,
these crude extracts were subjected to ethanol treatment to
Lipid quantification analysis purify WSPs. Furthermore, the experimental conditions of
these isolated and partially purified WSPs by acid, base and
The lipid quantification was performed according to a pre- water pretreatment were observed for various periods of
vious study with some modifications [18]. Briefly, 500 µL time at different temperatures to optimize suitable working
of sample was mixed with 200 µL of neutralizing agent conditions (Fig. 1). According to the results, we optimized
(100 mM Tris–HCL). Then this mixture was combined with the best working conditions in terms of total carbohydrate
200 µL of copper reagent (9:1:10 = 1 M triethanolamine:1 N for both T1 and T2 to be 37 °C for 24 h, respectively. Based
acetic acid:6.5 % of ­CuSO4) and 250 µL of chloroform. This on these primary findings, we selected 37 °C and 24 h as
mixture was centrifuged at 13,000 RPM for 5 min to remove the best experimental conditions for further experiments in
insoluble matter. 50 µL of this reaction mixture was mixed this study.
with 50 µL of color developer (1% diethyldithiocarbamate WSPs are one of the most promising BAS as they are
in butanol). The absorbance of the samples as measured at able to diffuse in the culture medium, free from cell debris,
440 nm compared with gallic acid control absorance. and are easy to collect and purify [23, 24]. However, after

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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
Fig. 1  Characterization of WSPs extracted from microalga Tet-
raselmis sp. 12236BP (T1) and 12432BP (T2) for its carbohydrate from defatted microalgal biomasses (Table 2).
content at various time and temperature points (optimization). a
Acid treatment, b base treatment, c water, acid 1 N H
­ 2SO4, base 1 M Chemical composition elucidation
NaOH
The FT-IR spectra of WSPs extracted from microalga Tet-
various pretreatments applied on these WSPs, the qual- raselmis sp. T1 and T2 was reconstructed by Microsoft
ity and recovery of WSPs comprise an important factor. Excel, using the spectra of the reference substances to
Therefore, after ethanol precipitation, the resulting WSPs determine their relative contribution to the overall constitu-
were freeze dried and ultimately the total % yield for WSPs ents of WSP from T1 and T2 (Fig. 2). The band spectrum
was calculated (Table 1). As shown in Table 1, the highest between 4000 and 3000 cm−1 showed an OH stretch that

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 T2
Acid Base Water Acid Base Water

Yield (%)a 7.0 31.5 10.0 4.5 34.5 9.1

a
 Yield = [dried biomass (g)/original biomass (g)] × 100

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

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Bioprocess and Biosystems Engineering (2018) 41:1611–1620 1617

assays, vitamin C (ascorbic acid) was used as positive con- whitening activity. Tyrosinase inhibition is the most com-
trol to evaluate the potency of these partially purified poly- mon methodology to attain skin whitening (hypopigmenta-
saccharides. The DPPH scavenging specific activities of tion), as tyrosinase is the rate-limiting enzyme in the mela-
WSP isolated from T1 were determined to be 7.23, 19.85, nin synthesis pathway [7, 8]. These facts help us find out
and 6.84 (µmol/g/min) after acid, base, and water pretreat- about tyrosinase inhibition by these WSPs extracted from
ments (Table 3), whereas DPPH radical scavenging specific T1 and T2 (Fig. 3). All samples from T1 and T2 showed
activity of WSP purified from T2, was observed to be 7.47, high relative inhibitory activity (Fig. 2a) with the highest of
60.33, and 8.67 (µmol/g/min) following acid, base, and water 86% for T1 and 73% for T2, following base (alkali)-extracted
pretreatments, respectively. These results clearly reveal that WSPs. T1 and T2 pretreated by acid showed approximately
a significant scavenging activity was observed after base 65 and 60% relative inhibitory activity, whereas 50 and 69%
treatment for both the strains of Tetraselmis sp., T1 and T2. relative inhibitory activity was observed for T1 and T2 after
Relative DPPH scavenging activity was varied between water extraction, respectively. On the contrary, the highest
3–15% as compared to ascorbic acid concentration, used specific activity was determined for WSP of T2, i.e., 243,499
as control (relative to ascorbic acid 100%) for T1; however U/min, whereas the T1 specific inhibitory activity ranged
non-significant change in relative DPPH scavenging activi- from 8787 to 52,815 U/min (Fig. 3b). These results were
ties for T2, i.e., 1–7% was observed. Next, FRAP antioxidant confirmed by the changes in the absorbed wavelength dur-
activity was observed and the reaction was established upon ing the experiment (Fig. 3c). Taken together, these results
the reaction of FRAP reagent with iron(II) and SH-groups demonstrate that WSPs from T1 and T2 are rich sources of
in antioxidants [28]. The reaction starts with the reduction tyrosinase inhibitors and hence can be potently applied in
of ­Fe3+ TPTZ complex (colorless) to F ­ e2+-tripyridyltriazine the cosmetics and therapeutic industry. 1U represents 0.1
(blue colored) involving the action of electron-donating absorbance change per gram of sample.
antioxidants. As a result, no significant specific activity was
observed among all treated groups of T1 and T2, indicating Antifungal activity
that WSP may not contain sulfate or other relative functional
groups in their structure. The study of antifungal agents has lagged far behind that
Next, ABTS antioxidant assay was performed and the of antibacterial agents, likely because fungi were not rec-
principle of the assay is based on the ability of a sample to ognized as important pathogens until several years ago [29,
scavenge ABTS cation by addition of potassium persulfate. 30]. The associated increase in fungal infections prompted a
This radical cation is blue in color and is highly reactive search for newer and safer agents to combat fungal infections
toward most of the antioxidants. The blue ABTS radical cat- [31]. These facts prompted us to find the antifungal activity
ion is converted to its original colorless form in the presence of these WSPs of microalga Tetraselmis sp. T1 and T2. Two
of an antioxidant. Table 3 shows ABTS scavenging relative different fungal strains were used in this study, i.e., C. albi-
and specific activities of WSPs of T1 and T2. Data show that cans and P. italicum to confirm the antifungal characteristics
the highest specific scavenging activity (65.67 µmol/g/min) of these extracted WSPs. We hypothesized that concentra-
was observed following base treatment. Taken together, all tion-dependent inhibition of growth would be observed, and
these results suggest that the total antioxidant activity can our experiments proved that the growth of C. albicans was
be related to higher yields of WSPs. inhibited up to 80% by base (alkali)-extracted WSPs from
T1, whereas the growth of C. albicans was inhibited up to
Tyrosinase inhibitory activity 70% following the same treatment for T2 (Fig. 4). However,
WSPs extracted with water and acid treatments from T1 and
To explore other potent biological activities of WSPs iso- T2 were unable to inhibit C. albicans growth; similar results
lated from T1 and T2, we investigated tyrosinase inhibitory/ were observed when WSP from T1 and T2 were applied to

Table 3  Antioxidant activity of T1 T2
WSP extracted from microalgae
Tetraselmis sp. 12236BP (T1) Acid Base Water Acid Base Water
and 12432BP (T2)
Specific ­activityb (µmol g−1 ­min−1)
 DPPH 7.23 ± 0.51 19.85 ± 0.91 6.84 ± 2.04 7.47 ± 1.11 60.33 ± 4.11 8.67 ± 1.51a
 FRAP 0.64 ± 0.02 0.64 ± 0.03 0.61 ± 0.01 0.16 ± 0.01 N/D 0.09 ± 0.01
 ABTS N/D 65.67 ± 4.12 24.15 ± 0.4 N/D 18.73 ± 3.13 N/D
a
 Mean values were calculated from three different experiments
b
 Specific activity was derived from calculated TC values of these WSPs

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1618 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

Fig. 3  Tyrosinase inhibitory activity of WSPs purified from micro-

alga Tetraselmis sp. 12236BP (T1) and 12432BP (T2). a Relative Conclusions
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- Our experimental findings suggest that WSPs are one of the
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
most important components of BAS and can be evolved as
value standard deviation (SD) with P < 0.05 indicating statistical sig- potent biological material in cosmeceuticals and therapeu-
nificance 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
P. italicum (Fig. 5). These results demonstrated that WSP enhance the properties of these BAS and WSPs from micro-
from T1 and T2 may have a broad spectrum of antimicrobial algal biomasses. Overall, these results evidently exhibit that
activity. BAS and WSP from the microalgal biomasses have potential
biological activities.

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Bioprocess and Biosystems Engineering (2018) 41:1611–1620 1619

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