Journal of Aquatic Food Product Technology

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Potential Cytotoxic Effects of Peptide Fractions

from Dunaliella salina Protein Hydrolyzed by
Gastric Proteases

Maliheh Darvish, Hasan Jalili, Seyed-Omid Ranaei-Siadat & Mahsa Sedighi

To cite this article: Maliheh Darvish, Hasan Jalili, Seyed-Omid Ranaei-Siadat & Mahsa
Sedighi (2017): Potential Cytotoxic Effects of Peptide Fractions from Dunaliella salina
Protein Hydrolyzed by Gastric Proteases, Journal of Aquatic Food Product Technology, DOI:
10.1080/10498850.2017.1414095

To link to this article: https://doi.org/10.1080/10498850.2017.1414095

Published online: 13 Dec 2017.

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 JOURNAL OF AQUATIC FOOD PRODUCT TECHNOLOGY
https://doi.org/10.1080/10498850.2017.1414095

Potential Cytotoxic Effects of Peptide Fractions from Dunaliella

salina Protein Hydrolyzed by Gastric Proteases
Maliheh Darvisha, Hasan Jalilia, Seyed-Omid Ranaei-Siadatb, and Mahsa Sedighia
a
Life Science Engineering Department, Faculty of New Sciences and Technologies, University of Tehran, Tehran, Iran;
b
Nano-Biotechnology Engineering Lab., Department of Biotechnology, Faculty of Energy Engineering and New
Technologies, Shahid Beheshti University, GC, Tehran, Iran

ABSTRACT KEYWORDS
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Dunaliella salina is a halotolerant microalga with high protein content. Until Dunaliella salina; hydrolyzed
now, there was no report on biological properties of peptides prepared by peptides; antimicrobial
enzymatic digestion of D. salina proteins. Therefore, antimicrobial and effect; antiproliferative effect
antiproliferative effects of the abundant protein of D. salina were investi-
gated. The extracted proteins were separated by fast protein liquid chro-
matography (FPLC), and protein hydrolysis was carried out with intestinal
proteases such as trypsin and chymotrypsin. The antimicrobial and antipro-
liferative activities of the hydrolyzed peptides were examined on Escherichia
coli, Staphylococcus aureus, and Helicobacter pylori, and colon cancer cell
lines, respectively. The results demonstrated that 63 kDa protein and its
derived peptides caused a decrease in bacteria growth, and <3 kDa peptide
fraction significantly reduced SW480 cell viability. Therefore, the peptide
fractions with antimicrobial and antiproliferative activities are worthy of
further investigation as functional food ingredients for health benefits.

Introduction
Microalgae are microscopic photosynthetic organisms with the largest primary biomass, which
covers almost three-quarters of the earth’s surface and organizes the base of the marine food web
through their photosynthetic activity (Chacón-Lee and González-Mariño, 2010; El Gamal, 2010;
Venugopal, 2008). In addition, microalgae are considered as nutrient supplements, food, and feed
additives for human consumption due to their high vitamin, lipid, protein, and carbohydrate
contents (Harun et al., 2010; Priyadarshani and Rath, 2012). Microalgae are able to promote the
nutritional value of food and animal feed due to their unique chemical composition, and they
comprise various useful compounds that make them living-cell factories for the production of
beneficial metabolites used in aquaculture, food, and pharmaceutical industries (Priyadarshani and
Rath, 2012).
In recent years, researchers have made intense efforts to find new protein sources as nutrient
supplements such as fungi, bacteria, yeasts, and microalgae that are known as single cell protein
(SCP). The importance of microalgae as SCP is its high protein content (more than 50% of biomass)
and particular amino acids composition vital for several biological and physiological functions. Due to
their abundance and amino acids profile, algal proteins are considered as an alternative protein source
in the food industry. Microalgal biomass displays favorable qualities without any negative effects as a
novel source of protein (Becker, 2007). The potential applications in foods and commercial value of
these proteins depend on the isolation process from the microalgae cells without any intense color and

CONTACT Hasan Jalili hjalili@ut.ac.ir Life Science Engineering Department, Faculty of New Sciences and Technologies,
University of Tehran, North Kargar Street, Tehran 1439957131, Iran
Color versions of one or more of the figures in the article can be found online at www.tandfonline.com/wafp.
© 2017 Taylor & Francis
 2 M. DARVISH ET AL.

taste and with intact molecular structure (Becker, 2007; Schwenzfeier et al., 2011). In this regard, marine
biotechnology is trying to improve the synthesis of secondary metabolites of microalgae by utilizing
different approaches and downstream processing (Ibañez and Cifuentes, 2013).
D. salina is a genus of unicellular and motile green algae belonging to the class Chlorophyta (Tang
and Suter, 2011). This species is one of the three major algae that produce a high content of valuable
compounds, such as proteins, lipids, and different types of pigments. In addition to a rich source of
deep yellow β-carotene, D. salina contains about 50% proteins, and its amino acid composition is
similar to some plants such as soybean (Hosseini Tafreshi and Shariati, 2009). The presence of high
levels of β-carotene, the lack of indigestible cell wall, the relatively good protein and fatty acid quality
of Dunaliella meal, as well as its generally identified as safe status make it an excellent nutritional
ingredient and remarkable protein source (Hosseini Tafreshi and Shariati, 2009; Priyadarshani and
Rath, 2012). In addition, D.salina is remarkable for its production of other bioactive compounds,
such as valuable vitamins, enzymes, and pharmaceuticals. Some studies showed that a crude extract
of this alga had antimicrobial activity against some microorganisms important for the food industry
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like E. coli and S. aureus (Hosseini Tafreshi and Shariati, 2009). Therefore, D. salina can be an
interesting source of bioactive compounds in nutritional and pharmaceutical industries because of its
valuable content and their diverse biological functions including antiproliferative, antioxidant,
antimicrobial, and immune-modulatory properties (Dipak and Lele, 2005).
The bioactive peptides are commonly produced through enzymatic hydrolysis of whole protein
content (Korhonen and Pihlanto, 2006; Zambrowicz et al., 2013). Some of the peptide fragments of
algal proteins normally inactive within the protein sequences can be released through enzymatic
digestion and represent their biological activity (Salami et al., 2008, 2009; Sedighi et al., 2016). To our
knowledge, no report is available dealing with functional properties of D. salina proteins. The
objective of this research was the investigation of digestibility of extracted proteins from D.salina
and evaluation of biological effects of released peptides on E.coli, S. aureus, H.pylori, and colon
cancer cell line to introduce them as a functional food.

Materials and methods

Materials
Microalgae, D. salina, were collected from the Persian Gulf. All chemicals were analytical grade
(Sigma–Aldrich, St. Louis, MO, USA and Merck, Darmstadt, Germany), and all solutions and
buffers, prepared with double-distilled water, were kept at 4ºC before use. Chymotrypsin (EC
3.4.21.1; activity 68.9 units.mg−1 protein) and trypsin (EC 3.4.21.4; activity 277 units.mg−1 protein)
were obtained from Worthington Biochemical Company (New Jersey, England). E. coli CETCT 434
and S. aureus ATCC 6538 were prepared from microbial collection of Azad University (Tehran,
Iran), and cagA-positive H. pylori, isolated from patients, were prepared from microbial collection of
University of Tehran (Tehran, Iran).

Microorganism cultivation conditions and biomass processing

The unicellular green algae D. salina was cultivated in Johnson medium (Johnson et al., 1968). The
inoculated flasks were incubated at room temperature under manual shaking and irradiated at 4000
Lux. Microalgae cells were harvested in mid-log growth phase by centrifuge (2500 × g, 15 min, 4 ºC)
and stored as pellets at −20 ºC.

Protein extraction and purification

Pellets were washed twice with extraction buffer including 50 mM Tris-HCl pH = 7.5, 10 mM
MgCl2 and 20 mM KCl and centrifuged at 6000 × g, 15 min, 4ºC. Then, the pellets were
 JOURNAL OF AQUATIC FOOD PRODUCT TECHNOLOGY 3

suspended in 5ml extraction buffer, followed by grounding under liquid nitrogen to lysis cell. The
cell lysate was separated by centrifugation at 10000 × g 60 min. Proteins in supernatant were
precipitated by cool acetone as follows: pre-cooled acetone was added to six volumes of super-
natant and keep at −20ºC overnight. Precipitated proteins were obtained at 14000 × g 20 min and
stored at −20ºC until used.
The Bradford method (Bradford, 1976) was utilized to determine protein content using
Coomassie Blue G-250 as a protein binding dye and spectrophotometric determination at 595nm
with bovine serum albumin (BSA) as standard.
The protein composition of the algal extracts was investigated by sodium dodecyl sulfate poly-
acrylamide gel electrophoresis (SDS-PAGE) (Bio-Rad Laboratories, Hercules, CA, USA) according to
the manufacturer’s instructions. Gels with 10% concentration were prepared, and samples mixed
with SDS sample buffer 5X were run at 120 V for 90 min. Finally, gels were stained with Coomassie
brilliant blue R-250.
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Size exclusion fast protein liquid chromatography analysis

The protein samples were filtered through a 0.22 µm filtration membrane (Millipore). An aliquot of
500 µL of obtained protein was injected manually on Superdex 75 10/100 GL size exclusion column
using 50 mM sodium acetate and 150 mM sodium chloride (pH 4.8). Analysis of proteinous
materials was performed in the 0.3 ml/min flow-rate and 2 ml injection size. Then, the column
eluent was monitored at 280 nm with spectrophotometer. The purity of the protein was checked by
SDS-PAGE and silver nitrate staining.

Preparation of peptides
Extracted and purified protein was hydrolyzed by intestinal proteases in 20 mM phosphate buffer
(pH 7.8). In this regard, the extracted protein was digested by trypsin in enzyme/substrate ratio of
1:50 (w/w) at 37 ̊ C for 18h (www.biochem.uwo.ca/wits/bmsl/protocols.html). Then, chymotrypsin
was added and proceeded with the same condition. Protein hydrolysates were centrifuged at 4000 × g
for 15 min to eliminate both enzymes and nonhydrolyzed proteins, then passed through 3 kDa and
10 kDa amicon filters (Billerica, MA, USA) to obtain smaller than 3 (<3 kDa), between 3 to 10 (3–10
kDa), and higher than 10 (>10 kDa) fractions. Each fraction was collected and examined for its
antimicrobial and antiproliferative properties.

Peptide concentration determination

Spectrophotometric assay using o-phthaldialdehyde (OPA) was used to investigate the proteolysis
degree (Church et al., 1983). A fresh OPA solution was prepared daily with 25 ml of 100 mM sodium
tetra hydroborate, 2.5 ml of 20% SDS solution (w/v), 1 ml of OPA reagent (40 mg OPA in 1 ml
methanol), and 100 µl of β-mercaptoethanol, then adjusted to 50 ml with distilled water. Then,
100 µl samples with 1 ml OPA solution were added to the test tube, and after 2 min, optical density
was read with spectrophotometer at 340 nm using distilled water as a control (peptone (25–2000 µg/
ml) was used as standard for OPA assay).

Antimicrobial property determination

The antimicrobial activity of the protein and resulting peptide fractions were examined against E.coli
and S.aureus. For this purpose, E.coli and S.aureus were cultured in Mueller Hinton Broth (MHB),
including 2.0 g beef infusion, 17.5 g acid casein peptone, 1.5 g corn starch, for 1L at 37°C, while the
bacterial turbidity adjusted to the 0.5 McFarland (0.08 to 0.13 absorbance). In order to measure the
bacterial growth rate in the presence of the samples, 50µl of each fraction was added to 200 µl of
 4 M. DARVISH ET AL.

bacteria culture medium in 96-well plate at 37°C for 16 h. The inhibitory effect was measured at
regular intervals based on optical density at 600 nm.
Antibiotic sensitivity of a bacterial strain is measured as the minimum inhibitory concentration
(MIC) that is defined as the lowest antimicrobial concentration that inhibits visible microorganism
growth. To investigate the MIC, dilution series of the peptide fraction with highest inhibition effect on E.
coli and S.aureus were prepared and added to separate 96-well plates containing 150 µl of MHB as the
microbe culture medium. Also, the MIC of the peptide fraction against H. pylori was calculated. The
procedure was quite similar to the method mentioned above, except the culture medium, which had
components such as Brucella agar, defibrinated sheep blood (7%), vancomycin (5mg.L−1), trimethoprim
(5 mg.L−1), polymyxin (50 μg.L−1), and amphotericin B (4 mg.L−1). Finally, the plates were incubated at
37°C for 18 h, and after that an ELISA reader was applied to investigate inhibitory values.

Cell culture and antiproliferative activity assay

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Human colon adenocarcinoma cell line (SW480) was seeded in appropriate culture medium (RPMI,
10% FBS, 1% Pen/Strep). Cells were seeded at a density of 2 × 103 cells/well in a 96-well microtiter
plate for 24 h and incubated at 37°C with humidified 5% CO2 atmosphere.
To evaluate the mitochondrial activity, the MTT assay was carried out according to the originally
described method (Gali et al., 2011). Briefly, the cells were incubated for 24 h at 37°C and 5% CO2
and then incubated for different time periods (24, 48, and 72 h) with each peptide fraction in
different concentrations. The MTT dye was added 4 h prior to completion of the incubation periods.
The medium from each well was discarded, and the resulting formazan crystals were solubilized by
adding 200 µl of dimethylsulfoxide (DMSO) and quantified by measuring absorbance at 570 nm with
TECAN Micro Plate Reader (Infinite® 200 PRO series, Switzerland). As a control, the appropriate
row or column of wells was left untreated at each time point. The hydrolysate concentration with
50% growth inhibition is referred to as the inhibitory concentration 50 (IC50). A simple method for
calculation of the IC50 is performed by linear interpolation between the concentrations above and
beneath 50% inhibition in the dose response curve (= two flanking points) that is calculated
according to:

IC50 = EXP (Ln (conc>50%) - ((signal>50%-50)/(signal>50%-signal<50%)× Ln(conc>50%/conc<50%)))

where conc is the effective concentration of the desired fraction, and signal is related to the inhibition
value of the considered sample.

Statistical analysis
In all experiments, the obtained values are reported as mean ± standard deviation (SD) from three
independent measurements. One-way analysis of variance (ANOVA) was used to statistically
evaluate the obtained values, with the level of significance set at probabilities of p < 0.05, p < 0.01,
or p < 0.001. SPSS 21.0 (SPSS, Chicago, IL, USA) software was used for the statistical analyses.

Results
Protein extraction and purification
The resistance of D.salina to extreme conditions and its high protein content were the reasons for
selecting this microalga as an unconventional protein source (Chen and Jiang, 2011; Emtyazjoo et al.,
2012). Several methods were examined to extract their total proteins (Gao et al., 2016; Tran et al.,
2009). According to protein concentration and SDS-PAGE results, among the tested methods (data
not shown), maximum protein fractions were obtained with liquid nitrogen and extraction buffer
(Tris-HCl, NaCl, and MgCl2) method (Figure 1A). From the obtained protein fractions, the fraction
 JOURNAL OF AQUATIC FOOD PRODUCT TECHNOLOGY 5
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Figure 1. Analysis of the extracted protein fraction (a) SDS-PAGE profile of whole proteins: (M) protein ladder (Prestained Protein
ladder, 10–250 kDa from Sinacolon), (A) dilution 1, (B) dilution 1/2 (C) dilution 1/4, (D) dilution 1/10. (b) SDS-PAGE after FPLC: (F8)
purified protein.

with higher concentration in 63 kDa molecular weight was separated for further experiments and
purified by using fast protein liquid chromatography (FPLC) system and DEAE column. For this
purpose, selected protein fraction was eluted with a buffer gradient (Tris-HCl 50mM and KCl 1M) in
F8 fraction. Figure 1B shows silver nitrate gel recorded for 9 FPLC peaks. According to our
observations, the considered extraction method provides a rapid, simple, and efficient approach to
isolate the highest fraction concentration protein of D. salina.

Enzymatic hydrolysis of the extracted protein

The algae protein hydrolysates were prepared by means of hydrolysis with commercial proteases
including trypsin and chymotrypsin. In this regard, the purified protein with 63 kDa molecular
weight most abundant in microalgae cell was hydrolyzed to produce bioactive peptides and differ-
entiated as follows: <3 kDa, 3–10, and >10 kDa. Due to the fact that the number of amino acids is
necessary in assessment of hydrolysates peptides functionalities, the fraction with the most signifi-
cant impact on cell viability was determined. The degree of hydrolysis measured by the OPA method
was raised to 17000 µg.mL−1 for all fractions. Optimal concentration of the efficient peptide fractions
was determined from the examination of the impact on cells of each fraction at concentrations
ranging from 0 to 160 µg.mL−1.

Cytotoxic activity of hydrolyzed peptide fractions

To meet the aim of this study, antibacterial property of peptides was investigated on E.coli, S.aureus,
and H.pylori compared to 63 kDa protein and D. salina biomass. As shown in Figure 2, the highest
inhibitory effect was ascribed to 63 kDa protein fraction with 59.4 and 42.9% cytotoxicity on E.coli
and S.aureus after 16 h, respectively. The comparison between proteins and their bioactive peptides
pointed out the significant role of bioactive peptides that can be considered as latent factors in parent
proteins before hydrolysis. The peptides with > 10 kDa molecular weights showed the highest
inhibitory effect on E.coli with 16.8% inhibitory effect, while the peptides with 3-10 kDa molecular
weights had 17.2% inhibition on S.aureus after 16h. Furthermore, the growth inhibitory effect of
protein against E.coli were 1.4 times greater than S.aureus, which can be due to the different
structures of the cell wall in gram positive and gram negative bacteria. Inhibitory yields are collected
 6 M. DARVISH ET AL.

1.2
(a)
1

0.8 Ref
OD 600 nm

Protein
0.6
Biopeptide A
0.4
Biopeptide B

0.2 Biopeptide C

0
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0 5 10 15 20
Time (h)
1.2
(b)
1

0.8
Ref
OD 600 nm

Protein
0.6
Biopeptide A
0.4
BiopeptideB

Biopeptide C
0.2

0
0 5 10 15 20
Time (h)

Figure 2. (a) E.coli and (b) S.aureus growth curve for the determination of the antimicrobial activity of 60 kDa protein and
biopeptides A, B and C that referred to hydrolysate peptides > 10, 3–10 and < 3 kDa respectively, compared to a control culture.

in Table 1, showing the growth inhibition percentage of each bacterium. Also, the MIC of the
peptide fraction with highest inhibition effect on the microorganisms were calculated as shown in
Table 2.
H. pylori is a gram negative bacterium able to induce gastric diseases. The bioactive peptides with
highest bacterial inhibition effect were on H. pylori culture. <3 kDa peptide fraction had MIC equal
to 0.175 mg against H. pylori (Table 2). Given that these bacterial species are resistant to most of the
antibiotics, this result can be promising to find a potent growth inhibitor for it. Hence, the <3

Table 1. Inhibitory effect of D. salina selected protein and produced bioactive peptides on E.coli and S.aureus cells in comparison
to their normal culture. Control: pure culture medium.
Control protein Peptide > 10 kDa Peptide 3–10 kDa Peptide < 3 kDa
Inhibition percent (E.coli) 0 59.4 16.8 7.8 10.3
Inhibition percent of (S.aureus) 0 42.9 4 17.2 7.2
 JOURNAL OF AQUATIC FOOD PRODUCT TECHNOLOGY 7

Table 2. MIC of the peptide fraction on the microorganisms.

Bacterial Species MIC (mg)
E. coli 0.58
S. aureus 0.81
H. pylori 0.175

peptide fraction was selected for the subsequent experiments. Since MIC is important in diagnostic
laboratories to confirm resistance of microorganisms to an antimicrobial agent and also to monitor
the activity of new antimicrobial agents, it was calculated for the most effective peptide fractions. For
both bacterial species, it had the highest value for <3 kDa peptide fraction.
The side effects of cancer treatment encourage investigation into alternative active agents like
antimicrobial peptides (AMPs). Some publications (Di Bernardini et al., 2011; Salami et al., 2008;
Udenigwe and Aluko, 2012) showed that AMPs are cytotoxic in cancer cells and able to prevent their
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undesired and uncontrolled growth. To demonstrate antiproliferative effect of peptide fractions in

comparison with purified protein, various concentrations of each fraction (0.1, 1, 10, 40, 80, and
160 µg.mL−1) were evaluated on the viability of the cultured SW480 cells at 24, 48, and 72 h time-point
(Figure 3). SW480 cell line is a human colon cancer cell line originated from primary tumors and
commonly used in vitro to test potential inhibitory substances. Among all three peptide fractions, the
most effective concentration was the peptides < 3 kDa in 0.1 µg.ml−1 concentration after 72h with
about 30% viability. Figure 4 demonstrates a dose- and time-dependent inhibition in the growth of
SW480 cells for <3 kDa peptide fraction. Peptide fractions had a better inhibitory effect on the viability
of SW480 cells in comparison to nonhydrolyzed protein even at low concentrations.
The IC50 is an efficient, measurable factor for the inhibitory effect of drugs. As shown in Figure 5,
compared with other samples, <3 kDa peptide fraction decreased cell viability to 50% in 17 µg.mL−1
bioactive peptides and 24 h time-points.
Treatment with the peptide fractions and extracted protein revealed significant growth inhibition
of SW480 cell line at 24 h time-point, especially with <3 kDa peptide fraction that not only
demonstrated a greater effect than other fractions but also that the IC50 was measurable after 24 h
treatment. However, there was an ignorable difference between various concentrations of peptide
fractions in inhibition effects after 24 and 48 h time-point, and all of them displayed 50% inhibitory
effect. As shown in Figure 4, 72 h after treatment, <3 kDa peptide fraction exhibited a remarkable
increase in growth inhibition of SW480 cells to 70%. Meanwhile, an increase in concentration of this
fraction did not show any effect on cell viability, and the lowest concentration (0.1 µg.ml−1) had a
quite similar inhibitory effect as the highest concentration. However, the 63 kDa protein demon-
strated enhanced growth inhibition after 24, 48, and 72h with increasing the concentration and
showed a time- and dose-dependent activity.

Discussion
Recently, a number of bioactive metabolites have been reported with antifungal, antiviral, antitumor,
and antiinflammatory properties (De Morais et al., 2015; Hasan et al., 2015; Kelecom, 1991; Sedighi
et al., 2016). Among these metabolites, peptides with antiproliferative and antimicrobial activity have
received more attention due to their remarkable characteristics such as high stability and sensitivity;
moreover, they display multifunctional and biological activities based on their properties like
structure, hydrophobicity, and charge.
The result of this study demonstrated that enzymatic digestion of protein caused significant
increase in antiproliferative properties of D. salina abundant protein. In this study, D. salina isolated
protein was hydrolyzed with food-grade proteolytic enzymes, including trypsin and chymotrypsin to
obtain its intrinsic peptides. For the initial screening of antiproliferative activity, the MTT assay
showed that the <3 kDa peptide fraction has a positive impact displaying more effective inhibition in
 8 M. DARVISH ET AL.
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Figure 3. Anti-proliferative activity of <3, 3–10, >10 kDa peptides against nonhydrolyzed protein on SW480 cell line at 24, 48 and 72h.

vitro than other protein fractions and nonhydrolyzed proteins on cancer cells. It should prove
beneficial in vivo since it does not require a complex delivery system because of its small size and
presumed ability to be readily absorbed through the gastro-intestinal epithelial cells.
 JOURNAL OF AQUATIC FOOD PRODUCT TECHNOLOGY 9

100

80

Cell viability (%)

60
24

40 48
72
20

0
0 0.1 1 10 40 80 160
Concentration (microgram/ml)
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Figure 4. Time-dependent activity of <3 kDa peptide fraction.

100 24 48 72
90
80
70
IC50 (µg. ml-1)

60
50
40
30
20
10
0
>10 kDa 3-10 kDa <3kDa Protein
samples

Figure 5. IC50 determination for anti-proliferative activity of peptide fractions and protein in 24, 48 and 72 h time-points on colon
cancer cells.

Moreover, the study of antimicrobial activity of peptide fractions and selected protein revealed
that protein could have more inhibitory effect on E.coli and S.aureus growth in comparison to
peptide hydrolysates. Taken together, the MIC results indicated that the E. coli had higher sensitivity
than the S. aureus. This could be due to differences in their cell membrane constituents and
structure. It is known that Gram-positive bacteria contain an outer peptidoglycan layer, while
Gram-negative bacteria contain an outer phospholipidic membrane, both of which undergo different
types of interaction when encountered by produced peptides. Also, MIC value of H. pylori revealed
that this Gram-negative microorganism is more resistant than others.
Our results suggest that further experiments should be done to explore whether the results
obtained here can be replicated in vivo. There has been a considerable rise in interest in potential
uses for antiproliferative peptides due to their multifunctional characteristics such as high sensitivity
and stability. There have been a few reports on antiproliferative peptides from food proteins,
including fish sauce, soy, mollusk, milk, and beef proteins (Ben, 2005; Jang et al., 2008; Kim et al.,
2000; Leng et al., 2005; Wakabayashi et al., 2006). Hence, its availability and cost effectiveness makes
D. salina attractive as a protein source to produce functional peptides in the future.
 10 M. DARVISH ET AL.

Conclusion
This study, which has been the first of its kind on Dunaliella salina, has shown that extracted and
hydrolyzed proteins from D. salina have inhibitory properties in vitro against three different types of
common pathogenic bacteria as well as against a colonic cancer cell line. Incorporation of these
extracts into nutritional foods could make use of these properties to provide health benefits. Further
work on product properties, manufacture, stability, suitable methods of incorporation, as well as
activity in vivo needs exploration.

Acknowledgments
This work was supported by the Nano-Biotechnology Engineering Laboratory in Shahid Beheshti University (Tehran,
Iran). The authors sincerely acknowledge the collaboration of Dr. Mojgan Emtiazjoo in developing this work.
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Declaration of interest
The authors have declared that there is no conflict of interest.

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