Nghiên cứu squalene từ vi tảo biển dị dưỡng schizochytrium mangrovei PQ6 định hướng làm nguyên liệu cho thực phẩm bảo vệ sức khỏe, mỹ phẩm và dược phẩm tt tiếng anh

MINISTRY OF EDUCATION AND

VIETNAM ACADEMY

TRAINING

OF SCIENCE AND TECHNOLOGY

GRADUATE UNIVERSITY SCIENCE AND TECHNOLOGY
----------------------------

NGUYEN CAM HA

RESEARCH ON SQUALENE FROM HETEROTROPHIC MARINE MICROALGA
SCHIZOCHYTRIUM MANGROVEI PQ6 ORIENTED AS FEEDSTOCK FOR
HEALTH FOOD, COSMETIC AND PHARMACEUTICAL
Major: Biochemistry
Code: 9 42 01 16

SUMMARY OF BIOLOGY DOCTORAL THESIS

Ha Noi - 2020


The doctoral thesis was completed at: Institute of Biotechnology, Graduate University of
Science and Technology, Vietnam Academy of Science and Technology.

Scientific supervisor: Prof. Dr. Dang Diem Hong
Institute of Biotechnology

Reviewer 1: …

Reviewer 2: …
Reviewer 3: ….

The doctoral thesis will be defended at the graduate university committee of doctoral
thesis evaluation at Graduate University of Science and Technology, Vietnam Academy of
Science and Technology on…………, date…….....month…….year 2020

See the detailed thesis at:
- Library of Graduate University of Science and Technology
- Vietnam National Library


LIST OF PUBLISHED WORKS (09)
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Nguyen Cam Ha, Hoang Thi Minh Hien, Nguyen Hoang Ngan, Dang Diem Hong, Safety
assessment and the effect of squalene isolated from Schizochytrium mangrovei PQ6 on
serum HDL - Cholesterol levels in animal models. Academia Journal of Biology, 2019,
41(2), 39-48 (in Vietnamese)
Hoang Thi Lan Anh, Nguyen Cam Ha, Le Thi Thom, Hoang Thi Huong Quynh Pham
Van Nhat, Hoang Thi Minh Hien, Ngo Thi Hoai Thu, Dang Diem Hong, Different
fermentation strategies by Schizochytrium mangrovei strain PQ6 to produce feedstock for
exploitation of squalene and omega-3 fatty acids. Journal of Phycology, 2018, 54 (4), 550556 (SCI, Q1; IF-3.0).
Nguyen Cam Ha, Hoang Thi Minh Hien, Dang Diem Hong, Hypocholesterolemic
mechanisms of squalene extracted from Schizochytrium mangrovei PQ6 in hepatocytes.
Proceeding of national Biotechnology conference 2018, Publishing house of natural
sciences and technology; 2018, 589-594 (in Vietnammese).
Nguyen Cam Ha, Hoang Thi Minh Hien, Le Thi Thom, Hoang Thi Huong Quynh, Dang
Diem Hong, Optimization of fermentation conditions for squalene production by
heterotrophic marine microalgae Schizochytrium mangrovei. Academia Journal of
Biology, 2017, 39(3), 449-458.
Hoang Thi Minh Hien, Nguyen Cam Ha, Le Thi Thom, Dang Diem Hong, Squalene
promotes cholesterol homeostasis in macrophage and hepatocyte cells via activation of
liver X receptor (LXR) α and β. Biotechnology Letters, 2017, 39 (8), 1101-1107 (SCI, Q2;
IF-1.7).
Dang Diem Hong, Nguyen Cam Ha, Le Thi Thom, Luu Thi Tam, Hoang Thi Lan Anh,
Ngo Thi Hoai Thu, Biofuel from Vietnam heterotrophic marine microalgae: Biodiesel and
salvaging co-products ((polyunsaturated fatty acids, glycerol and squalene) during
biodiesel producing process. Journal of Biology, 2017, 39 (1): 51-60. (in Vietnamese)
Nguyen Cam Ha, Le Thi Thom, Hoang Thi Huong Quynh, Pham Van Nhat, Hoang Thi
Lan Anh and Dang Diem Hong, Extraction of squalene from Vietnam heterotrophic
marine microalga. Proceeding of The 4 the Academic conference on natural Science for
Yong Scientists, Master & PhD. Student from Asian Countries. 15-18 December, 2015 Bangkok, Thailand, 2016, 46-56.
Hoang Thi Lan Anh, Nguyen Cam Ha, Le Thi Thom, Dang Diem Hong, Optimization of

culture conditions and squalene enrichment from heterotrophic marine microalga
Schizochytrium mangrovei PQ6 for squalene production. Research Journal of
Biotechnology, 2016, 14 (2), 337-346 (SCI-E).
Nguyen Cam Ha, Le Thi Thom, Đang Diem Hong, Hoang Minh Hien, Study on
Hypolipidic effect of squalene extracted from heterotrophic marine microalgal
Schizochytrium sp. on HepG2 cells. Journal of Pharmacutical, 2016, 21 (4), 270 -274.


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INTRODUCTION
1. The urgency of the thesis
Viet Nam has over 3200 km of coastline with a diversity of tropical
marine organisms, rich in species composition and natural compounds that can
be used in the food industry, agriculture, medicine… Microalgae with the main
advantages of being rich in nutrients, small cells (<10 µm), easy to digest, nontoxic, high growth rate and resistance to harsh environmental conditions are
considered is the first link, an important primary biomass source in the food
chain of aquatic ecosystems. In addition, microalgae biomass also contains
many important bioactive compounds for humans and animals such as
pigments, vitamins, minerals, proteins, polyunsaturated fatty acids - PUFAs) ...
especially squalene.
Squalene - a natural tripterpene synthesized in plants, animals as
a precursor of steroid commonly is used in nutritional products, health care,
cosmetics and medicine. Squalene is widely used in the cosmetic industry as
anti-dry and softening effects, protecting the skin against light and UV ray.
Recent epidemiological studies have indicated that squalene can effectively
inhibit chemically induced lung, colon and skin tumourigenesis on
experimental animals. Squalene is also natural antioxidant that protects cells
from free radicals and highly reactive oxygen species, capable of enhancing
the activity of the immune system. The daily supplement with a high squalene
content (about 500 mg/day) has been shown to be essential in promoting

human health and nutrition, significantly reducing cardiovascular disease and
cancer. Therefore, they are also commonly used in pharmaceuticals.
The current major commercial sources of squalene are deep-sea sharks
and plant seed oils. However, overexploitation affects the conservation of
marine fish resources in the wild, as well as the dependence on soil and season
of oil crops, leading to a decrease in squalene exploitation. Meanwhile,
the demand for squalene is now increasing in the food, cosmetic,
and pharmaceutical industries. Thus, finding alternative sources of commercial
squalene are being advanced for researchers.
Microorganisms in general and marine microalgae in particular are a
potential source for squalene production on a large scale. Recently, a number


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of heterotrophic marine microalgae species such as thraustochytrids have been
regarded as a promising cell factory for the production of high value
substances including squalene. However, the study on squalene in Vietnam has
been just started in 2012, the research results just stopped at providing the
initial scientific basis of squalene content from some microalgal strains, that
have not yet been obtained: optimal methods for squalene extraction, squalene
enrichment and purification; optimal cultivation conditions for potential
microalgae capable of producing high squalene; technological process to
produce biologically active squalene for use as health food, cosmetics and
pharmaceutical in Vietnam. Therefore, with the great goal on providing
squalene-rich products for public health from natural resources available in the
country, including microalgae, we wish to be carried out the project "Research
on squalene from heterotrophic marine microalgae Schizochytrium mangrovei
PQ6 oriented as feedstock in health food, cosmetics and pharmaceuticals”.
2. Research objectives of the thesis
- Screening and determining suitable culture conditions for strain of

Schizochytirum mangrovei PQ6 of Vietnam potential for squalene production.
- Determining suitable conditions for squalene extraction and
purification from selectable S. mangrovei PQ6 strain; evaluating the safety and
pharmacological/biological effects of extracted squalene oriented as feed stock
for human health food, cosmetics and pharmaceuticals.
3. The main research contents of the thesis
- Screening potential marine microalgal species/strains for squalene
production from some marine microalgal strains of Vietnam
- Optimizing the culture conditions to obtain algae biomass with high
squalene content in selected potential strains at flask and bioreactor 30 Liter
scales.
- Optimizing the conditions for squalene extraction, enrichment and
purification from selected marine microalgal strains; establishing the squalene
extraction and purification process; determining the purification and structure
of the extracted squalene.


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- Safety assessment of squalene including acute and subchronic
toxicity, bio-pharmacological effect on in vivo model (experimental animal
model).
- Initial study of cholesterol-lowering mechanism of squalene on in
vitro model (cell model).

Chapter 1: Literature review
Squalene (2,6,10,15,19,23-hexanmethyltetracosa 2,6,10,14,18,22 hexane) is a dehydro-triterpenic hydrocarbon (C30H50) with six double bonds,
containing six isoprene units that provide the backbone for the biosynthesis of
cholesterol, bile acids and steroid. Squalene plays an important role in immune
activity, antioxidant, cholesterol reduction, cardiovascular protection and liver
detoxification as feedstock for health food production, skin moisturizing and

softening in cosmetic industry. In addition, it is also highly effective treatment
of diseases and good applications in pharmaceuticals such as pronounced antitumor activity in experimental animal models.
In recent studies, microalgae have been explored as an alternative
source of squalene. Of all the microalgal groups, heterotrophic marine
microalgae Schizochytrium are regarded as a promising cell factory for the
production of high-value products such as squalene. According to the latest
publication of Martins et al. (2018), the species Aurantiochytrium sp. achieved
the highest squalene yield of 7.3 g / 100 g after 48 h of cultivation under the
conditions of 1.5% salinity, 3% initial glucose concentration at 26° C.
In Vietnam, strain S. mangrovei PQ6 isolated in Phu Quoc island,
Kien Giang province from 2006 to 2008, is a potential microalgal strain that
easy to grow in fermentation systems with volume of 5, 10, 30 and 150 liters,
fresh weight reached 70-100 g/L, equivalent to dry cell weight (DCW) of 2030 g/L, lipid content of up to 50-70% of DCW. Therefore, the marine
microalgae is considered as a potential source for production of biomass and
metabolites such as PUFAs and squalene on large - scale.
Chapter 2. Materials and methods
2.1. Materials
2.1.1. Samples


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- 40 strains heterotrophic marine microalgae including 31 strains of
Schizochytrium genus, 8 strains of Thraustochytrium genus isolated from
coastal areas and mangroves at Hai Phong, Nam Dinh, Quang Ninh, Thanh
Hoa, Nghe An and Binh Dinh in 2013-2014 , strain S. mangrovei PQ6 isolated
from Phu Quoc island, Kien Giang province in 2006-2008 were used for the
study.
2.1.2. Biology kits: PCR product purification kit (Genjet Purification, Thermo
ScientificTM (EU)), total RNA extraction kit RNAiso Plus (Takara, Tokyo,
Japan), cDNA synthesis kit (RevertAid First Strand cDNA -Thermo Fisher

Scientific Inc., Singapore) was used in this study. Sequence of primer pairs to
amplify genes (CYP7A1, LDL-R, ABCA-1, LXRα, ABCA-1, ABCG-1,
ApoE, LXRß, GADPH) is designed by Algae Technology Department,
Institute of Biotechnology.
2.1.3. Experimental animal: White rats, white mice are provided by the
Laboratory Animal Department – Vietnam Military Medical University.
2.1.4. Cell lines: Human liver cell line HepG2 and mouse macrophage cell line
RAW264.7 sourced from the Living Cell Bank, University of Seoul, Korean
were donated by Prof. Sung-Joon Lee, Korea University, Korea.
2.1.5. Chemicals: The used chemicals are common chemicals in the
laboratory, reaching the required purity for research.
2.1.6. Laboratory tools and equipments: Use common machines and
equipments in the laboratory.
2.1.7. Culture medium
- Stock medium and inoculated medium of Schizochytrium genus:
GPY medium includes glucose (2 g/L), polypepton (1 g/L), yeast extract (0.5
g/L), artificial seawater (17.5 g/L), agar (15 g/L).
- Strains of the genus Schizochytrium were cultured in 1,000 ml flasks
containing 300 mL of M1 medium, shaking mode of 200 rpm at 25-28°C.
- Strains of the genus Thraustochytrium were cultured in improved
Bajpai medium shaking mode of 200 rpm at 25-28°C.
- Culture medium of Schizochytrium spp. in bioreactor 30L using
M12 medium for batch fermentation (including 9% glucose, 1% yeast extract,
artificial seawater 17.5 g/L); first inoculum stock preculture medium (CNT1),


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second inoculum stock preculture (CNT2), for batch fermentation (CNT3)
were supplemented with substrates include: Medium CNT1 (%): glucose - 3,
yeast extract -0.4, monosodium glutamate - 6.42, NaCl - 1.25, MgSO4 - 0.4,

KCl - 0.05, CaCl2 - 0.01, NaHCO3 - 0.05, KH2PO4 - 0.4, vitamin mixture - 0.14
(vitamin B1 - 45 g/L, vitamin B6 - 45 g/L and vitamin B12 - 0.25 g/L) and trace
elements - 0.8; Medium CNT2 (%) include: glucose - 8.57, yeast extract 0.64, monosodium glutamate - 6.42, NaCl - 2, KH2PO4 - 0.64, MgSO4 - 2.29,
CaCl2 - 0.03, NaHCO3 - 0.03, Na2SO4 - 0.03, vitamin mixture - 0.14, trace
elements - 0.2; Medium CNT3 (%) include: glucose - 7.5, yeast extract - 1.2,
monosodium glutamate - 6.42, NaCl - 0.25, KH2PO4 - 0.96, MgSO4 - 1.2,
CaCl2 - 0.12, NaHCO3 - 0.12, KCl - 0.08, vitamin mixture - 0.4.
2.2. Research methods
2.2.1. Method group to determine strains/species; biological characteristics;
optimum culture conditions of potential strain/species for high squalene
production
2.2.1.1. Method for determining growth through cell density and dry biomass:
used Burker - Turk counting chamber (Germany), dried algal to a constant
weight biomass at 105ᵒC (Dang Diem Hong et al., 2011)
2.2.1.2. Method for taking photo of morfology: Cell morfology were taken by
Japanese Canon IXY 7.0 digital camera under Olympus CX21 optical
microscope.
2.2.1.3. Determination of total lipid content in algal biomass: according to the
method of Bligh and Dyer (1959) with some modification
2.2.1.4. Method for lipid staining with Nile Red (Doan và Obbard, 2010).
2.2.1.5. Method of residual sugar determination with DNSA: according to
Miller (1959).
2.2.1.6. Method for preliminary determination of squalene content: squalene
was quantitfied using colorimetric method (Rothblat et al., 1962).
2.2.1.7. Design experiments to study optimal culture conditions of potential
strains/species of genus Schizochytrium for high squalene production
Flask scale
Study on the effect of temperature, yeast extract concentration, initial
glucose concentration, and terbinafine (TBNF) concentration: Using 250 mL

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glass flask containing 100 mL of M1 medium, shaking at 200 rpm for 5 days
to study the effects of culture temperature (15°C, 20°C, 25°C, 30°C), initial
glucose concentration (15, 30, 40, 60 and 90 g/L), yeast extract (0.5, 1, 1.5, 2,
3, 4%); and TBNF (0, 0.1, 1, 10, 100, 150, 200 µg/mL), other ingredients were
remained the same as in the base medium.
Study on the effect of vitamin mixture (B1, B6, B12): Algae were
cultured in 250 mL flask containing 100 mL of CNT3 supplemented with 0;
0.2; 0.4; 0.6% vitamin mixture (vitamin B1- 45 g/L, vitamin B6- 45 g/L and B12
- 0.25 g/L). After 24, 48, 72, 96, 120, 144, 168 h of cultivation, carried out
collected, counted cell density, determined fresh biomass, DCW, and squalene.
Scale bioreactor 30L
Study on the effect of glucose
- The first inoculum stock: S. mangrovei PQ6 was cultured on GPY
agar medium, then transferred to 1 L flask containing 300 mL of M1 medium,
shaken at 200 rpm, 28ᵒC for 96 h.
- 2% of first inoculum stock was added to 30 L bioreactor containing 15
L of M12 medium with the glucose concentration varying from 3, 6, 9, 12, and
22%. After 24, 48, 72, 96 and 120 hours of fermentation, samples were taken,
observed the cell morphology, counted cell density, determined fresh biomass,
DCW, lipid and squalene.
Study on the effect of nitrogen sources: 2% of first inoculum stock were
added to 30 liter bioreactor containing 15 L of M12 medium with nitrogen
sources of 1% yeast extract or combination of 1.2% yeast extract (Y) and
6.42% monosodium glutamate (YM). The sample collection, taking image of
cell morphology, determinating of cell density, fresh biomass, DCW, residual
glucose and squalene content were carred out after 24, 48, 72, 96 and 120
hours of fermentation,
Study on effect of batch fermentation with substrate adding (fed-batch):

- The first inoculum stock: Colony S. mangrovei PQ6 was inoculated to
500 mL flask containing 200 mL of CNT1 medium, and shaken at 28°C, 200
rpm for 24 h.


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- The second inoculum stock: 1% of first inoculum stock was added to
2 L flask containing 1 L of CNT2 medium, and shaken at 28° C, 200 rpm for
24 h.
- 2% second inocolum stock was added to 30 L fermentor containing
15L of CNT3 medium. At 48 h, glucose was added to concentration of 22%.
The sample collection, determinating of cell density, fresh biomass, DCW,
residual glucose and squalene content were carred out at 12, 24, 36, 48, 60, 72,
84, 96 and 108 h of fermentation.
Study on effect of vitamin minxture for batch fermentation with substrate
adding:
- The first inoculum stock: Colony S. mangrovei PQ6 was transferred to
500 mL flask containing 200 mL of CNT1 medium, and shaken at 28°C, 200
rpm for 24 h.
- The second inoculum stock: 1% of first inocolum stock were added to
2 liter flask containing 1 L of CNT2 medium, with or without the addition of
0.14% vitamin mixture, shaken at 28°C, 200 v/p for 24 h.
- 2% second inoculum stock was added to 30 L bioreactor containing 15
L of CNT3 medium with or without the addition of 0.4% vitamin mixture.
After 12, 24, 36, 48 h, residual sugar content in the culture medium was
determined. When the residual sugar content is less than 2%, glucose was
added to concentration of 22%. After supplementing with glucose, sampling,
counting of cell density, determining of fresh biomass, dry cell weight,
residual glucose and squalene content were carried out at 12, 24, 36, 48, 60,
72, 84, 96, and 108 h.

2.2.2. Method group to determine conditions for squalene extraction and
purification from S. mangrovei PQ6
2.2.2.1. Method to determine the conditions for extraction and purification of
squalene small amount from S. mangrovei PQ6 biomass
- The method for unsaponified lipid extraction from total lipid: according to
the method of Lewis et al (2001). Unsaponified lipid is used to run thin layer
chromatography (TLC).
- Optimize the conditions for extraction of total lipid from S. mangrovei PQ6
biomass: study the effects of different solvents (n-hexane, chloroform,


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petroleum ether; temperature (0, 30, 50 and 80ᵒC); reaction time (1, 3, 4, 5
hours); stirring condition (no stirring, continuous stirring, intermittent stirring);
number of extraction time (1, 2, 3 times); ratio of biomass/solvent (1: 8, 1:10,
1:12), biomass drying temperature (60, 70, 80, 90ᵒC) and biomass moisture (3,
30, 50, 80%).
- Optimize extraction conditions for unsaponified lipid from total lipid: study
the effect of n-hexane/chloroform ratio is 1: 1; 2: 1; 3: 1; 4: 1 and 5: 1 while
the remaining steps are preserved.
- Squalene extraction, purification and enrichment using solvent method as
described in the report by Choo et al. (2005).
2.2.2.2. Method to determine conditions for squalene extraction and
purification at pilot scacle from S. mangrovei PQ6 biomass
- Extraction of crude squalene from biomass according to the method
of Lu et al. (2003). Study on the effect of solvent (ethanol and methanol),
biomass moisture (20% to 100%) on squalene extraction and content.
- Extraction of crude squalene from fermented solution as published
by Pora et al. (2015), purification of crude squalene using column
chromatography method.

2.2.2.3 Method for determining of squalene content and purity: using high
pressure liquid chromatography (HPLC) (Dinh Thi Ngoc et al., 2013)
2.2.2.4. Method for determining of squalene structure: by nuclear magnetic
resonance (NMR) spectroscopy using Bruker Avance-500 MHz spectrometer
machine (Poucher et al., 1993).
2.2.3. Method group to determine the quality parameters of squalene extracted
from S. mangrovei PQ6
Method to determine sensory: according to Vietnam standard (TCVN) 26271993
Method to determine physical and chemical properties: humidity: according to
TCVN 6120: 2007; acid, saponification, peroxide, iodine values according to
TCVN 6127: 2010; TCVN 6126: 2007; TCVN 6121: 2010 and TCVN 6122:
2010, respectively.
Method to determine total microbial numbers.
Method to determine metal properties


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2.2.4. Method group to evaluate the safety and pharmacological effects of
squalene extracted from S. mangrovei PQ6 on experimental animal model
(in vivo) and cell model (in vitro)
2.2.4.1. Method group to assess the safety and pharmacological effects of
squalene extracted from S. mangrovei PQ6 on in vivo model
- Study on acute toxicity of squalene according to the method of Litchfield Wincoxon (Do Trung Dam, 2014), regulations of the Vietnam Ministry of
Health (2018), guidelines of Organization for Economic Cooperation and
Development (OECD) (2002) and World Health Organization (2000).
- Evaluate semi-chronic toxicity: regulations of the Vietnam Ministry of
Health (2018) guidelines of Organization for Economic Cooperation and
Development (OECD) (2000) and World Health Organization (2000).
- Effect of squalene in The HDL-C increase on white mice was evaluated
according to the method described by Clara Gaba´s-Rivera et al (Do Trung

Dam, 2006).
2.2.4.2. Method group for initial evaluation on the mechanism of lipid
reduction effects of squalene extracted from S. mangrovei PQ6 on in vitro
model
- HepG2 and RAW264.7 cells were cultured on DMEM/high glucose medium
containing 10% FBS, 100 U/mL penicillin, and 0.1 mg/mL streptomycin in a
sterilized incubator at 37ᵒC, 5 % CO2.
- Toxicity of squalene extracted from S. mangrovei PQ6 on HepG2 cells was
analyzed by MTT method.
- Lipid staining with Oil red O (ORO) according to the method of Hoang et al
(2012).
- Extraction of intracellular lipids.
- Content of cholesterol and intracellular triglyceride was determined using
enzyme method.
- Total RNA extraction (according to kit RNAiso PlusTakara - Tokyo, Japan),
cDNA synthesis (according to RevertAid First Strand cDNA kit - Thermo
Fisher, Scientific Ins., Singapore). The cDNAs were then used as the template
for qPCR reaction. Glyceraldehyde-3-phosphate dehydrogenase was used to
normalize the gene expression data.


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2.2.5. Statistical analysis of the data
Data are presented as mean ± standard error. The difference is
considered to be statistically significant at P <0.05 level, data are statistically
processed according to Student's t-test method, compared with anova test
using SPSS 16.0 software.
2.2.6. Places to conduct experiments in research
Chapter 3. Results and disscusions
3.1. Screening of potential heterotrophic marine microalgal strains for

squalene production
Based on growth, lipid and squalene content, S. mangrovei PQ6 strain
was selected from 40 strains belonging to the genera Schizochytrium and
Thraustochytrium. Dry biomass, lipid and squalene content of strain PQ6 were
reached the highest of (12.38 ± 0.72) g/L, (39.61 ± 0.12)% DCW, (102.01 ± 1,
04) mg/g of DCW, respectively. In previous studies, strain PQ6 has been
studied carefully on biological characteristics and ability to grow on the large
scale. This is also a potential strain to produce PUFAs, including DHA (Dinh
Thi Ngoc Mai et al, 2013; Hoang et al, 2014).
3.2. Effects of culture conditions on growth and squalene content of S.
mangrovei PQ6
3.2.1. Optimization of culture conditions of S. mangrovei PQ6 for squalene
production at flask scale
3.2.1.1. Effect of temperature on growth and squalene content at flask sale
In the temperature range from 15-35ᵒC, growth and squalene content
of strain PQ6 cultured at 28ᵒC reached highest of (13.47 ± 0.53) g/L and
(61.42 ± 1.24) mg/g DCW after 4 and 5 days of culture, respectively. The
suitable temperature for growth and squalene synthesis of strain PQ6 was
higher compared with the publication of Lewis et al (2001), Nakazawa et al
(2012). Strain characteristics, natural conditions and climate may be
responsible for this difference.
3.2.1.2. Effect of yeast extract concentrations on growth and squalene content
at flask scale
In yeast extract concentration range 0.5-4%, DCW and squalene
content of strain PQ6 were reached nearly equivalent at concentrations of 1


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and 1.5%. At this concentration, DCW and squalene content of strain PQ6
reached up (13.56 ± 0.28) mg/g DCW and (61.02 ± 1.36) mg/g DCW after 5

days of culture, respectively. Research by Chen et al (2010) on strains of
Aurantiochytrium sp. showed that the growth of this strain increased with
increasing yeast extract concentrations from 0.5 to 3 g/L. The highest squalene
content and yield were reached 0.21 mg/g and 1.62 mg/L after 36 h culture
using 6 g/L yeast extract. Thus, the squalene content of strain PQ6 is higher
than that in the publication of other authors.
3.2.1.3. Effect of initial glucose concentration on growth and squalene content
at flask scale
When the glucose concentration increased from 1.5 to 9%, growth of
strain PQ6 reached maximum at glucose 4% after 5 days of culture of (13.06 ±
0.39) g/L. The highest lipid, squalene content and yield at 9% glucose after 6
and 5 days of culture were (51.02 ± 1.54) % DCW, (62.89 ± 2.59) mg/g DCW,
and (640.94 ± 10.04) mg/L, respectively. When this strain was cultured in
flask under optimal conditions, the squalene content reached up 6.3% of DCW
using TLC and HPLC methods. These values were several hundred times
higher than that of other thraustochytrid strains reported previously (0.0020.150 % of DCW) (Jiang et al, 2004; Li et al., 2009; Chen et al., 2010; Lewis
et al., 2001). ; Fan et al, 2010).
3.2.1.4. Effect of terbinafine concentration on growth and squalene content at
flask scale
TBNF is an inhibitor of the enzyme squalene monooxygenase in the
sterol biosynthesis pathway. This enzyme catalyzes the oxidation of squalene
to form 2,3 oxidosqualene in the presence of oxygen molecules and NADH
(Ono 2002; Ryder et al., 1992). The addition of TBNF from 0.1 to 200 µg/mL
into the medium significantly reduced cell density and DCW after 5 days of
culture. However, lipid and squalene content increased significantly. Squalene
content was almost unchanged when the TBNF concentration increased 10
times (0.1-1 µg / mL). With the increase of TBNF concentration in medium to
1,000-fold (100 µg/L), the squalene content reached maximum of (97.34 ±
1.97) mg/g DCW after 5 days of culture.

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3.2.1.5. Effect of vitamin mixture on growth and squalene content at the flask
scale
Research by Pora et al. (2014) showed that vitamins such as B1, B6
and B12 increased squalene production in Schizochytrium algae cultivation.
Research on the effect of the vitamin mixture at concentration of 0-0.6% on
the squalene synthesis of strain PQ6 showed that the growth and squalene
content was the highest at 0.4 and 0.6%. Because there was no significant
difference between 2 concentrations, the 0.4% concentration was selected for
the coutinous experiments. After 6 days of culture, DCW and squalene content
of strain PQ6 reached the highest at 0.4% vitamin mixture of (39.98 ± 0.35)
g/L and (76, 16 ± 2,34) mg/g of DCW, respectively (increase 34.43%
compared to control without vitamin supplementation).
3.2.2. Optimization of culture conditions for S. mangrovei PQ6 strain in
bioreactor 30 liter to obtain squalene-rich algae biomass
3.2.2.1. Effect of glucose concentration on cell growth and squalene
accumulation of S. mangrovei PQ6 strain in batch fermentation of 30 L
According to Fan et al. (2010), Nakazawa et al. (2012), initial glucose
concentration has great effect on the growth and squalene accumulation of A.
mangrovei (formerly S. mangrovei). In bioreactor 30 L, the initial glucose
concentration was set at 3-22%. Achieved results showed that cell density,
DCW and squalene yield of strain PQ6 increased strongly when the glucose
concentration increased from 3 to 9%. With initial glucose concentration of
9%, DCW and squalene yield reached the highest value of (35.5 ± 0.1) g/L and
(1.1 ± 0.1) g/L, respectively. Cell size at concentrations of 6% - 9% glucose
was uniform and larger than that of the high concentration of 12% and 22%
glucose. Therefore, initial glucose concentration of 9% was chosen for batch
cultivation of strain PQ6 in bioreactor 30L.
3.2.2.2. Effect of nitrogen sources on cell growth and squalene accumulation

of S. mangrovei PQ6 strain in batch fermentation of 30 L
With combining of yeast extract and monosodium glutamate sources
as nitrogen source, cell density, DCW, and squalene yield reached maximum
value of (3.3 x 108) cells/mL, 46.7 g/L and 1.6 g/L at 72 h of fermentation,
respectively. The squalene content did not change significantly with using only


13
yeast extract or combination of yeast extract and monosodium glutamate.
However, with combining the two nitrogen sources as mentioned above, the
squalene yield reached the highest value after 72 h (1605.5 mg/L), while using
only yeast extract reached 1195.2 mg/L. This may be due to the increase of
biomass in the medium with combination of nitrogen sources. Therefore, the
nitrogen source in our experiments only increased the growth rate, leading to
increased squalene yield, but not increased squalene accumulation. The result
is similar as described by Chen et al. (2010).
3.2.2.3. Effect of batch fermentation with substrate supplemention on cell
growth and squalene accumulation of strain S. mangrovei PQ6
Pora et al. (2014) reported that squalene content of some
thraustochytrid species increase 5-10 times in batch fermentation with adding
glucose up to 22% (fed-batch). For strain PQ6, fed-batch culture also increased
cell growth and squalene accumulation. Cell density and DCW were found to
increase maximal at 108 hours of fermentation after the addition of glucose
(392.53 ± 1.91) × 106 cells / mL and (100.41 ± 1.43) g/L, respectively. The
highest squalene yield achieved (4592.53 ± 0.3) mg/L at 84 h, increased in 2-3
times higher compared with batch cultivation.
3.2.2.4. Effect of vitamin mixture on cell growth and squalene accumulation
of S. mangrovei PQ6 strain
The addition of 0.4% vitamin mixture into the culture medium also
increased the biomass and squalene content of strain PQ6 cultured in fed-batch

fermentation. The highest DCW reached (105.25 ± 0.75) g/L after 96 h of
fermentation. Squalene content and yield reached the maximum of (91.53 ±
2.45) mg/g DCW and (6928.6 ± 14.6) mg/L after 48 h. The values were
increased in 2-3 times higher than by fed-batch culture in medium without the
addition of vitamins and in 4 - 6 times compared to batch culture. In the study
of Pora et al. (2014), squalene yield of strain PQ6 was higher than that of
strains Schizochytrium sp., Schizochytrium sp. ATCC 20888 and
Aurantiochytrium sp. ATCC PRA 276.
Nile Red is a lipophilic fluorescent dye used for intracellular lipid
determination in both prokaryotic and eukaryotic cells that is capable of
detecting neutral lipids (Cooksey et al., 1987). Since squalene is in the


14
unsaponifiable lipid, squalene accumulation can be observed qualitatively
through Nile Red staining (Figure 3.13).

Figure 3.13. Images of PQ6 strain cell morphology obtained during
different cultivation periods of fed-batch fermentation. A- Before adding
glucose; B- After adding glucose to 22%. (a) Light microscopy; (b) Nile
red; (c) Transmission electron microscop. L- Lipid bodies; Mimitochondria; N- nucleus; V- vacuoles. Scale bars: a-b=10 m; c=2 m
3.3. Establish procedure for squalene extraction and purification from S.
mangrovei PQ6
3.3.1. Optimization of extraction and purification conditions of squalene
small amount from S. mangrovei PQ6
3.3.1.1. Extract total lipid from S. mangrovei PQ6 biomass
The suitable conditions for total lipid extraction of S. mangrovei PQ6
are biomass dried at 80°C to 3% humidity, using n-hexane solvent, extraction
temperature at 70-75°C, continuous stirring for 4 hours, extraction 1 time with
biomass/solvent ratio of 1: 8 (w / v).

3.3.1.2. Extract unsaponifiable lipid from total lipid
In the n-hexane/chloroform ratios of 1: 1, 2: 1, 3: 1, 4: 1 and 5: 1,
squalene content in the unsaponifiable lipid at the ratio 2: 1 reached the highest
value of (8.1 ± 0.14)%.
3.3.1.3. Purify and quantify squalene using thin layer chromatography and
high pressure liquid chromatography
The results in Figure 3.15A showed that unsaponifiable lipid extracted
from strain PQ6 have similar to the standard squalene. Quantitative analysis by
HPLC, squalene content reached (50.10 ± 0.03) mg/g of DCW.


15
The extracted squalene has high purity (98.6%) (Figure 3.15 B). The
obtained squalene content is higher than that in Aurantiochytrium limacinum
9F-4a (0.6 mg/g DCW), 4W-1b (0.5 mg/g DCW), Schizochytrium limacinum
SR21 (0.2 mg/g) but lower than that in A. limacinum SR21 (171.1 mg/g DCW)
(Nakazawa et al., 2012).
A
B

Figure 3.15. Thin layer chromatography of unsaponifiable lipid (A) and typical
chromatography of purify squalene from biomass strain PQ6 (B)
(A: Lane 1: squalene standard - 2 mg; Lane 2, 3, 4, 5, 6, 7: unsaponifiable lipid
extracted from biomass of strain PQ6)
3.3.1.4. Extraction, purification and enrichment of squalene using solvent method
Since the obtained squalene content by the TLC and HPLC methods is
low, squalene enrichment was carried out as described by Choo et al. (2005) using
solvent method. The squalene content increased 1.7 times (from 61.76 ± 0.12 to
104.99 ± 0.34 mg/g of DCW). The obtained squalene has high purity (> 98%) and
was confirmed structural by 1H NMR 1H (500 MHz, CDCl3).

3.3.2. Establish process for squalene extraction and purification at pilot
scale from S. mangrovei PQ6
3.3.2.1. Effect of the agents on squalene extraction efficiency from S.
mangrovei PQ6 biomass
Effect of the solvent: KOH/ethanol mixture gave the best results with
crude squalene content of (123 ± 11) mg/g of DCW, of which the actual
squalene content of (0.32 ± 0.04) mg/mg crude squalene.
Effect of biomass moisture: Biomass moisture didn’t affect the
extracted squalene content. Actually and crude squalene content in biomass
with moisture content of 20 and 100% reached (128 ± 31) mg/g of DCW and


16
(0.30 ± 0.02) mg/mg crude squalene, respectively; (126 ± 14) mg/g of DCW
and (0.32 ± 0.03) mg/mg crude squalene.
3.3.2.2. Extract squalene from fermented solution of S. mangrovei PQ6 after
batch fermentation with substrate adding
The alkaline medium and high temperature can disrupt cell membrane
of species belong to genus thraustochytrids (Pora et al., 2014). Fermented
solution of strains PQ6 with cell density of 250-300 g/L was adjusted to pH 10
by 45% KOH, stirred at 60ᵒC, 150 rpm for 6 h. The obtained results showed
that there was no significant difference in squalene content between squalene
extracted from fermented solution ((0.29 ± 0.02) mg/mg crude squalene) and
from dry biomass ((0.28 ± 0.03) mg/mg crude squalene). Therefore, for
industrial production of squalene, extracting squalene directly from the
fermented solution was chosen.
3.3.2.3. Purification conditions of squalene in column chromatography
method
The squalene fractions obtained after passing the chromatographic
column with n-hexane elution solvent system. Obtained squalene has high

purity (accounting for 90-95% in comparing to the percentage peak area).
Besides, the recovery efficiency is about 50-60%. The research results of
Watanabe et al (2013) also showed that n-hexane allowed to collect squalene
with high purity and recovery efficiency up to 70-80%. Therefore, the nhexane elution solvent was chosen to purify squalene in column
chromatography method.
3.3.2.4. Establish the process of squalene extraction and purification from S.
mangrovei PQ6 biomass
Based on the studies of squalene extraction and purification with high
stability and repeatability, the process for squalene extracting and purifying
from the fermented solution after fed-batch cultivation of the PQ6 strain has
been established (Figure 3.26). With using this procedure, pure squalene has
been extracted in a colorless, odorless liquid with purity of 90-95%.


17

Figure 3.26. Squalene extraction procedure from S. mangrovei PQ6
3.3.3. Extraction and purification of squalene large amount from S.
mangrovei PQ6
With the established procedure (Figure 3.26), from 46 L of
fermentated solution (approximately (3.9 ± 0.1) kg of DCW), (1.08 ± 0.29) kg
of crude squalene were extracted and about 131 mL of pure squalene in
odorless, colorless liquid with squalene content of (305.23 ± 2.34) g were
purified (Figure 3.27). HPLC chromatographic analysis result showed that
obtained squalene has high purity (accounting for 90-95%; Figure 3.28) and
not been contaminated.
Analysis on 1H NMR spectrum (500 MHz, CDCl3), mass
spectrometry 13C NMR (125 MHz, 140 CDCl3) (Figure 3.29) and comparison
with standard squalene (Pouchert and Behnke, 1993) can confirm extraction of
squalene from heterotrophic marine microalgae S. mangrovei PQ6.

3.3.4. Analysis on sensory, physicochemical, microbiological and metallic
parameters of squalene extracted from S. mangrovei PQ6


18
The results on quality analysis of extracted squalene at the Vietnam
Certification Center (Quacert), Technical Center of Standards Metrology and
Quality, Ministry of Science and Technology showed that squalene meets the
quality requirements as material for production of health food, cosmetics and
pharmaceuticals: liquid, colorless, odorless; Acid index - 0.524 mg KOH/g;
peroxide index- 4.2 Meq O2/kg, no residue urea, within the permissible
limits of heavy metals
B
Figure 3.27. Illustrative image of
A
crude squalene (A), purified
squalene and fatty acid mixture
(B)
extracted
from
batch
fermentation
solution
with
substrate adding from biomass
PQ6

Figure 3.28. Chromatogram HPLC of Figure 3.29. Spectra 1H (A) and 13C
squalene standard (A) and purified squalene (B) NMR of the purified squalene
(B) from S. mangrovei biomass

from biomass S. mangrovei PQ6

PQ6


19
3.4. Safety and pharmacological effects of squalene extracted from S.
mangrovei PQ6 on experimental animal model (in vivo) and cell model (in
vitro)
3.4.1. Acute toxicity of squalene on experimental animal model
By oral administration of the squalene on white mice at the highest
dose of 50 mL/kg body weight (equivalent to 58.25 g/kg body weight) after
178 h, the LD50 was not determined. Our obtained results are also completely
consistent with publication of CTFA (1971) that the toxic and lethal effect was
not determined in mice with the highest squalene dose of 50 mL/kg of whole
body within 7 days. Therefore, squalene extracted from S. mangrovei PQ6 is
non-toxic.
3.4.2. Semi-chronic toxicity of squalene on experimental animal model
3.4.2.1. Effect of squalene on general condition and weight change in white
rats over long period of time
Squalene at doses of 400 mg/kg body weight/ 24 h and 1,200 mg/kg
body weight/24h for 30 and 60 consecutive days have allowed no change in rat
body weight, smooth hair, normal skin and mucous, normal behaviors of rat
include eating, drinking, urinating, defecating with solid feces.
3.4.2.2. Effect of squalene on hematological and biochemical indicators of rat
Squalene at doses of 400 mg/kg body weight/24 h and 1,200 mg/kg
body weitht/24h have allowed no change in hematological and biochemical
parameters after 30 days and 60 days of the study. However, after 60 days, the
HDL-C index was higher than that in initial time.
3.4.2.3. Evaluate the degree of damage to the liver and kidney functions of rat

using long-term squalene
Squalene at dose of 1,200 mg/kg body weght/day for 60 consecutive
days have allowed no change in the activity of enzymes AST and ALT, total
bilirubin indices, plasma albumin and total cholesterol, no liver damage, no
affect kidney and liver function of rat.
The anatomy of liver, kidney, spleen also showed no appearance any
damage to the organs in rats at doses of 400 and 1,200 mg/kg body weight/
day after 60 days of continuous use.


20
3.4.3. The pharmacological effects of squalene on experimental white mice
To explore the effects of squalene on the increase of HDL-C in mice
blood, body weight, liver weight, and blood lipid indices were evaluated. At
doses of 600 mg/kg/day and 1,200 mg/kg/day for 60 days of continuous use,
squalene has the effect in increasing HDL-C and ratio of HDL-C/total
cholesterol in the blood, reducing level of LDL-C and VLDL-C in the blood
and no effect in total cholesterol and blood TG, mice liver and body weight.
The obtained results were similar to studies of Pallavi et al. (2013), GabasRivena et al (2014).
3.5. Initial study on lipid lowering mechanism of squalene extracted from
S. mangrovei PQ6 on in vitro model
3.5.1. Evaluate toxicity of squalene on cell lines
Squalene toxicity assessment on two cell lines HepG2 and RAW264.7
showed that squalene was not toxic at all tested concentrations. About 95 to
100% cells growed normally after supplementing with squalene for 72 h.
3.5.2. The lipid-lowering effect of squalene on hepatocytes and macrophage
cells
3.5.2.1. Optimization of squalene incubation time and concentration on
changes in lipid content of cell HepG2
Study on the effect of squalene incubation time and concentration on

the changes in lipid concentration on hepG2 cell line with metabolic disorders
showed that TG content in HepG2 cells incubated with oleic acid (OA) and
palmitic acid (PA) was increased 85% compared with the control. After 72 h
of incubation with squalene at concentrations of 20, 50 and 100 µM, the
intracellular lipid accumulation of HepG2 cells was decreased by 20%
compared with the control. Therefore, squalene concentrations of 50 and 100
µM were used for cell treatment in subsequent experiments for 72 h.
3.5.2.2. The effect of squalene on the changes in intracellular lipid content of
HepG2 and RAW64.7 cells
Incubation of cell HepG2 with 100 µM squalene reduced cholesterol
and trigyceride from 100% in the control cell to 80% (P <0.05) and 65% (P
<0.01), respectively. Similarly, the cholesterol and trigyceride content in cell
RAW264.7 was decreased 100% in the control to 86% (P <0.05) and 52% (P


21
<0.01) with incubation at 100 µM squalene. Thus, squalene has the effect in
lowering cholesterol and TG in HepG2 liver and RAW264.7 macrophage
cells.
3.5.3. Molecular mechanism of cholesterol-lowering effect of squalene
extracted from S. mangrovei PQ6
- The expression level of LDL-R, CYP7A1 and LXRα genes in cell HepG2
incubated squalene was increased statistically significantly compared to the
control by 150%, 60% and 160%, respectively.
- Incubation of cell RAW264.7 with 50 and 100 µM squalene increased LXRα
receptor activation by 60% and 70% (P <0.05) and LXRβ receptors activation
by more than 30% compared to control, respectively. Thus, squalene has the
effect in activating the LXRs receptor. Cellular cholesterol content incubated
with 50 and 100 µM squalene was decreased by 10% and 14% compared to
the control (P <0.05). The cholesterol concentration in the culture medium of

cells incubated with squalene was 20% higher than that of the control (P
<0.05).
- At concentration of 100 µM, squalene increased expression level of gene
(46%) and protein (42%) of ABCA1 in RAW264.7 cell compared to the
control (P> 0.05). In addition, expression level of the genes involved in
reverse cholesterol transport that regulated by the LXR receptor of ABCG1
and ApoE was increased by 38% and 112%, respectively. Thus, the
cholesterol-lowering and HDL-C increase effects of squalene can be attributed
to the expression regulation of genes involved in cholesterol transport and
degradation such as LDL-R, CYP7A1, LXR receptor and target genes of the
LXR receptor.
3.5.4. Triglyceride lowering effect of squalene extracted from S. mangrovei PQ6

Intracellular TG content increased from 51 µg/mg protein in the
control to nearly 70 µg/mg protein in HepG2 cells incubated with T0901317.
In contrast, cells incubated with 100 µM squalene reduced the TG
concentration by 17% compared to the control. ORO staining also showed
similar results.
Thus, unlike the LXR receptor activators using chemical synthesis
method, squalene extracted from S. mangrovei PQ6 can selectively activate


22
target genes of LXR receptor without lipid synthesis in liver cells. Based on
the above results, the diagram of the cholesterol-lowering and the triglyceridelowering mechanisms of squalene have been established in Figure 3.39.

Figure 3.39. Summary diagram of the initial research on the molecular mechanisms
of cholesterol and lipid lowering effects of squalene extracted from S. mangrovei
PQ6. Note: LDL-R: The low - Density Lipoprotein Reccetor; CYP7A1: cholesterol
7a-hydroxylase; LXR: liver X receptor; ABC: ATP-binding cassette transporter

member 1; ApoE : apoliprotein; Insig-2a: insulin induced gene 2-a; SREBP-1c:
sterol regulatory element-binding protein 1c; FAS: Fatty acid synthase; FATP4: fatty
acid transport protein 4; SCD-1: stearoyl-Coenzyme A desaturase 1; CPT-1:
Carnitine palmitoyltransferase ; ACOX: acyl-CoA oxidase; LPL: lipoprotein lipase;
MCAD: medium-chain acyl CoA dehydrogenase; HMGCS2- hydroxymethylglutaryl
CoA synthase 2