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
Trang 1MINISTRY OF EDUCATION AND
TRAINING
VIETNAM ACADEMY
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
Trang 2Science and Technology, Vietnam Academy of Science and Technology
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
Trang 31 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)
2 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),
550-556 (SCI, Q1; IF-3.0)
3 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)
4 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
5 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)
6 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)
7 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
8 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)
9 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
Trang 4INTRODUCTION
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, non-toxic, 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
Trang 5of 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
Trang 6- Safety assessment of squalene including acute and subchronic
toxicity, bio-pharmacological effect on in vivo model (experimental animal
-of diseases and good applications in pharmaceuticals such as pronounced tumor activity in experimental animal models
anti-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 20-
30 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
Trang 7- 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),
Trang 8second 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
Trang 9glass 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 (B 1 , B 6 , B 12): 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
Trang 10- 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,
- Optimize the conditions for extraction of total lipid from S mangrovei PQ6
biomass: study the effects of different solvents (n-hexane, chloroform,
Trang 11petroleum 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
2010, respectively
Method to determine total microbial numbers
Method to determine metal properties
Trang 122.2.4 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
Trang 132.2.5 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