Research on squalene from heterotrophic marine microalga schizochytrium mangrovei pq6 oriented as feedstock for health food, cosmetic and pharmaceutical

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 6 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. 7 - 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, 8 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) 2627- 1993 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 9 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. 10 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 11 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 (0.002- 0.150 % 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. 12 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). 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. 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; Mi- mitochondria; N- nucleus; V- vacuoles. Scale bars: a-b=10 m; c=2 m 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). 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 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) 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 n- hexane 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 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