Nghiên cứu điều kiện lên men cordyceps sinensis tạo sinh khối giàu selen và khảo sát hoạt tính sinh học TT TIENG ANH

MINISTRY OF VIETNAM ACADEMY EDUCATION AND TRAINING OF SCIENCE AND TECHNOLOGY GRADUATE UNIVERSITY OF SCIENCE AND TECHNOLOGY - LÊ QUỐC PHONG STUDY ON FERMENTATION CONDITIONS OF SELENIUMENRICHED BIOMASS PRODUCTION BY Cordyceps sinensis AND EVALUATION OF ITS BIOACTIVITIES Major: Biotechnology Code: 42 02 01 SUMMARY OF DOCTORAL THESIS Ho Chi Minh City - 2021 The study was done at the Graduate University of Science and Technology – Vietnam Academy of Science and Technology Supervisor: Associate Prof NGÔ KẾ SƯƠNG Reviewer 1: … Reviewer 2: … Reviewer 3: … The thesis will be defended against the examination board of doctoral thesis at the academy level at Graduate University of Science and Technology – Vietnam Academy of Science and Technology on … 2021 This thesis can be found at: - The Library of the Graduate University of Science and Technology - The Vietnamese National Library INTRODUCTION The urgency of the thesis Cordyceps sinensis is well-known as a valuable medicinal mushroom It contains a variety of highly bioactive compounds such as adenosine, cordycepin, polysaccharide, β-glucan, and many other vitamins and minerals Previous studies have shown that C sinensis possesses many positive effects on human health such as immuneboosting, anti-aging, anti-inflammation, as well as supporting the treatment of diseases related to the lungs, kidneys, and chronic diseases such as diabetes, gout, or cancer So far, the natural yield of C sinensis has been increasingly reducing Therefore, studies on artificial cultivation of the fungus on liquid or semi-solid media are of great importance Especially, supplementing precursors into the culture media in order to improve the quality and stimulate the production of secondary metabolites by C sinensis fungus has been received great interest from scientists In this regard, selenium (Se) is one of the important precursors Se is a trace element involved in the structure of the 21st amino acid – selenocysteine It is also an important component of many antioxidant enzymes in humans such as glutathione peroxidase (GPx), iodothyronine deiodinases (DIOs), and thioredoxin reductase (Txnrd) Moreover, Se plays a vital role in the immune system and supports treatments of cardiovascular and musculoskeletal diseases However, it is toxic to humans in the inorganic form of Se To enhance the bioavailability of Se, biotransformation of inorganic to organic forms by C sinensis fungus could be feasible and applicable By doing this, one can increase the economic and medicinal value of this mushroom On the other hand, in Vietnam, several studies have been conducted on detection of Se in medicinal plants and mushrooms since 1965 Due to the highly medicinal impacts of Se, between 2003 and 2005, the Vietnamese Ministry of Health encouraged and directed studies on discovering new Se-supplying resources based on plants Some domestic medicinal herbs containing high Se levels such as garlic and Ganoderma lucidum have been used to produce Sesupplying functional products, but Se contents in these materials are often not stable Importantly, some internal assessments of Se in human blood and urine in some parts of Vietnam showed a significant deficiency of Se Therefore, to tackle these issues, this current project entitled "Study on fermentation conditions for selenium-enriched biomass production by Cordyceps sinensis and evaluation of its bioactivities" is timely and important The main objectives of the thesis This study aimed to produce selenium-enriched C sinensis biomass in order to (1) enhance the bioactivities of this medicinal mushroom and (2) provide a Se resource with high bioavailability The main contents of the thesis Content 1: Evaluation of impacts of inorganic Se forms on biomass production by Cordyceps sinensis Content 2: Optimization of culture medium components and conditions for production of Se-enriched Cordyceps sinensis biomass Content 3: Evaluation of biological activities of Se-enriched Cordyceps sinensis biomass CHAPTER – OVERVIEW 1.1 Cordyceps sinensis Cordyceps sinensis is a parasitic fungus on the larvae of the ghost moths Thitarodes (Hepialus) armoricanus belonging to the family Hepialidae residing underground It is endemic to the Tibetan plateau and the grasslands surrounding the Himalayan regions such as Bhutan, China, and Nepal at an altitude between 3500 and 5000m above sea level Its medicinal values have been recorded 2000 years ago in China as well as in Eastern countries with positive effects on human health such as improving liver and kidney functions, supporting the treatment associated with diabetes and fatty blood, helping health recovery, preventing heart diseases, rising life expectancy and improving physical health 1.2 Selenium and its biological roles Se was discovered in 1817 by Jacob Berzelius Jöns in the wastes of pyrite production In soil, Se mainly exists in the form of selenite (SeO32−) and selenate (SeO42−) Meanwhile, a small amount of organic Se forms are produced by microbial communities and plants So far, 25 genes encoding seleno-proteins have been identified in the human genome Seleno-proteins have various functions, including antioxidant activity, immune function, thyroid hormone metabolism, selenium transport, and redox homeostasis, and metabolism in bone and myocardium 1.3 Cordyceps sinensis artificial cultvation The natural yield and production of C sinensis are significantly decreasing due to climate change and the increasing demand Therefore, in order to maintain this valuable medicinal mushroom, scientists have taken efforts to artificially cultivate it so that it can bring long-term benefits to humans Currently, C sinensis cultivation can be carried out in two methods based on liquid fermentation and semi-solid cultivation Production of organic Se-enriched biomass by C sinensis has been attractive to mycologists and mushroom growers It is because the accumulation of Se in the fungal biomass not only increases the bioactivities of C sinensis but also provides an alternative organic Se resource to humans However, adding inorganic Se to the culture medium at high concentrations can cause negative impacts on the growth and biomass production of the fungus Hence, studies on the adaptability of inorganic Se, optimization of culture conditions and medium composition are needed to reduce the toxicity of inorganic Se In this concern, response surface method (RSM) has been widely applied to improve the biomass production and Se accumulation via evaluation of the interaction between multi-factors so that one can find the extreme point or optimal region for the experiment This method is beneficial to reduce timing and costs when compared to classical optimization methods CHAPTER - MATERIALS AND METHODOLOGY 2.1 Materials Cordyceps sinensis strain CS-YK2007 was provided by Dr Truong Binh Nguyen, Institute of High-Tech Agricultural Research and Application, Dalat University, Lam Dong Analytical chemicals were derived from Sigma Aldrich, Fisher Scientific, and culture chemicals were derived from domestic producers in Vietnam Equipment: Autoclave (Jibimed, China); Water incubator (Shinsaeng, Korea); Spectrophotometer (Phoenix, Germany); ICP-MS System 7700x (Agilent, USA); Microscope (China); Moisture meter (AND MX–50, Japan); Drying oven (MEMMRT, Germany); pH meter (HANNA, Romania); Micro centrifuge 17R (Germany); Technical balance (PioneerTM Ohaus, USA); Perkin Elmer System 350X (NexION, USA); Thermostatic tank F34 (Julabo, Germany) 2.2 Methodology 2.2.1 Evaluation of impacts of Se on the mycelial growth of Cordyceps sinensis on PDA plates and liquid medium 2.2.1.1 Impacts of inorganic Se on the mycelial growth of Cordyceps sinensis on PGA plates This work was conducted on potatoes glucose agar (PGA) plates Two inorganic Se salts Se+4 (Na2SeO3.5H2O) and Se+6 (Na2SeO4.12H2O) and selenourea (SeC(NH2)2 were used PDA plates containing Se at different concentrations of - 40mgSe/L were prepared Afterward, an agar plug of the fungus was put in the center The growth rate of the mycelium was observed at three different time points: 5, 10, and 15 days The diameter of the fungal colony was measured using an eyepiece micrometer 2.2.1.2 Impacts of inorganic Se on the mycelial growth of Cordyceps sinensis on liquid medium A liquid medium consisting of potatoes and saccharose (PS medium) was prepared Each Se salt was supplemented to the medium at different concentrations of - 30mgSe/L followed by injecting the inoculum of the fungus The fungus was cultivated at 22 ± 2°C for 40 days Lastly, yield of biomass production and total Se levels in biomass were determined 2.2.2 Modified Se-adaptive cultivation method In this work, we selected a Se salt which showed a lower toxicity to the growth of the fungus in section 2.2.1 To increase the adaptability of Se-rich medium, the fungus was adapted in Secontaining medium at ascending concentrations of – 25mgSe/L 2.2.3 Optimization of culture medium components and conditions for biomass production by Cordyceps sinensis on seleniumcontaining liquid medium 2.2.3.1 Screening of culture medium components using Plackett – Burman design To screen key factors for the optimization process with a variety of various culture medium components, Plackett-Burman’s design was applied Seven factors i.e potatoes (X1), saccharose (X2), peptone (X3), yeast extract (X4), KH2PO4 (X5), K2HPO4 (X6), and MgCl2 (X7) were assessed Each factor was run at levels (-1 and +1) In Plackett-Burman’s design X1, X2, X3, X4, X5, X6, and X7 are the independent variables A total of 12 experiments were done Yield of biomass and total Se levels in biomass were determined upon 40 days of cultivation 2.2.3.2 Optimization of culture medium components using Box – Behnken response surface methodology In this work, three main factors were determined based the Plackett-Burman experiment Afterward, to optimize these culture medium components for production of Se-enriched C sinensis biomass, a Box – Behnken response surface model was used Each factor was run at levels (-1, and +1) A total of 17 experiments were conducted Yield of biomass and total Se levels in biomass were determined upon 40 days of cultivation The outputs (Y) of the model were biomass yield (g/L) and total Se level in biomass (µg/g) A regression was computed as the following equation: Y = B0 + B1X1 + B2X2 + B3X3 + B12X1X2 + B13X1X3 + B23X2X3 + B11X12 + B22X22 + B33X32 In which, B1, B2, and B3 are coefficients of order 1; B11, B22, and B33 are quadratic coefficients; B12, B23, and B13 are the interaction coefficients of each pair of each factor; X1, X2, and X3 are the independent variables 2.2.3.3 Optimization of culture conditions using D-optimal response surface methodology In this experiment, a D-optimal response surface model was employed Three main factors including temperature (20, 25, and 30°C), light (red, white, and blue), and pH (6, 7, and 8) were optimized Each factor was run at levels (-1, and +1) A total of 19 experiments were conducted Yield of biomass and total Se levels in biomass were determined upon 40 days of cultivation The outputs (Y) of the model were biomass yield (g/L) and total Se level in biomass (µg/g) 2.2.4 Preparation of extracts Mushroom biomass was thoroughly extracted with 96% ethanol (EtOH) (a ratio of biomass powder : EtOH = 1:10, w/v) for 48h EtOH extract was obtained by evaporating the solvent using a vacuum evaporator The EtOH crude was fractionally extracted with different solvents in order of petroleum ether (PE), ethyl acetate (EtOAc), buthanol (Bu), and water (H2O) using liquid-liquid extraction technique Biomass residues were dried and then extracted with warm water at 65°C The water extract was filtered and concentrated followed by precipitating with 96% ethnol (extract: ethanol = 1:4, v/v) A polysaccharide crude (CPS) was obtained, dried at 50°C, and stored at 4°C Culture broth of C sinensis was concentrated and precipitated with cold 96% ethanol (broth:ethanol = 1:4, v/v) for 24h at 4°C An exopolysaccharide (EPS) crude was collected by centrifugation and washed times with 96% ethanol followed by drying and storing at 4°C For extraction of polysaccharide from biomass (IPS), biomass powder was extracted with 20mL of 0.4M NaOH at 60°C The aqueous extract was concentrated using a rotary evaporator at 60°C and precipitated with cold 96% ethanol (extract:ethanol = 1:4, v/v), for 24h at 4°C The IPS crude was redissolved with distilled water 15 Figure 3.3 (A) A comparison of biomass yields (g/L) and total Se levels between traditional (PP1) and adapted methods (PP2) upon 40 days of fermentation (B) The morphological characteristics of the fungus on PDA medium upon 14 days of incubation and on liquid medium (C) upon 40 days of fermentation The fungus was cultivated in Se-free medium (control) and in Se-supplementing medium according to the traditional method (PP1) and the adapted method (PP2) respectively Selenate salt was used at 25mgSe/L Concerning the accumulation of Se, the PP2 biomass showed a lower Se level when compared to the PP1 biomass, of 1355µgSe/g and 1645µgSe/g, respectively However, when fermenting in 1L of liquid medium, since PP2 method showed higher biomass yield, the total Se levels were not significantly different between PP2 and PP1 16 biomass of 23454.5 and 23882.9µgSe, respectively Collectively, the PP2 method enhanced the capability of biomass production by C sinensis on the Se-rich liquid medium HPLC-ICP/MS analyses illustrated that the PP2 biomass contained three types of Se forms including selanate (Se+6), selenomethionine (SE-MET), and selenocystine (SE-CYS2) Of those, Se was predominantly accumulated as selenate (991.9µgSe/g), followed by SE-MET (383.9µgSe/g) and SE-CYS2 (85.6µgSe/g) Meanwhile for PP1 biomass, SE-MET was predominant (645.6µgSe/g), followed by SE-CYS2 (93.5µgSe/g), selenite (84.2µgSe/g), and selenate (17.4µgSe/g) 3.2 Optimation of culture medium components and conditions for enhancing the biotransformation of Se in Cordyceps sinensis biomass From the screening data using Plackett – Burman modeling, we found that four medium components: saccharose, peptone, potatoes, and KH2PO4 significantly impacted the biomass production of C sinensis fungus Meanwhile, for the impact on the Se accumulation in biomass, the Plackett – Burman modeling showed no significant correlation between the surveyed components (i.e., potatoes, saccharose, peptone, yeast extract, KH2PO4, K2HPO4, and MgCl2) and the total Se levels in biomass (p>0.05) 3.2.1 Optimation of culture medium components In this work, to optimize the culture medium components, three components (saccharose, peptone, and KH2PO4) were used using the Box-Behnken surface response method Afterward, we predicted the optimal culture medium composition for the biomass production 17 of C sinensis used the DE software The parameters were verified as follows: saccharose 53.48g/L, peptone 11.17g/L, and KH2PO4 1.05g/L in which the model projected a biomass yield and total Se levels in biomass of 25.4g/L and 24343.9µg (approximately 960.3 µgSe/g biomass) To confirm the optimal parameters predicted by the Box – Behnken model, a trial was done Interestingly, the obtained results showed compatibility with the predicted outputs in which the biomass yields were 24.9g/L vs 25.4g/L, and the total Se levels were 1059µgSe/g vs 960.3 µgSe/g biomass In summary, for production of Se-enriched C sinensis biomass, the optimal culture medium components were 200g/L potatoes, 53.48g/L saccharose, 4g/L yeast extract, and 11,17g/L peptone, 1.05g/L KH2PO4, 0.5g/L K2HPO4, 0.1g/L MgCl2 3.2.2 Optimation of culture conditions For the production of Se-enriched C sinensis biomass, Doptimal model projected the optimal culture conditions as follows: temperature 20°C, white light, pH 6.1 The model predicted that biomass yield and total Se level in biomass were 24.8g/L and 1097µgSe/g iomass, respectively To confirm this, a trial was done on liquid medium As a result, the biomass yield reached 26.5g/L and the Se content was 1068µgSe/g biomass These results were compatible with the predicted outputs derived from the D-optimal model Nothwithstanding, these culture conditions did not significantly affect the accumulation of Se in biomass It is possible that a maximum tolerable uptake by C sinensis was between 1000 and 1200µgSe/g 18 3.3 Identification of Se-containing extracts in Se-enriched biomass Data showed that the extraction yields of the fractional extracts were different between Se-enriched biomass and Se-free control biomass All the extracts derived from the Se-enriched biomass contained Se at different levels Interestingly, a higher Se level was found in the highly polar extracts, especially in polysaccharide-rich extracts 3.4 Evaluation of bioactivities of Se-enriched Cordyceps sinensis biomass 3.4.1 In vitro antioxidant activities The overall ABTS+ free radical scavenging capacity was higher in the Se-rich extracts than in the control Se-free extracts Notably, the Se-rich EtOAc extract showed the highest activity with the lowest IC50 level of 2026 ± 23,45µg/mL In addition, the Se-rich EtOAc extract also had the ability to scavenging DPPH free radicals with the lowest IC50 value of 1200 ± 69.5µg/mL vs 1598 ± 17.5µg/mL of the Se-free EtOAc extract Likewise, the in vitro antioxidant property of all Se-rich extracts was observed in the reduction of ferric iron (Fe3+) to ferrous iron (Fe+2) Of those, the highest capacity was the Se-rich EtOAc extract with the highest OD value of 0.137) followed by the other Serich extracts such as EtOH extract (OD value = 0.120), H2O, BuOH, CPS, IPS, EPS, and PE extracts On the other hand, for Se-free extracts, the highest ferric reducing powder activity was the EtOH extract (OD value = 0.115) followed by H2O, EtOAc, BuOH, EPS, IPS, CPS, and PE extracts 19 In conclusion, we can conclude that the Se-rich EtOAc showed the highest in vitro antioxidant properties 3.4.2 Antioxidant activities through protection of DNA integrity Data indicated that all the Se-rich extracts were capable of effectively protecting the integrity of DNA at concentrations of 500 to 1500 µg/mL Remarkably, at 500µg/mL, the EtOAc, BuOH, and H2O extracts derived from the Se-enriched C sinensis biomass showed a high capacity of protection of DNA integrity when compared to the control By contrast, this capacity of extracts EtOH, IPS, PE, and CPS derived from the control Se-free biomass was low with higher DNA fragmentation (Figure 3.12) Figure 3.12 Protection of DNA integrity by extracts; (A,C) the activity is assessed on gel electrophoresis; (B,D) DNA integrity is showed as 3D bands; (+): undamaged DNA (with protectant); (-): damaged DNA (without protectant) Each extract was done at three concentrations of 1500µg/mL (left), 1000µg/mL(middle), and 500µg/mL (right) 20 3.4.3 In vitro xanthine oxidase inhibition activity Data indicated that the xanthine oxidase inhibitory activity of extracts derived from the Se-enriched C sinensis biomass was significantly higher than that of extracts derived from the control Sefree biomass For example, the Se-rich PE and BuOH extracts had the ability to inhibit this enzyme with IC50 values of 2558.49 ± 208.54 and 3017.41 ± 195.25µg/mL respectively Especially, the Se-rich EtOAc extract showed the highest inhibition capacity with the lower IC50 value of 1979.96 ± 225.73µg/mL Likewise, this property was observed in the Se-free EtOAc extract with IC50 = 1938.13 ± 165.40µg/mL 3.4.5 In vitro anti-inflammatory activity Data on albumin denaturation inhibition demonstrated that the in vitro anti-inflammatory activity was higher in extracts derived from the Se-enriched biomass than in extracts from the control Se-free biomass, suggesting that the accumulation of Se in biomass might result in an increase in this biological activity Evidently, the albumin denaturation inhibition capacity of the Se-rich EtOH extrac was 4.4 times higher that of the Se-free EtOH Similar trends were found in PE and EtOAc extracts between the Se-enriched and Se-free biomass In this regard, the highest inhibition capacity was observed in the Se-rich PE extract with the lowest IC50 value of 64,19 ± 0,22 µg/mL, followed by the Se-rich EtOH (IC50=195.58 ± 7.89 µg/mL), EtOAc (IC50=469.27 ± 16.57 µg/mL), CPS (IC50=1259.28 ± 46.20 µg/mL), and IPS extracts (IC50=1280.33 ± 135.22 µg/mL) Notwithstanding, the Se-rich remaining extracts such as BuOH and 21 water showed low inhibition capacity with the undetermined IC50 values in a range of to 1500µg/mL 3.4.6 In vitro α–glucosidase inhibition activity Most of the extracts showed low in vitro α–glucosidase inhibition activity In a range of to 5000µg/mL, none of the IC50 values was determined for all these extracts Notwithstanding, the inhibition percentage slightly increased when applied with the Se-rich extracts at higher concentrations but not exceeding 50% At 5000µg/mL, the inhibition rate of the Se-rich EtOAc was 23.76% vs 12.8% of the control Se-free EtOAc extract 3.4.7 Proliferation inhibition of cancer cell lines The cell toxicity assays showed that EtOH, PE, and EtOAc extracts from both Se-enriched and Se-free C sinensis biomass were capable of causing cytotoxicity to the MCF7 breast cancer cell line (Figure 3.14) Especially, the highest toxicity against MCF7 cells was observed in the Se-rich EtOAc with IC50 = 3.82 ± 0.12 g/mL that was 7.59 times higher than that of the Se-free EtOAc extract Similarly, the Se-rich EtOH extract showed a higher toxicity against the proliferation of MCF7 cells compared to the control Se-free EtOH extract (p < 0.05) All these results demonstrated that the presence of Se might increase the cytotoxicity of C sinensis biomass against the proliferation of MCF7 cells (Figure 3.14) 22 Figure 3.14 Cytotoxic activity of extracts against MCF7 breast cancer cells The lower the IC50 value presents the higher toxicity IC50 value is a concentration of each extract at which the inhibition rate of MCF7 cell proliferation was 50% Different letters above each column point out a significant difference between extracts at a significant value of α = 0.05 3.5 A proposal of a cultivation process for production of Seenriched Cordyceps sinensis biomass 3.5.1 Evaluation of acute toxicity on mice To evaluate the acute toxicity of the Se-enriched biomass on mice, we first determined that 2500 mg/kg was the highest tolerable dose via oral administration using a medical needle The results showed that there was no deaths and no abnormal symptoms upon 1h 23 and 24h of oral administration At days and 14 days after the oral administration, the animal health was still good without abnormal symptoms 3.5.2 Evaluation of food safety requirements In addition to the in vivo acute toxicity assay, we evaluated food safety requirements mandated by the Vietnamese Ministry of Health (No 46/2007/QD-BYT, QCVN 8-2:2011/BYT, and 46/2007/QD-BYT) All the criteria regarding heavy metals, mycotoxins, and bacterial contamination were met Lastly, we proposed a technological process for the production of Se-enriched C sinensis biomass (Figure 3.16) 24 Figure 3.16 A technological process for the production of Seenriched C sinensis mycelial biomass 25 CONCLUSION Based on the obtained findings, following conclusions can be drawn: Among the three types of Se salts: selenite, selenate and selenourea, selenate at a concentration of 25 mgSe/L was suitable for the production of Se-enriched C sinensis biomass The optimal composition and conditions for the cultivation of Se-riched C sinensis mycelial biomass were determined The optimal culture medium components consisted of 200g/L potatoes, 53.48g/L saccharose, yeast extract 4g/L, peptone 11.17g/L, KH2PO4 1.05g/L, K2HPO4 0.5g/L, MgCl2 0.1g/L, and 25mgSe/L selenate The optimal conditions for the fermentation process were at 20°C, under white light (12h light: 12h darkness), and pH 6.1 Consequently, the biomass yield reached 26.45 g/L and the total Se content in biomass was about 1068 µgSe/g Several organic Se forms converted from selenate have been discovered in biomass such as 0.6µgSe/g selenomethylcysteine, 383.9µgSe/g selenomethionine, 3.2µgSe/g selenocystamine, and 85.6µgSe/g selenocystine Additionally, Se was detected in polysaccharide and aqueous extracts suggesting that selenate could be transformed into seleno-polysaccharide by C sinensis Se-enriched C sinensis mushroom biomass showed a higher bioactivity when compared to the Se-free control biomass Especially, the ethyl acetate extract derived from the Se-riched biomass exhibited an increase in the antioxidant properties and the cytotoxic activity against two cancer cell lines MCF7 and Jurkat 26 A technological process for the production of Se-enriched C sinensis mycelial biomass at 10L pilot scale was proposed with a biomass yield of 26.45g/L and total Se levels of 1068 µgSe/g biomass Importantly, the Se-enriched biomass was safely evaluated with the food safety standards regulated by the Vietnamese Ministry of Health Lastly, further studies are needed such as (1) evaluation of in vivo anti-tumor, anti-inflammatory, and anti-oxidation activities of the Se-enriched biomass and (2) identification of metabolic profiles in the ethyl acetate extract of the Se-riched C sinensis biomass towards application 27 NEW FINDINGS OF THE THESIS This current work is the first to establish a production process of selenium-enriched Cordyceps sinensis biomass in Vietnam, aiming to not only enhance its medicinal values towards application, but also create an organic selenium-supplying resource with the high bioavailability Several seleno-proteins were discovered in the fungal biomass, such as selenomethylcysteine, selenomethionine, selenocystamine, and selenocystine These findings could be valuable hints to further elucidate the biotransformation pathway of selenium from inorganic to organic forms in fermentation conditions of Cordyceps sinensis fungus This study uncovered the impacts of nutritional parameters and cultivation conditions, especially temperature (20°C), pH 6.1, and white light on the growth and selenium accumulation of Cordyceps sinensis fungus The findings also demonstrated that the selenium-enriched biomass showed an increase in antioxidant activity Most importantly, we found that the ethyl acetate extract from the biomass was a promising extract in the proliferation inhibition of human breast cancer cell lines 28 LIST OF WORKS HAS BEEN PUBLISHED Trần Minh Trang, Phạm Tiến Dũng, Lê Quốc Phong, Đinh Minh Hiệp (2016), Nghiên cứu khả hấp thu selenium nấm Ophiocordyceps sinensis nuôi cấy lỏng, Tạp chí Phát triển Khoa học Cơng nghệ (ISSN 1859-0128), Đại học Quốc gia TP.HCM, tập 19, số T6, trang 53-61 Lê Quốc Phong, Nguyễn Tài Hoàng, Phạm Thị Mỹ Ninh, Trần Minh Trang, Đinh Minh Hiệp, Ngô Kế Sương (2017), Khảo sát ảnh hưởng thành phần môi trường dinh dưỡng đến sản xuất sinh khối nấm Ophiocordyceps sinensis có bổ sung selen, Tạp chí Cơng nghệ Sinh học (ISSN 1811-4989), Viện Hàn lâm Khoa học Công nghệ Việt Nam, tập 15, số 3A, trang 281-288 Lê Quốc Phong, Nguyễn Hoàng Đăng Khoa, Nguyễn Tài Hồng, Đinh Minh Hiệp, Ngơ Kế Sương (2020), Khảo sát thành phần hóa học hoạt tính úc chế xanthine oxidase nấm Ophiocordyceps sinensis giàu selen, Hội thảo khoa học Công nghệ Sinh học ứng dụng (lần 2), Khoa Công nghệ, Trường Đại học Văn Lang, tháng 7/2020, NXB Nông nghiệp trang 201-208 (ISBN 978604-60-3195-6) Lê Quốc Phong, Nguyễn Hoàng Đăng Khoa, Nguyễn Tài Hoàng, Đinh Minh Hiệp, Ngô Kế Sương (2020), Khảo sát hoạt tính kháng oxy hóa bảo vệ DNA in vitro nấm Ophiocordyceps sinensis giàu selen, Hội nghị Công nghệ Sinh học toàn quốc năm 2020, Thành phố Huế, tháng 10/2020, chủ đề “Công nghệ sinh học: Từ 29 nghiên cứu đến ứng dụng phục vụ công nghiệp hóa, đại hóa đất nước”; Kỷ yếu Hội nghị, trang 268-273, NXB Đại học Huế (ISBN 978-604-974-562-1) Lê Quốc Phong, Nguyễn Hoàng Đăng Khoa, Đặng Tú Quyên, Nguyễn Tài Hồng, Đinh Minh Hiệp, Ngơ Kế Sương (2020), Đánh giá hoạt tính kháng viêm, kháng khuẩn ức chế enzyme αglucosidase in vitro nấm Ophiocordyceps sinensis giàu selen, Tạp chí Khoa học Cơng nghệ Việt Nam (ISSN 1859-4794), Bộ Khoa học Công nghệ, series B, tập 62, số 8, trang 19-24 Le, Q.P., Nguyen, T.H., Huynh, V.P., Nguyen, T.T.M., Tran, M.T., Dinh, M.H., Ngo, K.S (2020), Optimization of culture parameters of selenium-enriched Ophiocordyceps sinensis biomass by response surface methodology, International Journal of Agricultural Technology (ISSN 2630-0192), 16(4):855-866 ... (2020), Khảo sát thành phần hóa học hoạt tính úc chế xanthine oxidase nấm Ophiocordyceps sinensis giàu selen, Hội thảo khoa học Công nghệ Sinh học ứng dụng (lần 2), Khoa Công nghệ, Trường Đại học. .. (2020), Khảo sát hoạt tính kháng oxy hóa bảo vệ DNA in vitro nấm Ophiocordyceps sinensis giàu selen, Hội nghị Cơng nghệ Sinh học tồn quốc năm 2020, Thành phố Huế, tháng 10/2020, chủ đề “Công nghệ sinh. .. (2017), Khảo sát ảnh hưởng thành phần môi trường dinh dưỡng đến sản xuất sinh khối nấm Ophiocordyceps sinensis có bổ sung selen, Tạp chí Cơng nghệ Sinh học (ISSN 1811-4989), Viện Hàn lâm Khoa học