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BỘ GIÁO DỤC VÀ ĐÀO TẠO MINSTRY OF EDUCATION AND TRAINING CAN THO UNIVERSITY SUMARY OF THE Ph D THESIS Major Biotechnology Code 62420201 NGUYEN THI NGOC NHI STUDY ON THE TECHNOLOGICAL PROCESS FOR BIOMA[.]

MINSTRY OF EDUCATION AND TRAINING CAN THO UNIVERSITY SUMARY OF THE Ph.D THESIS Major: Biotechnology Code: 62420201 NGUYEN THI NGOC NHI STUDY ON THE TECHNOLOGICAL PROCESS FOR BIOMASS PRODUCTION OF TERMITE MUSHROOMS (Termitomyces sp.) Can Tho, 2022 THIS STUDY WAS ACHIEVED AT CAN THO UNIVERSITY Main scientific supervisor: Assoc Prof Tran Nhan Dung The thesis was defended under the supervision of the grassroots doctoral dissertation grading council At The Room of Thesis Defend (Room 3), The Second Floor, Operation Building, Campus Can Tho University, on 14:00, December 18th, 2021 Reviewer 1:Assoc Prof Phan Thi Phuong Trang Reviewer 2: Assoc Prof Ngo Thi Phuong Dung Confirmation of review by the Chairman of the Board Assoc Prof Nguyen Van Thnh The thesis could be found in: - Learning Resource Center of Can Tho University - National Library of Vietnam PUBLICATIONS RELATED WITH THE PhD THESIS Nguyen Thi Ngoc Nhi and Tran Nhan Dung, 2016 Isolation of termite fungus Termitomyces sp Scientific report on Biology research and teaching in Vietnam, The 2nd National Science Conference, Da Nang, VNU Publishing House, 601-606 Nguyen Thi Ngoc Nhi and Tran Nhan Dung, 2016, Isolation of the termite fungus Termitomyces clypeatus Can Tho University Journal of Science, No 46: 17-22 Nguyen Thi Ngoc Nhi and Tran Nhan Dung, 2018 Determining the suitable culture medium and optimizing the composition of the medium for termite fungus biomass (Termitomyces clypeatus) Journal of Agriculture and Rural Development, No 350: 62-67 Duong Thi Kim Chi, Nguyen Thi Ngoc Nhi, Nguyen The Bao, Le Mau Long, Pham Cong Xuyen, 2018 Using machine learning to species identification of termite fungi The 11th Proceeding of the National Science and Technology Conference FAIR - Basic research and application of information technology Science and Technics Publishing House Nguyen Thi Ngoc Nhi and Tran Nhan Dung (2021) Biomass production of fungus Termitomyces clypeatus in a stirredtank bioreactor Academia Journal of Biology, 43(3), 9–17 https://doi.org/10.15625/2615-9023/15950 Nhi, Nguyen Thi Ngoc; Khang, Do Tan and Dung, Tran Nhan (2022) Termitomyces mushroom extracts and its biological activities Food Science and Technology Food Science and Technology 42, e125921, 2022 DOI: https://doi.org/10.1590/fst.125921 CHAPTER 1: INTRODUCTION 1.1 Rationale of the study Giant mushrooms, including edible and medicinal mushrooms, had been associated with human life for a long time Mushrooms played a significant role as a food source for processing daily dishes as well as medicinal herbs (Reishi mushrooms) for the production of functional foods and drug treatment in curing diseases Several mushrooms were determined to be affluent in essential elements, in particularly K, P, Ca, Mg, Mn, and Se; , and especially vitamins D, B, and fiber (Manzi et al., 1999; Sanmee et al., 2003; Kurtzman, 2005) Mushrooms provided quality protein containing all the essential amino acids needed for humans (Mattila et al., 2002; Colak et al., 2009) Mushrooms enclosed a small amount of cholesterol opposed to a large amount of unsaturated fatty acids and digestible carbohydrates (Breene, 1990), which possessed required properties of an ideal food supporting for obesity and diabetes prevention Some mushrooms were discovered as useful therapeutic foods in preventing diseases such as high blood pressure, hypercholesterolemia, atherosclerosis and cancer (Tidke et al., 2006) Termitomyces fungi were naturally endowed with high nutritional value It contains a full range of non-replaceable amino acids and minerals in high concentrations (Masamba et al., 2010; Davidson et al., 2012; Nakalembe et al., 2013; Nakalembe et al., 2015) and has a very delicious taste (Pegler Piearce, 1980) Some species of termite fungi were expended for medicinal purposes, such as T robustus, T striatus thanks for anti-aging effects (Adewusi et al., 1993); T heimii possessed fatty acid ergosterol, linoleic strengthens the immune system, prevents high blood pressure (Abd Malek et al., 2012) In addition, T heimii was considered as a tonic in wound healing and blood clotting (Chrawati et al., 2014); The nutrient-dense T microcarpus contains 40% of protein and 55% of dry weight carbohydrate, (Chra et al., 2009), especially it has β-D-glucan that had the ability to inhibit cancer cells (Villares et al ., 2012); T eurrhizus supported the treatment of arthritis, diarrhea and high blood pressure (Sachan et al., 2013); T clypeatus illustrated the resistant to Pseudomonas aeruginosa (Subrata et al., 2012); supporting for chickenpox treatment (Dutta et al., 2014); have high antioxidant activity (Mau et al., 2004, Pattanayak et al., 2015) Termitomyces clypeatus also contains AkP, which may be an effective biomolecule for killing cancer cells (Majumder et al., 2016) In India, T heimii and T microcarpus were used in the alleviation of fever, colds, and fungal infections (Paulsamy, 2016) In Cameroon, T titanicusis was used to treat gastric complications (Rosemary et al., 2017) In Tanzania, T microcarpus s was used to strengthen the immune system; T.titanicus, T letestui, T eurrhizus and T aurantiacus were considered to be supportive tonics in the treatment of gastrointestinal diseases such as colic, constipation, pain and stomach ulcers (Tibuhwa, 2012) Recently, the unfavorable weather together with environmental pollution caused by agricultural production had an effect on the lower growth of termites than in the past Without timely solution,we will no longer have the opportunity to witness and enjoy termite mushrooms someday Scientifically we can grow all living things (from the cellular level to the living organism) However, the termite fungus was a unique species with various physiological characteristics and nutritional mechanisms that differed from other fungi, so far, humans have not been able to grow it According to the research results of Ulziijargal and Mau (2011), the difference in the content of the essential components involving fruiting bodies and mycelia among fungal species of genera namelyAgaricus, Auricularia, Cordyceps, Trametes, Flammulina, Ganoderma, Lentinus, Pleurotus, and so on showed close resemblance Therefore, the study about the production process of termite mycelium applying liquid culture method that guide a premise for future studies on culturing other species Hence, the topic “Study on the technological process for biomass production of termite mushrooms (Termitomyces sp.)" was proposed 1.2 Objectives and Contents of the study 1.2.1 Objectives of the study (1) Isolation and identification of some naturally occurring Termitomyces sp strains (2) Investigation major components of the medium level and for the propagation of termite fungi in order to select effective environment for biomass production on a large scale (3) Producing Termitomyces sp biomass in Bioreactor 60 liters (4) Analysis the nutritional composition and investigation investigation activity of the termite mycelium (5) Testing the toxicity of termite mushroom biomass extract 1.2.2 Contents of the study The study was conducted with the following main contents: (1) Isolation, identification, and preservation of termite fungi (2) Surveying the liquid medium level and for the growth of Termitomyces sp (3) Research on the technological process of producing termite mycelium in Bioreactor 60 liters (4) Analysis of nutritional composition and investigation of biological activity of termite mycelium (5) Production and experiment of cytotoxicity, acute toxicity, and semi-permanent toxicity of the product 1.3 Object and scope of the study The object of the study: Pure Termitomyces sp strains were isolated from a species of termite fungus naturally occurred in Binh Duong, Vietnam; conditions for a submersion culture of termite mushroom mycelium Scope of the study: Source of Termitomyces sp samples was collected in Binh Duong provincce; production process of termite mushroom biomass in Bioreactor 60 liters 1.4 Novel contributions, scientific and practical significance The study had contributions in the field of research on termite fungi research in Vietnam, which which was obviously indicated through the results of this thesis: - Seven strains of Termitomyces sp were isolated and sequenced, of which two species were identified with scientific names: Termitomyces clypeatus and Termitomyces microcapus - Liquid medium levels and were studied in order to select effective conditions for a submersion culture of Termitomyces clypeatus - The technological process of producing termite mycelium in Bioreactor 60 liters were recommended for detecting the growth curve of Termitomyces clypeatus - The nutritional components were analyzed as well as the antibacterial and antioxidant activities of Termitomyces clypeatus mycelium were evalusted Simultaneously, Termitomyces biomas was produced for testing biological activity, cytotoxicity, acute toxicity, and semi-permanent toxicity of the extract In addition, the thesis results have opened the direction of research on the propagation of fungi by submersion culture method facilitates saving time, effort and providing a large number of highquality as well as uniform mushroom varieties The result has devoted practical significance in the biomass manufacturing of medicinal mushrooms, especially those precious mushrooms which fruiting bodies haven’t been growth Thus, bioreactor systems are a viable solution CHAPTER 2: LITERATURE REVIEWS 2.1 Origin and taxonomic position of termite fungi Termitomyces has an English name is Termite mushroom According to Trinh Tam Kiet (2011, 2012, 2013) and Le Xuan Tham (2013), the taxonomy of termite fungi is as follows: 2.2 Research situation on termite fungi in the world and in Vietnam 2.2.1 Research situation on termite fungi in the world Numerous research works on the role of termite fungi in humans had occurred in the world (Leung et al., 1997; Weis, 1999; Ashida et al., 2003; Mau et al., 2004; Lu et al., 2008; Giri et al., 2012; Chatterjee et al., 2013; Acharya et al., 2014; Pattanayak et al., 2015; Majumder et al., 2016…), symbiotic relationship between Termitomyces and termites (Noirot, 1958; Martin, 1978; Roul, 1991; Nobre Aanen, 2012), species composition (Otieno, 1968; Wei et al., 2006; Tibuhwa et al., 2010; Sridhar, 2013 ), genetic characteristics, distribution of termite fungi (Aanen, 2006; Aanen et al., 2007).… In 1982, the termite fungus T clypeatus was discovered in Malaysia in symbiosis with at least species of termites belonging to the genus Odontoterm: O dontotermes, O malaccensis and O sp., and O sarawakensis was located in Thailand (Pataragetvit, 1982) However, research on species composition was eliminated, mainly in some Asian and African countries Some publications on species composition include: species of termite fungi were described in South Africa (Eicker, 1990), in Belgium, seven species were described (Heim., 1951) In Thailand, many publications on species composition had been published, , especially two new species had been participated including Termitomyces floccose and Termitomyces upsilocystidiatus (Tang et al., 2020) In Malaysia, a new species of termite fungus had also been announced, Termitomyces gilvus (Seelan et al., 2020) According to published studies regarding the submersion culture for Termitomyces, each species required different liquid scale medium, (Hu et al., 2001; Zhang et al., 2002) ; Chen et al., 2002; Bose et al., 2007; Lu et al., 2008; Chatterjee et al., 2010; Ramrakhiani et al., 2011) In which, Lu et al (2008),cultured and collected the biomass of the termite fungus T albuminous in order to test the analgesic and anti-inflammatory effects in his research Furthermore, published by Zhao et al (2017) presented that the obtained extracellular polysaccharides after reproducing T albuminous in submersion culture help reducing histopathological damage in rat liver This is promising in the development of making drugs for the treatment of atherosclerosis while most of the secondary products have been acquired at the laboratory level There has not been any work on the submersionculture of termite fungi on a production scale for collecting filamentous biomass 2.2.1 Research situation on termite fungi in Vietnam In 2004, Kieu Huu Anh and his colleagues published results of isolation of termite fungi from spores This was the initial result showing the fungal colony image of a species of termite fungus Until 2013, Vietnam has recorded 10 species appertaining to Termitomyces, including species published in the Vietnam Big Mushroom Collection (Trinh Tam Kiet, 2013) together with species of Termitomyces mameliformis in the list of mushroom species in the Cat Tien national park (Le Xuan Tham and Pham Ngoc Duong, 2013) However, more data on Termitomyces are indispensable in order to construct identification keys in taxonomy for the genus Termitomyces in Vietnam instread of basing on few morphological characteristics Some large mushroom species have been studied for their nutritional composition as well as cultured so as to provide food for humans such as shiitake, abalone, shiitake, and so on while Termitomyces have not been published extensively There are no works relating to submersion culture of Termitomyces fungi in an attempt to create biomass CHAPTER 3: METHODOLOGY 3.1 Isolation and identification of termite fungi The studied mushroom varieties were identified based on morphological features combined with molecular biology through the identification keys as described by Mossebo et al (2009) This was modifsied and completed from the of the identification keys of Heim (1977) and Pegler (1977), depended on the published description of the shape, color, size of mushroom caps, mushroom stalks, mushroom body, spore size of many authors in the world as well as in Vietnam including Otieno (1968), Botha and Eicker, (1991), Tibuhwa (2012), Karun Sridhar (2013), Wong (2013), Trinh Tam Kiet (2011, 2013), Le Xuan Tham and Pham Ngoc Duong (2013) 3.2 Fungus reproduction level 1, level fungi and producing in Bioreactor Reproduction level 1: - Seed fungi activation: strains from the culture medium were activated in PDB medium The flasks were shaken at 150 rpm, temperature 280C, pH 5, culture time was 10 days - Propagation level 1: Seed rate 5%, shaking speed 150 rpm, temperature 280C, incubation time is 15 days Reproduction level 2: 2000mL of MTC1 media formulations were weighed, accurately measured, filtered, dispensed 2000 mL into a 5000 mL flask, pH was adjusted to 5, aeration mode 0.4 v/v/m, seed rate 10% Production of termite mycelium in Bioreactor 60 liters: The conditions for the growth and development of termite mycelium were prepared carefully Using equipment with automatic stirring system, aseptic gas supply system, temperature control, pH control to make sure that the optimal media was strictly controlled biomass is not degraded Both MF163445 and MF163446 samples have a high bootstrap index in a group, but the colony morphology has numerous differences, thus there are not enough data to confirm they are the same The remaining strains may be different species of termite fungi Figure 4.17 The phylogenetic tree was analyzed by the Maximum Likehood method in Mega X based on the sequences of the termite fungal strains, the bootstrap index was repeated 1000 times 4.2 The results of studying on the propagation environment of T clypeatus 4.2.1 Influence of medium and culture time on the accumulation of termite mycelium Experimental results revealed that termite fungi can progress on various culture media The mycelium dry weight after culturing 10 to 25 days from the inoculation day ranged about 2.05 g in the treatment applying MT1 medium at the date of 20 to 4.45 g utilizing MT3 medium at the culture date of 25 the mycelium of termite fungi developed slowly during the incubation period from 10 to 15 days This was the stage for collecting fungal biomass On the other hand, the mycelium biomass 12 grew gradually from the 25th day of culture and was not statistically significant Therefore, maintaining the culture time consumed abundantly effort, energy, and time as well as reducing product quality in this stage Research by Yin (2002) on the submersion culture of T eurrhizus indicated that the mycelium of T clypeatus grew gradually for a longer time than that of T eurrhizus The media granting the highest dry weight of mycelium was MT3 during 20 and 25 days of culture ( weighing 4.43 g and 4.46 g) as well as MT4 medium with the same duration ( weighing 4.07 g and 4.03 g) This result represented that the research media was efficient for the production of termite mycelium However, MT3 medium at 20 days of culture is optimal for commercial production of T clypeatus termite mycelium Figure 4.18 Effect of culture media and incubation time on dry weight of termite mycelium T clypeatus Note: The figures presented in each column arethe average of replicates Values that not have the same letters are statistically different at 5% of significance 13 4.2.2 Optimizing the concentration of major substances having an effect on the biomass accumulation of termite mycelium three factors including glucose concentration (X1), peptone concentration (X2), combined with mineral concentration KH2PO4 (X3) at different levels were set up in an experiment to evaluate their general effects on the biomass accumulation of termite mycelium Due to these selected values, the Box-Behnken response surface method was applied in the experimental layout The experimental results exposed that the lowest yield of fibrous biomass was 4.22 g/L and the highest was 5.85 g/L Biomass production: Y (g/L) = 5,82 + 0,4*X1 + 0,15*X2 + 0,19*X3 - 0,85*X12 0,098*X22 + 0,19X1*X2 + 0,23X2*X3 The R-Sq coefficient of the regression equation was 96.7%, proving that the equation had highly persuade in compare with the actual data the dry weight of the termite fungus biomass corresponding to the values of the experimental factors can be predicted from the regression equation with a confidence level of over 95% Minitab’s Response optimizer function selected three optimal values of the factors including glucose concentration, peptone concentration, and mineral concentration, respectively, 69.9 g/L; 5.96 g/L, and g/L along with the estimated dry biomass of termite mycelium of 6.128 g/L These values, together with the dry biomass of termite mycelium, are predicted to belong with the optimal range of the response surfaces presented above The actual dry biomass value after the experimental arrangement was 5.923 ± 0.124 g/L, lower than the optimal values as above This result was consistent with studies such as the submersion culture of Ganoderma lucidum (CAU5501) by Yuan et al (2012) using the optimal medium for EPS yield containing 70 g/L glucose, and the ratio C/N, 2.5 g/L KH2PO4, 0.75 g/L MgSO4.7H2O, 14