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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 2

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

1.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 NationalScience Conference, Da Nang, VNU Publishing House, 601-606

2.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.

3.Nguyen Thi Ngoc Nhi and Tran Nhan Dung, 2018.

Determining the suitable culture medium and optimizing thecomposition of the medium for termite fungus biomass

(Termitomyces clypeatus) Journal of Agriculture and Rural

Development, No 350: 62-67.

4 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 11 th

Proceeding of the National Science and Technology Conference.

FAIR - Basic research and application of information technology.Science and Technics Publishing House

5.Nguyen Thi Ngoc Nhi and Tran Nhan Dung (2021).

Biomass production of fungus Termitomyces clypeatus in a tank bioreactor Academia Journal of Biology, 43(3), 9–17.

stirred-https://doi.org/10.15625/2615-9023/15950

6.Nhi, Nguyen Thi Ngoc; Khang, Do Tan and

Dung, Tran Nhan (2022) Termitomyces mushroom

extracts and its biological activities Food Science and TechnologyFood Science and Technology 42, e125921, 2022.

DOI: https://doi.org/10.1590/fst.125921

CHAPTER 1:INTRODUCTION 1.1 Rationale ofthestudy

Giant mushrooms, including edible and medicinal mushrooms,had been associated with human life for a long time Mushroomsplayed a significant role as a food source for processing daily dishes

as well as medicinal herbs (Reishi mushrooms) for the production offunctional foods and drug treatment in curing diseases Severalmushrooms were determined to be affluent in essential elements, inparticularly 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 digestiblecarbohydrates (Breene, 1990), which possessed required properties

of an ideal food supporting for obesity and diabetes prevention.Some mushrooms were discovered as useful therapeutic foods inpreventing diseases such as high blood pressure,

hypercholesterolemia, atherosclerosis and cancer (Tidke et al., 2006).

Termitomyces fungi were naturally endowed with highnutritional 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 medicinalpurposes, 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 highblood pressure (Abd Malek et al., 2012) In addition, T heimii wasconsidered 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 ascolic, constipation, pain and stomach ulcers (Tibuhwa, 2012)

Recently, the unfavorable weather together with environmentalpollution caused by agricultural production had an effect on thelower growth of termites than in the past Without timelysolution,we will no longer have the opportunity to witness and enjoytermite mushrooms someday Scientifically we can grow all livingthings (from the cellular level to the living organism) However, thetermite fungus was a unique species with various physiologicalcharacteristics and nutritional mechanisms that differed from otherfungi, 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 involvingfruiting bodies and mycelia among fungal species of generanamelyAgaricus, Auricularia, Cordyceps, Trametes, Flammulina,Ganoderma, Lentinus, Pleurotus, and so on showed closeresemblance Therefore, the study about the production process oftermite mycelium applying liquid culture method that guide apremise 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 1and 2 for the propagation of termite fungi in order to select effectiveenvironment for biomass production on a large scale

(3) Producing Termitomyces sp biomass in Bioreactor 60

liters

(4) Analysis the nutritional composition and investigationinvestigation 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 1 and 2 for the

1.3 Object and scopeof thestudy

The object of the study: Pure Termitomyces sp strains were

isolated from a species of termite fungus naturally occurred in BinhDuong, Vietnam; conditions for a submersion culture of termitemushroom mycelium

Scope of the study: Source of Termitomyces sp samples

was collected in Binh Duong provincce; production process oftermite mushroom biomass in Bioreactor 60 liters

1.4 Novel contributions, scientific and practical significance

The study had contributions in the field of research ontermite fungi research in Vietnam, which which was obviouslyindicated 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 1 and 2 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 testingbiological activity, cytotoxicity, acute toxicity, and semi-permanenttoxicity of the extract

In addition, the thesis results have opened the direction ofresearch on the propagation of fungi by submersion culture methodfacilitates saving time, effort and providing a large number of high-quality as well as uniform mushroom varieties The result hasdevoted practical significance in the biomass manufacturing ofmedicinal mushrooms, especially those precious mushrooms whichfruiting bodies haven’t been growth Thus, bioreactor systems are aviable solution

CHAPTER 2: LITERATUREREVIEWS

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 insymbiosis with at least 3 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 onspecies composition include: 7 species of termite fungi weredescribed in South Africa (Eicker, 1990), in Belgium, seven specieswere described (Heim., 1951) In Thailand, many publications onspecies 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 submersionculture 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 histopathologicaldamage in rat liver This is promising in the development of makingdrugs for the treatment of atherosclerosis while most of thesecondary products have been acquired at the laboratory level Therehas 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 resultshowing the fungal colony image of a species of termite fungus.Until 2013, Vietnam has recorded 10 species appertaining toTermitomyces, including 9 species published in the Vietnam BigMushroom Collection (Trinh Tam Kiet, 2013) together with 1species of Termitomyces mameliformis in the list of mushroomspecies in the Cat Tien national park (Le Xuan Tham and PhamNgoc Duong, 2013) However, more data on Termitomyces areindispensable in order to construct identification keys in taxonomyfor the genus Termitomyces in Vietnam instread of basing on fewmorphological characteristics Some large mushroom species havebeen 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 extensivelyThere are no works relating to submersion culture of Termitomycesfungi in an attempt to create biomass

CHAPTER 3: METHODOLOGY

3.1 Isolation and identification of termite fungi

The studied mushroom varieties were identified based onmorphological features combined with molecular biology throughthe identification keys as described by Mossebo et al (2009) Thiswas modifsied and completed from the of the identification keys ofHeim (1977) and Pegler (1977), depended on the publisheddescription of the shape, color, size of mushroom caps, mushroomstalks, mushroom body, spore size of many authors in the world aswell as in Vietnam including Otieno (1968), Botha and Eicker,(1991), Tibuhwa (2012), Karun Sridhar (2013), Wong (2013), TrinhTam Kiet (2011, 2013), Le Xuan Tham and Pham Ngoc Duong(2013)

3.2 Fungus reproduction level 1, level 2 fungi and producing in Bioreactor

Reproduction level 1:

- Seed fungi activation: strains from the culture medium wereactivated 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 myceliumwere prepared carefully Using equipment with automatic stirringsystem, aseptic gas supply system, temperature control, pH control tomake sure that the optimal media was strictly controlled

Figure 3.1 Overall drawing of Bioreactor system 60 liters

3.3 Methods of analyzing the nutritional composition and testing active biological substances of termite mycelium

The nutritional composition was analyzed using high-pressureliquid chromatography (HPLC) The antioxidant activity wasinvestigated using DPPH (1,1-diphenyl-2-picrylhydrazyl) (Molyneux,2004; Mau et al., 2004, Mau et al., 2005; Tabart et al., 2009) Theantibacterial activity was estimated by the agar perforation method(Palaksha et al., 2010) Besides, The antibacterial activity of theextract was quantified by the MIC (Minimum Inhibit Concentration)method (broth dilution) on a 96-well plate (CLSI, 2010)

3.4 Toxicity testing of termite mushroom biomass extract

Method of investigation of cytotoxicity (MTT): Cytotoxicitywas determined by colorimetric method MTT (3-4,5-dimethylthiozol-2-yl)-2,5-diphenyltetrazolium bromide) (Brescia etal., 2009)

While the acute toxicity and semi-permanent toxicity wereevaluated according to the Lichtfield - Wilcoxon method (Do TrungDam, 2014)

CHAPTER4: RESULTS AND DISCUSSION

4.1 Isolation and identification of termite fungi

4.1.1 Isolation and identification of termite fungus T.

clypeatus

Characteristics: Specimens of termites collected in two

areas had similar morphological characteristics The mushroom caphad a diameter of 5-10 cm with conical shape and gray-brown color.The cap edge was light brown and bent inward while cracked edgeforming notches The mushroom blade was folded, thick, separated,and had white color The mushroom cap was usually gay-brownafter emerging from the ground, the larger it was, the lighter thiscolor became The mushroom cap was pointed The fruiting bodywithout the cap had white colors as well as the sweet taste ofchicken The average length of the mushroom body wasapproximately 8 to 16 cm, with Pseudorhiza enlarged near theground (Figure 4.1) the spores of T clypeatus observing under theoptical microscope at 400X magnification exposed the spherical orslightly oval form and had a uniform in size, about 8x10 µm, with

an opaque gray color

Figure 4.1 The shape of the T clypeatus

(a top view of mushroom cap., b: horizontal view of mushroombody and cap, c: underside - side of mushroom cap)

The growth of termite fungal tissue was monitored on fivemedia MT1, MT2, MT3, MT4, MT5 showed that MT5 medium was

a

morphology, thick folds together with sugars The diameter of theinitial tissue after culturing 20 days was 4.5 cm, while that tissue onMT4 medium was 3.5 cm and 2.5 cm on MT1 medium Thecolonies In both media MT1, MT4 colonies after 20 days of culture

at the same conditions possessed the rude, rough, thin, powderywhite surface (Figure 4.3) T clypeatus did not grow on both MT2,MT3 media; moreover, the explants were mushy, brown, possiblybecause of the presence a carbon source of CMC or soluble starch inthe nutrient medium that was difficult for termite fungi to assimilate This may also be related to the activity of amylase and cellulaseenzyme in termites The activities of these enzymes in termitesfungi was fragile, thus it was complicated for termites to expand

on media containing carbon sources of macromolecules such ascellulose or starch This was also consistent with Zeleke et al.’s(2013) results about termite fungal enzyme activity on differentmedia

The obtained results expressed that the growth of the myceliumdepended almost on the carbon source present in the medium Inparticular, T clypeatus required approximately 12-15 days to adaptand enlarge on the isolation medium in the laboratory

Figure 4.3 T clypeatus colony morphology after 20 days

The identification results based on DNA sequencing:Mushroom samples isolated in 2 regions were sequenced with DNA.The PCR products reached high quality manifesting a band-specific to the ITS1, 5.8S, ITS2, 28S rDNA fragments of termite

fungi on gel electrophoresis The obtained results underwent DNAsequencing of the ITS1, 5.8S, ITS2, 28S regions of the isolatedtermite fungi were compared with the sequences on the NCBI genebank The sequence 980 bp (KU569480) of the termite fungus in AnLinh commune, Phu Giao district together with the 938 bp sequence(MF163152) of the mushroom sample collected from Dinh Hiepcommune, Dau Tieng district achieved 99% of similarity and 100%

of coverage in comparison with T clypeatus species as the detection

of Sawhasan et al (2010), without deletion or extension mutationsoccurred (NCBI access codes: HQ702547.1 and GU5739599.1) TheDNA sequencing results on ITS1, 5.8S, ITS2, 28S regions of themushroom sample, combined with the morphological characteristics

of termite fungi demonstrated that botth fungal samples belong to

the T clypeatus species.

4.1.2 Isolation and identification of T microcarpus

Characteristics: The fruiting body had a slender stem The

mushroom cap was bell-shaped in the initial, then expanded into aflat cone, together with the creacked edge The cap edge wasstraight and lobed differently in each mushroom without followingany rules The hat was off-white while the color at the top wasslightly darker than the remaining parts of the mushroom The capdiameter was from 0.4 to 1.9 cm; the mushroom plate was lined,thick, along with the unequal serrated edge when cracking , and the

oval spores The T microcarpus propagated in clusters on termite

nests

Identification results based on DNA sequencing: The

obtained PCR products attained high quality because of a singleclear band on gel electrophoresis The band located around 700 to

800 bp Sequencing results of the isolated termite mushroom samplewere compared with sequences from the NCBI gene bank, showingthat the 721 bp sequence of this termite mushroom sample wasidentical to T microcarpus of Jannual et al (2020) Both of them

occur he results of DNA sequencing combined with themorphological characteristics represented that the isolated termitefungus was Termitomyces microcarpus (with accession No.MT672480).

Figure 4.7 Termite fungus T microcarpus

Figure 4.8 Colony morphology of T microcarpus

(a 15 days of isolation; b 20 days of subculture; c termite

c

Tthe tip of the mushroom was pointed The fruiting body without tipwas white and possessed mild taste The length of all over themushroom was from 7 to 13 cm.

The growth of termite fungal tissue on three medium MT1,MT4 and MT5 after monitoring showed that the reproduction onMT5 medium had the most robust transition The colonies hadsmooth, bright morphology as well as thick folds Tthe diameterunderwent culturing 20 days was 3.8 cm, while that in the MT1medium was 2.0 cm, and in MT4 medium was 3.1 cm The coloniescultured in both MT1, MT4 media after 20 days under the sameconditions had rude, rough, thin morphology together with whitepowdery spores

Identification results based on DNA sequencing: The

obtained PCR products attained high quality because of a singleclear band on gel electrophoresis The band located around 800 to

900 bp The 812 bp sequence of termite fungus samples collected inDinh Hiep commune, Dau Tieng district were compared with

sequences of Termitomyces sp.1 Group 7 as Taprab et al published in

2002 from the NCBI gene bank, showing that they had 100% ofsimilarity without deletion or extension mutation occurred.Depending on the results of DNA sequencing, it was initially

concluded that the termite fungus isolated was Termitomyces sp 1

(with access code on the NCBI Genbank: MF163149)

4.1.4 Isolation results of some other termite fungus

The growth of termite fungal tissue on MT5 medium after

10 days of isolation was monitored BD3 strain's colony was opaque,and possessed a rough surface, with the diameter about 2cm.Subsequently 30 days of subculture, the colonies became coarse,rough, thickly folded, white powdery surface, with certain brown

spots The result of sequencing strain BD3 was T sp 2 which code

was MF163445 on Genbank

The results of isolation the spores with regard to termitefungus strain BD5 from the nest on MT5 medium showed that the