Effects of ph on the growth and development of cordyceps militaris at institute forestry research and development thai nguyen province

THAI NGUYEN UNIVERSITY UNIVERSITY OF AGRICULTURE AND FORESTRY NGUYEN THI LAN ANH EFFECTS OF pH ON THE GROWTH AND DEVELOPMENT OF CORDYCEPS MILITARIS AT INSTITUTE FOR FORESTRY RESEARCH

THAI NGUYEN UNIVERSITY

UNIVERSITY OF AGRICULTURE AND FORESTRY

NGUYEN THI LAN ANH

EFFECTS OF pH ON THE GROWTH AND DEVELOPMENT OF CORDYCEPS MILITARIS AT INSTITUTE FOR FORESTRY RESEARCH

AND DEVELOPMENT, THAI NGUYEN PROVINCE

BACHELOR THESIS

Study Mode: Full - time

Major : Environmental Science and Management

Faculty : Advanced Education Program Office

Class : K46-N01

Batch : 2014 – 2018

Thai Nguyen, 24/09/2018

DOCUMENTATION PAGE WITH ABSTRACT

Thai Nguyen University Of Agriculture And Forestry

Degree Program: Bachelor of Environmental Science and Management

Student name: Nguyen Thi Lan Anh

Student ID: DTN1454290041

Thesis Title:

Effects of ph on the growth and development of cordyceps militaris

at institute forestry research and development, Thai Nguyen province

Supervisor’s:

Nguyen Huu Tho, PhD, Director Office of Research and International Affairs – Thai Nguyen University of Agriculture and Forestry, Vietnam

The biological characteristics of Cordyceps militaris include growth characteristics, nutrient media, pH of the medium, temperature and humidity However, this study evaluated the effect of different pH environments on the growth and development of C militaris within

50 days The results showed that when experimenting with 5 different pH treatments(3.5; 4.5; 5.5; 6.5 and 7.5), C.militaris had differnt growth and development in the ability to sprout the fruit body, the quantity, color, height and diameter of the fruit body Specifically, with

treatment 3 (pH = 5,5) and treatment 4 (pH = 6.5), the mushrooms had a good development and uniformity In contrast, C militaris had a slower development with treatment 5 (pH = 7.5) and the slowest development with treatment 1 at pH= 3.5 This research has found the optimal and most suitable pH treatment for the growth and development of C militaris that was treatment 3 with pH = 6.5

Key-words: Cordyceps militaris, fruiting body, experimental treatment, pH,

growth and development

Number of pages:

Date of submission:

ACKNOWLEDGEMENT

Firstly, I would like to express the deepest appreciation to teachers in Advanced Education Program Office as well as teachers in Thai Nguyen University of Agriculture and Forestry, who have dedicated teaching to me the valuable knowledge during study time in university and gave me a chance to work in this wonderful place

Formemost, I would like to express my gratitude and deep appreciation for my supervisor: Dr Nguyen Huu Tho has enthusiastically instructed me during my undergraduate thesis writing I also would like to thank Associate Prof Tran Thi Thu

Ha - Director of Forest Research and Development Institute and staffs at agency for creating the best conditions for me to have the opportunities to practice, study and write this thesis

I would like to thank those agencies again and I wish the Institute for Forestry Research and the Advance education program, Thai Nguyen university of agriculture and forestry will be achieved more successes in research, education and contribute more to the social development in the future as well In the process of implementing the project, I know that my thesis report got many mistakes so this report is invevitable shortcomings So, I would like to receive the attention and feedback from teachers and friends to this is more complete

Thai Nguyen, April 2018

Student

Nguyen Thi Lan Anh

TABLE OF CONTENTS

DOCUMENTATION PAGE WITH ABSTRACT i

ACKNOWLEDGEMENT iii

TABLE OF CONTENTS iv

LIST OF FIGURES AND GRAPHS vi

LIST OP TABLE vii

LIST OF ABBREVIATIONS viii

PART I INTRODUCTION 1

1 1 Research rationale 1

1.2 Research objectives 2

1.3 Research question and hypothesis 2

1.4 Limitations 3

PART II: LITERATURE REVIEW 4

2.1 The scientific basis of research topic 4

2.1.1 Definition and distribution of Cordyceps 4

2.1.2 Morphology of Cordyceps 5

2.1.3 Some bioactive compounds derived from Cordyceps 7

2.1.4 The values of C militaris for the human health 8

2.2 Research situation in the world and in Viet Nam 9

2.2.1 In the World 9

2.2.1 In Viet Nam 11

PART III METHOD 13

3.1 Materials 13

3.2 Methods 14

3.2.1 Data collection: 14

3.2.2 Experimental equipment preparation: 15

3.2.3 Data analysis 16

3.2.4 Conceptual framework 17

PART IV: RESULTS AND DISCUSSION 188

4.1 Effect of different pH levels on the ability to grow the fruit body of

C militaris 18

4.2 Effects of different pH levels on the average number and color of

C militaris’s fruit bodies 19

4.3 Effects of different pH levels on the average height (cm)

of C.militaris’s fruit bodies 20

4.4 Effects of different pH levels on the average diameter (mm)

of C.militaris’s fruit bodies 22

4.5 The quality of C militaris’s fruit bodies in the different experimental

treatments of pH 23

PART V: CONCLUSIONS AND RECOMMENDATIONS 25

5.1 Conclusions 25

5.2 Recommendations 26

REFERENCES 27

APPENDIX 31

LIST OF FIGURES

Figure 2.1 Natural morphology of the mushroom strains: C.sensensis,

C.gunnii, C barnessi, C gracilis, C liangshannesis, C militaris 7

Figure 2.2.The major chemical structures are purified from Cordyceps militaris

(A) Cordycepin, (B) Adenosine, (C) Ergrosterol 8

Figure 3.1 Conceptual framework 17

Figure 4.1 The different quantity and color of C.mitaris’s fruit boies at

5 different pH levels 20

Figure 4.2 Average height (cm) of C militaris’s fruit bodies in 5 diferent pH levels 21 Figure 4.3 The Difference between average height (cm) and diameter (mm) of C.militaris’s friut bodies at 5 different pH levels 22

Figure 4.4 Average diameter (mm) of C militaris’s fruit bodies in 5 diferent pH 22

LIST OF TABLE

Table 2.1 Description of some Cordyceps species’s morphology

(Hui-juan et al 2011) 6

Table 4.1 Effect of different pH levels on the ability to grow the fruit body

(flask number) of C militaris 18

Table 4.2 Effects of different pH levels on the average number and color

of C militaris’s fruit bodies 19

Table 4.3 Effect of pH level on the height of C.militari’s fruit bodies 20

Table 4.4 Effects of different pH levels on the average diameter (mm)

of C.militaris’s fruit bodies 22

Table 4.5 The quality of C militaris’s fruit bodies in pH = 6.5 23

LIST OF ABBREVIATIONS

Ave: Average

C militaris: Cordyceps militaris (mushroom strain)

FIPI: Forest Inventory and Planning Institute

IFRAD: Institute for Forestry Research and Development

Lux: Illumination of light

PART I INTRODUCTION

1 1 Research rationale

Cordyceps militaris L.ex Fr is a parasitic fungus in insects, considered to be a valuable medicinal product, because it contains many active substances (cordycepin, adenosine, polysaccharides, ergosterol, and mineral substances, etc.), it is good for human health, suitable for all ages and has the effect of hypoglycemia, anti-inflammatory, inhibit the growth of cancer cells, regulate the immune system, antioxidants, asthma and children rickets and growth retardation (Electronic Press, 2008) With high medicinal value, Cordyceps militaris that being over-harvested due

to extremely scarce and prices are extremely expensive Currently, the demand for this type of medicinal herbs is growing, while there are a lot of cordyceps products on the market with different quality and prices, almost uncontrollable In Vietnam, there have not been many publications of the C militaris cultivation The supply of this kind of herbal medicine from reputable, reliable production establishments is almost very few and the price is significantly high, therefore, it can not meet the needs of consumers, especially consumers with medium income Due to biological characteristics and life cycle of C militaris, it is very rare in nature Therefore, finding and using natural C militaris as a medicine or functional food is really difficult (Thuy, 2010) As a resuls, it

is important to determine the influence of some external factors on the growth and development of mycelium formation and fruiting bodies of C militaris With the aim

of finding the suitable culture environments for C militaris growth in Vietnam while still obtaining mushrooms with full medicinal values to meet the demand for use as medicine materials, improve human health and replicate the production of C militaris

at lower cost and not too expensive for middle income people to use Moreover, this is the basis for the conservation of this precious and rare gene source in the artificial

environment, the research project titled “Effects of pH on the growth and development of Cordyceps militaris at Institutue for Forestry Research and Development, Thai Nguyen Province” have been conducted at Thai Nguyen

University of Agriculture and forestry

1.2 Research objectives

General objective: This study aimed to assess the effect of pH on the growth and

development of Cordyceps militaris D, belongs to subfamily Clavicipitaceae in the laboratory of the Institutute for Forestry Research and Development (IFRAD), Thai Nguyen Province

- How does the pH impact the growth and development of C militaris?

- Which pH experimental treatment is the best for culturing C militaris?

Research hypothesis:

- Ho: Different pH levels do not impact the development of C militaris

- H1: Different pH levels impact the development of C militaris

1.4 Limitations

Because the laboratory lack of equipment, therefore some of the effects of

external environment on the development of C militaris are difficult to control

PART II: LITERATURE REVIEW

2.1 The scientific basis of research topic

2.1.1 Definition and distribution of Cordyceps

According to John et al (2005), for thousands of years, Cordyceps has been regarded as a pioneer in traditional medicine in Vietnam and China Cordyceps Sacc Link (4cm to 11 cm) and Cordyceps ophiglossoides Her - Fr (2cm to 6 cm) Cordyceps

is a fungi, it was first discovered by Chinese scientists in the Tibetan Plateau This medicinal plant is actually larvae of the Thitarodes or Hepialus species of parasitic Cordyceps sinensis (Berk) Sacc The most common Cordyceps are the young worms

of the Hepialus armoricanus More than 400 Cordyceps subspecies have been found and described, but only about 36 species are cultured under artificial conditions to produce fruit bodies (Sun et al., 2006) Among these species, only C militaris were grown on a large scale because it has very good medicinal properties and short production time In China, 60 species have been found, including C lilacsensis C., C grasspara zang, C kangdingenis zang, C guizhouensis Liu, etc There are also C militaris, C nutans Pat, C Tricentri Yasuda, C.gunnuii Berk However, so far, only two species of Cordyceps sincensis and Cordyceps militaris Link have been studied The fungus is widely distributed in China, the Tibetan Plateau, Bhutan, Nepal and North-East India with the average height from 3500m to 5000m above sea level Other studies have also shown that members of Cordyceps can also be found in Thailand, Korea, Japan, and Vietnam Cordyceps belongs to:

Kingdom: Fungus

Phylum: Ascomycota

Class: Ascomyceptes

Order: Hypocreales

Family: Clavicipataceae

Genus: Cordyceps

Species: C.sinensis (Berk) Sacc

Cordyceps is a fungus found naturally in the form of fruit bodies The formation process produces a large fruit body yield in artificial conditions that cannot be achieved as expected The cause is that the Cordyceps has a slow growth cycle, the growth process is often associated with or can be symbiotic with other fungi, hence, the purity of the wild-type strains then cultured under artificial conditions is rather difficult

2.1.2 Morphology of Cordyceps

According to Hui-juan et al (2011), books in traditional medicine describe the formation of dermatophytes as follows: Ascomyceptes host fungi are parasitic caterpillars of a butterfly (Caterpillar) scaly wings, especially in the moth Hepialus armoricanus, which spawns, hatching eggs and become young worms In winter, the larvae crawl into the soil, and in humid environments, Ascomycestes spore on the insect larvae undergoes appropriate conditions to germinate and grow into the mycelium Initially the fungal mycelium was white color, and later on they grew rapidly into a thick, solid block, some of which turned into dark yellow to brown, some still developing a faint pink, to orange Finally, when the nutrients are consumed completely, the mycelium is rapidly darkened after 30 - 60 days Then the entire worm body is just a cover of hyphae In the summer, from the mouth of the worms, the mushrooms grow on the surface of the stalk there are sprouts and grow into mushroom bodies about 10 cm Below is a table describing the different mushroom forms:

Table 2.1 Description of some Cordyceps species’s morphology (Hui-juan et al 2011)

C sinensis

( A )

The body of the larvae is like a silkworm, with a length of 3-5 cm, diameter from 3-8mm, dark yellow

to brown-yellow

Slender cylindrical substrate, 4-7cm in length, 3mm in diameter with sterile spikes

C gunnii

( B )

The body parts of the larvae are like silkworms, 3-6cm in length, 3-10mm in diameter, yellow-brown

to brown

Cylindrical substrate, stout and rough, 4-12cm in length, with sterile or branched first peak

C barnesii

( C )

The body of the kidney is curved, measuring 1.5cm-2 cm in length, with a small tip, with a pair

short-of teeth

Single substrate, slender and curved, 2-6cm in length and about 2mm in diameter

C gracilis

( D )

The body of larvae is like a silkworm, slender, 2 -4 cm long and 2-5 mm in diameter; brown, brown or reddish brown, with 20-

40 nodes; legs 8 pairs; sclerotium

of hard and solid larvae

Usually no substrate, substrate slivers, 2-3 cm in length and a diameter of about 2 mm, with sterile tip completely

C.liangshannesis

( E )

The larval body is like a silkworm, 3-6cm in length, 6mm-10 mm in diameter The outer surface is dark brown color

Substrate like filament, branched or non-branched, 10-30cm in length and 1-2mm in diameter

Figure 2.1 Natural morphology of the mushroom strains: C.sensensis, C.gunnii,

C barnessi, C gracilis, C liangshannesis, C militaris

2.1.3 Some bioactive compounds derived from Cordyceps

According to Lo et al (2013), the chemical composition of Cordyceps fungi, the most important group are cordycepic acid, cordycepin and polysaccharide, are effective in the treatment of cancer and viral diseases Besides the three components

on the analysis of fungi, scientists have also discovered many valuable compounds, such as cordycepic acid, N-acetylgalactosamine, adenosine, ergosterol and ergosterol esters, bioxanthracenes, hypoxanthine, polysaccharide and exopolysaccharide, chitinase, cadeapeptins and myriocin, are saccharides (trehalose and polysaccharides), nucleosides (adenosine, inosine and cordycepin), manitol and sterols (ergosterol) (Lu

et al., 2014)

Figure 2.2.The major chemical structures are purified from Cordyceps militaris

(A) Cordycepin, (B) Adenosine, (C) Ergrosterol

2.1.4 The values of C militaris for the human health

- Anti-cancer: The ability to inhibit the growth of tumors is found in many

genera of the Cordyceps Biologically active ingredients with anti-cancer effects are mainly polysaccharides, sterols and adenosine In particular, sterols and adenosine are the hottest research topics in cancer resistance (Zhoua et al 2009) The anti-cancer mechanism of C militaris is:

+ Strengthen the function of the immune system and natural immunity

+ Selective inhibition of RNA synthesis, thereby affecting protein synthesis + Active antioxidant and anti-free radicals

+ Anti-mutation

+ Interference with the replication process of tumor-inducing virus

+ Induction of methylation of nucleic acid

- Immune regulation: According to a study by Shashidhara et al (2013),

Cordyceps' main effects on immune system into cells such as lymphocyte response, natural killer (NK), and stimulate into: interleukin-2 (IL-2) and tumor necrosis factor (TNF-α) inhibitors on human mononuclear cells, this is the result of the production of

cytokines The therapeutic effects of mushrooms, such as preventing autoimmune diseases, allergies and related impacts immunomodulatory

- Protect other organs: Cordyceps also shows impact on other organ systems

For example, the nerve center, Cordyceps relieves pain, anticonvulsants and heat pumps On the respiratory system, Cordyceps has the effect of dilating the lungs, namely the increase in adrenaline from the adrenal glands and also the role of windpipe by histamine It also has anti-cough effect, eliminate sputum and asthma, and prevent pneumothorax In the endocrine system, Cordyceps affects the men's hormones and increases corticosterone levels (Zhoua et al., 2009)

In recent years Cordyceps has become a very important source of medicine and functional foods Most biologically active substances extracted from Cordyceps are anti-aging agents and effects that help regulate sleep, enhance immunity, and increase appetite

2.2 Research situation in the World and in Viet Nam

to create two main active ingredients Cordycepin and Adenosine are made from mycelium Countries such as China, South Korea, the United States, Japan, and Malaysia have produced industrial capital investments on an industrial scale since

1995, the 20th century (Wang et al., 2010)

At the Shanghai Endocrine Research Institute (China), the white spot fungus has been used to treat impotence effectively Nan et al (2001) demonstrated that Cordyceps was effective in treating hepatic dysfunction Anti-cancer effects are mentioned in many studies by authors in many countries around the world Mushroom fruit body extracts have anti-cancer effects, which are effective against two types of umbilical vein membranes, the HT1080 and B16-F10, which are resistant to the formation of new blood vessels by reducing expression The present of bFGF, one of the factors that stimulate this process Due to its role in suppressing the formation of blood vessels that inhibit the metastasis and growth of cancer cells (Yoo et al., 2009), extract from Cordyceps can inhibit the growth of lung, and breast cancer cells (Ahn et al., 2014) Also, warm water extracts can inhibit the growth of human blood cancer cell lines by causing cell death through caspase-3 activation (Lee et al., 2006)

The experiment of Kim et al (2006) showed similar results to the blood cancer treatment of this fungus extract as well Scientists have also used extracts from the fungus for testing on normal cell lines and cancer cell lines The results showed that two K562 (leukemia) and Du145 (prostate cancer cell carcinoma) cell lines were strongly inhibited by solvent extraction with butanol Anti-aging, anti-inflammatory effects are shown in research by Won and Park (2005) Ahn et al (2014) argued that Cordyceps has anti-inflammatory activity that inhibits the growth of certain viruses, bacteria, and fungi

2.2.2 In Viet Nam

In Viet Nam, research on Cordyceps is in the new stage to achieve some results The new study purposes only thing out, discovered and collected in the conditions of Vietnam

In 2009, the Forest Science Institute of Vietnam and the Viet Nam National Forestry University conducted surveys on Cordyceps nutans in Tay Yen Tu - Son Dong - Bac Giang Author Pham Quang Thu has announced the discovery of Cordyceps nutans This is the first described and documented mushroom species distributed in Vietnam (Thu, 2009)

In the Tam Dao National Park, Vinh Phuc, author Pham Quang Thu discovered Cordyceps Gunnii Cordyceps (Thu, 2009)

In 2009, Pham Thi Thuy collected Cuc Phuong National Park, Ninh Binh Province, Cordyceps fungus (Newspaper, 2008)

The Cordyceps militaris was first discovered and described in Vietnam (Thu, 2009) These fungi are distributed in natural forests of 1,900m to 2,100m above sea level The host of this species is the pupa of the Lepidoptera, a mushroom of 2 to 6.5

cm in length, the shape of the mace, small body and stalk, the head (reproduction) is enlarged up to 0.6 cm wide Mushroom’s stalk and reproduction are different colors, the fungus has smooth orange color, the reproduction is dark orange and many small bumps Bottles are plugged in very loosely or are partially plugged into the tissue of the fungus in the reproduction part The sporulation bags sized 300 - 510µ x 3,5 – 5µ, the capsule on the fruit body is 3.5 - 5 μm (Thu & Ha, 2009)

Other areas are also being researched for Cordyceps, usually Cordyceps in

m In the research program of the Vietnam-China Protocol on Cordyceps militaris, Associate.Prf Pham Thi Thuy, The plant protection research institute has developed Cordyceps as a functional food ingredient for humans As a result, there were three Cordyceps species:

+ Cordyceps nutans in Cuc Phuong, Ninh Binh and Tam Dao, Vinh Phuc

+ Cordyceps militaris in Vu Quang, Ha Tinh

+ Cordyceps sp 1 in Son Dong, Bac Giang

The author also identified some medicinal properties of C militaris, including Cordycepin, HEAA, some vitamins and some trace elements Above are some initial results of research on Cordyceps in Vietnam Therefore, research on this fungus is necessary to continue to deepen and develop the Cordyceps as a functional food for humans (Thuy, 2010)

PART III METHOD 3.1 Materials

Research material

3.1.1 Strains: Cordyceps militaris D is a genus from the Vietnamese Genetics

Institute

3.1.2 Chemicals and raw materials:

+ Peptone, high yeast

+ Refrigerator JEIO Tech -SI300 (Made in China)

+ pH meter HANNA (Made in Japan)

+ Telstar sterile culture cabinet (Made in Vietnam)

+ SA-300VF Sturdy Sterilizer (Made in Germany)

+ Analytical balance (Made in Switzerland)

+ Refrigerator (Made in Vietnam)

+ Thermo Scientific-Max Q 4000 shaker (Made in Japan)

+ Compact fluorescent lighting system (Made in Vietnam)

3.2 Methods

3.2.1 Data collection:

Before conducting this research, information was accessed from two data sources include primary data and secondary data:

+ Primary data: Collaborate with staffs of the Institute to collect data

+ Secondary data is collected from previous studies and reports, available in newspapers, magazines or scientific websites

Research is also considered based on two variables: dependent and independent variables:

+ Dependent variables: the number of fruit boddies inside the experimental flasks: height, diameter, color of C militaris’s fruit bodies

+ Independent variable: different pH levels

Based on the results of the analysis, the most effective pH experimental treatment for the growth and development of C militaris would be determined Referring consultants is also an important method for objectively assessing the best application

of the environment for the growth and development of Cordyceps militaris, enabling subsequent researchers to make informed decisions It is appropriate to complete the