INTRODUCTION
Research objectives
This study evaluated the impact of pH levels on the growth and development of Cordyceps militaris D, a member of the Clavicipitaceae subfamily, conducted at the Institute for Forestry Research and Development (IFRAD) in Thai Nguyen Province.
- To determine the optimal of pH environment for C militaris growth and development
- To determine the quality of C militaris’s fruit bodies in the different experimental treatments of pH.
Research question and hypothesis
- How does the pH impact the growth and development of C militaris?
- Which pH experimental treatment is the best for culturing C militaris?
- Ho: Different pH levels do not impact the development of C militaris
- H1: Different pH levels impact the development of C militaris
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
LITERATURE REVIEW
The scientific basis of research topic
2.1.1 Definition and distribution of Cordyceps
Cordyceps has been a staple of traditional medicine in Vietnam and China for thousands of years (John et al., 2005) This unique fungus, first discovered by Chinese scientists on the Tibetan Plateau, includes species such as Cordyceps sinensis and Cordyceps militaris, with the latter being cultivated extensively due to its excellent medicinal properties and short production time (Sun et al., 2006) Over 400 subspecies of Cordyceps have been identified, but only about 36 are grown artificially In China alone, 60 species have been documented, including C lilacsensis and C guizhouensis Cordyceps fungi thrive at altitudes between 3,500 and 5,000 meters, with additional populations found in Thailand, Korea, Japan, and Vietnam.
Cordyceps is a naturally occurring fungus known for its fruit bodies, but achieving a high yield in artificial cultivation is challenging due to its slow growth cycle This growth often relies on symbiotic relationships with other fungi, making it difficult to maintain the purity of wild-type strains in controlled environments.
Traditional medicine literature, as noted by Hui-juan et al (2011), describes the life cycle of dermatophytes originating from parasitic caterpillars of the moth Hepialus armoricanus After hatching, the larvae burrow into the soil, where, in humid conditions, Ascomycetes spores germinate and develop into white fungal mycelium This mycelium rapidly expands into a dense mass, changing colors from dark yellow to brown, and sometimes faint pink to orange After 30 to 60 days, as nutrients are depleted, the mycelium darkens, eventually enveloping the caterpillar's body In summer, mushrooms emerge from the caterpillar's mouth, growing into bodies approximately 10 cm in height A table detailing various mushroom forms follows.
Table 2.1 Description of some Cordyceps species’s morphology ( Hui-juan et al 2011)
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
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
The body of the kidney is short- curved, measuring 1.5cm-2 cm in length, with a small tip, with a pair of teeth
Single substrate, slender and curved, 2-6cm in length and about 2mm in diameter
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
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
Environment nutrients the body no larvae
Flat substrate, slightly curved, about 5cm in length, orange-yellow to orange-red
Figure 2.1 Natural morphology of the mushroom strains: C.sensensis, C.gunnii,
2.1.3 Some bioactive compounds derived from Cordyceps
Cordyceps fungi contain several key compounds, including cordycepic acid, cordycepin, and polysaccharides, which have been shown to be effective in treating cancer and viral diseases (Lo et al., 2013) In addition to these primary components, researchers have identified a variety of other valuable compounds within Cordyceps, such as N-acetylgalactosamine, adenosine, ergosterol, and various saccharides, nucleosides, and sterols (Lu et al., 2014).
Figure 2.2.The major chemical structures are purified from Cordyceps militaris
2.1.4 The values of C militaris for the human health
Cordyceps exhibits anti-cancer properties by inhibiting tumor growth, primarily due to its biologically active components such as polysaccharides, sterols, and adenosine Among these, sterols and adenosine are currently the focus of extensive research on cancer resistance (Zhoua et al 2009) The specific anti-cancer mechanism of C militaris plays a crucial role in its effectiveness against cancer.
+ 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
+ Interference with the replication process of tumor-inducing virus
+ Induction of methylation of nucleic acid
A study by Shashidhara et al (2013) highlights that Cordyceps significantly influences immune regulation by enhancing lymphocyte responses and natural killer (NK) cell activity It stimulates the production of interleukin-2 (IL-2) and tumor necrosis factor (TNF-α) inhibitors in human mononuclear cells, showcasing its potential benefits for immune system health.
9 cytokines The therapeutic effects of mushrooms, such as preventing autoimmune diseases, allergies and related impacts immunomodulatory
Cordyceps offers protective benefits to various organ systems, particularly by alleviating pain and acting as an anticonvulsant in the nervous system In the respiratory system, it aids in lung dilation by increasing adrenaline from the adrenal glands and enhancing bronchial function through histamine, while also providing anti-cough effects and helping to eliminate sputum, asthma, and prevent pneumothorax Additionally, Cordyceps influences the endocrine system by impacting male hormones and boosting corticosterone levels (Zhoua et al., 2009).
In recent years, Cordyceps has emerged as a significant source of medicinal and functional foods, known for its biologically active compounds These compounds are recognized for their anti-aging properties and beneficial effects on sleep regulation, immune enhancement, and appetite stimulation.
Research situation in the world and in Viet Nam
Cordyceps has garnered significant attention from scientists worldwide, leading to valuable advancements in its study Through cutting-edge biotechnology, various countries have successfully cultivated Cordyceps substrates, fostering the growth of the Cordyceps manufacturing industry Notably, Biofact Life in Malaysia has effectively utilized advanced technology from Japan and other nations to cultivate Cordyceps mycelium in artificial environments, producing key active compounds such as Cordycepin and Adenosine Countries including China, South Korea, the United States, Japan, and Malaysia have made substantial industrial capital investments in the Cordyceps sector on a large scale.
1995, the 20th century (Wang et al., 2010)
Research from the Shanghai Endocrine Research Institute in China highlights the effectiveness of white spot fungus, particularly Cordyceps, in treating impotence and hepatic dysfunction (Nan et al., 2001) Numerous studies globally indicate its anti-cancer properties, with mushroom fruit body extracts showing efficacy against resistant cancer cell lines, such as HT1080 and B16-F10, by reducing bFGF expression and inhibiting new blood vessel formation (Yoo et al., 2009) Furthermore, Cordyceps extracts have been shown to suppress the growth of lung and breast cancer cells (Ahn et al., 2014), while warm water extracts can induce cell death in human blood cancer cell lines through caspase-3 activation (Lee et al., 2006).
Research by Kim et al (2006) demonstrated that extracts from a specific fungus exhibit promising results in treating blood cancer Further studies have shown that butanol solvent extraction significantly inhibits the growth of K562 (leukemia) and Du145 (prostate cancer) cell lines Additionally, Won and Park (2005) highlighted the anti-aging and anti-inflammatory properties of this fungus, while Ahn et al (2014) confirmed its ability to inhibit the growth of various viruses, bacteria, and fungi.
Research on Cordyceps in Vietnam is currently in its early stages, focusing on discovering and collecting samples specific to the country's unique conditions.
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, leading to the discovery of this unique mushroom species by author Pham Quang Thu This marked the first documentation of Cordyceps nutans 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,
The fungi in question are found in natural forests at elevations between 1,900m and 2,100m They primarily infect the pupae of Lepidoptera and grow as mushrooms measuring 2 to 6.5 cm in length, featuring a mace-like shape with a small body and stalk The reproductive head can expand to 0.6 cm in width, displaying distinct color variations: the stalk is smooth and orange, while the reproductive part is dark orange with numerous small bumps The sporulation bags range in size from 300 to 510 μm by 3.5 to 5 μm, and the capsule on the fruit body measures 3.5 to 5 μm (Thu & Ha, 2009).
Other areas are also being researched for Cordyceps, usually Cordyceps in Vietnam distributed in evergreen tropical forests, ranging in height from 800 to 2000
In the Vietnam-China Protocol on Cordyceps militaris research program, Associate Professor Pham Thi Thuy from the Plant Protection Research Institute has successfully developed Cordyceps as a functional food ingredient for human consumption, leading to the identification of three distinct 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
C militaris possesses several medicinal properties, including Cordycepin, HEAA, various vitamins, and trace elements These initial findings highlight the importance of further research on Cordyceps in Vietnam to enhance its potential as a functional food for human consumption (Thuy, 2010).
METHOD
Materials
3.1.1 Strains: Cordyceps militaris D is a genus from the Vietnamese Genetics
+ Sterile needles (needle diameter Φ = 1.2 mm)
+ 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)
+ Thermo Scientific-Max Q 4000 shaker (Made in Japan)
+ Compact fluorescent lighting system (Made in Vietnam)
Methods
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
The analysis will identify the optimal pH level for the growth and development of C militaris Consulting experts is crucial for objectively evaluating the best environmental conditions for Cordyceps militaris, providing valuable insights for future researchers to make informed decisions.
15 research and propose appropriate consultations with the laboratory to improve the quality and save time and costs of developing C militaris
3.2.2 Experimental equipment preparation: All strains were maintained according to the procedures provided by the IFRAD laboratory Silkworms were bought in households with a tradition of silkworms The pupae were bought with their cocoons to ensure they were not contaminated during transport and storage The equipment used in this study includes: incubator (JEIO Tech -SI300), biological safety cabinet (Telstar), pH meter (Hanna), autoclave (SA-300VF Sturdy) air-conditioners, needle syringes, sterile needles (needle diameter Φ = 1.2 mm), disposable syringes (5 ml), fluorescent lighting systems (capacity per luminaire 0,05W) and samples are illuminated by fluorescent yellow lamps
Cordyceps militaris D 4% (v/v) spore solution was prepared and incubated at 20°C for 7 days in a basic environment Following trituration, the spore solutions were inoculated into 540 mL test bottles containing 100 mL of a nutrient-rich medium, which included 1.5% glucose, 0.5% peptone, 0.3% KH2PO4, 0.1% K2HPO4, 0.05% MgSO4, and 0.05% NaCl, and incubated at 25°C using a Thermo Scientific-Max Q shaker.
For optimal growth, incubate the inoculated test tubes at 4000 RPM for three days under humidity levels of 80-90% After approximately 4-5 days post-transplant, mycelium development will commence, with the fruit bodies of C militaris emerging around 15 days later.
- Research design: All experimental treatments were arranged in a completely randomized design (CRD) There were 5 different experimental treatments of pH under the homogeneous environment of light, temperature and humidity; each
The experimental treatment involved 20 bottles, repeated 5 times, resulting in a total of 500 bottles Over a period of 50 days, the growth and development of fungi were monitored, with each stage measured three times at 15-day intervals To assess the impact of five different pH treatments on the growth and development of C militaris, various parameters were evaluated.
To identify the optimal pH for the production of C militaris D, a study was conducted using a 540 ml flask containing 100 ml of a basic medium, with pH levels adjusted between 3.5 and 8.5 The pH was modified using HCl to increase acidity or NaOH to decrease acidity The cultures were maintained in a static environment for a duration of 50 days.
+ Ability to grow of C militaris’s fruit bodies (number of flasks)
+ Average number of C militaris’s fruit bodies
+ The color of the C militaris’s fruit bodies
+ Average height of C militaris fruit bodies (cm)
+ Average diameter of C militaris fruit bodies (mm)
The data was analysed by Excel in Microsoft to calculate the mean of experiment treatments’ iterations Moreover, research also use Excel to analyze a
17 factor variance (ANOVA) to test the hypothesis with an error of only 5%, and find out the impacts or differences among experimental treatments
RESULTS AND DISCUSSION
Effects of different pH levels on the average number and color of C militaris’s fruit bodies
Table 4.2 Effects of different pH levels on the average number and color of C militaris’s fruit bodies
Average number of fruit bodies inside the flasks
Table 4.2 presents the average number of fruit bodies and their colors across five different pH treatments Treatment 4 (pH = 6.5) yielded the highest average of 122.00 fruit bodies, surpassing treatment 3 (pH = 5.5) with 100.80 and treatment 5 (pH = 7.5) with 92.60 Notably, treatment 1 (pH = 3.5) showed a significantly lower average of only 33.40 fruit bodies In terms of color, the fruit bodies of C militaris in treatment 4 exhibited the most vibrant fresh orange hue, while treatments 1 and 3 displayed lighter yellow and dark yellow colors, respectively The accompanying figure further illustrates the quantity and color variations of C militaris's fruit bodies.
Figure 4.1 The different quantity and color of C.mitaris’s fruit boies at 5 different pH levels
Effects of different pH levels on the average height (cm)
Table 4.3 Effect of pH level on the height of C.militari’s fruit bodies
Table 4.3 demonstrates the specific data on average height of C militaris after
After 50 days of follow-up with five replications for each treatment, the growth of C militaris fruit bodies varied significantly across different pH levels In treatment 1 (pH = 3.5), the fruit bodies reached an average height of only 2.48 cm This increased to 2.59 cm in treatment 2 (pH = 4.5) and nearly 4 cm in treatment 5 (pH = 7.5) However, the most impressive growth was observed in treatment 4 (pH = 6.5), where the fruit bodies achieved a height of 4.45 cm, nearly doubling the height observed in treatment 1.
21 below would show clearly the difference in average height (cm) of fruit bodies among
Figure 4.2 Average height (cm) of C militaris’s fruit bodies in 5 diferent pH levels
Figure 4.3 The Difference between average height (cm) and diameter (mm) of C.militaris’s friut bodies at 5 different pH levels
Effects of different pH levels on the average diameter (mm)
Table 4.4 Effects of different pH levels on the average diameter (mm) of C.militaris’s fruit bodies
Table 4.4 presents the average diameter of fruit bodies after 50 days of implantation, with five replications conducted in the laboratory The results indicate that the average diameter of C militaris fruit bodies in treatment 4 (pH=6.5) exceeded 2 mm, nearly doubling the diameter observed in treatment 1, which measured only 1.25 mm Additionally, the average diameters in treatment 2 (1.85 mm) and treatment 3 (2.01 mm) showed minimal differences.
The graph and figure below would show a clearer difference in the average diameter of C militaris’s fruit bodies affected when tested on 5 different pH treatments:
Figure 4.4 Average diameter (mm) of C militaris’s fruit bodies in 5 diferent pH treatments
The quality of C militaris’s fruit bodies in the different experimental
Table 4.5 The quality of C militaris’s fruit bodies in pH = 6.5
Number Name of target unit Test method Result
1 Adenosine content mg/g H.HD.QT.223
2 Cordycepin content mg/g H.HD.QT.223
3 Total number of microorganisms CFU/G ISO 4833-1:2013
6 Total of spore fermentation- mold CFU/G TCVN 8275-2: 2010
8 Copper content mg/kg H.HD.QT.176
9 Arsenic content mg/kg H.HD.QT.429
10 Cadmi content mg/kg H.HD.QT.429
11 Lead content mg/kg H.HD.QT.429
12 Aflatoxin B1 content àg/kg H.HD.QT.011
13 Aflatoxin total content(B1, B2, G1, G2) àg/kg H.HD.QT.011
Table 4.5 outlines the targets for assessing the quantity of C militaris fruit bodies at a pH level of 6.5, which is optimal for its growth Notably, there are two primary active compounds present in various types of Cordyceps However, the levels of Adenosine and Cordycepin can vary depending on the specific type of Cordyceps and its age The content of these beneficial compounds ranges from 8 to 15 mg/g, which is favorable for human health Additionally, findings from the National Institute for Food Control (NIFC) reveal no harmful factors in C militaris fruit bodies when evaluated against other variables.
CONCLUSIONS AND RECOMMENDATIONS
Conclusions
Throughout the process of designing experiements, culturing C militaris and tracking the indicators of C militaris growth and development, this research had some main following conclusions:
The pH level plays a crucial role in the formation of mycelium and the growth of C militaris fruit bodies Analysis of tables and charts in the report clearly demonstrates significant differences in the growth rates, quantity, color, average height, and diameter of C militaris based on varying pH levels.
Scientific analysis indicates that Cordyceps fungi thrived best at pH levels of 5.5 and 6.5 (treatments 2 and 4), showing robust growth and development In treatment 5 (pH = 7.5), C militaris continued to grow, albeit at a reduced rate and yield compared to the previous treatments Conversely, treatment 1 (pH = 3.5) resulted in the slowest growth for C militaris, failing to meet standards for quantity, color, height, and diameter.
The optimal pH level for the growth and development of C militaris, under controlled conditions of light, temperature, and humidity, has been identified as treatment 4, with a pH of 6.5 This specific treatment resulted in vibrant, fresh orange-colored fruit bodies and an increased quantity of fruit bodies in the flasks.
26 more than 122 fruit bodies/bottle, the average height (nearly 4.5 cm) and diameter (more than 2 mm) developed nearly maximum of quantity and quality standards as well.
gunnii, barnessi, gracilis, liangshannesis, militaris
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
The body of the kidney is short- curved, measuring 1.5cm-2 cm in length, with a small tip, with a pair of teeth
Single substrate, slender and curved, 2-6cm in length and about 2mm in diameter
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
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
Environment nutrients the body no larvae
Flat substrate, slightly curved, about 5cm in length, orange-yellow to orange-red
Figure 2.1 Natural morphology of the mushroom strains: C.sensensis, C.gunnii,
2.1.3 Some bioactive compounds derived from Cordyceps
Cordyceps fungi contain key chemical components such as cordycepic acid, cordycepin, and polysaccharides, which have been shown to be effective in treating cancer and viral diseases (Lo et al., 2013) In addition to these primary compounds, researchers have identified various other valuable substances, including N-acetylgalactosamine, adenosine, ergosterol, and bioactive compounds like bioxanthracenes and chitinase, as well as saccharides like trehalose and nucleosides such as inosine (Lu et al., 2014).
Figure 2.2.The major chemical structures are purified from Cordyceps militaris
2.1.4 The values of C militaris for the human health
Cordyceps species possess anti-cancer properties by inhibiting tumor growth, primarily due to their biologically active compounds such as polysaccharides, sterols, and adenosine Among these, sterols and adenosine are currently the focus of extensive research in cancer resistance (Zhoua et al 2009) The anti-cancer mechanism of C militaris plays a crucial role in its therapeutic potential.
+ 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
+ Interference with the replication process of tumor-inducing virus
+ Induction of methylation of nucleic acid
A study by Shashidhara et al (2013) highlights the immune-regulating effects of Cordyceps, demonstrating its influence on lymphocyte responses and natural killer (NK) cells The research indicates that Cordyceps stimulates the production of interleukin-2 (IL-2) and tumor necrosis factor (TNF-α) inhibitors in human mononuclear cells, showcasing its potential role in enhancing immune function.
9 cytokines The therapeutic effects of mushrooms, such as preventing autoimmune diseases, allergies and related impacts immunomodulatory
Cordyceps has a significant impact on various organ systems, including the nervous system, where it alleviates pain and acts as an anticonvulsant In the respiratory system, Cordyceps promotes lung dilation by increasing adrenaline from the adrenal glands and modulating histamine in the windpipe, while also providing anti-cough benefits, reducing sputum, managing asthma, and preventing pneumothorax Additionally, in the endocrine system, Cordyceps influences male hormones and boosts corticosterone levels (Zhoua et al., 2009).
Cordyceps has emerged as a significant source of medicinal and functional foods in recent years, known for its biologically active compounds These compounds primarily serve as anti-aging agents, while also promoting better sleep regulation, boosting immunity, and stimulating appetite.
2.2 Research situation in the World and in Viet Nam
Cordyceps has garnered significant attention from scientists worldwide, leading to valuable advancements in its study With the help of advanced biotechnology, several countries have successfully cultivated Cordyceps substrates, fostering the growth of the Cordyceps manufacturing industry Biofact Life in Malaysia has effectively integrated advanced technology from Japan and other nations to cultivate Cordyceps mycelium in artificial environments, resulting in the production of key active ingredients such as Cordycepin and Adenosine Major industrial investments in Cordyceps production have been made by countries including China, South Korea, the United States, Japan, and Malaysia.
1995, the 20th century (Wang et al., 2010)
Research at the Shanghai Endocrine Research Institute in China has shown that white spot fungus is effective in treating impotence, while studies by Nan et al (2001) highlight the efficacy of Cordyceps in addressing hepatic dysfunction Numerous global studies indicate that mushroom fruit body extracts possess anti-cancer properties, particularly against HT1080 and B16-F10 umbilical vein membranes, by reducing the expression of bFGF, a factor that promotes new blood vessel formation This suppression is crucial in inhibiting cancer cell growth and metastasis (Yoo et al., 2009) Additionally, Cordyceps extracts have been found to hinder the growth of lung and breast cancer cells (Ahn et al., 2014), and warm water extracts can induce cell death in human blood cancer cell lines through caspase-3 activation (Lee et al., 2006).
Research by Kim et al (2006) demonstrated that extracts from a specific fungus exhibit promising results in treating blood cancer Further studies have confirmed that butanol solvent extraction significantly inhibits K562 leukemia and Du145 prostate cancer cell lines Additionally, Won and Park (2005) highlighted the anti-aging and anti-inflammatory properties of this fungus, while Ahn et al (2014) emphasized its ability to inhibit the growth of various viruses, bacteria, and fungi.
Research on Cordyceps in Vietnam is in its early stages, focusing on discovering and collecting specimens under local conditions.
In 2009, the Forest Science Institute of Vietnam and the Vietnam National Forestry University conducted surveys on Cordyceps nutans in Tay Yen Tu, Son Dong, Bac Giang Author Pham Quang Thu announced the discovery of this mushroom species, marking it as the first documented species of Cordyceps in Vietnam.
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,
This species of fungi, found in natural forests at elevations between 1,900m and 2,100m, primarily hosts the pupa of Lepidoptera The mushrooms range in size from 2 to 6.5 cm, featuring a mace-like shape with a small body and stalk The reproductive head can expand up to 0.6 cm in width, displaying a smooth orange color, while the reproductive structures are dark orange with numerous small bumps The sporulation bags measure between 300-510 μm in length and 3.5-5 μm in width, with the fruit body capsule measuring 3.5-5 μm (Thu & Ha, 2009).
Other areas are also being researched for Cordyceps, usually Cordyceps in Vietnam distributed in evergreen tropical forests, ranging in height from 800 to 2000
In the Vietnam-China research program on Cordyceps militaris, Associate Professor Pham Thi Thuy from the Plant Protection Research Institute has successfully developed Cordyceps as a functional food ingredient for human consumption, resulting in the identification of three distinct species of Cordyceps.
+ 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
C militaris possesses several medicinal properties, including Cordycepin, HEAA, vitamins, and trace elements These initial findings from research on Cordyceps in Vietnam highlight the importance of further studies to enhance the understanding and development of Cordyceps as a functional food for human health (Thuy, 2010).
3.1.1 Strains: Cordyceps militaris D is a genus from the Vietnamese Genetics
+ Sterile needles (needle diameter Φ = 1.2 mm)
+ 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)
+ Thermo Scientific-Max Q 4000 shaker (Made in Japan)
+ Compact fluorescent lighting system (Made in Vietnam)
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
The analysis will identify the optimal pH level for the growth and development of Cordyceps militaris Consulting experts is crucial for objectively evaluating the best environmental conditions for cultivating C militaris, which will aid future researchers in making informed decisions.
15 research and propose appropriate consultations with the laboratory to improve the quality and save time and costs of developing C militaris
3.2.2 Experimental equipment preparation: All strains were maintained according to the procedures provided by the IFRAD laboratory Silkworms were bought in households with a tradition of silkworms The pupae were bought with their cocoons to ensure they were not contaminated during transport and storage The equipment used in this study includes: incubator (JEIO Tech -SI300), biological safety cabinet (Telstar), pH meter (Hanna), autoclave (SA-300VF Sturdy) air-conditioners, needle syringes, sterile needles (needle diameter Φ = 1.2 mm), disposable syringes (5 ml), fluorescent lighting systems (capacity per luminaire 0,05W) and samples are illuminated by fluorescent yellow lamps