INTRODUCTION
Research objectives
This study aimed to evaluate how pH levels influence 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 cornerstone of traditional medicine in Vietnam and China for centuries (John et al., 2005) This medicinal fungus, first discovered by Chinese scientists on the Tibetan Plateau, includes species like Cordyceps sinensis and Cordyceps militaris, which are known for their significant health benefits While over 400 subspecies have been identified, only about 36 are cultivated for their fruit bodies, with C militaris being the most widely grown due to its excellent medicinal properties and short production time (Sun et al., 2006) Cordyceps species are predominantly found in regions such as China, Bhutan, Nepal, and North-East India, thriving at altitudes between 3,500m and 5,000m Additionally, they have been discovered in Thailand, Korea, Japan, and Vietnam, highlighting their extensive distribution and importance in herbal medicine.
Cordyceps is a naturally occurring fungus known for its fruit bodies, but achieving a substantial yield in artificial cultivation is challenging This difficulty arises from the fungus's slow growth cycle and its tendency to symbiotically associate with other fungi, making it hard to maintain the purity of wild-type strains in controlled environments.
According to Hui-juan et al (2011), traditional medicine literature outlines the lifecycle of dermatophytes, which originate from parasitic caterpillars of the butterfly species Hepialus armoricanus After hatching from eggs, the larvae burrow into the soil during winter In humid conditions, Ascomycetes spores infect the larvae, leading to the growth of white fungal mycelium that rapidly develops into a dense block, changing color from dark yellow to brown and occasionally faint pink or orange Once nutrients are depleted, the mycelium darkens within 30 to 60 days, leaving the larval body encased in hyphae By summer, mushrooms emerge from the larvae's mouth, growing into bodies approximately 10 cm in height.
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 key chemical compounds such as cordycepic acid, cordycepin, and polysaccharides, which have shown effectiveness in treating cancer and viral diseases (Lo et al., 2013) In addition to these primary components, researchers have identified numerous valuable compounds, including N-acetylgalactosamine, adenosine, ergosterol, bioxanthracenes, hypoxanthine, and various saccharides like trehalose, as well as 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 species possess significant anti-cancer properties, primarily due to their biologically active compounds, including polysaccharides, sterols, and adenosine Notably, sterols and adenosine are at the forefront of research focused on cancer resistance (Zhoua et al 2009) The anti-cancer mechanism of C militaris highlights its potential in tumor growth inhibition.
+ 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 properties of Cordyceps, demonstrating its ability to enhance lymphocyte responses and activate natural killer (NK) cells Additionally, Cordyceps stimulates the production of interleukin-2 (IL-2) and tumor necrosis factor (TNF-α) inhibitors in human mononuclear cells, showcasing its potential in modulating immune function.
9 cytokines The therapeutic effects of mushrooms, such as preventing autoimmune diseases, allergies and related impacts immunomodulatory
Cordyceps positively influences various organ systems, demonstrating pain relief and anticonvulsant properties in the nervous system It enhances respiratory function by dilating the lungs through increased adrenaline and histamine activity, while also providing anti-cough benefits and alleviating conditions such as asthma and pneumothorax Additionally, Cordyceps impacts the endocrine system by modulating male hormones and elevating corticosterone levels (Zhoua et al., 2009).
In recent years, Cordyceps has emerged as a significant source of medicinal and functional foods, renowned for its biologically active compounds These substances are primarily recognized for their anti-aging properties, as well as their ability to regulate sleep, boost immunity, and stimulate appetite.
Research situation in the world and in Viet Nam
Cordyceps has garnered significant attention from scientists worldwide, leading to remarkable advancements in its study and cultivation Utilizing cutting-edge biotechnology, various countries have successfully developed Cordyceps substrates, fostering a robust manufacturing industry Notably, Biofact Life in Malaysia has integrated advanced technology from Japan to cultivate Cordyceps mycelium in artificial environments, resulting in the production of key active ingredients such as Cordycepin and Adenosine Countries like China, South Korea, the United States, Japan, and Malaysia have made substantial industrial capital investments in the Cordyceps sector, driving its growth on an industrial scale.
1995, the 20th century (Wang et al., 2010)
The Shanghai Endocrine Research Institute in China has found that white spot fungus effectively treats impotence, while research by Nan et al (2001) indicates that Cordyceps can address hepatic dysfunction Numerous global studies highlight the anti-cancer properties of mushroom fruit body extracts, particularly against HT1080 and B16-F10 umbilical vein membranes, by reducing bFGF expression, which is crucial for new blood vessel formation This suppression plays a vital role in inhibiting cancer cell growth and metastasis (Yoo et al., 2009) Additionally, Cordyceps extracts have been shown to inhibit lung and breast cancer cell proliferation (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 effectively treat blood cancer, with further studies indicating strong inhibition of K562 (leukemia) and Du145 (prostate cancer) cell lines when using butanol extraction Additional findings by Won and Park (2005) highlighted the anti-aging and anti-inflammatory properties of this fungus, while Ahn et al (2014) supported these claims by asserting that Cordyceps exhibits anti-inflammatory activity that can inhibit the growth of certain viruses, bacteria, and fungi.
Research on Cordyceps in Vietnam is in its early stages, focusing on discovering and collecting findings specific to the country's unique 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 species, marking it as the first documented mushroom species found 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,
In natural forests at elevations between 1,900m and 2,100m, a specific species of fungi thrives, primarily parasitizing the pupae of Lepidoptera This mushroom typically measures 2 to 6.5 cm in length and features a mace-like shape with a small body and stalk, while its reproductive head can expand to 0.6 cm in width The fungus showcases a smooth orange color, while its reproductive structures are dark orange with numerous small bumps The sporulation bags range from 300 to 510 µm in size, with the fruit body capsule measuring between 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 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 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 notable medicinal properties, including cordycepin, HEAA, various vitamins, and trace elements These initial findings on Cordyceps in Vietnam highlight the need for further research to enhance and promote its potential as a functional food for human health (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 treatment for the growth and development of C militaris Consulting experts is crucial for objectively evaluating the ideal environmental conditions for Cordyceps militaris, allowing future researchers to make well-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
The preparation of the Cordyceps militaris D 4% spore solution involved creating a basic environment and incubating the mixture at 20°C for 7 days Following this, the spore solution was inoculated into 540 mL test bottles containing 100 mL of a nutrient-rich basic environment, which included 1.5% glucose, 0.5% peptone, 0.3% KH2PO4, 0.1% K2HPO4, 0.05% MgSO4, and 0.05% NaCl The inoculated bottles were then placed in a Thermo Scientific-Max Q shaker and maintained at 25°C for optimal growth conditions.
For optimal growth, incubate the inoculated test tubes at 4000 RPM for three days under humidity levels of 80-90% Mycelium development will commence approximately 4-5 days post-transplant, with the fruit bodies of C militaris beginning to emerge after 15 days.
- 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 five times, resulting in a total of 500 bottles Over a span of 50 days, the growth and development of fungi were closely monitored, with measurements taken at three different stages, each 15 days apart The study focused on assessing the impact of five distinct pH treatments on the growth and development of C militaris.
To identify the optimal pH for the production of C militaris D, a study was conducted in which the fungus was cultured in a 540 ml flask containing 100 ml of a basic medium with pH levels varying from 3.5 to 8.5 The pH of the culture media was meticulously adjusted using HCl to raise the pH or NaOH to lower it The flasks 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 displayed the most vibrant fresh orange hue, while treatments 1 and 3 exhibited lighter or darker yellow shades The accompanying figure further illustrates the quantity and color variations of C militaris 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
In a study involving 50 days of follow-up and five replications for each treatment, the growth of C militaris fruit bodies varied significantly with pH levels The fruit bodies measured only 2.48 cm in treatment 1 (pH = 3.5), while treatment 2 (pH = 4.5) yielded slightly larger fruit bodies at 2.59 cm Notably, treatment 5 (pH = 7.5) produced fruit bodies nearing 4 cm in height The most impressive growth was observed in treatment 4 (pH = 6.5), where the fruit bodies reached a height of 4.45 cm, nearly doubling the size of those 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 illustrates that after 50 days of cultivation with five replications, the average diameter of C militaris fruit bodies was significantly influenced by pH levels The fruit bodies in treatment 4 (pH=6.5) exhibited an average diameter exceeding 2 mm, nearly doubling the size of those in treatment 1, which measured only 1.25 mm Meanwhile, treatments 2 and 3 showed similar average diameters of 1.85 mm and 2.01 mm, respectively, indicating minimal variation between these two treatments.
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 optimal growth conditions for C militaris, highlighting that a pH level of 6.5 is ideal for the development of its fruit bodies Notably, C militaris contains two primary active compounds, Adenosine and Cordycepin, which vary based on the type and age of the Cordyceps The concentration of these beneficial compounds, which are known to support human health, typically ranges from 8 to 15 mg/g Additionally, findings from the National Institute for Food Control (NIFC) confirm that C militaris fruit bodies do not contain any harmful substances.
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 the data reveals significant differences in growth rates, quantity, color, average height, and diameter of C militaris, as illustrated by the tables and charts in the report.
Scientific analysis indicates that Cordyceps fungi thrived best in treatment 2 (pH = 5.5) and treatment 4 (pH = 6.5), demonstrating robust growth and development In treatment 5 (pH = 7.5), C militaris still exhibited growth and yielded some results, but it was less than the first two treatments Conversely, treatment 1 (pH = 3.5) showed the slowest growth for C militaris, failing to meet standards in quantity, color, height, and diameter compared to the other treatments.
The optimal pH level for the growth and development of C militaris under consistent light, temperature, and humidity conditions is pH 6.5 This specific treatment results in vibrant, fresh orange 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 compounds 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 components, researchers have identified a variety of other valuable substances, including N-acetylgalactosamine, adenosine, ergosterol, bioxanthracenes, hypoxanthine, and various saccharides and nucleosides, which further enhance the therapeutic potential of these fungi (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 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) Specifically, the anti-cancer mechanism of C militaris contributes to its potential effectiveness in combating 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 the immune-regulating effects of Cordyceps, demonstrating its ability to enhance lymphocyte response and stimulate natural killer (NK) cells Additionally, Cordyceps promotes the production of interleukin-2 (IL-2) and tumor necrosis factor (TNF-α) inhibitors in human mononuclear cells, showcasing its potential in immune system modulation.
9 cytokines The therapeutic effects of mushrooms, such as preventing autoimmune diseases, allergies and related impacts immunomodulatory
Cordyceps is known for its protective effects on various organ systems It alleviates pain and acts as an anticonvulsant in the nervous system In the respiratory system, Cordyceps promotes lung dilation by increasing adrenaline from the adrenal glands and enhancing windpipe function through histamine, while also providing anti-cough benefits, reducing sputum, alleviating asthma, and preventing pneumothorax Additionally, Cordyceps influences the endocrine system by affecting male hormones and elevating corticosterone levels (Zhoua et al., 2009).
In recent years, Cordyceps has emerged as a significant source of medicinal and functional foods, primarily due to its biologically active compounds These compounds are known for their anti-aging properties and benefits, which include regulating sleep, 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 aid of advanced biotechnology, several countries have successfully cultivated Cordyceps substrates, fostering the growth of the Cordyceps manufacturing industry In Malaysia, Biofact Life has effectively combined cutting-edge technology from Japan and other nations to cultivate Cordyceps mycelium in artificial environments, resulting in the production of two key active ingredients: Cordycepin and Adenosine Major players in the industry, including China, South Korea, the United States, Japan, and Malaysia, have made substantial industrial capital investments to scale up production.
1995, the 20th century (Wang et al., 2010)
Research at the Shanghai Endocrine Research Institute has shown that white spot fungus effectively treats impotence and that Cordyceps demonstrates significant benefits in addressing hepatic dysfunction (Nan et al., 2001) Numerous studies worldwide highlight the anti-cancer properties of mushroom fruit body extracts, particularly against resistant cancer cell lines such as HT1080 and B16-F10, by reducing bFGF expression, which is crucial for new blood vessel formation This suppression plays a vital role in inhibiting cancer cell growth and metastasis (Yoo et al., 2009) Additionally, Cordyceps extracts have been found to inhibit lung and breast cancer cell proliferation (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 involving both normal and cancer cell lines revealed that butanol solvent extraction significantly inhibited the growth of K562 (leukemia) and Du145 (prostate cancer) cells 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 due to its anti-inflammatory activity.
Research on Cordyceps in Vietnam is in its early stages, focusing on discovering and collecting findings specific to the country's unique conditions.
In 2009, researchers from 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 Pham Quang Thu announced the discovery of this mushroom species, marking it as the first documented and described 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,
The fungi studied are found in natural forests at elevations between 1,900m and 2,100m, specifically targeting the pupa of Lepidoptera as their host These mushrooms range from 2 to 6.5 cm in length and have a mace-like shape, featuring a small body and stalk, with the reproductive head expanding to 0.6 cm in width The stalk and reproductive parts exhibit different colors, with the fungus displaying a smooth orange hue and the reproductive section being dark orange with numerous small bumps The sporulation bags measure between 300-510 µm in size, while the capsule on the fruit body ranges from 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 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, various vitamins, and trace elements These findings represent the preliminary research on Cordyceps in Vietnam, highlighting the need for further investigation into this fungus to enhance its development as a functional food for human consumption (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 aims to identify the optimal pH treatment for the growth and development of C militaris Consulting experts is crucial for objectively evaluating the ideal environmental conditions for Cordyceps militaris, which will assist 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