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 Effects of ph on the growth and development of cordyceps militaris at institute forestry research and development, Thai Nguyen province Nguyen Huu Tho, PhD, Director Office of Research and International Affairs – Thai Nguyen University of Agriculture and Forestry, Vietnam Thesis Title: Supervisor’s: Supervisor’s signature’s: Abstract: In recent years, Codyceps militaris species have become increasingly popular as a health food supplement In addition, they are noted by pharmacists for the diversity of biological compounds According to traditional Chinese medicine, the Codyceps militaris is used to successfully treat blood lipid disorders, chronic bronchitis, bronchial asthma, chronic kidney disease, kidney failure, arrhythmias, hypertension, allergic rhinitis, chronic hepatitis B and sexual dysfunction It is also used in treating immune disorders and supporting cancer treatment 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 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 i treatment (pH = 5,5) and treatment (pH = 6.5), the mushrooms had a good development and uniformity In contrast, C militaris had a slower development with treatment (pH = 7.5) and the slowest development with treatment 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 with pH = 6.5 Key-words: Cordyceps militaris, fruiting body, experimental treatment, pH, growth and development Number of pages: Date of submission: ii 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 iii 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 Research rationale 1.2 Research objectives 1.3 Research question and hypothesis 1.4 Limitations PART II: LITERATURE REVIEW 2.1 The scientific basis of research topic 2.1.1 Definition and distribution of Cordyceps 2.1.2 Morphology of Cordyceps 2.1.3 Some bioactive compounds derived from Cordyceps 2.1.4 The values of C militaris for the human health 2.2 Research situation in the world and in Viet Nam 2.2.1 In the World 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 iv 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 v LIST OF FIGURES Figure 2.1 Natural morphology of the mushroom strains: C.sensensis, C.gunnii, C barnessi, C gracilis, C liangshannesis, C militaris Figure 2.2.The major chemical structures are purified from Cordyceps militaris (A) Cordycepin, (B) Adenosine, (C) Ergrosterol Figure 3.1 Conceptual framework 17 Figure 4.1 The different quantity and color of C.mitaris’s fruit boies at different pH levels 20 Figure 4.2 Average height (cm) of C militaris’s fruit bodies in diferent pH levels 21 Figure 4.3 The Difference between average height (cm) and diameter (mm) of C.militaris’s friut bodies at different pH levels 22 Figure 4.4 Average diameter (mm) of C militaris’s fruit bodies in diferent pH 22 vi LIST OF TABLE Table 2.1 Description of some Cordyceps species’s morphology (Hui-juan et al 2011) 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 vii 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 R: Repetition Rpm: Shaking speed number of cycles/ minute T: Treatment % v/v: Volume of a mixture is calculated as a percentage of the total volume of the mixture viii PART I INTRODUCTION 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, antiinflammatory, 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 4.5 The quality of C militaris’s fruit bodies in the different experimental treatments of pH Table 4.5 The quality of C militaris’s fruit bodies in pH = 6.5 Number Name of target unit Test method Result Adenosine content mg/g H.HD.QT.223 (HPLC) 3.34 Cordycepin content mg/g H.HD.QT.223 (HPLC) 8.52 Total number of microorganisms CFU/G ISO 4833-1:2013 Coliforms CFU/G TCVN 6848: 2007 E.coli CFU/G TCVN 7924-2:2008 Total of spore fermentation- mold CFU/G TCVN 8275-2: 2010 Humidity g/100g Copper content mg/kg Arsenic content mg/kg 10 Cadmi content mg/kg 11 Lead content mg/kg H.HD.QT.062 H.HD.QT.176 (ICP-OES) H.HD.QT.429 (ICP-MS) H.HD.QT.429 (ICP-MS) H.HD.QT.429 (ICP-MS) 12 Aflatoxin B1 content µg/kg H.HD.QT.011 (LC-MS/MS) 13 Aflatoxin total content(B1, B2, G1, G2) µg/kg H.HD.QT.011 (LC-MS/MS) KPH (LOD: 10 CFU/g) KPH (LOD: 10 CFU/g) KPH (LOD: 10 CFU/g) KPH (LOD: 10 CFU/g) 7.08 15.3 0.19 0.62 0.061 KPH (LOD:0 µg/kg) KPH (LOD:0 23 µg/kg) Table 4.5 show tagets to assess the quatity of C militaris’s fruit bodies in the level of pH=6.5 which is best for growth of C militaris To be specific, there are two main active materials in almost types of Cordyceps Howerver, deping on different type of Cordyceps or in different age are different Adenosine, Cordyceps content Beside, this results of content of two main active materials are good because the content of that which is good for human health fluctuating around to 15 mg/g Finally, the result from National institute for food control (NIFC) indiscover any harmful factor in C militaris’s fruit bodies from other factors 24 PART V: CONCLUSIONS AND RECOMMENDATIONS 5.1 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 factor had significantly influences to the formation of mycelium as well as the growth and development of C militaris’s fruit bodies Specifically, the difference in rates on grow fruit bodies; quantity; color; average height and diameter of C militaris had been demonstrated clearly through above analysis of tables and charts in the report Based on the results of scientific analysis above, it can be recognized that with treatment (pH = 5.5) and treatment (pH = 6.5), Cordyceps fungi grew and developed really well In the ftreatment (pH = 7.5), C militaris still can develop and gave a certain yield but grew less than above formulas When it was tested on the treatment (pH =3.5), C militaris had a slowest growth compared to all the other treatments and did not meet the standard in terms of quantity, color, height as well as diameter Therefore, the most ideal pH treatment for the growth and development of C militaris within a homogeneous condition of light, temperature and humidity have been found, which was the treatment (pH = 6.5) With this treatment, the color of the fruit bodies was very beautiful (fresh orange), the number of fruit bodies in the flasks was 25 more than 122 fruit bodies/bottle, the average height (nearly 4.5 cm) and diameter (more than mm) developed nearly maximum of quantity and quality standards as well 5.2 Recommendations For the Institute for Forestry and Research Development: Cordyceps militaris is a new species of fungus that has variety of usage, which are really good for human health, therefore scientists and researchers of IFRAD should continue to focus on the study of the optimum environment for culturing and quickly propagating this fungi strain as a functional food for human consumption Besides, the Institute for Forestry and Research Development should be fit up some equipment which can control the influence of external environmental factors on the laboratory For the Advanced Education Program Office – Thai Nguyen University: More opportunities and more time should be created so that students can access more interesting research during their internship course to write more intensive theses, provide prerequisite and valuable reference materials for the next course Therefore, students would feel more excited in scientific research and improving their knowledge of their major as well as promoting their own capacity in the training process, and after graduating, students will not be surprised by new challenges 26 REFERENCES Ahn YJ, Loganathan J, Jiang J, Smith A, Jedniak A, Thyagarajan – Sahu A, Sandusky GE, Nakshatri H, Sliva D (2014) “The mushroom Ganoderma lucidum suppresses breast – to – lung cancer metastasis through the inhibition of pro – invasive genes” US National Library of Medicine National Institue of Health; 2014 Jun, 44(6) E-Newspaper (2008), "Vietnam Standards and Consumer Protection Association Timo Cordyceps King", April 1, 2008 Hui-juan L, Hao-bin H, Chu C, Quin L, Ping L (2011) “Morphological and microscopic indentification studies of Cordyceps and its counterfeits” Acta Phamarceutica Sinica B (3): 189 – 195 John H, Matt C, Aloha M Inc, SantaCruz, California U.S.A (2005) Cordyceps “Wasser SP Institue of Evolution” Uviversity of Haifa, Mt Carmel, Haifa, Israel Kim G.Y, Ko W.S, Lee J.Y, et al, editors (2006) “Water extract of Cordyceps militaris Bio Pharm maturation of murine bone narroe – derived dendritic cells in vitro” Bio Pharm Bull; 29: 354 – 60 Lee K.W, Wang Y, Chan F.L, Chen S, Leung L.K (2006) “The red wine polyphenol resveratrol displays bilevel inhibition on aromatase in breast cancer cells” Toxicol Sci; 92; 71 – 77 Li S.P., Yang F.Q., Tsim K.W.K., 2006 “Quality control of Cordyceps sinensis, a valued traditional Chinese medicine” Journal of Pharmaceutical and Biomedical Analysis; 41: 1571–84 27 Lo HC, Chienyan H, Fang YL and Hsu TH (2013) “A systematic review of the mysterious caterpillar fugus Ophio Cordyceps sinesis in DongChongXiaXao and Related Bioactive ingredients” Journal of Traditional and Complement Medicine 3(1): 16 – 32 Lu W.J, Chang N.C, Jayakumar T, Liao J.C, Lin M.J, Wang S.H, Chou D.S, Thomas P.A, Sheu J.R (2014) “Ex vivo and in vivo studies of CME – 1, a novel polysaccharide purified from the mycelia of Cordyceps sinesis that inhibits human platelet activation by activating adenylate cyclase/cyclic AMP)” Thromb Res 134 (2014), pp 1301 – 1310 Nam JX, Park EJ, Yang BK, Song CH, Ko G, Sohn DH (2001) “Anti – fibrotic effect of extracellular biopolymer from submerged mycelial cultures of Cordyceps militaris on liver fibrosis incluced by bile duct ligation and scission in rats” Arch Pharm Res; 24:327 – 332 Nguyen Thi Bich Thuy (2010), "Research on the breeding and cultivating of Dendrocalamus" Vietnam Forestry Academy, 24 / 4-25 / 4/2010 Agricultural publisher Pp 224-231 Pham Quang Thu (2009), "Detection of Cordyceps gun (Berk) in Tam Dao National Park, Vinh Phuc" Journal of Agriculture and Rural Development, - June 2009 Pham Quang Thu (2009), "Detection of Cordyceps mitans patus in Tay Yen Tu - Son Dong - Bac Giang Nature Reserve" Journal of Agriculture and Rural Development, - April 2009 28 Pham Quang Thu and Nguyen Manh Ha (2009), "Detection of Cordyceps militaris (L: FK) in Hoang Lien National Park, Lao Cai Province" Journal of Agriculture and Rural Development, - September 2009 Shashidhara MG, Gridhare P, Udaya Sk, Manohar B India (2013) “Bioactive of principles from Cordyceps sinesis: Apotent food supplement – A review” Journal of functional foods, p.p.1013 – 1030 Sun HC, Jong CK, Jung SL, Chul SY, Jong HK, Hyo IC, Seung WK (2006) “Morpholigical characteristics of Cordyceps sinesis 16 and production of mycelia and exo – biopolymer from molasses in sub merged culture” Journal of Industrial and Engineering Chemistry, 12(1): 115 – 120 Wang H.J., Chu Z., Feng L (2010) “A comparative study on effect of two plant growth promoters on the growth of Cordyceps militaris Lishizhen Medicine And Material Medical Research”; 21: 541–542 Wang Y, Che Z.M, Zhou L.L (2004) “Study breeding of a new variety of Cordyceps militaris by mutated with ultraviolet radiation” Food Ferment Industry 30 (8): 35 – 38 Won S.Y, Park E.H (2004), “Anti – inflammatory and related pharmacological activities of cultured mycelia and fruiting bodies of Cordyceps militaris” J Ethnopharmacol; 96: 555 – 561 Yoo H.S, Park SE, Kim J, Lee Y.W, Cho CK (2009) “Antiumor activity of water extracts from Cordyceps militaris in NCI – H460 cell xenografted nude mice” Acupunct Meridian Stud; 2: 294 – 300 29 Zhoua X.W, Zhenghua G, Ying S, Juan L, Tang K (2009) “Cordyceps fungi: natural products, pharmacological functions and developmental products” Journal of Pharmacy and Pharmacology: 61(3): 279 – 91 30 APPENDIX 1.The ability to grow fruit bodies of C militaris on different pH levels Number of flasks cannot grow Number of flasks grow Treatmen AV t R1 R2 R3 R4 R5 AVE R1 R2 R3 R4 R5 E 21 14 17 16 15 17 79 2 17 18 16 16 17 16 83 3 18 20 16 18 20 92 0 19 20 18 20 20 97 13 17 16 17 18 19 87 2.Quantiy of C militaris’s fruit bodies with different pH levels Number of repetitions Treatment AVE F1 F2 F3 F4 F5 30.78 32.47 31.45 34.35 36.00 33.01 68.23 70.43 71.18 66.21 71.05 69.42 100.08 99.35 102.15 103.05 98.60 100.65 120.08 122.35 124.38 122.57 120.72 122.02 90.00 92.37 94.06 95.75 91.11 92.66 31 Color of C militaris’s fruit bodies with different pH levels Number of repetitions Treatment AVE R1 R2 R3 R4 R5 Light Light Light Light Light Light yellow yellow yellow yellow yellow yellow Dark Dark Dark Dark Dark Dark yellow yellow yellow yellow yellow yellow Orange Orange Orange Orange Orange Orange yellow yellow yellow yellow yellow yellow Fresh Fresh Fresh Fresh Fresh Fresh orange orange orange orange orange orange Dark Dark Dark Dark Dark Dark orange orange orange orange orange orange 3.The height of C miliaris’s fruit bodies (cm) with different pH levels Number of repetitions Treatment AVE R1 R2 R3 R4 R5 2.60 2.62 2.55 2.60 2.62 2.60 3.49 3.50 3.52 3.46 3.53 3.50 4.13 4.14 4.15 4.12 4.15 4.14 4.38 4.43 4.48 4.50 4.51 4.46 3.96 3.95 3.97 4.00 3.95 3.97 32 ANOVA results of average height (cm) of C militaris’s fruit bodies with different formulas SUMMARY Groups Count Sum Average Variance T1 (3.5) 12.99 2.598 0.00082 T2 (4.5) 17,50 3.500 0.00075 T3 (5.5) 20.69 4.138 0.00017 T4 (6.5) 22.30 4.460 0.00295 T5 (7.5) 19.83 3.966 0.00045 ANOVA Source of Variation SS df MS F P-value F crit Between Groups 10.3952 2.598814 2874.794 2.73E-27 2.866081 Within Groups 0.01808 20 0.000904 Total 10.4133 24 α = 0.05 33 The diameter of C miliaris’s fruit bodies (mm) with different pH levels Number of repetitions Treatment AVE R1 R2 R3 R4 R5 1.30 1.31 1.26 1.32 1.26 1.29 1.55 1.56 1.48 1.54 1.57 1.54 1.95 1.99 2.02 2.01 2.00 2.00 2.21 2.23 2.22 2.22 2.23 2.22 1.86 1.85 1.82 1.84 1.83 1.84 ANOVA results of average diameter (mm) of C militaris’s fruit bodies with different formulas SUMMARY Groups Count Sum Average Variance T1 (3.5) 6.5 1.290 0.00108 T2 (4.5) 7.7 1,540 0,00125 T3 (5.5) 9.97 1.994 0.00077 T4 (6.5) 11.11 2.222 7.5E-5 T5 (7.5) 9.2 1.840 0.00025 34 ANOVA Source of Variation SS df MS Between Groups 2.74906 0.687266 Within Groups 0.01404 20 0.000702 Total 2.76310 24 F 979.0114 P-value F crit 1,26E-22 2,866081 α = 0, 05 35 Images of doing experiments Prepareing the environment for culturing C.militaris Sterizating the prepared culturing environment by using a sterile autoclave 36 Implanting C.miltaris inside the flasks Cultivating C.militaris after 50 days in laboratory 37 ... DTN1454290041 Effects of ph on the growth and development of cordyceps militaris at institute forestry research and development, Thai Nguyen province Nguyen Huu Tho, PhD, Director Office of Research and. .. 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. .. 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