MINISTRY OF EDUCATION MINISTRY OF AGRICULTURE AND TRAINING AND RURAL DEVELOPMENT VIETNAM ACADEMY OF AGRICULTURAL SCIENCES NGUYEN THI BICH THUY STUDY ON BIOLOGICAL CHARACTERISTICS AND T
Trang 1MINISTRY OF EDUCATION MINISTRY OF AGRICULTURE AND TRAINING AND RURAL DEVELOPMENT
VIETNAM ACADEMY OF AGRICULTURAL SCIENCES
NGUYEN THI BICH THUY
STUDY ON BIOLOGICAL CHARACTERISTICS AND TECHNOLOGY OF SPAWN PRODUCTION, CULTIVATION OF KING OYSTER MUSHROOM
(PLEUROTUS ERYNGII) AND YUNZHI MUSHROOM (TRAMETES VERSICOLOR) IN VIETNAM
Branch of study: Crop Science Code : 62.62.01.10
SUMMARY OF DOCTORAL THESIS
Hanoi - 2014
Trang 2This thesis is completed at:
VIETNAM ACADEMY OF AGRICULTURAL SCIENCES
Science supervisor:
1 Prof Ph.D Trinh Tam Kiet
2 Ass Dr Nguyen Trung Thanh
Critic 1: Critic 2: Critic 3:
The thesis will be defended before Council for thesis assessment at Institutional level held at Vietnam Academy of Agricultural Sciences
at hour minute, day month year
This Thesis can be referred to at:
1 National Library
2 Library of VietnamAcademy of Agricultural Sciences
3 Library of
Trang 3INTRODUCTION
1 The urgency of the topic
King Oyster Mushroom (Pleurotus eryngii (DC.) Quél ) is a delicious,
edible fungus, high nutritional and medicinal values King oyster mushroom’s fruiting bodies are of large size, pretty shape, so for these advantages, this fungus is called "King Oyster mushroom” Yunzhi
mushroom (Trametes versicolor (L.) Pilat) is a medicinal fungus of very
high medicinal value, that has been preferred by consumers in countries like China, Japan, European, American countries, In Yunzhi mushroom,
there are polysaccharide compounds associated with protein, consisting of
two main types: PSP (polysaccharide peptide) and PSK (polysaccharide krestin) PSP and PSK have the effect to inhibit various types of cancer cells such as carcinoma cell (carcinoma), cancer of the blood cells (leukemia), stomach cancer, colon cancer, throat cancer, lung cancer and breast cancer … (P.M.Kidd, 2000)
Together with the development of society, the demand for people’s
is not only good, but also nutritious, safe and valuable health promoting food Now, on the market, there are many types of edible and medicinal mushrooms imported from China into Vietnam with quantity of hundreds
of tonnes/year, by many different ways, without origins, long time of transportation, using preservatives, thus causing bad psychological attitudes for consumers Currently, the edible mushroom - medicinal mushrooms cultivated in Vietnam, especially the King Oyster and Yunzhi mushrooms are still interested by consumers
The production of edible - medicinal mushrooms is growing strongly, the units of breeding and cultivating mushrooms in our country are all applying traditional breeding technologies (solid), so there exist some disadvantages as: high rate of disease - infected spawns, long incubation time for one spawn level with an average duration from 10-25 days, the time from implanting spawns into farming materials to commercial mushroom picking is long, within 2 months that lead to high prices for spawn and commercial mushrooms While research and seed production
of liquid spawn in world has reached some remarkable achievements The application of liquid spawn production show remarkable effectiveness as compared to solid spawn production, that help shortening growth duration
of one spawn level down to only 3-5 days, with high purity, good quality, reducing infection rates, appropriate for spawn production and mushroom cultivation in industrial scale
Trang 4In order to contribute to the overcoming of the above limitations, edible - medicinal mushrooms in general and King Oyster -Yunzhi mushrooms in particular to develop effectively, the research theme: "Study on biological characteristics and technology of spawn production, cultivation of the King Oyster Mushroom (Pleurotus eryngii) and Yunzhi mushroom (Trametes versicolor) in Vietnam" has been conducted
2 Objective of topic
To conduct research on some biological characters of strains of
King Oyster and Yunzhi mushrooms to identify best developing strains in climatic condition of Vietnam, and at the same time, to set up spawn
production technologies for mushroom strains of liquid spawn in order to
improve production efficiency for edible and medicinal mushrooms
3 Scientific and practical significances
3.1 Scientific significance
The findings of the study have provided scientific data on the basic growth indicators of King Oyster mushroom (P eryngii) and Yunzhi mushroom (T versicolor), as well as the need for nutrition and optimal
environmental conditions for growth and development of two mushroom varieties on liquid cultivation; pointing out the genetic differences of the currently being stored strains and correlation with the biological characteristics in the cultivation, and at the same time, to provide the data observed by the electron microscope
3.2 Practical significance
Through the results of the research subject, two mushroom varieties
of King Oyster mushroom (P eryngii) and Yunzhi mushroom (T.versicolor), suitable for growing conditions in Vietnam, have been
released to production; at the same time, propagation technologies in liquid types for these two strains have been set up, improving their growth capacity, shorterning incubation time, increasing productivity of commercial mushrooms This technology is highly feasible, capable of applicating in mushroom production in industrial scale
3.3 The novelty of the thesis
Having studied the morphological characteristics basidiospores, indicating the growth characteristics and development of mushroom mycelium and fruiting bodies; simultaneously assess the genetic differences between strains of King oyster mushroom and Yunzhi
Trang 5mushroom On that basis, edible mushrooms and medicinal mushrooms adaptable to production conditions of Vietnam have been selected
Having build up successful propagation processes for liquid spawns
of King oyster and Yunzhi mushrooms (from propagation of the first grade spawn → intermediate spawn → commercial spawn and using for cultivation); replacing traditional technologies, shortening incubation time, increasing mushroom yield, enhancing economic efficiency
4 The layout of the thesis
The main content of the thesis are presented in 134 pages (excluding
references and appendices) Introduction: 4 pages; Chapter 1: Overview document: 36 pages; Chapter 2: Materials, content and research methodology: 17 pages; Chapter 3: Research Results and Discussion: 75 pages; Conclusions and recommendations: 2 pages The thesis consists of
26 tables, 65 figures, 18 Vietnamese documents, 78 documents in English The appendix includes: Process Mapping; Calculation of the economic efficiency of liquid spawn multiplication technologies in comparison with traditional technologies; Images of mushroom spawns and mushroom fruiting bodies in some experiments; Results of statistical analysis and experimental data processing and content related to the thesis
Chapter 1 OVERVIEW
1.1 Status of research and production of mushrooms in the world and
in Vietnam
1.1.1 Status of research and production of mushrooms in the world
1.1.1.1 Research situation on multiplication of liquid spawns and
cultivation of King Oyster and Yunzhi mushrooms
As King oyster and Yunzhi mushrooms are of high medicinal value, they are interested to be studied
on by many concerned countries Scientists around the world have conducted further studies in the growth capacity of King oyster and Yunzhi mushroom by the effects of additional nutrient supply such as sources of carbon, nitrogen, mineral as well as their best additional contents
Authors also studied the effect of pH, temperature, nutrients to the
growth of King oyster and Yunzhi musrooms Cultivating materials and nutritional supplement components, nutrient contents were also mentioned
Deep layer nurturing fermentation method (liquid spawn
multiplication/propagation) is applied to produce different varieties of
Trang 6edible and medicinal mushrooms (Liu, 2010; Diamantopoulou P, 2012; Jonathan, 2009
At present, technology for liquid spawn propagation has been developed in many countries over the world, there have been many studies
to point out optima additional nutrition component, aeration rate, affects of liquid medium pH to the growth of fiber systems, advantages and disadvantages of liquid spawn propagation
1.1.1.2 Research on the genetic diversity of some strains
1.1.1.3 The situation of mushroom production in the world
1.1.2 Status of research and production of mushroom in the country
1.1.2.1 Research on the genetic diversity of some strains
In Vietnam, the RAPD technique has much been applied in the genetic analysis of plants Some authors have used RAPD technique to analyze genetic diversity in the different objects but genetic research on diversity of
edible mushrooms and medicinal mushrooms have not been many
1.1.1.2 The situation of mushroom research and production in Vietnam
In recent years, there have been many research works of authors such
as Trinh Tam Kiet, Nguyen Thi Chinh, Phan Huy Duc, Le Xuan Tham, Le Thi Hoang Yen These authors focused on the study of different areas on various different objects There are several studies on liquid spawn but mainly in the laboratory with the aim of extracting biomass, not any research work has been applied in farming sector
Centre for Mushroom Research and development and Reishi Co., Ltd has successfully produced many products with preventive and curative effects, deeply focuses on scientific research to create food functional products Along with the studies on the effect of Yunzhi mushroom, our company has also conducted studies on manufacturing processes for products from Yunzhi mushroom There are 2 main types of products, that are mycelia biomass powder and fruit bodies from Yunzhi mushroom At present, annually, the company provides about 10 tons of
bioproducts-mycelia biomass powder/year and 2-3 tons of fruit bodies /year Mushroom cultivation is being transferred to multiple production facilities Materials used for cultivation are sawdust supplemented with cultivating nutritional additives; the most suitable temperature for mushroom fruit bodies to grow and develop is from 20 - 250C and the most appropriate time for fruit bodies to grow is from february to may (Solar calendar) (Nguyen Thi Chinh , 2011 )
Trang 7Some new species of mushrooms imported into Vietnam, including King oyster mushroom, and fresh mushroom products currently on the market are mainly imported from China King oyster mushroom, also called chicken leg mushroom, is now being studied and farmed by the Center for Plant Biotechnology Research by a traditional method, with mixing farming materials of 43 % cotton by-product + 43 % sawdust + 6
% corn starch + 7 % rice bran + 1 % CaCO3 The cultivation stages of King oyster mushroom are described in detail: using plastic bags size 19 x
33 cm as packages, weight of material bag: 0.8 kg / bag Authors also identified suitable environmental conditions for cultivation of King oyster mushroom: temperature from 12 -150C; light intensity from 800 - 1200lux
; air humidity: 85-95%; good ventilation condition The findings noted that, with the above growing conditions, the actual yield of King oyster mushrooms reached 35-40 % (Dinh Xuan Linh et al, 2012)
In general, a number of scientists in our country focussed deeply on the study of edible mushrooms, especially medicinal mushrooms (Reishi mushroom, Yunzhi mushroom, Pupae cordyceps mushroom ) This trend
of study was performed in liquid medium but intensivly concentrated on mycelia biomass extraction technology to produce functional products; as mushroom cultivating for obtaining fruiting bodies mainly used solid spawn sources (seed spawns), the cultivation time usually lasts longer
1.2 General introduction of King oyster and Yunzhi mushrooms
King oyster mushroom (English name is also King oyster mushroom)
has a scientific name as Pleurotus eryngii (DC Fr.) This is a specy of edible mushroom that has high medicinal and nutritional values
Yunzhi mushroom has the scientific name as Trametes versicolor
(L.Fr.) Pilat, with its English name as Yunzhi or Turkey tails Previously,
they had different scientific names as Coriolus versicolor, Polyporus versicolor Yunzhi is a medicinal mushroom which is used in many Asian
countries
During the processes of growth, development, King oyster and Yunzhi mushrooms are influenced by many factors Agents that affect the formation of fruiting bodies of the fungi can be divided into 3 types: external factors such as temperature, humidity, light, pH, CO2 concentration and wind speed as well as the role played by other microorganisms coexisting in the ecosystem; necessary nutrients
1.3 Factors affecting the growth of King oyster and Yunzhi mushrooms
During the processes of growth, development, King oyster and Yunzhi mushrooms are influenced by many factors Agents that affect the
Trang 8formation of fruiting bodies of the fungi can be divided into 3 types: external factors such as temperature, moisture, humidity, light, pH, CO2 concentration and wind speed as well as the role played by other microorganisms coexisting in the ecosystem; necessary nutrients
1.4 The role of King oyster and Yunzhi mushrooms
King Oyster and Yunzhi are 2 types of mushrooms that have high
medicinal and nutritional values Especially Yunzhi, a kind of mushroom that is studied deeply on its medicinal properties by a lot of scientists around the world There have been many published articles that announced the uses of Yunzhi such as for cancer treatment, anti HIV virus, immune enhancing effects Besides, the cultivation of King oyster and Yunzhi mushrooms have contributed to the use of all by-products from agricultural waste, contributing to environmental protection
Chapter 2 RESEARCH MATERIALS AND METHODOLOGY 2.1 Research Materials
2.1.1 Mushroom spawn : Including 6 spawns of King oyster mushroom
and 2 Yunzhi mushroom spawns of different origins, currently stored at the Center for Plant Biotechnology, Agricultural Genetics Institute
2.1.2 Materials and chemicals
* Materials used in DNA extraction
- 23 RAPD primers were used in the study of biodiversity of King oyster mushrooms, 10 RAPD primer used for Yunzhi mushroom
* The chemicals used in propagation experiments of spawns level 1 and intermediate level
+ Glucose, yeast extract, peptone, MgSO4.7H2O, KH2PO4, thiamine, originating from Merck Company
* Materials used in commercial spawn propagation experiments and
Trang 92.2.3 Propagation of liquid spawns of King oyster and Yunzhi mushrooms 2.2.4 Using liquid spawns to cultivate King oyster and Yunzhi mushrooms
2.3.2.3 Methods of data analysis
2.3.3 The research to propagate liquid spawns of King oyster and Yunzhi mushrooms
2.3.3.1 Experiments to propagate the first grade liquid spawns of King
oyster and Yunzhi mushrooms
Experiment 3 : Effect of temperature to the growth of King oyster and
Yunzhi Mushroom in the liquid medium
Experiment 4 : Effect of pH to the growth of King Oyster and Yunzhi mushrooms in the liquid medium
Experiment 5 : Effect of the liquid medium components to the growth
of the first grade liquid spawns of King oyster and Yunzhi mushrooms
Experiment 6 : Effect of shaking mode to the growth of King oyster and Yunzhi mushrooms in the liquid medium
Experiment 7 : Effect of base rate of original spawns to the growth of King oyster and Yuanzhi mushrooms in the liquid medium
Experiment 8 : Effect of incubation time to the growth of King oyster and Yuanzhi mushrooms - liquid spawn types
2.3.3.2 Research to propagate King oyster and Yunzhi mushroom Liquid spawns at intermediate Grade
Experiment 9: Effect of aeration mode to the growth of King oyster
and Yunzhi mushrooms of intermediate grade
Experiment 10: Effect of the spawn implantation rate to the growth of King oyster and Yunzhi mushrooms of intermediate grade
2.3.3.3 Research to propagate commercial liquid spawn of King oyster and Yunzhi mushrooms
Trang 10Experiment 11: Effect of liquid environmental components to the growth of King oyster commercial mushrooms
2.3.4 The experiment using liquid spawns to the cultivate King oyster and Yunzhi mushrooms
Experiment 12: Evaluation of the impact of the original source of spawns and cultivating materials to the growth of King oyster and Yunzhi mushrooms
Experiment 13: Effect of age of liquid spawns to the growth of King oyster and Yunzhi mushrooms on farming materials
Experiment 14: Effect of quantity of commercial spawns to the growth and development of King oyster and Yunzhi mushrooms on farming materials
2.4 Methods of environment preparation
2.5 Experimental designing method
- The experiment was repeated 3 times arranged and carried out as follows: + For the first grade spawns: 5 flasks of 500ml /treatment, each flask contains 200ml liquid nutrient medium A total of 15 flasks /1 treatments + For the spawns of intermediate grade: 5 Duran flasks of 5000ml/treatment, each Duran flask containing 3000 ml liquid nutrient medium A total of 15 Duran flasks/1 treatment
+ For commercial spawn: 30 liters liquid nutrient medium / 1 bioreator / treatment
+ In the breeding period: each treatment, 100 bags/ experimental layout A total of 300 bags of materials / treatments; tracking targets growth of 50 bags / treatments, taking samples at 5 diagonal points, 10 bags /each point
* Observation criteria
- Monitoring the growth characteristics in pure culture
- Monitoring the growth characteristics in the liquid spawn period + Mycelia pellet size (KLC) (mm)
+ Mycelia biomass (gr/1000ml)
+ Mycelia pellet density
* King Oyster Mushrooms
- Monitoring the growth characteristics on their cultivation phase .
Trang 112.6 Methods of data processing
The study results are processed by biostatistics methods by Excel and software of IRRISTAT 4.0
2.7 Location and time of the experiments
- The experiment was conducted at the Center for Plant Biotechnology; Division of Genetic Technique - Agricultural Genetics Institute; Institute
of Microbiology and Biotechnology - Hanoi National University; Van Giang Mushroom Research Center, Van Giang - Hung Yen
- Duration of experiment: from 4/2008 - 4/2013
CHAPTER 3 RESULTS AND DISCUSSION 3.1 Study on some biological characteristics of King oyster and Yunzhi mushrooms
3.1.1 The growth and formation of fruiting bodies of King oyster mushrooms in pure culture
In this study, six strains of King oyster mushroom are incubated in
pure culture in different temperature ranges
Table 3.1 Effect of temperature on the growth of King oyster
mushroom mycelia system in pure culture
*Note:
I Thickness of mycelia system (unit)
3 unit thickness: very thick mycelia system
2.5 units thickness : thick mycelia system
Growth rate of the mycelia system (µm/h)
2 unit thickness : average mycelia system 1.5 units thickness : thin mycelia system
1 unit thickness : very thin mycelia system
The results showed that 5 varieties of King oyster mushrooms from E1 - E5 have many similar characteristics when cultured on agar in the studied temperature range The growth rate of mycelia system of E1 strain
is the fastest The features of strain E6 are clearly different with the above
Trang 125 strains on the growth duration and characteristics of fiber systems, the formation of fruiting bodies
3.1.2 The growth and formation of fruiting bodies of King Oyster mushroom on cultivating materials
Table 3.3 Some growth characteristics of the King oyster
(mm)
Length of
stalk (mm)
No of fruiting bodies / bag
Average weight
of fruiting
body (gr)
Yield /bag
(gr)
E1 34,6 41,6 148,8 1,6 141,6 197,6 E2 29,4 39,8 145,6 1,4 118,2 165,4 E3 22,7 31,4 126,4 2,1 62,7 145,7
3.1.3 Characteristics of King oyster mushroom spores
Spores of the fungus species were observed on a scanning electron microscope with magnification of 500-15,000 times In 7,500 times magnification, King oyster mushroom spores have ovoid shape put closely together attached to tapered basidium
3.1.4 The growth and formation of fruit bodies of Yunzhi mushroom in pure culture
The growth of the Yunzhi mushroom mycelium in pure culture were
recorded in Figure 3.32
Trang 13Figure 3:32: growth rate of Yunzhi mushroom mycelia system
in different temperature conditions
In different temperature range of the culture, the growth rate of Yunzhi
mushroom mycelia system differs, characteristics of mycelia system are also different During the whole process of rooting and the after the mycelium covered the medium surfaces, at any temperature, lighting conditions, there are not signs of formation of fruiting bodies in pure culture of the two Yunzhi mushroom strains
3.1.5 Studies on the growth and fruitingbody formation of Yunzhi on farming materials
The results show that: from February to April: The mycelia systems and fruiting bodies of 2 Yunzhi mushroom strains have good growth, less affected by diseases Two Yunzhi strains Tra-1 and Tra-2 have a lot of similiar characteristics: mycelia sprouting time, fruiting body formation time, fruiting body size, color, yields of fresh fruiting bodies of 2 strains in different times of the year of the study…
Table 3.6: Affects of cultivating time to fruiting body size and yield of
of mushroo
m cap
(mm)
Horizontal size of mushroom cap
(mm)
No of mushroom cap layers /penicle
Weight of fresh mushroom/ bag
400
Tốc độ sinh trưởng Tra
1 (µm/h)