MINISTRY OF EDUCATION AND
TRAINING CAN THO
UNIVERSITY
SUMMARY OF DOCTORAL THESIS
Specialization: Biotechnology
Code: 94 20 201
HO THI THU BA
COLLECTING, SELECTING AND THE
RESEARCHING THE CULTIVATION OF A WILD
EDIBLE MUSHROOM AND A WILD MEDICINAL
MUSHROOM IN THE THAT SON REGION,
AN GIANG PROVINCE
Can Tho, 2019
THIS STUDY WAS COMPLETED AT
CAN THO UNIVERSITY
Scientific supervisor: Assoc. Prof. Dr. TRAN NHAN DUNG
Dr. BUI THI MINH DIEU
The dissertation was defended at the university
examination committee
At.……………………………………….,
University
Can
Tho
At… . hour ….… ,on date……..month…..…. year……
Referee 1:
Referee 2:
Referee 3:
The dissertation is available at Libraries:
1. Central library of Can Tho University.
2. National library of Vietnam.
3.
PUBLISHED PAPERS
1. Ho Thi Thu Ba, Tran Nhan Dung, Trinh Tam
Kiet and Truong Tran Thuan, 2017. Propagation of
Ganoderma applanatum mushroom originated from Tinh
Bien, An Giang Journal of Vietnam Agricultural Science
and Technology. 102-105 (in Vietnamese).
2. Ho Thi Thu Ba, Tran Nhan Dung and Truong
Tran Thuan, 2018. Surveying subchronic toxic of
meshima wild mushroom (Phellinus sp.) in white mouse.
Journal of Vietnam Agricultural Science and Technology.
50-53 (in Vietnamese).
Chapter I
INTRODUCTION
1.1 Necessity of the dissertation
All of useful microorganisms are used, the closest to
humans being mushrooms. Mushroom is an eukaryote, nonchlorophyll, heterotrophic. In the five-gender classification
system, fungi rank the third in line with plants and animals
(Tran Van Mao, 2004). The technology of breeding, cultivating
and processing mushrooms have now imported from Japan,
Taiwan and other European countries. In process of research
and production, there have been changes to suit the natural and
social conditions of Vietnam (Nguyen Huu Dong et al., 2000).
Vietnam is an agricultural country, and there are some
conditions for developing mushroom cultivation, especially in
the southern provinces. Materials source, abundant labor and
weather, the climate is almost constant throughout the year,
mushroom can be provided throughout the four seasons
(Unesco Center for Knowledge, Culture and Community
Education, 2004). In addition, Vietnam is a mountainous
country with diverse biological resources. In the rainy season,
especially there are many kinds of mushrooms growing. The
Mekong Delta is blessed with That Son mountains, cooling
climate through the year with many kinds of edible and
medicinal mushrooms are valuable but have not been
cultivated. The facing global climate change, conservation of
this precious resource is necessary to do right. Due to the above
reasons, the thesis “collecting, selecting and the studying the
cultivation of a wild edible mushroom and a wild medicinal
mushroom in the That Son region, An Giang province” was
conducted.
1
1.2 The objective of the dissertation
1.2.1 The general objective of the dissertation:
Finding how to cultivate cultivateof edible and
medicinal mushroom collected in the That Son region, An
Giang province.
1.2.2 The specific objective of the dissertation:
(1) The collection of large mushroom species in That
Son region, An Giang province.
(2) The acute toxicity testing, fungal isolation on PDA
media, molecular identification of non-toxic fungal species.
(3) Determination of nutrient ingredient, medicinal
value of wild mushroom. Analysis of semi-chromic toxicity
and try out anti-cancer effect in vitro on blood cancer cells and
colon cancer of new wild herb mushroom.
(4) The studing of complete cultivation process process
for one edible mushroom and one selected medicinal
mushroom.
1.3 The object and limit of the study
The edible and wild mushrooms have been used by
local people but have not been researched and raised from That
Son region, An Giang province.
1.4 Time and place of study
Performed from October 2013 to August 2017 at Can
Tho University
1.5 The new contributions of the dissertation
The novelty of the thesis is the study in two local
mushroom, Lentinus Squarosolus and Phellinus sp. In which,
2
Inside Lentinus Squarosolus is a new edible mushroom that has
been successfully cultivated from wild mushrooms in nature,
which has led to the step by step establishment of the brand
name mushroom by Vietnam. The thesis also shows the
successful cultivation process of the high value medicinal
mushrooms without semi-chromic toxicity in test mouse.
Which has great scientific and community significance,
because of the wild mushroom that was first collected, isolated
and studied. In particular, Thuong Hoang fungus has the
potential to inhibit the proliferation and death of the colon
cancer cell line.
1.6 The layout of the dissertation
The dissertation is 193 pages, including an introduction,
a review of the literature, research methods, discussion results,
conclusions, recommendations and appendix. The thesis has 56
tables, 43 images and 150 references.
3
Chapter 3
RESEARCH METHODS
3.1 Experimental materials
The wild mushrooms were collected from That Son
region, An Giang province.
3.2 Content and method research
The dissertation had performed 4 general research
contents. In each main contents with the small contents, every
small content was corresponding to the method. Details are
given in below sections.
3.2.1 Collection of large mushroom in the That Son
mountain, An Giang province. Then, a local investigate and
find out which mushroom are usable and identify
mushroom.
3.2.1.1 The collecting large mushroom
Purpose: collect over 20 species of mushrooms on The
That Son mountain, An Giang province, set up a collection of
varieties mushroom.
The primary identification is based on mushroom
morphology, the basic procedure for the investigation of wild
mushrooms according to international standards (Trinh Tam
Kiet, 2011, Trinh Tam Kiet, 2012, Trinh Tam Kiet, 2013).
*Monitoring criteria: The substratum imagine where
large mushrooms are found; classification of mushrooms
collected (mushrooms are classified into one of four groups:
edible mushrooms medicinal mushroom, poisonous mushroom
and uncategorized mushrooms)
4
3.2.1.2 The investigation mushroom species that
people have ever used
Purpose: Primary selection of new mushroom was used
by local people but not yet cultivation in Vietnam
3.2.1.3 The identified based on the mushroom morphology
Collecting mushrooms were identified by external
morphological, the basic procedure for the investigation of
wild mushrooms according to international standards (Trinh
Tam Kiet, 2011, Trinh Tam Kiet, 2012, Trinh Tam Kiet, 2013).
3.2.2 Determination of acute toxicity levels, isolation and
sequencing ITS of edible and medical mushrooms
3.2.2.1 Determination acute toxicity of edible or
medicinal mushroom was selected by the local population
The process of determining acute toxicity level was
conducted according to toxicology determination procedure
issued by the Institute of Medicinal Materials, Ministry of
Health: methods of pharmacological effects of herbs drugs
(2006) and method of determining the acute toxicity of the
drug, Do Trung Dam in 1996
Selection of non-toxic mushrooms for next research
3.2.2.2 Isolation of selected non-toxic mushrooms
The mushroom without acute toxicity were isolated and
examining mycelium system on PDA media (Dung, 2003).
3.2.2.3 Identification of selected mushrooms by
molecular biology method
3.2.3 Determination of nutrient, pharmaceutical,
semi-chromic toxicity, testing the anti-cancer effects of
medicinal mushrooms with blood and colon cancer cells.
5
Purpose: selecting a high nutritional value edible
mushrooms and a highly medicinal mushroom. All
experiments in this content was conducted at the Institute of
Ginseng and Medicinal Materials of Ho Chi Minh City.
3.2.3.1 Determine nutritional ingredients of selected
mushrooms
Purpose: Determine nutritional ingredients of the four
mushroom that were selected. Thereby, selecting an edible and
a medicinal mushroom have high nutrition.
Procedure: Quantify the value components in
mushroom by thin layer chromatography and high pressure
liquid chromatography. Quantification of substances: fat,
protein, vitamins A, vitamin C, amino acids and trace minerals
(calcium, iron, zinc, potassium).
3.2.3.2 Determine of medical mushroom quantify
Purpose: Determine medical ingredients of the two
mushroom that were selected in content 2. Thereby, selecting a
medicinal mushroom have high nutrition.
Procedure: Quantify the value components in
mushrooms such as triterpenoid, sugar-free, reducing sugars
and polysaccharide. Compare triterpenoie, polysaccharide,
reducing sugar, sugar-free of some mushrooms to pick one
with higher levels to assessment semi-chromic toxicity.
3.2.3.3 Determine of mushroom semi-chromic toxicity
Purpose: Determine new wild mushroom was selected
without semi-chromic toxicity to study the culture process.
Procedure:
Determine
basic
nutritional
and
pharmacological criteria according to “Methods of studying the
effects of drugs from pharmacological Herbs” (Institute of
6
Medicine Materials - Ministry of Health, Science and
Technology Publishing, 2006).
3.2.3.4 Determine of cancer cell growth inhibitory of
mushroom extracts
Purpose: Determine the ability to inhibit the growth and
death of blood cancer cells and colorectal cancer cell by the
mushroom extracts
Procedure: The experiments were carried out on 96well plates. Monitoring criteria: the morphology of cancer cells
after treatment with mushroom extracts; cellular viability when
treated with different drug concentrations.
3.2.4 Contents 4: Research on the technology of
cultivating 2 mushroom were selected
Cultures were bred grade I, grade II, raise by Nguyen
Lan Dung process (2003)
Purpose: Complete the cultivation of high nutrition
edible mushroom and medicinal mushroom without semichromic toxicity. Procedure: examine various of environments
and different substrates in order to find the suitable
environment and substrate for the growth, filaments
development, forming the fastest and most effective fruit of the
two mushrooms were selected.
3.2.4.1 The procedure of cultivating edible mushrooms
a) Experiment 1: Determine the best environment for
passing edible mushrooms
Purpose: Select the best cultural medium to domesticate
edible mushrooms.
Monitoring criteria: observation the filaments
development on the medium and survey average rate of
7
filaments development on the medium by measure the depth of
the spread (cm) every Tuesday, Thursday, Saturday from the
date of the passes. Select the environment for fast and thick
filaments spread the most, continue to the next experiment
b) Experiment 2: Identify edible mushrooms propagation
environment.
Purpose: Selection of the most suitable propagation
medium
Monitoring criteria: dates spread 50% and 100%
mushrooms filaments on the spread environment. Select the
environment for fast and thick filaments spread most.
Experiment 3: Choose the best edible mushroom
cultivation environment
Purpose: Selection of suitable substrate for cultivating
edible mushrooms.
Monitoring criteria: dates spread 50% and 100%
mushrooms filaments on the suitable substrate. Select the
environment substrate for fast and thick filaments spread most.
3.2.4.2 The procedure of cultivating medical
mushrooms (The experiment replicate quite the edible
mushrooms)
a) Experiment 1: Determine the best environment for
passing medical mushrooms
b)Experiment 2: Identify medical mushrooms
propagation environment.
c) Experiment 3: Choose the best medical mushroom
cultivation environment
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Chaper IV
RESULT AND DISCUSSION
4.1 Collection of some mushrooms from Than Son
mountain (An Giang), investigate to local people to find
what mushrooms can be use, identification of the
mushrooms.
4.1.1 Mushroom collection
We have collected 28 wild mushroom sample at Bay
Nui religion in An Giang. These mushroom samples were
preliminarily identifyed and classifyed: 11 species are unknow,
7 medicinal mushrooms, 5 edible mushrooms and 5 poisonous
mushrooms.
Five edible mushrooms including Caesar's mushroom
Amanita caesarea (code 01), snow mushroom Tremella
fuciformis (code 09), Lentinus squaroslus (code 14); Tremella
cinnabarina (code number 18); bitter tylopilus mushroom
Tylopilus felleus (code 19).
Seven species of medicinal mushrooms include
Pycnoporus
sanguineus
(code
02),
Amauroderma
subresinosum (code 05), Van Chi mushroom-Trametes sp.
(Code 10), Thuong Hoang mushroom-Phellinus givus (Code
15), Linh Chi Tang mushroom-Ganoderma apllanatum (Code
20), Phellinus sp. (code 22), Amauroderma niger (code 25).
The unclassified fungal species (11 species) included
Gymnopilus penetrans (code 07), Lycogala epidendrum (code
08), Mycena niveipes (code 11), Xylaria polymorpha (code 12),
Thelephora genus (code 13) Cyathus striatus (code 16),
Daldinia concentrica (code 21), Polyporus ciliatus (code 24),
Cookeina sinensis (code 26), Moc Ba Hue unidentified
mushroom (code 27) and genus Coprinopsis (code 28).
9
4.1.2 Investigation of selected mushroom samples
28 collected wild mushroom samples were investigate
from local people. Five edible/medicinal mushrooms is
Ganoderma apllanatum, Trametes sp., Phellinus sp., Lentinus
squarosolus and Moc Ba Hue.
4.1.3 Identification of mushroom using morphological
characters
4.1.3.1 Ganoderma apllanatum Local Name: Linh Chi
Tang. Live on trunk, the filamentous system grows in fresh
dead plant tissue, was found in the Cam Mountain. The spore is
slightly rounded egg-shape.
Figure 4.1: Spore and Linh Chi Tang mushroom (code 20)
Linh Chi Tang is a mushroom has some layers, fruiting
body has fan-shape or circle, diameter about 6-100 cm, 3-8 cm
thick. The mushroom has no gloss bark on the pileus surface,
no stem, it has brown or dark brown color, concentric striated
lines can be prominent clearly or not, forming rough
protrusions on pileus surface. Ganoderma apllanatum is wood
mushrooms, keratinized, ruffled, the edge has the same color as
pileus and is curved with thickness about 0.5-1 cm.
4.1.3.2 Trametes sp.
10
Local name: Van Chi mushroom. Van Chi is a
mushroom without stem, growing on one side. Young fruiting
bodies are tumor rounded up shape, forming a rim with creamy
white canopy. Developed mushroom has accessory fruiting
form. Fruiting body has kidney-shape and concentric striated
lines which are stacked alternately like roofing tiles, pileus is
thin, smooth or lightly wizen, grows in clusters which are
about 40-50cm wide. There are no fungal fimbriae and
concentric opalescent rings on cap surface. Lamella has tiny
2
tubes whose density is about 4-5 tube/mm . The tubes are
round or lightly round.
Figure 4.2: Van Chi mushroom (code 10)
4.1.3.3 Phellinus sp. Local name: Thuong Hoang
Thuong Hoang mushroom grows on alive tree trunk, at
a height of about 5-10m. It only grows deep in the forest and
o
high up in mountain where the temparature is about 24-27 C.
Fruiting body has tube layers, tissue, rusty iron-color tube.
There is no stem attach to fruiting body.
Figure 4.3: Thuong hoang mushroom and its spore (code
22)
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The mushroom doesn’t form a cluster and it’s soft
wood mushroom. Upper surface of the mushroom is dark
brown or black, has drain, hard bark with dark brown color and
some deep crack. The cap is initialy covered by a yellow velvet
coat, becoming darker to black with many small bulges,
cracking into small areas.Edge of pileus is yellow-brown, and
young fruiting body has yellow color inside, becoming brown
rusty-iron-color latterly. Based on the external characteristics,
the species is similar to Phellinus linteus, Phellinus igniarius
and Phellinus lamaensis.
4.1.3.4 Lentinus squarosolus Local name: Xoai
Figure 4.4: Dai mushroom (code 14) and its spores
Dai mushroom has white funnel-shaped cap, whose
diameter is about 2-15cm, its upper surface is covered by light
brown bristly coat. The mushroom’s white inside. The
mushrooms’ white gills embraces thin stems. The stems are 35cm long, opaque white, covered by scale, the mushroom
doesn’t have ring and volva. L. squarosolus usually appears in
from March to November. This mushroom grows individually
or in large clusters on the trunk or punk mango. The
mushrooms grow year-round, especially after rain, thrive in the
summer with wet weather. The spore has stick-shaped.
4.1.3.5 Moc Ba Hue Local name: Moc Ba Hue
12
Moc Ba Hue grow on plant roots, stems is in soil,
yellow heart-shaped fruiting bodies form a cluster on lie
ground. There are black dots on fruiting body. Mushroom
mycelium is also yellow. This is a relatively new species of
mushroom, there is no any description of the external
morphology as well as identification of the mushroom.
Figure 4.5: Moc Ba Hue mushroom (code 27)
4.2 Acute toxicity study, isolation of the mushrooms onf
PDA medium, identification of no-toxic mushroom species
by molecules technique.
4.2.1 Acute toxicity study of valuable collected mushrooms
Table 4.1: Maximum dose of mushrooms
Name
g dry mushroom/kg weight of
mice
Dmax(g)
Water extract
Alcohol extract
Linh chi tang
23,41
28,66
137,7
391,9
Thuong hoang
22,86
27,97
134,5
383,4
Van chi
23,42
28,80
132,9
388,2
Dai
23,51
29,72
111,1
338,9
Moc ba hue
23,26
29,27
111,3
332,1
13
Water extract
Alcohol extract
Treated of mushrooms did not show any visual
symptoms of toxicity after using these extracts in 72 hours.
Observing mice in 14 day following, no unusual
symptoms of the mice has been noted. The results show that
high concentrated extracts of Linh Chi Tang, Van Chi, Dai,
Moc Ba Hue mushroom are devoid of any adverse effects in
mice. Next, we isolated these mushroom in order to preserve
their breed.
4.2.2 Isolation of selected mushroom in 4.2.1
PDA medium was used for isolation of mushroom in
the same temperature and time conditions. The result indicated
that PDA medium has potential for isolating Linh Chi Tang,
Thuong Hoang, Van Chi, Dai mushroom, but the medium isn’t
appropriate for isolation of Moc Ba Hue mushroom. We
sequenced ITS to identify four species correctly in next
experiment.
4.2.3 ITS sequencing, species identification
4.2.3.1 Van Chi mushroom
The ITS sequence of Van Chi mushroom, which has
length 666 bps, share 93% of the DNA sequence with some
mushroom speices in Tramates genus according to datebase on
NCBI. According to Trinh Tam Kiet’s morphological
description (2012). Initial evaluation of the collected
mushroom sample is a species in Trametes genus.
4.2.3.2 Dai mushroom
The 567bps ITS sequence of Dai mushroom is 96%
similar to IST sequence of Lentinus squarrosulus, according to
database on NCBI. Initial evaluation of the mushroom
collected was one of the Lentinus squarrosulus species.
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According to Trinh Tam Kiet’s morphological description
(2012), we suggested that the collected mushroon in An Giang
is Lentinus squarrosulus
4.2.3.3 Linh Chi Tang mushroom
The 631bps ITS sequence of Linh Chi Tang mushroom
is 97% similar to IST sequence of Ganoderma applanatum,
according to database on NCBI. According to morphological
description of Trinh Tam Kiet (2012), we suggested the
collected mushroom sample in An Giang province is
Ganoderma applanatum speices.
4.2.3.4 Thuong Hoang mushroom
The ITS sequence of Thuong Hoang mushroom, which
has length 666 bps, share 92% of the DNA sequence with
Phellinus linteus according to datebase on NCBI. Combine
with morphological analysis, we suggested that the collected
mushroom sample is a species belong to Phellinus genus, the
mushroom is temporarily called Phellinus sp.
Based on two researches, four species of mushrooms
were selected for further study: Dai mushroom - Lentinus
squarrosolus, Linh Chi Tang mushroom Ganoderma
applanatum, Thuong Hoang mushroom Phellinus sp. and Moc
Ba Hue mushroom.
4.3 Determination of nutrient composition, pharmacology,
subchronic toxicity and anticancer effect of these
mushrooms on blood cancer and colon cancer cell line
4.3.1 Determination of nutrient
pharmocology of selected mushroom
composition
and
We used HPLC for determining chemical compounds
in these selected mushrooms.
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Table 4.2: Nutrient composition and pharmocology of
mushroom
Composit
ion
Name
h m l ng x c
nh g
mushroom
Moc ba
hue
Dai
Thuong
hoang
Linh chi
tang
Lipid
7,01
vt
indefined
indefined
mg/kg
Protein
0,79
0,75
indefined
indefined
mg/kg
4Hydroxyprolin
193
undetected
376
186
mg/kg
Acid aspartic
624
695
2510
891
mg/kg
Acid glutamic
759
693
1780
767
mg/kg
Alanin
68,8
72,8
1460
623
mg/kg
Cystine
1470
undetected
355
338
mg/kg
Glycin
419
331
1810
566
mg/kg
Histidin
176
169
520
259
mg/kg
Isoleucin
106
113
829
421
mg/kg
Leucin
439
432
1480
671
mg/kg
Lysin
172
309
540
349
mg/kg
Methionine
237
262
undetected
103
mg/kg
Phenylalanin
244
245
658
470
mg/kg
Prolin
649
495
1470
550
mg/kg
Serin
245
313
1550
623
mg/kg
Threonin
507
537
1610
563
mg/kg
Tyrosin
117
165
158
193
mg/kg
Valin
298
248
1190
494
mg/kg
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