Pharmacology & Pharmacy, 2013, 4, 1-5
doi:10.4236/pp.2013.45A001 Published Online August 2013 (http://www.scirp.org/journal/pp)
1
Optimization of Extraction Technology of Polysaccharide
of Tricholoma giganteum
Meihua Mo*, Shenghua Hu, Xuefeng Xu, Ziying Ma, Yan Ni, Yaowu Wei, Jiang Nie
College of Food Science, South China Agricultural University, Guangzhou, China.
Received May 11th, 2013; revised June 11th, 2013; accepted July 15th, 2013
Copyright © 2013 Meihua Mo et al. This is an open access article distributed under the Creative Commons Attribution License,
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
ABSTRACT
Tricholoma giganteum also being called Tricholoma lobayense, belongs to Basidiomycotina, Hymenomycetes, Agari-
cales, Tricholomataceae and Tricholoma. It has high economic and medicinal values and is a kind of rare edible mush-
room. Modern pharmacological studies have shown that T. giganteum have antitumor, antioxidant, antimicrobial and
free radicals scavenging activity, and it can inhibit high blood pressure and HIV-1 reverse transcriptase. In this study,
the technologies of isolation and purification of polysaccharides of T. giganteum were systematically studied. T. gigan-
teum polysaccharides were obtained by hot-water extraction method. The infection of the extraction temperature, the
extraction time, the solvent-solid ratio and the volume of solvent used during precipitation on the polysaccharides ex-
traction ratio were studied. The extraction process was optimized by the uniform design (UD) method on the basis of
the results of the single-factor experiments. The optimum extraction conditions of the mushroom polysaccharides (MP)
were as follows: Extraction temperature was 100˚C; extraction time was 3 hours; solvent-solid ratio was 20:1 and 5
times volume of 95% ethanol. The yield of polysaccharides of T. giganteu m was 12.92%.
Keywords: Polysaccharide; Tr i c holoma gig a nt e um; Extraction; Uniform Design
1. Introduction
Tricholoma giganteum, also known as T. lobayene and
Macrocybe giganteum, distributes in the pantropical re-
gions such as Asia, Africa, etc. and mainly distributes in
USA, Japan, China, India, and Korean etc. [1-5]. It be-
longs to Basidiomycotina, Hymenomycetes, Agricales,
Tricholomataceae, Tricholoma. It has high economic and
medicinal values. Its fruit bodies are rich in protein, poly-
saccharides, dietary fiber, mineral salts, vitamins and
other healthful substances [6]. Modern pharmacological
studies have shown that T. giganteum have antitumor, an-
tioxidant, antimicrobial and free radicals scavenging ac-
tivity. It has a novel angiotensin I-converting enzyme in-
hibitory peptide and can inhibit HIV-1 reverse transcrip-
tase [7-15]. T. giganteum has received considerable at-
tention in recent years because of the nutritional and
health protective value.
Macromolecular compounds with important biological
function. MP has the biological effects of detoxification,
anti-oxidation, reducing blood pressure, reducing blood
lipids, enhancing immune and lowering cholesterol levels
[9,16-20]. Currently, the extraction methods of mush-
room polysaccharides (MP) mainly include hot water
extraction, ultrasonic extraction, enzyme extraction, al-
kaline extraction, acid extraction and microwave extrac-
tion [16,21-23]. Ultrasonic extraction technology has
been widely used in the extraction of plant active ingre-
dients. The main mechanism of ultrasonic extraction is
due to cavitation generated by ultrasound [24]. The ad-
vantage is to greatly increase the extraction yield of ac-
tive ingredients, reduce the extraction time and avoid the
destruction of the active ingredients [25]. But it is not
suitable for practical application because of its high cost.
Enzyme extraction can hydrolyze the cellulose, pectin
and crude protein, and break down the cell walls. It is a
more advanced and effective extraction method [26].
Water extraction is the traditional method of polysaccha-
ride. This method is simple. But the parameters of its ex-
traction only remain in the single factor tests and ignore
the process of MP interaction between the factors. The
extraction of polysaccharides from mushroom may be af-
fected by various factors, such as the extraction tem-
perature, the extraction time, the solvent-solid ratio and
the volume of solvent used during precipitation. When
*Corresponding author.

Optimization of Extraction Technology of Polysaccharide of Tricholoma giganteum
2
many factors and interactions affect the desired result,
the uniform design (UD) method is an effective tool for
finding their optimal values. UD is a statistical-mathe-
matical method which uses quantitative data in an ex-
perimental design to determine and to solve multivariable
equations in order to optimize processes or products. It
was proposed by Fang [27], based on quasi-Monte Carlo
method or number-theoretic method. It is precisely such
a technique for experimental designs, emphasizing the
uniformity of space filling in experimental domain. More
recently, theoretical results from the relationship between
the uniformity of the experimental points in the domain
and some advanced properties of experimental designs
have also provided strong support to this concept. One
can even simply use uniformity as a criterion to obtain
better orthogonal designs. These processes are affected
by numerous variables and it is necessary to select those
that have major effects as well as to identify their levels.
It is widely applied for different purposes in chemical
and biochemical processes [27-29]. The aim of the pre-
sent work is to study the effects of the temperature of
extraction, the time of extraction, the solvent-solid ratio
and the volume of solvent used during precipitation on
the MP extraction ratio as well as to optimize extraction
conditions by using the UD.
2. Materials and Methods
2.1. Tricholoma giganteum
Tricholoma giganteum was provided by Guangzhou Yue-
wang Agricultural Limited Company, Guangzhou City,
Guangdong Province, China.
2.2. Preparation of Mushroom Powder
The fruit-bodies of T. giganteum were collected from the
mushroom plant and were cleaned. The mushroom head
was cut. The rest of the fruit-bodies were washed and
dried at 50˚C, then ground in a grinder and sieved with
80 mesh. The mushroom powder was kept at the tem-
perature of 4˚C.
2.3. Extraction of Polysaccharides
One gram of mushroom powder was weighed for differ-
ent optimization tests including extraction temperature,
extraction time, solvent-solid ratio, Crude water-soluble
polysaccharides were extracted with pure hot water and
precipitated with 95% ethanol, yielding the MP. The ex-
traction was dried to calculate the yield.
2.4. Experimental Design and Statistical Analysis
The effect of the four variables at five variation levels
(Table 1) on MP extraction ratio was studied to deter-
mine the optimum combination of the variables, using
the U5 (54) uniform design table (Table 2) to arrange the
experiment (Table 3).
The model proposed for the target function Y (extrac-
tion ratio) is given below:
22
011223344516
22
7384912 10131114
122 313 24143 4
Ybbxbxbxbxbx bx
bx bx bxxbxxbxx
bxx bxx bxx
 
 

2
(1)
where b0 was offset term, b1, b2, b3 and b4 were related
to the linear effect terms, b5, b6, b7, and b8 were con-
nected to the quadratic effects and b9, b10, b11, b12, b13 and
b14 were associated with the interaction effects, x1, x2, x3,
x4 were the four variables.
The adequacy of the polynomial model was expressed
by the multiple coefficient of determination, R2. The sig-
nificance of each coefficient was determined by using F
Table 1. Levels of the variables tested in the Uniform
design.
Levels of the variables
Levels No.Extraction
temperature
(˚C)
x1
Extractio
n time
(h)
x2
solvent-
s
olid
Ratio
(ml/g)
x3
the volume ratio
of 95% ethanol
(ml/mg)
x4
1 80 3 20:1 3:1
2 85 4 30:1 4:1
3 90 5 40:1 5:1
4 95 6 50:1 6:1
5 100 7 60:1 7:1
Table 2. U5 (54) Uniform design table.
Levels No.
Tests x1 x
2 x 3 x 4
1 1 2 3 4
2 2 4 1 3
3 3 1 4 2
4 4 3 2 1
5 5 5 5 5
Table 3. Experimental scheme of UD and results.
Levels of the variables
Tests
No.
Extraction
temperature
(˚C)
x1
Extraction
time
(h)
x2
solvent-
s
olid
ratio
(ml/g)
x3
Volume
of 95%
ethanol
x4
Extraction
ratio
(%)
Y
1 80 4 40:1 6:1 10.55
2 85 6 20:1 5:1 10.93
3 90 3 50:1 4:1 11.90
4 95 5 30:1 3:1 12.34
5 100 7 60:1 7:1 11.92

Optimization of Extraction Technology of Polysaccharide of Tricholoma giganteum 3
value and P value. The optimum condition was verified
by conducting experiments under these conditions. The
result was compared with the model predictions and the
test which the extraction ratio was the highest in Table 3.
Statistical analysis was performed by using the DPS data
processing system [30].
3. Results and Discussion
3.1. Results of UD
The scheme and results of MP extraction are listed in the
last column in Table 3. The data were analyzed using the
DPS data processing system. It is suggested that a quad-
ratic regression analysis be used to identify the signifi-
cant variables, relationship coefficient and the model
parameters. The model can be used to gain the optimum
conditions of the extraction process and predict the MP
extraction ratio [28].
The regression equation representing the relationship
between MP extraction ratio and the test variables de-
rived from UD were as follows:
123
2.8932 0.10390.00030.0254Yxxx 24 xx (2)
Y is a coded fitting equation. x1, x2, x3, x4 were the four
variables. When the p value is less than 0.05, it shows
that this term is significant, on the other hand, when the p
value is less than 0.01, it shows that the term is highly
significant and that means this term had a greater influ-
ence than other variables [13]. F and p value, which de-
termines the significance of each coefficient. The multi-
ple coefficient of correlation (R = 0.99996) and the mo-
dified coefficient (Ra = 0.99994) indicated high agree-
ment between experimental and predicted values of the
MP extraction ratio in this experiment. The p value was
0.0018 and less than 0.01, F value was 166666.4. This
proved that the regression equation was reliable.
The corresponding variables will be more significant if
the absolute t value becomes larger and p value becomes
smaller. According to the regression terms coefficient t
analysis result (Table 4) indicated that the most signifi-
cant variable was linear the temperature of extraction (x1)
and the interaction between the time of extraction (x2)
and Solid-liquid ratio (x3). Their p values were 0.0001
which seem highly significant. That was followed by the
interaction between the time of extraction (x2) and Vol-
ume ratio of 95% ethanol (x4). The p value was 0.0106
and less than 0.05, which seem significant. According F
and p values other terms of b2, b3, b4, b5, b6, b7, b8, b9, b10,
b11 and b14 did not have the statistical significance.
The experimental values, predicted values and error of
fitting were listed in Tab le 5. The biggest error of fitting
was just 0.0017, it means that the regression equation fit
very well.
Mathematic model simulation analysis was conducted
to find out the maximum extraction ratio of MP (12.92%)
Table 4. Regression terms coefficient t analysis result.
Regression
terms Partial Correlation Coefficient t value Level of Significance (p)
x1 1 377.8397 0.0001
x2 × x3 −0.9947 9.6466 0.0106
x2 × x4 −1 133.9125 0.0001
Table 5. Experimental values, predicted values and error of
fitting.
Tests No.Experimental
values
Predicted
values Error of fitting
1 10.55 10.5510 −0.0010
2 10.93 10.9293 0.0007
3 11.90 11.8983 0.0017
4 12.34 12.3415 −0.0015
5 11.92 11.9199 0.0001
was obtained when the temperature is 100˚C, the time is
3 hours, solvent-solid ratio 20:1, precipitated with 5
times volume of 95% ethanol.
3.2. Verification of Results
In order to examine whether the equation gained from
UD could fit the relationship between extraction ratio
and each variable well, confirmatory experiment was
carried out. The extraction ratio gained from the optimal
extraction conditions reached 12.92%, comparing to the
predicted extraction ratio 13.00% was not different sig-
nificantly (p > 0.05) and comparing to the extraction ra-
tio 12.35% gained from test No. 4 in Table 3, they are
different significantly (p < 0.05). This result showed that
the technology of MP extraction obtained from UD was
accurate and reliable.
4. Concluding Remarks
The present study had shown that the extraction ratio of
the MP was optimized by using UD. The four independ-
ent variables, involved in the optimization, were the tem-
perature of extraction, the time of extraction, the solvent-
solid ratio and the volume of 95% ethanol in precipita-
tion. The UD result indicated that the variable with the
largest effect was the temperature of extraction and the
interaction between the time of extraction (x2) and the
solvent-solid ratio (x3). That was followed by the inter-
action between the time of extraction (x2) and the volume
of 95% ethanol (x4). Moreover, the optimal MP yield of
0.1292 g from 1 g mushroom powder of T. giganteum
was obtained.
5. Acknowledgements
We gratefully acknowledge the financial support receiv-

Optimization of Extraction Technology of Polysaccharide of Tricholoma giganteum
4
ed in the form of a research Grant (Grant No. 31272218)
from the National Natural Science Foundation of China;
the Planned Scientific and Technological Project of Guang-
dong Province (Grant No. 2011B020- 303007); The Ap-
plied Basic Research Foundation of Guangzhou (Grant
No. 2012J4100125); The national Spark Program project
(Grant No. 2012GA780042); and Guangdong Province
Agricultural Standards (Amendment) and standardization
research project.
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