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Enhanced ganoderic acid Me (GAMe, an important antitumor triterpene) yield was attained with the medicinal fungus
Modern chemistry studies show that
Currently, submerged fermentation of
According to the culture media and fermentation conditions used for the maximum production of total crude GAs in our previous work [
In our earlier study, glucose, peptone and culture time were the key factors for GA production [
A central composite design (CCD) was used in the optimization of GAMe production. The ranges and the levels of the variables investigated in this study are given in
Experimental range and levels of the independent variables of ganoderic acid Me (GAMe) optimization.
Independent variables  Range and levels  

−1.682  −1  0  1  1.682  
Glucose,

36.59  40.0  45.0  50.0  53.41 
Peptone,

3.32  4.00  5.0  6.00  6.68 
Fermentation time,

265.91  300  13  400  434.09 
The results of CCD experiments for GAMe production are presented in
The central composite design matrix and the response of ganoderic acid Me (GAMe) of
Runs  Coded values  





1  −1  −1  −1  4.9 
2  −1  −1  1  9.9 
3  −1  1  −1  4.1 
4  −1  1  1  7.4 
5  1  −1  −1  6.9 
6  1  −1  1  10.5 
7  1  1  −1  5.1 
8  1  1  1  6.7 
9  −1.682  0  0  6.1 
10  1.682  0  0  8.1 
11  0  −1.682  0  7.0 
12  0  1.682  0  6.7 
13  0  0  −1.682  3.1 
14  0  0  1.682  11.2 
15  0  0  0  8.9 
16  0  0  0  8.8 
17  0  0  0  8.8 
18  0  0  0  8.5 
19  0  0  0  7.9 
20  0  0  0  8.2 
Analysis of variance (ANOVA) for the full quadratic model for optimization of ganoderic acid Me (GAMe) production in
Regression  DF  Sum of Squares  

Linear  3  63.2143  0.7614  34.16  <0.0001 ** 
Quadratic  3  10.0708  0.1213  5.44  0.0177 * 
Crossproduct  3  3.5738  0.0430  1.93  0.1886 
Total model  9  76.8589  0.9257  13.84  0.0002 ** 
Analysis of variance (ANOVA) for three factors for optimization of ganoderic acid Me (GAMe) production of
Factor  DF  Sum of Squares  Mean Square  

x1  4  8.3684  2.0921  3.39  0.0534 
x2  4  13.8737  3.4684  5.62  0.0123 * 
x3  4  60.1597  15.0399  24.38  <0.0001 ** 
** Significant at 0.01 level, * Significant at 0.05 level.
The significance of each coefficient was determined by Student’s
Results of regression analysis of a predictive polynomial model for optimization of ganoderic acid Me (GAMe) of
Parameter  DF  Coefficients estimated  Standard error  

Intercept  1  8.5169  0.3203  26.59  <0.0001 ** 

1  0.4586  0.2125  2.16  0.0563 

1  −0.6886  0.2125  −3.24  0.0089 ** 

1  1.9860  0.2125  9.34  <0.0001 ** 

1  −0.5021  0.2069  −2.43  0.0356 * 

1  −0.2875  0.2777  −1.04  0.3249 

1  −0.5905  0.2069  −2.85  0.0171 * 
1  −0.3875  0.2777  −1.40  0.1931  

1  −0.4625  0.2777  −1.67  0.1268 

1  −0.4844  0.2069  −2.34  0.0412 * 
** Significant at 0.01 level, * Significant at 0.05 level.
Equation (1) also reveals that culture time (
The 3D response surface is generally the graphical representation of the regression equation.
From the response surface plots, it is easy to understand the interactions between two nutrients and also to locate their optimum levels. It can be seen from
The surface plot of the combined effects of glucose and peptone on Y_{1} (GAMe) production. Fixed level: culture time = 0 (coded value).
The surface plot of the combined effects of glucose and culture time on Y_{1} (GAMe) production by
The surface plot of the combined effects of peptone and culture time on Y_{1} (GAMe) production by
Triplicate experiments were carried out to verify the availability and accuracy of the model (Equation (1)) for GAMe production in flask shake culture. The results are shown in
Verification results for ganoderic acid Me (GAMe) production under the optimized culture conditionby
Culture systems  Cell density (g·L^{−1})  GAMe content in Cell (μg/100 mg DW)  GAMe production in Cell (mg·L^{−1})  GAMe production in fermented broth (mg·L^{−1})  Final glucose concentration (g·L^{−1}) 

Flask culture  11.3 ± 0.2  42.7 ± 0.4  4.8 ± 0.7  7.6 ± 0.2  4.7 ± 0.3 
Bioreactor culture  9.1 ± 0.5  33.9 ± 0.6  3.1 ± 0.2  8.3 ± 0.7  4.3 ± 0.6 
Until now, GAs were mainly extracted from the solid cultivated fruiting bodies of
Stock cultures of
The bioreactor culture was carried out by a similar method described in our previous work [
Samples collected from flasks were filtered using a 40mesh stainless sieve and the mycelium was harvested. Mycelial biomass was collected by centrifuging the mycelium at 8,000 rpm for 15 min, washing the precipitated cells for three times with distilled water, and drying at 60 °C until it reached to a constant weight [
The GAMe yield (mg·L^{−1}) includes the contents of GAMe from both mycylia and fermented broth. The determination of GAMe was made by a similar method described in a previous work [
The preliminary trails indicated that glucose, peptone and fermentation time were the significant variables for GA production. Hence, these three variables were chosen to obtain the optimum levels. A central composite design (CCD) was used in the optimization of GAMe production.
The accuracy and general ability of the above polynomial model could be evaluated by the coefficient of determination
The SAS statistical package [
Enhanced production of individual GAMe, an important bioactive triterpene, by
Thanks the financially support by the National “948” Project of China (Project No. 2012411) and the National Natural Science Foundation of China (Project No. 30700552).