Optimization of Extraction Technology of Polysaccharide of Tricholoma giganteum

Tricholoma giganteum also being called Tricholoma lobayense, belongs to Basidiomycotina, Hymenomycetes, Agaricales, Tricholomataceae and Tricholoma. It has high economic and medicinal values and is a kind of rare edible mushroom. 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. giganteum 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 extraction 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. giganteum was 12.92%.

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][17][18][19][20].Currently, the extraction methods of mushroom polysaccharides (MP) mainly include hot water extraction, ultrasonic extraction, enzyme extraction, alkaline extraction, acid extraction and microwave extraction [16,[21][22][23].Ultrasonic extraction technology has been widely used in the extraction of plant active ingredients.The main mechanism of ultrasonic extraction is due to cavitation generated by ultrasound [24].The advantage is to greatly increase the extraction yield of active 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 polysaccharide.This method is simple.But the parameters of its extraction 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 affected by various factors, such as the extraction temperature, the extraction time, the solvent-solid ratio and the volume of solvent used during precipitation.When 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-mathematical method which uses quantitative data in an experimental 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][28][29].The aim of the present 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.

Tricholoma giganteum
Tricholoma giganteum was provided by Guangzhou Yuewang Agricultural Limited Company, Guangzhou City, Guangdong Province, China.

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 temperature of 4˚C.

Extraction of Polysaccharides
One gram of mushroom powder was weighed for different 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 extraction was dried to calculate the yield.

Experimental Design and Statistical Analysis
The effect of the four variables at five variation levels (Table 1) on MP extraction ratio was studied to determine the optimum combination of the variables, using the U 5 (5 4 ) uniform design table (Table 2) to arrange the experiment (Table 3).
The model proposed for the target function Y (extraction ratio) is given below: The adequacy of the polynomial model was expressed by the multiple coefficient of determination, R 2 .The significance of each coefficient was determined by using F  Copyright © 2013 SciRes.PP 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].

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 quadratic regression analysis be used to identify the significant 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 derived from UD were as follows: Y is a coded fitting equation.x 1 , x 2 , x 3 , x 4 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 influence than other variables [13].F and p value, which determines the significance of each coefficient.The multiple coefficient of correlation (R = 0.99996) and the modified coefficient (Ra = 0.99994) indicated high agreement 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 significant variable was linear the temperature of extraction (x 1 ) and the interaction between the time of extraction (x 2 ) and Solid-liquid ratio (x 3 ).Their p values were 0.0001 which seem highly significant.That was followed by the interaction between the time of extraction (x 2 ) and Volume ratio of 95% ethanol (x 4 ).The p value was 0.0106 and less than 0.05, which seem significant.The experimental values, predicted values and error of fitting were listed in Table 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%) 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.

Verification of Results
In

Concluding Remarks
The present study had shown that the extraction ratio of the MP was optimized by using UD.The four independent variables, involved in the optimization, were the temperature of extraction, the time of extraction, the solventsolid ratio and the volume of 95% ethanol in precipitation.The UD result indicated that the variable with the largest effect was the temperature of extraction and the interaction between the time of extraction (x 2 ) and the solvent-solid ratio (x 3 ).That was followed by the interaction between the time of extraction (x 2 ) and the volume of 95% ethanol (x 4 ).Moreover, the optimal MP yield of 0.1292 g from 1 g mushroom powder of T. giganteum was obtained.

Acknowledgements
We gratefully acknowledge the financial support receiv-

Y b b x b x b x b x b x b x b x b x b x x b x x b x x b x x b x x b x
b 0 was offset term, b 1 , b 2 , b 3 and b 4 were related to the linear effect terms, b 5 , b 6 , b 7 , and b 8 were connected to the quadratic effects and b 9 , b 10 , b 11 , b 12 , b 13 and b 14 were associated with the interaction effects, x 1 , x 2 , x 3 , x 4 were the four variables.
According F and p values other terms of b 2 , b 3 , b 4 , b 5 , b 6 , b 7 , b 8 , b 9 , b 10 , b 11 and b 14 did not have the statistical significance.

Table 5 . Experimental values, predicted values and error of fitting.
PP 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 Guangdong Province (Grant No. 2011B020-303007); The Applied 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.