Optimization of enzyme assisted extraction of polysaccharides from Poria cocos

Water-soluble polysaccharide was isolated from Poria cocus by enzyme-assisted method using orthogonal methodology. By single factor test and orthogonal group, the extraction conditions of watersoluble polysaccharide were investigated, such as liquid-solid ratio, temperature, time and pH. The optimum conditions for single factor were as follows Enzyme concentration (%), Extraction temperature oC, Extraction time/h, Extraction pH. The result revealed that 2% complex enzyme, remained most important factor of polysaccharide extraction, followed by temperature and pH. The liquid-solid ratio was 1:50, temperature was 40°C, time was 3.0 h, and pH was 5. The highest extraction rate of crude polysaccharide remained 4.14%. Results indicated that the complex-enzyme assisted remained best technique for extracting polysaccharide from P. cocus. It proved to be as highly effective as well as energy and time saving extraction techniques.


INTRODUCTION
Enzyme-assisted extraction (EAE) has been demonstrated to be effective in improving the yield of the model component (Li et al., 2006).Mushrooms have long been used as oriental medicine, as well as dietary supplements and in natural cosmetics (Kaneno et al., 2004).Poria cocus is a fungus that grows on the roots of pine trees, and used as most important traditional medicines in China and other Asian countries; moreover, it consists of culinary and medicinal properties such as anti-inflammatory, antitumor, complement activating, and immune stimulating activities (Kanayama et al., 1983;Lee and Jeon, 2003;Yasukawa et al., 1998;Yu and Tseng, 1996).P. cocus mainly contain polysaccharide (Jia et al., 2016;Wu et al., 2016).The major chemical constituents of P. cocus are polysaccharides, triterpenoids, ergosterol and proteins (Tai et al., 1992(Tai et al., , 1995a, b;, b;Wang and Wan, 1998;Yang and Bao, 2005).
Polysaccharide is a vital bioactive substance with some *Corresponding author.E-mail: huangwen@mail.hzau.edu.cnAuthor(s) agree that this article remain permanently open access under the terms of the Creative Commons Attribution License 4.0 International License physiological functions, such as immune, regulating cell growth and senescence (Jin and Xu, 2002).It can be widely used in medicine, health products and functional foods.Many mushrooms revealed a great range of the healthy properties of polysaccharides, being the most significant modulation of the immune system.Bioactive carbohydrates, including β-glucans, are generally examined as biological response modifiers (BRM) (Wasser, 2002).P. cocus useful in health products it can be used as raw material to produce various kinds of health products, such as a P. cocus polysaccharide capsule, tablet, electuary and so on.Hot water extraction of several mushrooms used in traditional Chinese medicine believed to be effective in the treatment of different diseases including many forms of cancer.
The enzymatic extraction process is manipulated by several factors, including enzyme concentration, temperature, pH, activators and/or inhibitors (Dam, 2004).While an enzyme is a protein-based catalyst, its rate of reaction depends on its concentration and has an optimum temperature at which shows maximum.Stirring speed also facilitates contact and the reaction between the substrate and the catalyst.An optimization of reaction time is important to ensure maximum extraction without degrading product quality.In order to get the pharmacological composition of mushroom presently, the extraction methods of mushroom polysaccharides (MP), generally include hot water extraction, ultrasonic extraction, enzyme extraction, alkaline extraction, acid extraction and microwave extraction (Jiang et al., 2008;Ghosh et al., 2005;Huang and Ning, 2010;Yu et al., 2009).These methods are regularly associated with higher temperature, extended extraction time and excessive energy consumption and lower yield.Whereas the enzyme assisted extraction with lower temperature, less time, less power consumption and high extraction yield is arising technology in the food industry.
It is therefore necessary to evaluate on the extraction method of polysaccharides; the comparative analysis about extraction method of P. cocus polysaccharides has seldom been studied.
Orthographic graph is a statistic method to make the complex work simple.In this investigation, (A) complex enzyme amount (B) extraction temperature (C) extraction time and (D) extraction pH were selected as four independent variables, combined by three levels which formed the orthographic graph (Table 1).Furthermore, all three Taka-diastase, alpha amylase and cellulose enzyme were separately investigated; also, four independent variables (A) enzyme amount (B) extraction temperature (C) extraction time and (D) extraction pH were selected (Tables 2, 3 and 4).This diagram was used to optimize the amount of extract and polysaccharides yield.The aim of this work is to examine the performance of a combination of cellulose, alpha amylase, Taka-diastase, and complex-enzyme-hydrolysis-assist) extraction from P. cocus.After a set of prospective tests, four parameters (enzyme ratio, extraction temperature, extraction time and extraction pH) were optimised in order to improve the yields of polysaccharides extraction.

MATERIALS AND METHODS
The samples of P. cocus and enzymes were obtained from Wuhan, Hubei Province, China.Other analytical grade reagents were obtained from the Sinopharm Chemical Reagent Co. Ltd (Shanghai, China).

Isolation of polysaccharides from Poria cocos using complex enzyme
The P. cocus were cut into pieces, ground by electric mill, and sieved through screens 60-80 mesh (LD-Y500A, Ding Shuai Hardware Products Co., Ltd.Zhejiang, China).Ten grams of the powder were mixed in 200 mL distilled water respectively, of 9 bottles in which the experiments were carried out.The amounts of complex enzyme were also added corresponding to Table 2.The bottles were labelled and pH for extraction were adjusted for all samples shown in Table 2.The bottles were placed in a water bath at the temperature of 40, 50 and 60°C at extraction time of 3 h, 2 h and 1.5 h, respectively.
The sample was filtered and concentrated to about one-fifth of the original volume in a rotary evaporator (Model RE-2000A Yarong Bio-instrument Shanghai, China) at 40°C.One part of the solution was dried into solid and powdered to calculate the amount of extract.Ethanol was added into the remaining part and kept at room temperature for 24 h.The precipitate was collected after centrifugation (4,000 r/min, 15 min), and then dried to obtain the desired crude polysaccharides (Thulasi et al., 2008).Furthermore, the same methods as mentioned above were applied for the Takadiastase, alpha amylase and cellulose enzyme and their amount of enzyme and pH were computed in Tables 3, 4 and 5 respectively.

Determination of amount of extract and polysaccharides yield
Glucose was accurately weighted 0.0200 g and dried in 105°C prior to dissolving in 250 mL and the standard solution was diluted to different concentrations making up the final concentration with pure water.Following that, 1.0 mL phenol and 5 mL sulfuric acid were added in 1.0 mL of glucose solution for each concentration, mixed by vortex and reacting samples for 10 min in boiling water bath and cooled at room temperature.The absorbance of each tube was measured at 490 nm with an ultraviolet-visible spectrophotometer (1.0 mL); distilled water was used as blank; standard curve was obtained with x-coordinate for concentration of glucose and ycoordinate for absorption value, and finally, based on the equation of glucose standard curve, the glucose contents of the samples were determined.Considering the dilution ratio, the amount of extract and polysaccharides yield was calculated by the following equation.

Complex enzyme extraction method
Results of amount of extract and polysaccharides yield with complex enzyme are shown in Table 5.On the basis of single factor experiments, four factors and three levels of L9 (3 4 ) orthogonal test were selected.Conditions of pH and temperature were modified in each experiment to match the optimum conditions of the measured enzyme.The experiments were carried out for complex enzyme 1.5%, extraction temperature at 40°C, extraction time of 3 h and extraction pH at 5. After this condition, the amount of extract was 4.14% and polysaccharides yield was 5.3%.Furthermore, the results of 9 samples demonstrated in Table 5 and Sample 4 shows the highest amount of extract with Sample 1 showing the higher polysaccharides yield, that is, 7.6%.In order to get the consequence of different independent variables and their levels on polysaccharides yield and amount of extract, the K factor along with R factor were calculated are presented in Table 5.

Cellulose enzyme extraction method
The results of the polysaccharides yield and the amount of extract with cellulose enzyme are shown in Table 6.On the basis of single factor experiments, four factors and three levels of L9 (3 4 ) orthogonal test were selected.The factor levels are revealed in Table 2.The result showed that highest amount of extract was 8.4%, polysaccharides yield of 2.60% was observed at 50°C with the cellulose enzyme amount of 0.835.Furthermore, the results of 8 samples showed that sample 4 show lowest amount of extract 5.6% and sample 3 show lowest polysaccharides yield 1.76%.In addition, to get the result of different independent variables and their levels on polysaccharides yield and amount of extract, the K factor and R factor were calculated and presented in Table 6.Whereas, K is the standard value of related results of every independent variable.The factors which have an impact on the polysaccharides yield in appropriate order were extraction complex enzyme amount >pH>extraction time > extraction temperature.

Alpha amylase enzyme extraction method
The results for the polysaccharides yield and amount of extract with alpha amylase are shown in Table 7.On the basis of single factor experiments, four factors and three levels of L9 (3 4 ) orthogonal test were selected (Table 3).The highest amount of the extract 7.6% was in Sample 2, moreover the higher polysaccharides yield 4.24% was found in sample 9 and extract were 6.5%.In order to get the effect of different independent variables and their levels on polysaccharides yield and amount of extract, the K factor along with an R factor were calculated as found in Table 7.The factors which have an impact on the polysaccharides yield in appropriate order were extraction enzyme amount >extraction temperature > extraction time > pH.

Taka-diastase enzyme extraction method
The results of the polysaccharides yield and the amount of extract with Taka-diastase are shown in Table 8.On the basis of single factor experiments, four factors and three levels of L9 (3 4 ) orthogonal test was selected.The factor levels were revealed in Table 4.The experiments were carried out, for example, condition of extraction Sample 1 was a complex enzyme amount of 1.5%, extraction temperature at 40°C extraction time of 3 h and extraction pH at 5. The result shows that the highest amounts was found in Sample 3 with extract of (7.4%), however, the high yield of polysaccharides (4.07%) was found in Sample 8.In order to get the significance of different independent variables and their levels on polysaccharides yield and amount of extract, the K and R factors were calculated as found in Table 8.The factors which have an impact on the polysaccharides yield in appropriate order were extraction temperature > extraction enzyme amount < extraction time > pH.

DISCUSSION
The use of medicinal mushroom extracts for the medicinal use is recognized and familiar in China, Japan, Korea, Russia and now increasingly in the USA (Mizuno et al., 1995).A number of methods have been developed to extract anti-cancer polysaccharides from mushroom fruitbodies, mycelium and liquid media (Mizuno, 1999).In this study, extraction condition of total polysaccharides for effective yield enzyme amount, extraction temperature, extraction time and extraction pH is investigated.According to results of the orthogonal trial, these four methods were evaluated.Recently, several methods of extracting polysaccharides from mushrooms are reported, such as microwave assisted extraction, water extraction, ultrasonic assisted extraction and some enzymatic assisted methods (Boroushaki et al., 2010;Dkhil et al., 2011;Radheed et al., 2003).The literature reported that the function of enzyme is to degrade cell wall constituents and discharge intracellular contents.Generally, a plant cell wall comprises cellulose, hemicelluloses and pectin, while flesh is main content for pectin and proteins.
Cellulases and pectinases can therefore be used for degradation of cell structure in the extraction process (Fernandes, 2010).It is observed from the results, that the extraction yield was decreased with the increasing of temperature from 40 to 50°C whereas there was no significant change found amongst all samples.According to Chunhua et al. (2014), enzyme assistant extraction method in polysaccharide extraction is an innovative research in recent years and has advantages over the conventional hot water extraction.Furthermore, complex enzyme hydrolysis method can be performed under low bath temperature (47°C) and the yield of the polysaccharide significantly improved to microwave-assist extraction and hot water extraction method (Chunhua et al., 2014).Enzyme assisted extraction is an advanced and efficient extraction method (Jiang et al., 2010).Moreover, the study was carried out by the single enzyme assist extraction method, the results of the cellulose, alpha amylase and Taka-diastase enzymes experiment as were shown in Tables 6, 7 and 8.However, in present study, the highest extraction yield amongst the three enzymes was 4.24% in the optimal conditions (50°C, 120 min,) with alpha amylase.In complex enzyme assay, extraction method and pH are the main factors which manipulated the effective extraction both for amount of extract and polysaccharides yield.pH can affect enzyme activity because every enzyme possesses their optimal pH, while the change of pH affects the structure of enzyme and enzymatic activity (Yin et al., 2011).Further study of Yin et al. (2011) explained the pH can affect enzyme activity as different enzymes have their own optimal pH.Study further revealed that extraction time remained a significant factor to affect the polysaccharides yield; similar findings are reported by Chunhua et al. (2014) in the extraction of polysaccharide from ganoderma lucidum.
The study further demonstrated that yield of polysaccharide increased with increase in temperature and amount of the extract enhanced with different enzymes.The correlated result was reported that due to a higher temperature, the polysaccharides matter leaved out from the mushroom particles into the solution (Li et al., 2006).Long extraction time observed plays a positive role in the production of polysaccharides (Ros et al., 2004).Multi enzymatic hydrolysis helps to split linkage and assists in releasing the polysaccharide, increasing the rate of extraction.Furthermore, the enzymehydrolysis-assist extraction showed its advantages in extraction conditions and great specificity for application.
In the meantime, enzyme hydrolysis assists extraction demonstrating its advantages in consideration for extraction conditions and extremely specific application.
As it seldom damages the molecular three-dimensional structures, it consequently maintains the bioactivity of mushroom polysaccharide.Furthermore, it also helps to progress the purity of polysaccharide.This result will be consistent with that previous experiment in which the polysaccharide was extracted from the fruit body of mushroom C. maxima (Yang et al., 2011).Multi enzymatic hydrolysis, help to break down the linkage and assist in discharging the polysaccharide and raise the extraction rate as reported by Chen et al. (2013).

Conclusions
Extraction pH is the most important factor which influenced the extraction efficiency both for amount of extract and polysaccharides yield.Enzyme-hydrolysisassist extraction remained effective.It can hydrolyze the cellulose, pectin and break down the cell walls.This study reports an effective extraction method for polysaccharide, the best optimal conditions were determined: complex enzyme amount of 2%, extraction temperature at 40°C, extraction time is 3 h and extraction pH is 5.While the best optimal conditions for the amount of extract were as follow: complex enzyme amount of 2%, extraction temperature at 60°C, extraction time 1 hr and extraction pH is 6.Furthermore best optimal conditions with alpha amylase assist extraction as follows for enzyme amount of 0.03896%, extraction temperature at 50°C, extraction time of 2 h, and extraction pH is 6.Further studies on the complex enzyme hydrolysis assist extraction and single enzyme necessitates is suggested for finding out the best extraction condition.
R refers to the result of extreme analysis.
R refers to the result of extreme analysis.