Xiaoqiong Ql，Li CHEN

School of Chemistry and Life Science，Hubei University of Education/Hubei Key Laboratory of Purification and Application of Plant Anti-cancer Active Ingredients，Wuhan 430205，China

An Orthogonal Experiment of Papain-Assisted Extraction of Polysaccharides from Agrocybe aegirit

Xiaoqiong Ql*，Li CHEN

School of Chemistry and Life Science，Hubei University of Education/Hubei Key Laboratory of Purification and Application of Plant Anti-cancer Active Ingredients，Wuhan 430205，China

［Objective］This study aimed to explore optimum conditions for papain-as-sisted extraction of polysaccharides from Agrocybe aegirit.［Method］Because enzyme is capable of destroying organizational structure of cells，papain was selected to cat-alyze cells to let polysaccharides out.An orthogonal experiment was carried out to investigate the effects of hydrolysis temperature，hydrolysis time，enzyme concentra-tion and solid to liquid ratio on extraction of polysaccharides from A.aegirit.［Result］Through the orthogonal experiment，the optimum papain-assisted extraction conditions were obtained：hydrolysis temperature，45℃；hydrolysis time，2 h；enzyme concen-tration 2%；solid to liquid ratio，1∶80.［Conclusion］This study will provide a reference for extraction of polysaccharides from A.aegirit.

Agrocybe aegirit；Papain；Orthogonal experiment；Polysaccharide extrac-tion

F ungal polysaccharides are a class of active substances iso-lated from fungi，mycelia and broth.Internationally，polysaccharides of edible fungi are known as"Biologi-cal Response Modifier"（BRM）［1］.Fun-gal polysaccharides have many physi-ological functions，such as reducing blood sugar，immunoregulation，anti-tumor，anti-oxidation，anti-virus，anti-mutagen，anti-aging and anti-inflam-matory［2-3］.In some edible fungi，such as Lentinus edodes，Agrocybe aegirit and Hericium erinaceus，polysaccha-rides are widely distributed.Xiong et al.［4］found that A.aegirit has higher content of polysaccharides compared with other edible fungi.The polysac-charides account for 30%-40%of dry weight of A.aegirit，and they are mainly composed of α-（1，3）-D-glucan，as well as mannose，galactose，rham-nose，xylose and arabinose［4］.Fungal polysaccharides have very extensive pharmacological effects，so they have great development prospects.

Currently，more than 200 kinds of bioactive polysaccharides have been isolated from nearly 300 kinds of fungi at home and abroad［5］.Experts predict-ed that the 21stcentury will be the era of polysaccharides.Obviously，how to improve the extraction rate and quality of polysaccharides from fungi has be-come the primary problem.Now the commonly used methods for extrac-tion of fungal polysaccharides include hot water extraction，acid extraction，alkaline extraction and enzymolysis method.Among them，hot water ex-traction method is simple but has low efficiency；although acid and alkaline extractions have high extraction rates but acid and alkaline have great ef-fects on polysaccharide activity；en-zymolysis method is relatively mild with higher yield，shorter extraction time and less damage.In addition to polysaccharides，A.aegirit also con-tains proteins and other components.Enzyme can destroy organizational structure of cell wall in vitro，and then the exposed cells could be broken up due to differentosmotic pressure. Next，polysaccharides can be extract-ed from water because they are solu-ble in water.In present study，papain was selected to extract polysaccha-rides from A.aegirit cells.

Jiang et al.［6］carried out an or-thogonal experiment to explore the optimum conditions for papain-assist-ed extraction of polysaccharides from A.aegirit using hot water method.The results showed that the extraction ratio was 1.47%under conditions of solid to liquid ratio of 1∶60，water temperature of 60℃and extraction time of 2 h；un-der the assistance of papain，the aver-age extractionratioreached 3.08% when the pH，solid to liquid ratio，mass ratio between A.aegirit and papain，hydrolysis temperature were set up to 5.0，1∶80，100∶2 and 45℃，respec-tively.Obviously，enzymolysis method is better than hot water extraction method.However，enzymatic reaction is affected by many factors，such as reaction time，reaction temperature，enzyme concentration，solid to liquid ratio and so on.Moreover，there are interactions among these factors. Therefore，an orthogonal experiment was carried out to determine the opti-mum reaction conditions for extracting polysaccharides from A.aegirit under the assistance of papain in this study. The results will provide a reference for the improvement of extraction ratio of polysaccharides and practical applica-tion of papain-assisted extraction method.

Materials and Methods

Materials and reagents

The used materials and reagents included dry A.aegirit，glucose，dis-tilledwater，ethanol（density：0.79g/ml），chloroform，n-amyl alcohol，concen-trated sulfuric acid，phenol and papain（3 U/mg）.The concentrated sulfuric acid is for the excellent level of pure，and the other reagents are all of ana-lytical grade.

Instruments and equipment

The used instruments and equip-ment included electric oven（202-type），high-speed multi-function mill（LFP-2500A）， visible spectrophotometer（722S，Shanghai Jinghua Science& Technology Instrument Co.，Ltd.），low speed automatic balance centrifuge（TD5-II，Changsha Pingfan Instru-ments and Meter Co.，Ltd.），circulating water pump （SHZ-D（III），Shang-hai Qingpu Huxi Instrument Factory）and digital thermostatwith three water tanks（HH-42）.

Methods

Preparationofstandardglucose solution The standard glucose was dried at 105℃ to constant weight. Subsequently，the dried standard glu-cose weighing 100.0 mg was dis-solved in distilled water to 100 ml. Thus the standard glucose solution with concentration of 1 mg/ml was prepared.

Preparation of standard curve

Certain amounts（0.5，1.0，1.5，2.0，2.5 and 3.0 ml）of standard glucose solu-tion were transferred to six volumetric flasks and diluted to 50 ml respective-ly.A certain volume （2.0 ml）of new standard glucose solution ateach concentration was mixed with a certain volume（1.0 ml）of 5%phenol.Subse-quently，a certain amount（5.0 ml）of concentrated sulfuric acid was added rapidly.After shaken for 5 min，the mixtures were bathed in boiling water for 15 min.And then，they were bathed in cold water for 30 min.The distilled water was used as the blank control. The absorbance of the mixtures was determined at 490 nm.

Orthogonal experiment of papain-assisted extraction of polysaccha-rides from A.aegirit According to the previous information，the optimum temperature for papain was 45-55℃，the optimum hydrolysis time was about 1.5 h，the solid to liquid ratio ranged from 1∶80 to 1∶40［7-8］，and the enzyme concentration was about 1.5%.Refer-ring to the data above，an orthogonal experiment was designed as follows（Table 1）.

Deproteinization The extracted coarse polysaccharides also contain proteins and other ingredients，which can be removed with Sevag method. The chloroform and n-amyl alcohol were mixed （v∶v，4∶1）.Subsequently，equivalent-volume coarse polysac-charides solution was added.Then the mixture was centrifuged.After that，the mixture was divided into three layers：polysaccharides layer （upper），pro-teins layer（middle）and organic sol-vent layer（lower）.Thus the polysac-charides were separated from proteins and other ingredients.

Determination of polysaccharides content A certain volume（2.0 ml）of each supernatant was transferred to a tube.Subsequently，a certain volume（1.0 ml）of 5%phenol was added to the tube and was mixed together.And then，a certain volume（5.0 ml）of con-centrated sulfuric acid was added to the tube.After mixed，the mixture was bathed in boiling water for 15 min，and then bathed in cold water for 30 min. The distilled water was used as the blank control.The absorbance of the mixtures was determined at 490 nm.

Table 1 Factors and levels of orthogonal experiment

Results and Analysis

Standard curve of glucose

The absorbance of the standard glucose solutions at various concen-trations was determined.With glucose concentration as the abscissa and ab-sorbance as the ordinate，the standard curve was fitted：

Table 2 An orthogonal experiment of papain-assisted extraction of polysaccharides from A.aegirit

Orthogonal experiment

Based on the absorbance，the yields of polysaccharides were calcu-lated.As shown in Table 2，the highest yield of polysaccharides was 5.5%，while the lowest was 1.4%.Based on the range values，the effect intensities of the four influencing factors ranked as enzyme concentration＞hydrolysis temperature＞solid to liquid ratio＞hy-drolysis time.The optimum combina-tion of the four factors was as follows：hydrolysis temperature of 45℃，hy-drolysis time of 2 h，enzyme concen-tration of 2%and solid to liquid ratio of 1∶80.Under the optimum extraction conditions，the yield of polysaccha-rides reached 5.5%.

Discussion

According to the results of previ-ous studies，the influencing factors were chosen as enzyme concentra-tion，hydrolysis temperature，hydroly-sis time and solid to liquid ratio，ex-cluding pH.On one hand，referring to previous study，the pH for papain is 3.5-9.0，and the optimum pH is around 7.The pH will not greatly affect experi-ment results.On the other hand，if the pH is adjusted，not only the follow-up separation and purification of polysac-charides will be harder，but also the activity of polysaccharides might be affected.The ranges of the four influ-encing factors were determined refer-ring to previous studies.Thus，an or-thogonal experiment was designed.

The effect intensities of the four factors ranked as enzyme concentra-tion＞hydrolysis temperature＞solid to liquid ratio＞hydrolysis time，indicating that the enzyme itself showed the greatest effect of experiment results. The main function of enzyme is to hy-drolyze cell wall and to let cellular con-tents out.Suitable enzyme concentra-tion will play an important part in cell disruption，which is an important pre-requisite for polysaccharides extrac-tion.So enzyme concentration has the greatest effect.Hydrolysis temperature determines the activity of enzyme.Un-der the optimum temperature，the ac-tivity of enzyme is highest，and the cell wall will also be broken best.The ef-fect intensity of solid to liquid ratio ranks third.At the solid to liquid ratio of 1∶80，A.aegirit shows the strongest absorption for hot water，thus the os-motic pressure is highest and the dam-age to cell is severest，providing favor-able conditions for the hydrolysis.Cer-tainly，the higher solid to liquid ratio is not necessarily ideal than the lower ra-tio.In the study，the solid to liquid ratio was ever enlarged by 10-20 times，but the polysaccharides were prone to be-ing lost，resulting in larger errors. Moreover，the longer dilution time also had higher requirements for experi-mental instruments and equipment，as well as concentration conditions.Since enzymatic reaction is very rapid，the hydrolysis time shows the weaker ef-fect.As the enzymatic reaction is pro-ceeding，the enzyme concentration becomes smaller and smaller，so the reaction speed also becomes slower.

Conclusions

In this study，the optimum condi-tions for papain-assisted extraction of polysaccharides from A.aegirit were explored.Under condition of constant pH，the effects of solid to liquid ratio，hydrolysis temperature，enzyme con-centration and hydrolysis time on ex-traction rate of polysaccharides from A.aegirit were discussed.The results showedthatenzymeconcentration had the greatest effect on polysac-charides yield，followed by hydroly-sis temperature，solid to liquid ratio and hydrolysis time.The determined optimum conditionsforpapain-as-sisted extraction of polysaccharides from A.aegirit were as follows：hydrol-ysis temperature of 45℃，hydrolysis time of 2 h，enzyme concentration of 2%and solid to liquid ratio of 1∶80.Un-der the optimum extraction conditions，the polysaccharides yield reached 5.5%.

［1］FANG YH（方一泓），YU P（余萍），LI W（李薇），et al.Isolation，purification and im-munological activities of a polysaccha-ride fraction from Agrocybe aegerita fruit bodies（茶樹菇子實(shí)體多糖的分離純化和免疫活性研究）［J］.Acta Edulis Fun-gi（食用菌學(xué)報），2006，13（4）：63-68

［2］ZHAO Y（趙艷）.Study on extraction and properties of polysaccharides（菇類多糖提取及性能研究）［D］.Shijiazhuang：Hebei University of Science and Tech-nology（石家莊：河北科技大學(xué)），2013.

［3］LI L（李磊），WANG WG（王衛(wèi)國）.Re-search progress of fungal polysaccha-rides on the pharmacological actions and its extraction and purification（真菌多糖藥理作用及其提取、純化研究進(jìn)展）［J］.Journal of Henan University of Technology（Natural Sciences）（河南工業(yè)大學(xué)學(xué)報 （自然科學(xué)版）），2008，29（2）：87-92.

［4］XIONG F（熊芳），SUN SJ（孫淑靜），XIAO Y（肖穎），et al.The comparison of the polysaccharides obtained from six dif-ferent edible fungus mycelia（6種食用菌菌絲體多糖含量的比較）［J］.Food Re-search and Development（食品研究與開發(fā)），2012，33（8）：27-31.

［5］MAO HL（毛慧玲），SHU YH（束盈慧），YANG W（楊文），et al.Study on the ex-traction process of polysaccharide from Agrocybe cylindracea（茶樹菇多糖提取工 藝 的 優(yōu) 選 ）［J］.Chinese Herbal Medicines（中藥材），2007，30：217-220.

［6］JIANG N（姜寧），LIU XP（劉曉鵬），XIAO Q （肖強(qiáng)），et al.Papayotin-assisted ex-traction of polysaccharides from Agrocybe aegirit（木瓜蛋白酶酶解輔助提取茶樹菇多糖的研究）［J］.China Brewing（中國釀造），2012，31（1）：109-121.

［7］ZHOU Y（周瑤），MAO SH（毛淑紅），SUN Q （孫祺），et al.Study on screening of composite enzyme in extraction of polysaccharides from Agrocybe aegirit（茶樹菇多糖提取的復(fù)合酶制劑篩選及工藝研究）［J］.Food Research and De-velopment（食品開發(fā)與研究），2013，34（2）：73-76.

［8］MAO HL（毛慧玲），SHU YH（束盈慧），YE ZJ（葉子堅），et al.Extraction methods of polysaccharides from Agrocybe aegirit（茶樹菇多糖提取方法）［J］.Journal of Nanchang University（Science&Tech-nology）（南昌大學(xué)學(xué)報 （理科版）），2006，30（1）：50-72.

Responsible editor：Tingting XU

Responsible proofreader：Xiaoyan WU

木瓜蛋白酶提取茶樹菇多糖的相關(guān)因素的正交試驗(yàn)研究

齊小瓊*，陳 麗 （湖北第二師范學(xué)院化學(xué)與生命科學(xué)學(xué)院/植物抗癌活性物質(zhì)提純與應(yīng)用湖北省重點(diǎn)實(shí)驗(yàn)室，湖北武漢430205）

［目的］探索木瓜蛋白酶提取茶樹菇多糖的最佳反應(yīng)條件。［方法］利用酶能夠破壞細(xì)胞的組織結(jié)構(gòu)的原理，選用木瓜蛋白酶酶解細(xì)胞獲取多糖。同時考慮到影響酶作用的幾個因素，利用正交法研究了木瓜蛋白酶在4個因素（酶解溫度、酶解時間、酶濃度和料液比）3水平下的提取效果。［結(jié)果］正交試驗(yàn)確定了木瓜蛋白酶提取茶樹菇多糖的得率最高的是：酶解溫度為45℃、酶解時間2 h、酶解濃度為2%、料液比1∶80，在該提取條件下，茶樹菇多糖的得率為5.5%。［結(jié)論］正交試驗(yàn)結(jié)果確定了木瓜蛋白酶法提取茶樹菇多糖的最佳反應(yīng)條件。

茶樹菇多糖；木瓜蛋白酶；正交實(shí)驗(yàn)；多糖提取

國家自然科學(xué)基金青年科學(xué)基金項(xiàng)目（31201242/C130405）；湖北省教育廳科學(xué)研究計劃項(xiàng)目（Q20143004）。

齊小瓊（1981-），女，山東昌邑人，講師，博士，主要從事分子進(jìn)化遺傳研究。*通訊作者，E-mail：108816564@qq.com。

2015-05-24

Supported by Youth Fund of National Natural Science Foundation of China（31201242/ C130405）；Science Research Program of Education Department of Hubei Province（Q20143004）.

*Corresponding author.E-mail：108816564@qq.com

Received：May 24，2015 Accepted：September 7，2015

修回日期 2015-09-07