Enhanced polysaccharide production in mycelium of Ganoderma atrum by solid-state fermentation

Transcription

1 Mycosphere 7(6): (2016) ISSN Artcle Do /mycosphere/7/6/5 Copyrght Guzhou Academy of Agrcultural Scences Enhanced polysaccharde producton n mycelum of Ganoderma atrum by sold-state fermentaton Wen TC 1, Kang C 2, Wang F 2, Lang DQ 1 and Kang JC 1* 1 The Engneerng Research Center of Southwest Bo-Pharmaceutcal Resources, Mnstry of Educaton, Guzhou Unversty, Guyang, , Guzhou, Chna 2 Insttute of Bology, Guzhou Academy of Scences, Guyang, , Guzhou, Chna Wen TC, Kang C, Wang F, Lang DQ, Kang JC Enhanced polysaccharde producton n mycelum of Ganoderma atrum by sold-state fermentaton. Mycosphere 7(6), , Do /mycosphere/7/6/5 Abstract Polysacchardes are some of the most mportant boactve compounds produced by speces of Ganoderma. In ths study, effects of rato of materal to water, the lqud supplement medum ncludng carbon sources, ntrogen sources, and norganc salts were also studed for the polysaccharde producton n mycelum of Ganoderma atrum by sold-state fermentaton. The seed of G. atrum was noculated n 300 ml cylndrcal glass bottles contanng 60 g wheat gran at 28 C for 20 days. One-factor-at-a tme and an orthogonal desgn method were used to establsh the optmal lqud supplement medum for maxmum polysaccharde producton n mycelum. The results showed that the hghest polysaccharde producton was acheved wth rato of materal to water of 1:3, and the optmal lqud supplement medum contaned 15 g/l maltose, 10 g/l peptone, 1 g/l KCl and 1.5 g/l K 2 HPO 4 3H 2 O. The maxmum polysaccharde yeld n mycelum was 1.8 mg/g on 20 days. Ths method frst provdes an effectve way for obtanng polysacchardes n mycelum by sold-state fermentaton n G. atrum. The strateges used n ths study could be wdely practcal to other fermentaton processes. Key words culture condton mycelum polysaccharde Orthogonal desgn optmzaton Introducton Ganoderma (Polyporales, Basdomycota) s one of the most mportant Tradtonal Chnese Medcnal mushrooms wth a long hstory of use n the Orent (Gao et al. 2004, Zhang et al. 2014, Rchter et al. 2015). Pharmaceutcally actve compounds n Ganoderma frutng bodes and mycelum nclude adenosne, ganoderc acd, lectns, peptdes, protens, polysacchardes, terpenods and sterols (L & Wang 2006, Zong et al. 2012, Ne et al. 2013, Zhang et al. 2014, Hapuarachch et al. 2015). Polysacchardes and trterpenods are the major source of bologcal actvty and therapeutc (Lu & Zhang 2007). Ganoderma has been used as a medcnal agent responsble for mmunologcal regulaton (Chen et al. 2014, Sh et al. 2014) and antcancer/anttumor (L & Wang 2006, Zhao et al. 2010, Zhang et al. 2013, Yu et al. 2014, Hapuarachch et al. 2016), antoxdant (Chen et al. 2008), ant-fungus (L et al. 2012), ant-hepatts (L & Wang 2006), and ant-hyperlpdema (Meng et al. 2011, Ne et al. 2013). Submerged culture, sold-state fermentaton (SSF) and statonary lqud fermentatons are currently used for producng polysacchardes and trterpenods from Ganoderma (Tang & Zhong 2002b, Lee et al. 2003, Hseh & Yang 2004). Submerged fermentaton was consdered as a good Submtted 18 September 2016, Accepted 31 October 2016, Publshed onlne 14 November 2016 Correspondng Author: J-Chuan Kang e-mal jckang@gzu.edu.cn 757

2 method to ncrease mycelum, valuable metabolte, polysaccharde, and trterpenod producton (Lu & Zhang 2007). In prevous studes, ethyl acetate extracts from Eupolyphaga snenss and Catharsus molossus, methanol, ethanol, 1-propanol, 2-propanol, whey permeate and corn ol, were added to the medum for mproved polysaccharde producton by G. lucdum (Lu & Zhang 2007, Song et al. 2007, Huang et al. 2009). In sold-state fermentatons, t was consdered that several months were needed to cultvate the Ganoderma frutng bodes and that yeld of bomass would be low (Douanla-Mel & Langer 2009). Sources for sold substrate fermentatons, such as gran, sawdust or wood, straw, and tea waste are abundant (Yang & Lau 1998, Peksen & Yakupoglu 2009), SSF has therefore become an attractve alternatve to submerged culture fermentaton for specfc applcatons due to recent mprovements n reactor desgns (Couto & Sanromán 2006). In ths study, we optmzed fermentaton medum nutrents to mprove polysaccharde producton n mycelum of G. atrum for the frst tme. Ths s one of the most mportant speces of Ganoderma used for polysaccharde producton. We used a one-factor-at-a tme approach and an orthogonal desgn to mprove yelds. The effects of rato of materal to water, carbon sources, ntrogen sources, and norganc salts were studed by sold-state fermentaton n 300 ml cylndrcal glass bottles. Materals & Methods Mcroorgansm The stran of G. atrum was obtaned from the culture collecton of Huazhong Agrcultural Unversty (Hube, Chna). The stock culture was mantaned on potato dextrose agar (PDA) slants. The slants were noculated wth mycela and ncubated at 28 C for 10 days. Seed culture on wheat by sold-state culture The seed medum was prepared by mxng 60 g of wheat (It was soaked n dstlled water for 12h, and was draned for water on wheat gran surface) and 20 ml of dstlled water n a 300 ml cylndrcal glass bottle (nner dameter 7 mm, heght 12 mm) and the bottle was sealed by flm of polypropylene plastc. The medum was autoclaved for 30 mn at 121 C. The medum was cooled to room temperature and noculated wth three dscs (1 cm 2 ) from the PDA slants and ncubated at 28 C for 10 days n the dark. Sold-state fermentaton for mycelum The fermentaton medum was prepared by mxng 60 g of wheat (It was soaked n dstlled water for 12h, and was draned for water on wheat gran surface) and the lqud supplement medum (carbon sources, ntrogen sources and norganc salts) n a 300 ml cylndrcal glass bottle (nner dameter 7 mm, heght 12 mm) and the bottle was sealed by flm of polypropylene plastc. The medum was autoclaved for 30 mn at 121 C. The medum was cooled to room temperature and noculated wth 1 spoon wheat gran seed (The bomass weght of mycelum and wheat gran are 10 g) and ncubated at 28 C for 20 days n the dark. Extracton of polysaccharde n G. atrum mycelum and assay The experments were performed n 300 ml cylndrcal glass bottles for 20 days. Total fermentaton substrates contanng 60 g wheat and mycelum of G. atrum were mxed wth deonzed water, and were extracted twce by hot water extracton for one hour each tme. All extracts were fltered through gauze of sx-layer and fnally cooled. The cooled extracts were precptated wth two tmes volume of 95% (v/v) ethanol overnght n refrgerator at 4 C. All precptated samples were centrfuged at 2810 g for 10 mn. The rough polysaccharde n mycelum at the bottom of tubes were collected, and dred to a constant dry weght at 60 C. Fnally, the content of polysaccharde n mycelum was measured usng a phenol sulfurc acd method (Douanla-Mel & Langer 2009). 758

3 Orthogonal array for medum optmzaton In order to evaluate the nfluence of lqud supplement medum component (maltose, peptone, KCl and K 2 HPO 4 3H 2 O) on mycelum polysaccharde producton (MPP), L 9 (3 4 ) orthogonal array was used to evaluate. The L 9 (3 4 ) orthogonal array was selected to examne the effects of four factors on mycelum polysaccharde producton (Table 1). The orthogonal arrays, data analyss and ANOVA were obtaned usng Desgn Expert Verson 8.0.5b software package (Stat-Ease Inc., Mnneapols, USA) based on the Taguch method. Table 1 Orthogonal experment factors and levels. Level 1 Level 2 Level 3 Factors Symbols (g/l) (g/l) (g/l) Maltose A Peptone B KCl C K 2 HPO 4 3H 2 O D Statstcal analyss The dry weght of G. atrum mycelum polysaccharde (GAMP) producton was expressed as means ± SD. An Analyss of Varance (ANOVA) followed by Tukey s test was appled for multple comparsons of sgnfcant analyses at P < Orthogonal desgn experments and statstcal data analyses were performed n Statstc Package for Socal Scence (SPSS) verson 17.0 software packet. Results and dscusson Effects of rato of materal to water for polysaccharde In order to nvestgate the effect of rato of materal to water on the polysaccharde producton n mycelum, dfferent ratos of materal to water (1:1 1:5, m/v) were prepared n ths study. The hghest polysaccharde producton of 1.48 ± 0.10 mg/g was acheved wth rato of materal to water of 1:3 (Fg. 1). Fg. 1 Effects of rato of materal to water on the polysaccharde producton n G. atrum mycelum by sold-state fermentaton. 759

4 Fg. 2 Effect of carbon sources on the polysaccharde producton n G. atrum mycelum by soldstate fermentaton. Effect of carbon sources To choose a sutable carbon source for polysaccharde n mycelum by G. atrum, sx carbon sources ncludng glucose, sucrose, lactose, soluble starch, maltose and fructose were prepared at concentratons of 30 g/l n the basal medum. Maltose and fructose were the best carbon sources for polysaccharde producton n mycelum (Fg. 2). Polysaccharde producton n mycelum reached 1.48 ± 0.09 mg/g wth maltose and ± 0.15 mg/g wth fructose. But there s a dfference n ths result compare to prevous studes (Tang & Zhong 2002a). Possble reasons are mparty of sold substrate or the cultvaton method. Maltose was selected as the man carbon source n the remanng experment. Effects of ntrogen sources To fnd a sutable ntrogen source for polysaccharde producton n mycelum, we added varous ntrogen sources (soybean meal, peptone, yeast extract, beef extract, NH 4 NO 3, NH 4 Cl) at a concentraton of 20 g/l to the ntrogen-free basal medum (Fg. 3). Peptone and yeast extract dramatcally mproved the mycelum growth rate of G. atrum, and the growth rate was faster when addng peptone as compared to yeast extract. Fermentaton substrates of wheat gran were covered wth mycelum of G. atrum at 15 days. However, growth rate of mycelum was slow when addng NH 4 Cl or NH 4 NO 3. Organc ntrogen was advantageous to both growth and bosynthess of metaboltes. The result s consstent wth the expermental data reported n other medcnal mushroom (Wen et al. 2008, Kang et al. 2012). The results suggested that maxmum mycelum polysaccharde producton of 1.39 ± 0.08 mg/g was produced wth peptone as a ntrogen source. Inorganc on s an mportant nutrtonal component n medum for mycelum growth (Bae, et al. 2000). In order to nvestgate the effect of norganc salt for the polysaccharde producton n mycelum by G. atrum, varous norganc salts (MgSO 4 7H 2 O, ZnSO 4, KH 2 PO 4, KCl, CaSO 4 2H 2 O, and K 2 HPO 4 3H 2 O) were added to basal medum at a concentraton of 1 g/l, ndvdually. All norganc salts mproved polysaccharde producton (Fg. 4). The hghest polysaccharde producton (1.36 ± 0.09 mg/g) was observed n medum, when K 2 HPO 4 3H 2 O was used. At last, K 2 HPO 4 3H 2 O and KCl were recognzed as the best two norganc salts for mycelum polysaccharde producton. 760

5 Fg. 3 Effect of ntrogen sources on the polysaccharde producton n G. atrum mycelum by sold-state fermentaton. Effects of norganc salt Orthogonal expermental desgn To nvestgate the roles of dfferent lqud supplement medum components and optmze ther concentratons for polysaccharde producton n mycelum, the orthogonal layout L 9 (3 4 ) method was used. Accordng to the above results acheved usng the one-factor-at-a-tme method, we selected and separated three levels as shown n Table 1. Table 2 shows the detaled expermental desgn and results. Nne experments were carred out n trplcate. The experment desgn and ANOVA for the expermental results obtaned by SPSS Verson software package, and optmal levels of each factor for obtanng hgher polysaccharde producton n mycelum are gven n Table 2 and Table 3, respectvely. And Fg. 5 shown the mycelum growth state for polysaccharde of G. atrum mycelum at 20 days by L 9 (3 4 ) orthogonal desgn. Fg. 4 Effect of norganc salt on the polysaccharde producton n G. atrum mycelum by soldstate fermentaton. 761

6 Table 2 The L 9 (3 4 ) orthogonal array appled for the polysaccharde producton n G. atrum mycelum by sold-state fermentaton. Run Varables Code Polysaccharde producton ( mg/g, n=3) A B C D Y 1 Y 2 Y 3 Y 1 a b K c K K k d k k R e Optmal level a : The arrangements of column A D were decded by orthogonal desgn for L 9 (3 4 ). b : Average of trple determnatons. c : A K mycela polysaccharde yeld at A. Values are mean of trple determnatons. d A A : k K / 3. Values are mean of trple determnatons. e A A : R max K mn k A. Values are mean of trple determnatons. Table 3 The ANOVA of the results of L 9 (3 4 ) orthogonal desgn for the polysaccharde producton n G. atrum mycelum. Source Sum of quares df Mean Square F-value p-value Prob > F A ** B ** C ** D Error Total Pred-R 2 = 0.961; Adj-R 2 = 0.944; ** 1% sgnfcance level. 762

7 Fg. 5 The mycelum growth state for polysaccharde of G. atrum mycelum at 20 days by L 9 (3 4 ) orthogonal desgn. As shown n Table 2, t s evdent that maxmum polysaccharde producton of mycelum by G. atrum of 1.54 mg/g could be acheved usng a combnaton of coded levels (A, maltose, level 1; B, peptone, level 1; C, KCl, level 2; D, K 2 HPO 4 3H 2 O, level 3), and actual values for varous factors were 15 g/l maltose, 10 g/l peptone, 1 g/l KCl, 1.5 g/l K 2 HPO 4 3H 2 O, respectvely. Accordng to the magntude order of R (Max Df), the order of effect of all factors on polysaccharde producton could be determned, and was peptone > maltose > KCl > K 2 HPO 4 3H 2 O. Analyss of ANOVA (Table 3) showed that peptone, maltose, and KCl should be effectve ngredents for mproved polysaccharde producton (P < 0.05). Nevertheless, K 2 HPO 4 3H 2 O was not sgnfcant (P > 0.05). Peptone was more sgnfcant than all other ngredents. Conclusons In ths work, the one-factor-at-a tme method and orthogonal desgn method were employed to establsh the key factors and dentfy optmal culture condtons to mprove the polysaccharde producton n G. atrum mycelum by sold-state fermentaton for the frst tme. The results showed that the hghest polysaccharde producton was acheved wth rato of materal to water of 1:3, and the optmal lqud supplement medum contaned 15 g/l maltose, 10 g/l peptone, 1 g/l KCl and 1.5 g/l K 2 HPO 4 3H 2 O. The maxmum polysaccharde yeld n mycelum was 1.8 mg/g on 20 days. Ths method frst provdes an effectve way for ncreasng polysacchardes producton by soldstate fermentaton n G. atrum. The strateges used n ths study could be wdely practcal to other fermentaton processes. 763

8 Acknowledgements Ths work was supported by the project of Karst Scence Research Center of Natonal Natural Scence Foundaton of Chna - the People's Government of Guzhou Provnce, the Scence Foundaton of Guzhou Unversty (No ) and the Youth Foundaton of Guzhou Academy of Scences (No ). References Bae JT, Snha J, Park JP, Song CH, Yun JW Optmzaton of submerged culture condtons for exo-bopolymer producton by Paeclomyces japonca. Journal of Mcrobology and Botechnology 10(4), Chen Y, Xe MY, Ne SP, L C, Wang YX Purfcaton, composton analyss and antoxdant actvty of a polysaccharde from the frutng bodes of Ganoderma atrum. Food Chemstry 107(1), , Do /j.foodchem Chen Y, Zhang H, Wang YX, Ne SP, L C, Xe MY Acetylaton and carboxymethylaton of the polysaccharde from Ganoderma atrum and ther antoxdant and mmunomodulatng actvtes. Food Chemstry 156, , Do /j.foodchem Couto SR, Sanromán MA Applcaton of sold-state fermentaton to food ndustry a revew. Journal of Food Engneerng 76(3), , Do /j.jfoodeng Douanla-Mel C, Langer E Ganoderma carocalcareus sp. nov., wth crumbly-frable context paraste to saprobe on Anthoclesta nobls and ts phylogenetc relatonshp n G. resnaceum group. Mycologcal Progress 8(2), , Do /s Gao YH, Chan E, Zhou SF Immunomodulatng actvtes of Ganoderma, a mushroom wth medcnal propertes. Food Revews Internatonal 20(2), , Do /FRI Hapuarachch KK, Wen TC, Deng CY, Kang JC, Hyde KD Mycosphere Essays 1: Taxonomc confuson n the Ganoderma lucdum speces complex. Mycosphere 6(5), , Do /mycosphere/6/5/4. Hapuarachch KK, Wen TC, Jeewon R, Wu XL, Kang JC, Hyde KD Mycosphere Essays 7: Ganoderma lucdum - are the benefcal ant-cancer propertes substantated? Mycosphere 7(3), , Do /mycosphere/7/3/6. Hseh C, Yang FC Reusng soy resdue for the sold-state fermentaton of Ganoderma lucdum. Boresource Technology 91(1), , Do /S (03) Huang HC, Chen CI, Hung C, Lu YC Expermental analyss of the ol addton effect on mycela and polysaccharde productons n Ganoderma lucdum submerged culture. Boprocess and Bosystems Engneerng 32(2), , Do /s z. Kang C, Wen TC, Kang JC, Qan YX, Le BX Effcts of addtves and dfferent culture condtons on cordycepn producton by the medcnal fungus Cordyceps mltars (n Chnese). Mycosystem 31(3), , Lee HY, Song MY, Hwang SH Optmzng boconverson of deprotenated cheese whey to mycela of Ganoderma lucdum. Process Bochemstry 38(12), , Do /S (02) L WJ, Ne SP, Lu XZ, Zhang H, Yang Y, Yu Q, Xe MY Antmcrobal propertes, antoxdant actvty and cytotoxcty of ethanol-soluble acdc components from Ganoderma atrum. Food and Chemcal Toxcology 50(3), , Do /j.fct L YQ, Wang SF Ant-hepatts B actvtes of ganoderc acd from Ganoderma lucdum. Botechnology Letters 28(11), , Do /s Lu GQ, Zhang KC Enhancement of polysacchardes producton n Ganoderma lucdum by the addton of ethyl acetate extracts from Eupolyphaga snenss and Catharsus molossus. Appled Mcrobology and Botechnology 74(3), , Do /s Meng GL, Zhu HY, Yang SJ, Wu F, Zheng HH, Chen E, Xu JL Attenuatng effects of 764

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