College of Bioscience and Bioengineering,Jiangxi Agricultural University,Nanchang 330045,China

Solid Culture of Lactarius deliciosus

Li NIE,Huiling PENG,Wei XIA,Saijin WEI*

College of Bioscience and Bioengineering,Jiangxi Agricultural University,Nanchang 330045,China

The research explored the effects of carbon,nitrogen and inorganic salt nutritional factors onLactarius deliciosushyphae as per solid culture.The optimal recipe of culture medium was selected by observing hypha density and computing the growth index,which is the most suitable for hypha growth.The results indicated that glucose and peptone were optimal carbon and nitrogen forLactarius deliciosus; MgSO4,CuSO4and VB2promoted hypha growth;FeSO4and CoCl2had inhibition effects.The orthogonal test concluded the optimal recipe contained glucose of 20 g/L, peptone of 1 g/L,KH2PO4of 0.3 g/L,MgSO4of 0.3 g/L,VB2of 2 mg/L,and agar of 20 g/L,with original pH value adopted.After optimization,growth index of hyphae cultured at 28℃was 4 055.9.

Lactarius deliciosus;Optimal medium;Growth index of hypha

L actarius deliciosus,commonly known as red pine mushroom and saffron milk cap,is of the large milk-cap genusLactariusin the order Russulales.It is a delicious wild edible mushroom[1].Furthermore, it performs well in controlling tumour and the produced lactaroviolin has an antibacterial action[2-3].Besides,the sporocarps would produce sphingolipids and lectins[4],of which polysaccharides plays the role of antineoplastic activity and immunoregulatory activ ity[5-6].BecauseLactarius deliciosuskeeps symbiotic relationship with plants,it has a special living condition, nutrition-absorption way and fruitbody differentiation condition.Therefore,it is difficult for domestication and the cultivation at large-scale is not available at home and abroad yet[7-8].On the other hand,researches are few on recipe of solid medium ofLactarius deliciosusand the cultivation conditions.The research selected the optimal cultivation medium recipe and condition as per solid culture ofLactarius deliciosusbased on nutrient factors and cultivation conditions in order to further developLactarius deliciosusand provide theoretical references for artificial cultivation ofLactarius deliciosus.

Materials and Methods

Materials

Test strainsLactarius deliciosuswas provided by Yichun Vocational Technical College.

Culture mediaMediumⅠcontained potato of 200 g,glucose of 20 g, KH2PO4of 1 g,MgSO4of 0.5 g,agar of 20 g,and distilled water of 1 000 ml, with original pH value adopted.

MediumⅡcontained glucose of 20 g,urea of 2 g,KH2PO4of 1 g,Mg-SO4of 0.5 g,agar of 20 g,and distilled water of 1 000 ml,with original pH value adopted.

MediumⅢcontained soluble starch of 20 g,sodium nitrate of 2 g, agar of 20 g,and distilled water of 1 000 ml,with original pH value adopted.

MediumⅣcontained soluble starch of 20 g,sodium nitrate of 2 g, KH2PO41 g,Mg SO40.5 g,agar of 20 g,and distilled water of 1 000 ml,with original pH value adopted.

Carbon sourceGlucose,fructose, saccharose,lactose,maltose,and soluble saccharide,with the same mass,were used to replace glucose in mediumⅡ,and the treatment without

Nitrogen sourcePeptone,yeast cream,beef extract,glutamic acid, ammonium nitrate,ammonium sulfate, with the same mass,were used to take place of urea in mediumⅡ,and the treatment without nitrogen as a control group.

Inorganic saltKH2PO4,MgSO4, Fe SO4and NaCl were added into basic mediumⅢat 0,300,600,900 and 1 200 mg/L,respectively;ZnSO4, SO4,SO4and CoCl2were added at 0, 20,40,60 and 80 mg/L were added into basic mediumⅢ,with mediumⅢas a control group.

VitaminOf vitamins,VB1,VB2and VB6have the most significant effects on edible mushrooms,which were added to basic mediumⅣat 0,2,4,6, 8 and 10 mg/L respectively,with mediumⅣas a control group.

Methods

Test of nutrient factorsAfter twice actications of culture,bacterial mass was collected with a puncher(diameter of 9 mm)and inoculated to media. It is notable that every treatment was set with 4 repetitions at(28±1)℃.The diameter of bacterial colony was signed every two days with the method of delimiting lines.

Orthogonal experimental design

On basis of single-factor experiment, the culture media was further optimized as per orthogonal experimental design with three factors and three levels(Table 1).Furthermore,the lower contents of vitamins and trace elements require further exploration.

Test methodThe diameter of bacterial colony was signed every two days with the method of delimiting lines and growth vigor ofLactarius deliciosuswas measured.In addition, growth index of hypha was introduced for comparisons[9],as follows:

Growth index of hypha=Assessment score of hypha growth vigor× Colony area(1)

Growth vigor of hypha can be divided into sparse,sparser,ordinary, denser and dense,and the scores were 1,2,3,4 and 5,respectively.

Data analysis and processingThe test data were analyzed with Excel, Origin 8.5 and DPS.

Table 1Experimental factors and levels

Results and Analysis

Effects of carbon sources onLactarius deliciosushyphae

The use rates ofLactarius deliciosushyphae on 6 carbons tended to be volatile,represented by glucose, followed by fructose,saccharose and lactose(Table 2).

Effects of nitrogen source onLactarius deliciosushyphae

As shown in Table 3,the effects of 6 nitrogen sources were diversified, of which the treatments with peptone, yeast extract,and beef extract showed insignificant differences,and the three materials can be all taken as optimal nitrogen sources,with ammonium nitrate the poorest.

Effects of mineral elements ofLactarius deliciosushyphae

As shown in Fig.1,the inhibition of FeSO4onLactarius deliciosushyphae was the most strongest,and the differences kept decreasing upon concentration.Specifically,KH2PO4and NaCl at low concentrations promote the hyphae growth a little and the advancement would change to inhibition upon concentration.Furthermore, 300 mg/L MgSO4has the promotion effects,but the promotions of CuSO4and MnSO4on hyphae are growing upon concentrations.For example, when CuSO4concentration was at 80 mg/L,the promotion effect reached the peak,and when ZnSO4concentration was at 80 mg/L,inhibition effects reached the highest.Additionally,Co-Cl2also had inhibition effects.

Effects of vitamin onLactarius deliciosushyphae

As shown in Fig.2,Lactarius deliciosushyphae were not so sensitive to VB6;the promotion effects were the highest with VB2at 2 mg/L and the inhibition effects were the strongest with concentration at 6 mg/L;the inhibition on the hyphae kept increasing upon concentration of VB1.

Analysis of orthogonal experiment results

The optimal treatment combination was A1B1C1,containing glucose of20%,protein of 1%and MgSO4of 0.3%,which indicated that the theoretical combination results were in consistent with the optimal combination results.

Table 2Effects of carbon sources onLactarius deliciosushyphae

Table 3Effects of nitrogen source onLactarius deliciosushyphae

Fig.2Effects of different vitamins on the growth ofLactarius deliciosushypha

Conclusions and Discussions

According to the use effects ofLactarius deliciosushyphae on carbons,the use rate ofLactarius deliciosuson lactose was the least.For instance,the hyphae grew slowly and sparsely.Therefore,monosaccharide, glucose and fructose were top priority at using saccharides,followed by maltose,and the use rates of lactose and saccharose maintained lower,which suggested that the research was not in consistent with Zhou’s[10].

The use effects ofLactarius deliciosuson carbons tended to be volatile,which suggested that nitrogen from different sources has diversified effects on growth of hyphae in growing period.Specifically,for organic nitrogen,the use effects of peptone byLactarius deliciosusproved the best, followed by yeast extract,beef extract, and glutamic acid.For inorganic nitrogen,it is(NH4)2SO4that promoted hyphae growth the most,and NH4NO3the least.In general,organic nitrogen is the most suitable forLactarius deliciosushyphae growth,followed by inorganic nitrogen,which coincided with the research of Zhou[11].

According to the effects of inorganic salt on hyphae growth,MgSO4, CuSO4and Mn SO4were the most suitable for growth ofLactarius deliciosushyphae;the inhibition was growing upon FeSO4concentration.It can be concluded that during culturing medium,Fe2+of FeSO4has changed to Fe3+,affecting the absorption ofLactarius deliciosusand preventing hyphae growth.

Lactarius deliciosusis not so sensitive to VB6;the promotion of VB2was the highest;the inhibition effects were growing as the concentration of VB1increased.On basis of trace elements and the allowed concentrations,the effects onLactarius deliciosushyphae were not so significant, and the research conducted by Longet al.showed that VB1had the promotion effects onLactarius deliciosushyphae[12].

The single factor experiment determined that peptone and Mg SO4were the major factors affecting growth ofLactarius deliciosushyphae.The orthogonal test concluded the optimal recipe contained glucose of 20 g/L, peptone of 1 g/L,KH2PO4of 0.3 g/L,

Table 4Range analysis of orthogonal experiment

MgSO4of 0.3 g/L,VB2of 2 mg/L,and agar of 20 g/L,with original pH value adopted.After optimization,growth index of hyphae cultured at 28℃was 4 055.9.

[1]ZHOU GY(周國英),LI QR(李倩茹),LIU JA(劉君昂).A study on the nutritive factors and fermented process of the submerged culture ofLactarius deliciosus(松乳菇菌絲深層培養(yǎng)營養(yǎng)因子及發(fā)酵條件研究)[J].Acta Agriculturae Universitatis Jiangxiensis(江西農(nóng)業(yè)大學(xué)學(xué)報),2003,25(2):246-249.

[2]GUO JM(郭嘉銘),SHANGGUAN ZJ(上官舟建),CHEN JC(陳景潮).Research and development of medical fungi(藥用真菌的研究與開發(fā)概述)[J].Edible Fungi of China(中國食用菌),1994,13(3): 7-10.

[3]ONDRU?EK V,PRO?TENIK M.Sphingolipids in fruit bodies of the basidiomyceteLactarius deliciosus[J].Experimental Mycology,1978,2(2):156-160.

[4]GUILLOT J,GIOLLANT M,DAMEZ M,et al.Isolation and characterization of a lectin from the mushroom,Lactarius deliciosus[J].Journal of biochemistry, 1991,109(6):840-845.

[5]CHEN YQ(陳楊瓊),DING X(丁祥),WU CL(伍春蓮),et al.Anti-tumor and immunomodulatory activity of polysaccharide fromLactarius deliciosusfruit bodies(松乳菇多糖抗腫瘤和免疫調(diào)節(jié)活性研究)[J].Acta Edulis Fungi(食用菌學(xué)報),2012,19(3):73-78.

[6]LI H(李河),ZHOU GY(周國英),LAN GH (蘭貴紅).Crude polysaccharide fromLactarius deliciosusfruit bodies:optimization of extraction conditions and polysaccharide composition(松乳菇子實(shí)體多糖提取工藝及其組分的研究)[J]. Acta Edulis Fungi(食用菌學(xué)報),2007, 14(3):58-61.

[7]TANG C(唐超),CHEN YL(陳應(yīng)龍),LIU RJ(劉潤進(jìn)).Research progress of edible mycorrhizal fungi(菌根食用菌研究進(jìn)展)[J].Mycosystema(菌物學(xué)報),2011, 30(3):367-378.

[8]XIONG T(熊濤),XIAO M(肖滿).Research progress ofLactarius deliciosus(松乳菇研究進(jìn)展)[J].Food and Fermentation Industries Editorial Staff(食品與發(fā)酵工業(yè)),2005,31(5):84-86.

[9]MA L(馬璐),DU ST(杜雙田),JIN LY(金凌云),et al.Studies on the nutritional physiology ofPleurotus eryngii(杏鮑菇營養(yǎng)生理研究)[J].Journal of Northwest Sci-Tech University of Agriculture and Forestry(Natural Science Edition)(西北農(nóng)林科技大學(xué)學(xué)報(自然科學(xué)版)),2010, 38(9):129-134.

[10]ZHOU GY(周國英),LIU JA(劉君昂),LI QR(李倩茹).Tissue isolation and mycclium growth characteristics ofLactarius deliciosus(松乳菇菌種分離及菌絲生長特性的研究)[J].Journal of Zhejiang Forestry College(浙江林學(xué)院學(xué)報),2003,20(2):50-53.

[11]ZHOU CY(周傳云),LIAO XH(廖興華), TAN ZJ(譚周進(jìn)),et al.Study on the strain breeding and cultivating conditions of wildLactarius deliciosus(野生松乳菇菌種分離與培養(yǎng)特性的研究)[J]. Food Science(食品科學(xué)),2004,25(8): 66-69.

[12]LONG F(龍芳),ZHANG WP(張萬萍). Effects of growth regulatory substances on growth ofLactarius deliciosus(不同生長調(diào)節(jié)物質(zhì)對松乳菇菌絲生長的影響)[J].Edible Fungi of China (中國食用菌),2014,33(3):27-30.

Responsible editor:Xiaoxue WANG

Responsible proofreader:Xiaoyan WU

松乳菇固體培養(yǎng)條件的研究

聶麗，彭惠玲，夏薇，魏賽金*
（江西農(nóng)業(yè)大學(xué)生物科學(xué)與工程學(xué)院，江西南昌330045）

采用固體培養(yǎng)的方法，研究不同碳源、氮源和無機(jī)鹽營養(yǎng)因子對松乳菇菌絲的影響。通過觀察菌絲密度和計算菌絲生長指數(shù)篩選出最適合松乳菇菌絲生長的培養(yǎng)基配方。結(jié)果表明：松乳菇的最適碳、氮源分別為葡萄糖與蛋白胨；MgSO4、CuSO4、VB2促進(jìn)菌絲生長，F(xiàn)eSO4、CoCl2則有較明顯的抑制作用。正交試驗(yàn)，確定松乳菇菌絲生長的最佳培養(yǎng)基配方為：葡萄糖20 g/L，蛋白胨1 g/L，KH2PO40.3 g/L，MgSO40.3 g/L，VB22 mg/L，瓊脂20 g/L，pH自然。經(jīng)過優(yōu)化以后，在28℃下培養(yǎng)，菌絲生長指數(shù)為4 055.9。

松乳菇；最適培養(yǎng)基；菌絲生長指數(shù)carbon as a control group.

江西農(nóng)業(yè)大學(xué)博士啟動基金（09004659）。

聶麗（1991-），女，江西萍鄉(xiāng)人，碩士生，主要從事微生物學(xué)的研究，E-mail：530041190@qq.com。*通訊作者，魏賽金，博士，教授，E-mail：weisaijin@126.com。

2015-04-04

修回日期 2015-05-16

Supported by the Doctoral Scientific Research Foundation of Jiangxi Agricultural University(09004659).

*Corresponding author.E-mail:weisaijin@126.com

Received:April 4,2015 Accepted:May 16,2015