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.

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Responsible editor:Xiaoxue WANG

Responsible proofreader:Xiaoyan WU

松乳菇固体培养条件的研究

聂丽，彭惠玲，夏薇，魏赛金*
（江西农业大学生物科学与工程学院，江西南昌330045）

采用固体培养的方法，研究不同碳源、氮源和无机盐营养因子对松乳菇菌丝的影响。通过观察菌丝密度和计算菌丝生长指数筛选出最适合松乳菇菌丝生长的培养基配方。结果表明：松乳菇的最适碳、氮源分别为葡萄糖与蛋白胨；MgSO4、CuSO4、VB2促进菌丝生长，FeSO4、CoCl2则有较明显的抑制作用。正交试验，确定松乳菇菌丝生长的最佳培养基配方为：葡萄糖20 g/L，蛋白胨1 g/L，KH2PO40.3 g/L，MgSO40.3 g/L，VB22 mg/L，琼脂20 g/L，pH自然。经过优化以后，在28℃下培养，菌丝生长指数为4 055.9。

松乳菇；最适培养基；菌丝生长指数carbon as a control group.

江西农业大学博士启动基金（09004659）。

聂丽（1991-），女，江西萍乡人，硕士生，主要从事微生物学的研究，E-mail：530041190@qq.com。*通讯作者，魏赛金，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