Response Surface Optimization of Medium Formulation for Culture of Lyophyllum decastes
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摘要:目的 以提高鹿茸菇液体菌种菌丝生物量为目的，通过优化液体菌种发酵配方，制备优良液体菌种，推动鹿茸菇工厂化生产快速发展。方法 以鹿茸菇为研究材料，采用摇瓶培养方式，通过单因素设计分析6种碳源、8种氮源、10种金属离子对鹿茸菇菌丝生长的影响。采用Box-Behnken响应面法进一步优化单因素筛选出的碳源、氮源与金属离子，比较分析液体菌种与固体菌种应用于鹿茸菇生产的优劣性。结果 适合鹿茸菇生长的营养要素分别为全麦粉、花生饼粉、KH2PO4与MgSO4·7H2O，营养要素间的最优配方为全麦粉47 g·L−1、花生饼粉22 g·L−1、KH2PO4 2.00 g·L−1、MgSO4·7H2O 2.00 g·L−1，当发酵培养至第8 d时，菌丝生物量达到最大值（32.81±1.10 g·L−1），以优化配方制备的鹿茸菇液体菌种菌丝生物量较初始配方提高了7.34倍。在生产试验中液体菌种较固体菌种，制种时间缩短48 d，菌丝萌发时间缩短2 d，单袋产量提高15.47%。结论 本研究研发的液体菌种发酵配方可获得高质量的生产用种，以液体发酵方式制备鹿茸菇生产用种适合鹿茸菇工厂化生产。Abstract:Objective To improve the mycelium biomass of Lyophyllum decast liquid seeds, high-quality liquid seeds are prepared by optimizing the liquid fermentation formula, to promote the rapid development of industrial production of L. decastes mushrooms.Methods Taking L. decastes as the research material, the effects of 6 kinds of carbon sources, 8 kinds of nitrogen sources, and 10 kinds of metal ions on the mycelium growth were analyzed by single-factor design. The carbon source, nitrogen source, and metal ions screened out by single-factor design were further optimized by the Box-Behnken response surface method, and the advantages and disadvantages of liquid seeds and solid seeds in the production of L. decastes mushrooms were compared.Results The nutritional elements suitable for the growth of L. decastes were whole wheat flour, peanut meal, K2HPO4, and MgSO4·7H2O, and the optimal formula was whole wheat flour 47 g·L−1, peanut meal 22 g·L−1, K2HPO4 2.00 g·L−1, MgSO4·7H2O 2.00 g·L−1. When the fermentation culture reached the 8th day, the dry cell mass (DCM) was 32.81±1.10 g·L−1. Compared with the DCM by the initial formula, the DCM prepared with the optimized formula was increased by 7.34 times. Compared with solid seeds, the production time of liquid seeds was shortened by 48 days, the time of mycelium germination was shortened by 2 days, and the yield per bag increased by 15.47%, in industrial production.Conclusion The liquid fermentation formula developed in this study can obtain high-quality production seeds, and the liquid fermentation strategy for the preparation of seeds is suitable for the industrial production of L. decastes mushrooms.
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Keywords:
- Lyophyllum decastes /
- Liquid seeds /
- Liquid fermentation formula /
- Mycelium biomass
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图 1 鹿茸菇液体菌种生长动力学曲线
不同字母表示均值之间差异显著（P＜0.05），下同。
Figure 1. The growth dynamics curve of L. decastes liquid strain
Data with different letters indicate significant differences at P＜0.05. Same for the following figures and tables.
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图 2 3类营养要素对鹿茸菇液体菌丝生长的影响
A、B与C分别代表6种碳源、8种氮源与10种金属离子对鹿茸菇液体菌丝生长的影响。
Figure 2. Effects of 3 type nutrients on growth of L. decastes in liquid medium
A, B, and C represent the effects of 6 carbon sources, 8 nitrogen sources, and 10 metal ions on the growth of L. decastes liquid strain, respectively.
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图 3 单因素试验
A、B、C与D分别代表全麦粉、花生饼粉、K2HPO4与MgSO4·7H2O剂量梯度对鹿茸菇液体菌丝生物的影响。
Figure 3. Results of single-factor design experiment
A, B, C, and D: Effects of dose-dependent whole wheat flour, peanut cake flour, K2HPO4, and MgSO4·7H2O, respectively, on growth of L. decastes in liquid medium.
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图 5 子实体的外观形态
a)：接种液体菌种的菌包；b)：接种固体菌种的菌包。
Figure 5. Appearance of mushroom fruiting bodies
a): Mushroom packet for inoculation of mycelia cultured in liquid medium; b): mushroom packet for inoculation of mycelia cultured in solid medium.
表 1 Box-Behnken设计因素水平及编码
Table 1 Codes and levels of factors in Box-Behnken experiment
水平
Level因素 Factors/(g·L−1） A 全麦粉
Whole wheat flourB 花生饼粉
Peanut mealC 磷酸氢二钾
K2HPO4/D七水硫酸镁
MgSO4·7H2O/+1 55 24 2.25 2.25 0 50 22 2.00 2.00 −1 45 20 1.75 1.75 
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表 2 Box-Behnken试验设计及试验结果
Table 2 Design and results of Box-Behnken experiment
试验号
NumberA全麦粉
Whole wheat
flourB花生饼粉
Peanut
mealC磷酸氢
二钾
K2HPO4D七水
硫酸镁
MgSO4·7H2O菌丝生物量
Mycelium
biomass/
(g·L−1)1 −1 −1 0 0 29.61±1.18 2 +1 −1 0 0 29.90±1.45 3 −1 +1 0 0 32.27±1.11 4 +1 +1 0 0 28.77±2.01 5 0 0 −1 −1 30.79±1.28 6 0 0 +1 −1 29.41±1.88 7 0 0 −1 +1 28.00±0.30 8 0 0 +1 +1 30.21±1.09 9 −1 0 0 −1 31.72±2.12 10 +1 0 0 −1 29.37±1.63 11 −1 0 0 +1 30.20±2.00 12 +1 0 0 +1 29.11±2.51 13 0 −1 −1 0 29.29±4.29 14 0 +1 −1 0 30.00±0.68 15 0 −1 +1 0 30.20±1.45 16 0 +1 +1 0 30.11±4.80 17 −1 0 −1 0 30.00±2.97 18 +1 0 −1 0 30.17±4.12 19 −1 0 +1 0 32.48±0.21 20 +1 0 +1 0 28.73±4.01 21 0 −1 0 −1 30.09±4.38
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表 3 二次回归方程方差分析
Table 3 ANOVA on quadratic regression equation
来源
Source平方和
SS自由度
DS均方
MSF P 显著性
Significance模型
Model0.7362 14 0.0526 98.95 ＜0.0001 ** A 0.0878 1 0.0878 165.28 ＜0.0001 ** B 0.0127 1 0.0127 23.85 0.0002 ** C 0.0074 1 0.0074 13.96 0.0022 ** D 0.0248 1 0.0248 0.31 ＜0.0001 ** AB 0.0358 1 0.0358 0.02 ＜0.0001 ** AC 0.0371 01 0.0371 6.12 ＜0.0001 * AD 0.0044 1 0.0044 0.32 0.0118 * BC 0.0015 1 0.0015 0.08 0.1114 BD 0.0011 1 0.0011 0.02 0.1702 CD 0.0327 1 0.0327 0.03 ＜0.0001 ** A2 0.1299 1 0.1299 169.26 ＜0.0001 ** B2 0.1974 1 0.1974 9.19 ＜0.0001 ** C2 0.2045 1 0.2045 127.92 ＜0.0001 ** D2 0.2302 1 0.2302 37.54 ＜0.0001 ** 残差
Residual0.0074 14 0.0005 失拟项
Lack of fit0.0047 10 0.0005 0.69 0.7126 纯误差
Pure error0.0027 4 0.0007 总和
Cor total0.7436 28 R2=0.9900 R2adj=0.9800 *表示差异显著（P＜0.05），**表示差异极显著（P＜0.01）。
* indicates significant difference at P＜0.05; ** extremely significant at P＜0.01.
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表 4 液体菌种和固体菌种栽培过程及出菇比较
Table 4 Comparison of fermentation processes andmushroom fruiting using liquid and solid media
指标
Index液体菌种
Liquid strain固体菌种
Solid strain原种培养时间 Original culture time/d 8 24 栽培种培养时间 Culture spawn incubation time/d 8 40 制种周期 Seed production cycle/d 16 64 菌丝萌发时间 Time of mycelium germination/d 2 4 满袋时间 Bags full time/d 42 45 污染率 Pollution rate/% 8.33 29.17 现蕾时间 Budding time/d 7 9 采收时间 Harvest time/d 25 27 单包产量 Single package output/g 500.89±20.12 432.00±25.36 子实体整齐度 Fruiting body uniformity +++ ++ 菇长 Length/cm 12.82±1.19 11.76±0.83 菇径 Size/mm 12.37±1.66 17.36±1.71 盖高 Height/mm 6.86±1.35 10.72±1.85 盖径 Diameter/mm 20.06±3.77 26.10±3.94 +++表示子实体整齐度较一致； ++表示子实体整齐度一致。
+++shows uniform fruiting body formation; ++acceptable fruiting body uniformity.
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