响应面法优化鹿茸菇液体菌种发酵配方

单灿灿; 叶典章; 郭宇; 吴斌; 李佳欢; 程泳春; 孙淑静; 胡开辉; 金文松

doi:10.19303/j.issn.1008-0384.2023.06.011

响应面法优化鹿茸菇液体菌种发酵配方

doi: 10.19303/j.issn.1008-0384.2023.06.011

单灿灿^1,,
叶典章¹,
郭宇¹,
吴斌¹,
李佳欢^{1, 2},
程泳春³,
孙淑静^{1, 2},
胡开辉^{1, 2, 4, ,},
金文松^{1, 2, 4, ,}

1.
福建农林大学生命科学学院，福建　福州　350007
2.
福建农林大学（古田）菌业研究院，福建　宁德　352299
3.
福建珍菌子生物科技有限公司，福建　宁德　352256
4.
福建省食用菌产业技术创新研究院，福建　宁德　352299

基金项目: 国家自然科学基金项目（81803667）；中国博士后科学基金面上项目（132300346）；福建省科技厅对外产业化合作项目（2020I1008）；中央引导地方科技发展资金项目（2022L3005）

详细信息

作者简介:
单灿灿（1994 —），女，硕士研究生，研究方向：食用菌工厂化栽培（E-mail：shancancan123@163.com）

通讯作者:
胡开辉（1962 —），男，教授，研究方向：食用菌的应用、开发与工厂化栽培（E-mail: Hukh@fafu.edu.cn）

金文松（1983 —），男，博士，副研究员，研究方向：食药用真菌次级代谢产物合成途径解析（E-mail:jinws@fafu.edu.cn）

中图分类号: S646
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出版历程
- 收稿日期: 2022-03-12
- 修回日期: 2022-12-06
- 网络出版日期: 2023-06-02
- 刊出日期: 2023-06-28

Response Surface Optimization of Medium Formulation for Culture of Lyophyllum decastes

SHAN Cancan^1
,,
YE Dianzhang¹,
GUO Yu¹,
WU Bin¹,
LI Jiahuan^{1, 2},
CHENG Yongchun³,
SUN Shujing^{1, 2},
HU Kaihui^{1, 2, 4
, ,},
JIN Wensong^{1, 2, 4
, ,}

1.
College of Life Sciences, Fujian Agriculture and Forestry University, Fuzhou, Fujian　350007, China
2.
Edible Fungi Research Institute (Gutian), Fujian Agriculture and Forestry University, Ningde, Fujian　352299, China
3.
Fujian Zhen Jun Zi Biological Technology Co., Ltd., Ningde, Fujian　352256, China
4.
Edible Fungi Industry Technology Innovation Research Institute of Fujian, Ningde, Fujian　352299, China

摘要

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、KH₂PO₄2.00 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。
- 鹿茸菇 /
- 液体菌种 /
- 发酵配方 /
- 菌丝生物量
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.
- Lyophyllum decastes /
- Liquid seeds /
- Liquid fermentation formula /
- Mycelium biomass

HTML全文

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.

下载: 全尺寸图片幻灯片

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.

下载: 全尺寸图片幻灯片

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.

下载: 全尺寸图片幻灯片

图 4 两因素之间的交互作用对菌丝生物量的影响

Figure 4. Effect of interaction between two factors on DCM

下载: 全尺寸图片幻灯片

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.

下载: 全尺寸图片幻灯片

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）
水平 Level	A 全麦粉 Whole wheat flour	B 花生饼粉 Peanut meal	C 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

下载: 导出CSV

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 Number	A全麦粉 Whole wheat flour	B花生饼粉 Peanut meal	C磷酸氢二钾 K₂HPO₄	D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

下载: 导出CSV

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 MS	F	P	显著性 Significance
模型 Model	0.7362	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 Residual	0.0074	14	0.0005
失拟项 Lack of fit	0.0047	10	0.0005	0.69	0.7126
纯误差 Pure error	0.0027	4	0.0007
总和 Cor total	0.7436	28
R²=0.9900			R²_adj=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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响应面法优化鹿茸菇液体菌种发酵配方

doi: 10.19303/j.issn.1008-0384.2023.06.011

作者简介:
单灿灿（1994 —），女，硕士研究生，研究方向：食用菌工厂化栽培（E-mail：shancancan123@163.com）

通讯作者:
胡开辉（1962 —），男，教授，研究方向：食用菌的应用、开发与工厂化栽培（E-mail: Hukh@fafu.edu.cn）

金文松（1983 —），男，博士，副研究员，研究方向：食药用真菌次级代谢产物合成途径解析（E-mail:jinws@fafu.edu.cn）

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Response Surface Optimization of Medium Formulation for Culture of Lyophyllum decastes

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响应面法优化鹿茸菇液体菌种发酵配方

doi: 10.19303/j.issn.1008-0384.2023.06.011

作者简介: 单灿灿（1994 —），女，硕士研究生，研究方向：食用菌工厂化栽培（E-mail：shancancan123@163.com）

通讯作者: 胡开辉（1962 —），男，教授，研究方向：食用菌的应用、开发与工厂化栽培（E-mail: Hukh@fafu.edu.cn） 金文松（1983 —），男，博士，副研究员，研究方向：食药用真菌次级代谢产物合成途径解析（E-mail:jinws@fafu.edu.cn）

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Response Surface Optimization of Medium Formulation for Culture of Lyophyllum decastes

计量

出版历程

目录

作者简介:
单灿灿（1994 —），女，硕士研究生，研究方向：食用菌工厂化栽培（E-mail：shancancan123@163.com）

通讯作者:
胡开辉（1962 —），男，教授，研究方向：食用菌的应用、开发与工厂化栽培（E-mail: Hukh@fafu.edu.cn）

金文松（1983 —），男，博士，副研究员，研究方向：食药用真菌次级代谢产物合成途径解析（E-mail:jinws@fafu.edu.cn）