樟芝-太子参双向液体发酵培养基优化及效应分析

赵生月; 吴小环; 姜春玲; 张余; 郑世仲; 刘盛荣; 叶祖云

doi:10.19303/j.issn.1008-0384.2024.01.010

樟芝-太子参双向液体发酵培养基优化及效应分析

doi: 10.19303/j.issn.1008-0384.2024.01.010

赵生月^{1, 2,},
吴小环³,
姜春玲^{3, 4},
张余^{1, 2},
郑世仲³,
刘盛荣^3, ,,
叶祖云^{2, 3}

1.
福建中医药大学药学院，福建　福州　350122
2.
福建省特色药用植物工程技术研究中心，福建　宁德　352100
3.
宁德师范学院生命科学学院，福建　宁德　352100
4.
福建农林大学食品科学学院，福建　福州　350002

基金项目: 福建省科技厅对外合作项目（2021I0046）；福建省科技计划高校产学合作项目（2021N5005）；宁德师范学院科研项目（2022ZX01）

详细信息

作者简介:
赵生月（1996 —），女，硕士研究生，主要从事中药发酵及活性成分分析研究，E-mail：1870770064@qq.com

通讯作者:
刘盛荣（1973 —），男，博士，教授，主要从事食药用菌发酵及育种研究，E-mail：fjhost@163.com

中图分类号: Q93
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- 文章访问数: 260
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- 被引次数: 0
出版历程
- 收稿日期: 2023-06-28
- 修回日期: 2023-10-25
- 网络出版日期: 2024-01-25
- 刊出日期: 2024-01-28

Formulation of Antrodia camphorata Culture Medium Incorporated with Pseudostellaria heterophylla By-product

ZHAO Shengyue^{1, 2
,},
WU Xiaohuan³,
JIANG Chunling^{3, 4},
ZHANG Yu^{1, 2},
ZHENG Shizhong³,
LIU Shengrong^{3
, ,},
YE Zuyun^{2, 3}

1.
College of Pharmacy, Fujian University of Traditional Chinese Medicine, Fuzhou, Fujian　350122, China
2.
Research Center of Fujian Province Characteristic Medicinal Plants, Ningde, Fujian　352100, China
3.
College of Life Science, Ningde Normal University, Ningde, Fujian　352100, China
4.
College of Food Science, Fujian Agriculture and Forestry University, Fuzhou, Fujian　350002, China

摘要

摘要: 目的有效利用太子参根须副产物资源，提高樟芝（Antrodia camphorata）菌丝产量，促进活性成分合成。方法以太子参根须（粉末）作为培养基组分，Plackett-Burman试验筛选影响樟芝菌丝生长的关键成分，正交试验优化培养基组成。结果筛选试验结果表明，葡萄糖、酵母粉、蛋白胨及KH₂PO₄对樟芝菌丝生长有重要影响，正交试验优化的培养基组成为：太子参6 g·L⁻¹，葡萄糖10 g·L⁻¹，酵母粉4 g·L⁻¹，蛋白胨6 g·L⁻¹，KH₂PO₄ 1.5 g·L⁻¹。利用优化培养基深层发酵，樟芝生物量达到4.73 g·L⁻¹，与对照培养基及PDB培养基相比，胞内三萜含量分别提高2.75%和24.85%，胞外多糖分别提高161.11%和113.64%；发酵液中高密度无性孢子含量达1.8×10⁷ 个·mL⁻¹。结论太子参适合作为樟芝双向液体发酵的药性成分，可有效促进樟芝菌丝生长、活性成分合成和无性孢子形成，是太子参根须副产物资源利用新途径。
- 樟芝 /
- 太子参 /
- 正交试验 /
- 培养基优化 /
- 活性成分
Abstract: Objective To utilize a Pseudostellaria heterophylla by-product for increasing Antrodia camphorata yield and bio-effectiveness, formulation of a culture medium was optimized. Methods Powder of P. heterophylla fine roots was included in formulating a medium to maximize the mycelial growth of A. camphorata by a Plackett-Burman orthogonal experimental design. Results In addition to 6 g·L⁻¹ of P. heterophylla powder, the medium was optimized to contain the ingredients critical to the mushroom growth, which included glucose 10 g·L⁻¹, yeast extract 4 g·L⁻¹, peptone 6 g·L⁻¹, and KH₂PO₄1.5 g·L⁻¹. The A. camphorata culture on the formulated medium produced 4.73 g·L⁻¹ biomass, which yielded 2.75% more intracellular triterpenoids than control and 24.85% more than PDB medium, and 161.11% more exopolysaccharides than control and 113.64% more than PDB medium. The fermentation broth also contained a high asexual spore at a density of 1.8×10⁷ spores·mL⁻¹. Conclusion P. heterophylla root powder could plausibly be added to the medium for A. camphorate culture as it promoted the mycelial growth, bioactive constituents yield, and asexual spore production of the mushroom.
- Antrodia camphorata /
- Pseudostellaria heterophylla /
- orthogonal experiment /
- medium formulation /
- bioactive composition

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图 1 樟芝在不同培养基发酵形成的无性孢子

Figure 1. Asexual A. camphorata spores produced in various culture media

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表 1 Plackett-Burman试验变量、水平及编码值

Table 1. Level and code of variables in Plackett-Burman design for medium formulation

变量 Variables	代码 Codes	水平 Levels/ (g·L⁻¹)
变量 Variables	代码 Codes	低 Low (−1)	高 High (+1)
葡萄糖 Glucose	A	0	10
酵母粉 Yeast extract	B	0	5
蛋白胨 Peptone	C	0	5
蔗糖 Sucrose	D	0	10
玉米粉 Corn flour	E	0	5
KH₂PO₄	F	0	1
MgSO₄·7H₂O	G	0	0.50

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表 2 L₁₆ (4⁵) 正交试验因素及水平

Table 2. Factors and levels in L₁₆(4⁵) Taguchi array optimization experiment

水平 Levels	因素 Factors/(g·L⁻¹)
水平 Levels	太子参（X₁）	葡萄糖（X₂）	酵母粉（X₃）	蛋白胨（X₄）	KH₂PO₄ （X₅）
1	2	5	2	2	0.5
2	4	10	4	4	1.0
3	6	15	6	6	1.5
4	8	20	8	8	2.0

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表 3 培养基成分筛选的PB试验设计及其生物量

Table 3. Plackett-Burman design matrix of variables for medium formulation and resulting yield of A. camphorata biomass

试验号 No.	因素 Factors									生物量 Biomass / (g·L⁻¹)
试验号 No.	葡萄糖（A）	酵母粉（B）	蛋白胨（C）	蔗糖（D）	虚拟变量（D_m1）	玉米粉（E）	KH₂PO₄（F）	MgSO₄·7H₂O（G）	虚拟变量（D_m2）	生物量 Biomass / (g·L⁻¹)
1	−1	−1	1	−1	1	1	−1	1	1	0.87
2	−1	1	1	1	−1	−1	−1	1	−1	1.81
3	1	−1	1	1	1	−1	−1	−1	1	1.45
4	1	1	1	−1	−1	−1	1	−1	1	3.26
5	1	−1	−1	−1	1	−1	1	1	−1	1.39
6	−1	−1	−1	−1	−1	−1	−1	−1	−1	1.88
7	−1	1	1	−1	1	1	1	−1	−1	1.43
8	1	1	−1	1	1	1	−1	−1	−1	2.10
9	1	1	−1	−1	−1	1	−1	1	1	1.04
10	−1	1	−1	1	1	−1	1	1	1	2.39
11	−1	−1	−1	1	−1	−1	1	−1	1	1.41
12	1	−1	1	1	−1	1	1	1	−1	1.92
D_m1和D_m2为虚拟变量。 D_m1 and D_m₂ were dummy variables.

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表 4 不同培养基成分对樟芝生物量的效应、贡献率及重要性排序

Table 4. Main effects, contribution rates, and orders of importance of medium ingredients on A. camphorata mycelial growth in submerged culture

因素 Factors	效应 Effects	贡献率 Percent contribution/%	重要性排序 Importance order
葡萄糖（A）	0.23	3.42	5
酵母粉（B）	0.52	17.61	2
蛋白胨（C）	0.088	0.51	7
蔗糖（D）	0.20	2.67	6
玉米粉（E）	−0.57	21.17	1
KH₂PO₄（F）	0.44	12.78	3
MgSO₄·7H₂O（G）	−0.35	8.11	4

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表 5 培养基优化的正交试验结果及极差分析

Table 5. Orthogonal experiment results and range analysis on medium optimization

试验号 Run	因素 Factors					生物量 Biomass/ (g·L⁻¹)
试验号 Run	太子参（X₁）	葡萄糖（X₂）	酵母粉（X₃）	蛋白胨（X₄）	KH₂PO₄ （X₅）	生物量 Biomass/ (g·L⁻¹)
1	1	1	1	1	1	2.90
2	1	2	2	2	2	4.41
3	1	3	3	3	3	4.52
4	1	4	4	4	4	2.53
5	2	1	2	3	4	2.92
6	2	2	1	4	3	4.10
7	2	3	4	1	2	3.34
8	2	4	3	2	1	4.51
9	3	1	3	4	2	2.67
10	3	2	4	3	1	4.62
11	3	3	1	2	4	3.93
12	3	4	2	1	3	3.82
13	4	1	4	2	3	3.26
14	4	2	3	1	4	3.62
15	4	3	2	4	1	2.48
16	4	4	1	3	2	3.17
K₁	3.592	2.940	3.423	3.527	3.630
K₂	3.717	4.188	4.027	3.408	3.397
K₃	3.760	3.567	3.807	3.830	3.925
K₄	3.132	3.507	2.945	3.437	3.250
极差R	0.628	1.248	1.082	0.422	0.675
优化水平 Optimal level	3	2	2	3	3

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表 6 正交试验方差分析

Table 6. Variance analysis on orthogonal experimental results

因素 Factors	平方和 Sum of squares	自由度 Degree of freedom	均方 Mean square	F值 F value	显著性 Significance
太子参（X₁）	0.992	3	0.331	2.214
葡萄糖（X₂）	3.135	3	1.045	6.998	*
酵母粉（X₃）	2.709	3	0.903	6.047	*
蛋白胨（X₄）	0.448	3	0.149	1.000
KH₂PO₄（X₅）	1.040	3	0.347	2.321
误差 Error	0.45	3	0.150
表示差异显著（P＜0.10）。 indicates significant difference (P＜0.10).

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表 7 不同培养基发酵樟芝菌丝产量以及菌丝和发酵液主要活性成分含量

Table 7. A. camphorata mycelia biomass and polysaccharide and triterpenoids contents in fermentation broth under submerged culture with different media

培养基 Medium	生物量 Biomass/(g·L⁻¹)	胞内三萜 Intracellular triterpenoids/(mg·g⁻¹)	胞内多糖 Intracellular polysaccharides/(mg·g⁻¹)	胞外多糖 Exopolysaccharides/(g·L⁻¹)
优化培养基 Optimum	4.73±0.07 a	20.15±0.78 a	69.21±3.84 a	0.94±0.49 a
对照培养基 Control	1.96±0.05 b	19.61±0.17 a	70.27±1.31 a	0.36±0.03 c
PDB培养基 PDB medium	1.34±0.08 c	16.14±1.51 b	56.39±0.86 b	0.44±0.04 b
不同小写字母表示不同培养基之间差异显著（P＜0.05）。 Data with different letters on same column represent significant differences at P＜0.05.

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