Isolation of Pure Species of Wild Boletaceae and Optimization of Inducer for Triterpenoid Synthesis in Liquid Culture
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摘要: 目的：为获得牛肝菌人工纯菌株，提高液态培养菌丝及产三萜类物质的能力。方法：以云南鲜野生牛肝菌子实体为研究对象，采用组织分离法进行母体纯菌株分离并鉴定；以金属离子Ca2+、亚油酸及香菇水提物作为诱导剂，研究其对液态菌丝体中三萜类物质含量的影响。结果：经多次分离纯化获得1株纯菌种NG-3，经 rDNA-ITS分子生物学鉴定，NG-3为暗褐网柄牛肝菌（Phlebopus portentosus）；对NG-3菌丝体进行SEM观察，在培养8~16 d时，出现具有结实能力的锁状联合形态菌丝。对3种诱导剂进行了响应面法优化，得到的结果为Ca2+浓度12 mmol/L、亚油酸量4%、香菇水提物浓度150 mg/100 mL，此时三萜含量为6.77 mg/g，比未加诱导剂三萜含量提高了76.00%。结论：3种诱导剂复合使用能够促进三萜类物质合成，提高其含量，为解决野生资源不足、实现牛肝菌功能产品产业化提供技术参数。Abstract: Objective: To obtain artificial pure strain of porcini and improve the ability of liquid culture mycelium and production of triterpenoid. Methods: The fresh wild Boletaceae fruit bodies from Yunnan were used for the study, and the parent pure strain was isolated and identified by tissue isolation method; Ca2+, linoleic acid, and mushroom aqueous extract were used as inducing agents to study their effects on the content of triterpenoids in liquid mycelium. Results: A pure strain of NG-3 was obtained by multiple purifications and identified by rDNA-ITS molecular biology as Phlebopus portentosus; the mycelium of NG-3 was observed by SEM, and the lock-like joint morphology mycelium with the ability of fruiting appeared at 8-16 d of cultivation. Three inducers were optimized by response surface methodology. The results showed that the concentration of Ca2+ was 12 mmol/L, the amount of linoleic acid was 4%, and the concentration of mushroom aqueous extract was 155 mg/100 mL, at which time the triterpene content was 6.77 mg/g, which was 76% higher than the triterpene content without inducer. Conclusion: The combination of three inducers can promote the synthesis of triterpenoids and increase their content, which can provide technical parameters to solve the shortage of wild resources and realize the industrialization of functional products of Boletaceae.
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Key words:
- wild Boletaceae /
- pure parent strain /
- liquid mycelium /
- triterpenoids /
- inductor
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图 1 NG-3牛肝菌的菌落、菌丝、孢子形态
Figure 1. The morphology of colony, mycelium and spore of NG-3 strains
注： a菌落形态；b菌丝形态；c孢子形态。
图 2 不同培养时间NG-3菌落的菌体形态
Figure 2. The morphology of NG-3 strains in different culture days
注：a、b、c分别为培养第8、16、24 d。
图 3 菌株NG-3 的ITS-PCR扩增电泳图
Figure 3. Electrophoresis pattern of ITS-PCR amplification of strain NG-3
注：5为暗褐网柄牛肝菌条带；M为Marke。
图 4 基于rDNA-ITS序列构建NG-3的系统发育树（Bookstrap≥50%）
Figure 4. Construction of the phylogenetic tree of NG-3 based on rDNA-ITS sequences （Bookstrap≥50%）
图 6 亚油酸对菌丝量及三萜含量的影响
Figure 6. Effect of linoleic acid on mycelium quantity and triterpene content
图 7 香菇水提物对菌丝量及三萜含量的影响
Figure 7. Effect of water extract of Lentinus edodes on mycelium quantity and triterpene content
图 8 各因素交互作用对三萜含量的影响
Figure 8. Effects of interaction of various factors on triterpenoid content
表 1 响应面试验因素与水平
Table 1. Response surface experiment factors and levels
因素 水平 −1 0 1 A Ca2+（mmol/L） 8 12 16 B 亚油酸（%） 3 4 5 C 香菇水提物（mg/100 mL） 100 150 200 
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表 2 响应面试验设计及结果
Table 2. Response surface experimental design and results
序号 A B C 三萜含量（mg/g） 1 0 1 1 5.63 2 1 0 1 4.03 3 0 0 0 6.69 4 1 −1 0 3.91 5 0 0 0 6.66 6 0 1 −1 4.24 7 −1 −1 0 4.31 8 −1 0 −1 4.29 9 0 −1 1 4.25 10 0 0 0 6.71 11 0 0 0 6.97 12 −1 0 1 4.79 13 0 −1 −1 4.68 14 1 1 0 4.58 15 −1 1 0 4.54 16 0 0 0 6.77 17 1 0 −1 4.28
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表 3 响应面模型方差分析
Table 3. Response surface model variance analysis
方差源 平方和 自由度 均方 F值 P值 显著性 模型 20.78 9 2.31 110.85 <0.0001 *** A 0.16 1 0.16 7.66 0.0278 * B 0.42 1 0.42 20.31 0.0028 ** C 0.18 1 0.18 8.79 0.0210 * AB 0.048 1 0.048 2.32 0.1713 AC 0.14 1 0.14 6.75 0.0355 * BC 0.83 1 0.83 39.75 0.0004 *** A2 8.12 1 8.12 389.81 <0.0001 *** B2 4.52 1 4.52 217.04 <0.0001 *** C2 4.41 1 4.41 211.83 <0.0001 *** 残差 0.15 7 0.021 失拟误差 0.084 3 0.028 1.82 0.2829 Not significant 纯误差 0.062 4 0.015 总和 20.93 16 R2=0.9983 R2Adj=0.9841 变异系数=2.81% 注：***差异高度显著（P<0.001）；**差异极显著（P<0.01）；*差异显著（P<0.05）。
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