基于Box-Behnken法的马桑香菇菌株培养条件优化

Optimization of culture conditions for Lentinula edodes based on Box-Behnken design

摘要:

【背景】 野生香菇菌种资源的保护和开发将是稳固我国粮食安全的重要举措。【目的】 明确马桑香菇菌株不同状态的培养条件，为开发应用奠定基础。【方法】 基于菌株生物学特性进行单因素筛选、正交试验及响应面法分析，对菌株的固体培养及液体发酵的各参数进行优化。【结果】 采集的野生菌株Le.Msy鉴定为香菇，在24 ℃时生长状态良好，菌丝生长偏好于pH 5.0−6.0的弱酸性环境，培养基营养因子中对碳源没有强选择性，氮源中除尿素外均可生长，碳氮比为20:1−30:1时菌丝生长最好，并且无机盐中对镁离子有嗜好性；正交试验表明麦芽糖、麸皮、硫酸镁的组合最适宜菌株的生长；通过响应面优化的菌种培养基中主要因子的用量为碳源23.05 g/L、氮源6.14 g/L和无机盐5.48 g/L，7 d菌落直径达(50.10±1.02) mm；基于优化的培养基配比做液体发酵参数优化，生物量可作为稳定的预测指标，最佳发酵培养参数为温度26.71 ℃、转速127.21 r/min、接种量9.69块/100 mL，此时生物量干重可达(5 505.67±93.22) mg/L。【结论】 菌株Le.Msy对营养因子的适应性广，固体培养时菌落直径能作为精准预测生长发育模型的响应指标；而液体发酵时菌丝球密度、平均直径及有效接触面积只能在非极端温度范围内反应菌株状态，菌丝球生物量干重在任何参数变化时均能精准反映菌丝生长发育状态。

Abstract:

[Background] The conservation and development of wild Lentinula edodes germplasm resources is a crucial measure to ensure China’s food security. [Objective] To clarify the culture conditions for different states of L. edodes and lay a foundation for the development and application. [Methods] We optimized the parameters of solid culture and liquid fermentation by single factor tests, orthogonal design, and response surface methodology based on the biological characteristics of the strain. [Results] The wild strain Le.Msy collected was identified as L. edodes. It exhibited good growth at 24 ℃, with mycelial growth favoring a weakly acidic environment of pH 5.0–6.0. The strain showed no strong preference to carbon sources among the nutrients in the culture medium and could grow with other nitrogen sources except urea. The optimal carbon to nitrogen ratio for mycelial growth was within the range of 20:1 to 30:1, and the strain preferred magnesium ions among inorganic salts. The orthogonal experiment results indicated that the combination of maltose, wheat bran, and magnesium sulfate was most suitable for the strain growth. The medium formula optimized by the response surface methodology was composed of 23.05 g/L carbon source, 6.14 g/L nitrogen source, and 5.48 g/L inorganic salt, in which the colony diameter reached (50.10±1.02) mm after 7 days. Based on the optimized culture medium formula, liquid fermentation conditions were further optimized with biomass as the indicator. The optimal fermentation conditions were a temperature of 26.71 ℃, a shaking speed of 127.21 r/min, and an inoculum amount of 9.69 pieces per 100 mL, under which the dry biomass reached (5 505.67±93.22) mg/L. [Conclusion] Le.Msy exhibits broad adaptability to nutrients. In solid culture, colony diameter can serve as an accurate indicator of growth and development. In liquid fermentation, the density, average diameter, and effective contact area of the mycelial pellets can only reflect the strains status within a non-extreme temperature range. The dry mycelial biomass can accurately reflect the growth and development status under any parameter changes.