Propagating Stropharia rugosoannulata in Liquid Medium
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摘要:目的 优化大球盖菇液体菌种培养基，探究液体培养过程中的生长规律，确立液体菌种繁育栽培种工艺参数。方法 以大球盖菇8号为试验菌株，以菌丝体生物量为评价指标，采用单因素和正交试验L9(34)优化液体菌种培养基。测定液体菌种菌丝体生物量，还原糖和氨基氮含量，羧甲基纤维素酶、淀粉酶、酸性蛋白酶、漆酶胞外酶酶活生理生化指标，确立优化配方的液体菌种最优培养时间。以平均满袋时间为指标，确立液体菌种扩繁栽培种接种量、培养基配方颗粒度和碳氮比。结果 优化得到大球盖菇液体菌种最优配方为葡萄糖20 g·L−1、小麦粉30 g·L−1、酵母粉0.75 g·L−1、磷酸二氢钾1.00 g·L−1、硫酸镁0.50 g·L−1、起始pH 5。培养第8 天时，大球盖菇菌丝体生物量最大，为1.66 g·hmL−1；液体培养过程中还原糖含量由12.23 mg·mL−1降至1.38 mg·mL−1，氨基氮含量由0.09 mg·mL−1降至0.06 mg·mL−1；羧甲基纤维素酶和淀粉酶酶活在第4 天最高，酶活分别为6.49 、5.16 U，酸性蛋白酶酶活在第2 天 最高，酶活为1.80 U，漆酶酶活在第6 天 最大，酶活为1.63 U。液体菌种扩繁栽培种生产工艺参数：接种量为15 mL，菌包培养基配方颗粒度的粗细木屑比为7∶3，碳氮比为50∶1。结论 大球盖菇液体菌种活性与上述指标具有一定的相关性，结合发酵液生理生化指标，判定第7天的液体菌种活力最高。利用大球盖菇液体菌种扩繁栽培种，平均满袋时间为23.7 d，缩短生长周期2.7 d。本研究建立配套的制种工艺，为大球盖菇栽培种工厂化生产技术奠定基础。Abstract:Objective PropagatingStropharia rugosoannulata in liquid culture medium was studied.Methods S. rugosoannulata No. 8 was cultivated in experimental media to determine the mycelial biomass increase in a single factor and orthogonal test L9(34) for formulation and culture conditions optimization. Mycelial biomass, reducing sugars, and amino nitrogen content as well as the extracellular enzyme activities of carboxymethyl cellulase, amylase, acid protease, and laccase of the culture were monitored to determine cultivation end point. Average full-bag filling time was used as indicator for the inoculation amount, substrate particle size, and carbon-to-nitrogen ratio in a maximized reproduction and yield.Results The optimized liquid culture medium was formulated with 20 g·L−1 glucose, 30 g·L−1 wheat flour, 0.75 g·L−1 yeast powder, 1.00 g·L−1 potassium dihydrogen phosphate, and 0.50 g·L−1 magnesium sulfate at an initial pH of 5. On the 8th day of culture, the mycelial biomass reached a maximum at 1.66 g·h mL−1. As the mycelia grew, the reducing sugar in the medium decreased from 12.23 mg·mL−1 to 1.38 mg·mL−1 and the amino nitrogen from 0.09 mg·L−1 to 0.06 mg·L−1 during the culture process. The activities of carboxymethyl cellulase and amylase were the highest on the 4th day with the activities of 6.49 U and 5.16 U, respectively. The activity of acid protease peaked at 1.80 U on the 2nd day; and that of laccase, at 1.63 U on the 6th day. The inoculum production was best carried out with an inoculation of 15 mL, a coarse-to-fine ratio of 7∶3 on the substrate particle size, and a carbon-to-nitrogen ratio of 50∶1.Conclusion Since the viability of the mushroom propagation correlated with some of the physiological and biochemical indicators of the liquid medium, 7 d was determined to be the peak for the cultivation. For filling a hyphal bag, 23.7d of culture was required, which was 2.7d shorter than what needed without the optimization. The results provided the basis for the development of a scale-up industrial operation.
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Keywords:
- Stropharia rugosoannulata /
- liquid culture /
- medium /
- optimization /
- application
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图 1 不同碳源对大球盖菇菌丝体生物量的影响
不同小写字母表示差异显著水平（P＜0.05）。图2~8、10~12同。
Figure 1. Effect of carbon sources on mycelial biomass of S. rugosoannulata
Data with different lowercase letters indicate significant difference at P＜0.05. Same for Figs. 2-8, and 10-12.
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图 2 不同氮源对大球盖菇菌丝体生物量的影响
Figure 2. Effect of nitrogen sources on mycelial biomass of S. rugosoannulata
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图 3 不同小麦粉添加量对大球盖菇菌丝体生物量的影响
Figure 3. Effect of amount of wheat flour on mycelial biomass of S. rugosoannulata
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图 4 不同酵母粉添加量对大球盖菇菌丝体生物量的影响
Figure 4. Effects of adding amount of yeast powder on mycelial biomass of S.rugosoannulata
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图 5 不同起始pH对大球盖菇菌丝体生物量的影响
Figure 5. Effect of initial pH on mycelial biomass of S. rugosoannulata
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图 7 不同培养时间的还原糖和氨基氮
Figure 7. Changes on reducing sugar and amino nitrogen at different culture time
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图 8 不同培养时间的胞外酶活性
Figure 8. Changes on extracellular enzyme activities at different culture time
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图 9 不同菌种类型扩繁栽培种生长情况
左边：固体原种，右边：液体原种
Figure 9. Mycelial growth under different culture type
Left: solid culture; right: liquid culture
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图 10 不同接种量对平均满袋时间的影响
Figure 10. Effect of inoculation dose on average fill time of hyphal bag
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图 11 不同颗粒度对平均满袋时间的影响
Figure 11. Effect of particle size on average fill time of hyphal bag
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图 12 不同碳氮比对平均满袋时间的影响
Figure 12. Effect of carbon-nitrogen ratio on average fill time of hyphal bag
表 1 正交试验因素和水平
Table 1 Orthogonal factors and levels on factors affecting mycelial biomass
水平
Levels因素 Factors/(g·L−1) A葡萄糖
A GlucoseB小麦粉
B Wheat flourC酵母粉
C Yeast powderD 起始pH
D Initial pH1 10 20 0.75 5 2 15 25 1.00 6 3 20 30 1.25 7 
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表 2 不同碳氮比的菌包培养基配方
Table 2 Culture media with varied carbon-nitrogen ratio in bag
编号
No.杂木屑
Wood
chips/%麸皮
Wheat
bran/%花生粕
Peanut
meal/%碳酸钙
calcium
carbonate/%碳氮比
Carbon
nitrogen
ratio1 83 14 2 1 70∶1 2 81 14 4 1 60∶1 3 80 14 5 1 50∶1 4 77 14 8 1 40∶1 5 73 14 12 1 30∶1
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表 3 培养基优化的正交试验结果
Table 3 Orthogonal experiment results and range analysis for medium optimization
试验号
No.因素 Factors 菌丝体
生物量
Mycelial
biomass/
( g·hmL-1)A葡萄糖
A GlucoseB小麦粉
B Wheat
flourC酵母粉
C Yeast
powderD起始pH
D Initial
pH1 1 1 1 1 1.23 2 1 2 3 3 1.12 3 1 3 2 2 1.29 4 2 1 3 2 1.18 5 2 2 2 1 1.34 6 2 3 1 3 1.44 7 3 1 2 3 1.28 8 3 2 1 2 1.56 9 3 3 3 1 1.59 K1j 3.64 3.68 4.23 4.17 K2j 3.96 4.02 3.91 4.03 K3j 4.43 4.33 3.89 3.84 k1j 1.21 1.23 1.41 1.39 k 2j 1.32 1.34 1.30 1.34 k 3j 1.48 1.44 1.30 1.28 极差 R 0.26 0.21 0.12 0.11 优化水平
Optimal level3 3 1 1
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表 4 正交试验方差分析
Table 4 Variance analysis on orthogonal experiment results
方差来源
Source平方和
Sum of
squares自由度
Degree of
freedom均方
Mean squareF值
F value显著性
SignificanceA葡萄糖 0.41 2 0.20 80.13 ** B小麦粉 0.28 2 0.14 54.28 ** C酵母粉 0.06 2 0.03 11.01 ** D起始 pH 0.09 2 0.05 18.39 ** 误差 Error 0.05 18 0.01 总和 Sum 49.92 27 **表示差异显著（P＜0.01）。
**means significant differences at P＜0.01.
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