大球盖菇液体菌种繁育工艺研究

曾志恒; 戴建清; 陈文智; 曾辉; 郭仲杰; 蔡志欣

大球盖菇液体菌种繁育工艺研究

福建省农业科学院食用菌研究所/特色食用菌繁育与栽培国家地方联合工程研究中心，福建　福州　350014

基金项目: 福建省属公益类科研院所基本科研专项（2021R1035002）；宁夏回族自治区重点研发计划项目子课题（2022BBF02022-04）；福建省农业科学院东西部协作项目（闽农科政[2023]8号）

详细信息

作者简介:
曾志恒（1984 —），男，硕士，助理研究员，主要从事食用菌工厂化制种技术研究，E-mail：67847563@qq.com

通讯作者:
蔡志欣（1983 —），男，硕士，副研究员，主要从事食用菌遗传育种及配套栽培技术研究，E-mail: 181351945@qq.com

中图分类号: S646.1
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出版历程
- 收稿日期: 2024-04-12
- 修回日期: 2024-06-14
- 网络出版日期: 2024-07-10

Study on liquid spawn reproductive technology of Stropharia rugosoannulata

Edible Fungi Institute of Fujian Academy of Agricultural Sciences/National and Local Joint Engineering ResearchCenter for Breeding & Cultivation of Featured Edible Fungi, Fuzhou, Fujian　350014, China

摘要

摘要: 目的本研究优化了大球盖菇液体菌种培养基，探究液体培养过程中生长规律，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。通过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 d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·L⁻¹降至1.38 mg·L⁻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 d最高，酶活分别为6.49 U和5.16 U，酸性蛋白酶酶活在第2 d 最高，酶活为1.80 U，漆酶酶活在第6 d 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7d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了基础。
- 大球盖菇 /
- 液体菌种 /
- 培养基 /
- 优化 /
- 应用
Abstract: Objective To optimize the liquid culture medium for Stropharia rugosoannulata and investigate its growth patterns during the liquid culture process, thereby establishing the parameters for the reproduction and cultivation of the liquid spawn. Methods Using S^. rugosoannulata No.8 as the experimental strain, the mycelial biomass was taken as the evaluation index. A single factor and orthogonal test L₉(3⁴) were employed to optimize the liquid culture medium. The 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 optimized liquid culture were measured to determine the end point of liquid culture. The average full-bag time was used as the indicator to establish the inoculation amount, particle size of the substrate formulation, and carbon-to-nitrogen ratio for reproduction and cultivation of the liquid spawn. Results The optimal formulation for the liquid spawn of S. rugosoannulata was determined to be 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, with an initial pH of 5. On the 8th day of culture, the mycelial biomass reached a maximum at 1.66 g/100mL. The content of reducing sugar decreased from 12.23 mg·L⁻¹ to 1.38 mg·L⁻¹, and the content of amino nitrogen decreased from 0.09 mg·L⁻¹ to 0.06 mg·L⁻¹ during the liquid culture process. The activities of carboxymethyl cellulase and amylase was the highest on the 4 th day with the activity of 6.49 U and 5.16 U, respectively. The activity of acid protease was the highest on the 2 nd day, and the enzyme activity was 1.80 U. The laccase activity was the highest on the 6 th day, and the enzyme activity was 1.63 U. The production parameters for the breeding and cultivation of the liquid inoculum were: an inoculation amount of 15 mL, a coarse-to-fine ratio of 7:3 for the particle size of the substrate formulation, and a carbon-to-nitrogen ratio of 50:1. Conclusion The viability of liquid spawn of S. rugosoannulata correlated with some of selected physiological and biochemical indices. Combining the physiological and biochemical indicators of the fermentation fluid, the highest vitality of the liquid spawn was judged to be on the 7th day. Using of liquid spawn of S. rugosoannulata for reproduction and cultivation, the average hyphal pocketful time is 23.7 d, which can shorten the growth cycle by 2.7 d. The research results have established the formulation of the liquid culture medium and the accompanying spawn-making process for S. rugosoannulata, laying the foundation for the industrial spawn production technology.
- Stropharia rugosoannulata /
- liquid spawn /
- 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2～6，8，10～12同。

Figure 1. Effects of different carbon sources onmycelial biomass of S.rugosoannulata

The different lowercase letters indicate the significant level of difference at P＜0.05. The same as Fig.2-6, 8, 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. Effects of different nitrogen sourceson 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. Effects of adding 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 不同培养时间菌丝生物量的变化

Figure 5. Changes on mycelial biomass at different culture time

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图 6 不同时间培养液还原糖和氨基氮的变化

Figure 6. Changes on reducing sugar and amino nitrogen at different culture time

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图 4 不同起始pH对大球盖菇菌丝体生物量的影响

Figure 4. Effects of initial pH on mycelial biomass of S.rugosoannulata

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图 7 不同时间培养液胞外酶菌丝活性的变化

Figure 7. Changes on extracellular enzyme activities at different culture time

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图 8 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 8. The growth of cultivation expanded by different spawn type (Left: solid spawn, right: liquid spawn)

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图 9 不同接种量对平均满袋时间的影响

Figure 9. Effect of different inoculation dose on the average hyphal pocketful time

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图 10 不同颗粒度对平均满袋时间的影响

Figure 10. Effect of different granularity on the average hyphal pocketful time

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图 11 不同碳氮比对平均满袋时间的影响

Figure 11. Effect of different carbon nitrogen ratio on the average hyphal pocketful time

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表 1 不同因素对菌丝体生物量影响的正交因素和水平

Table 1. Orthogonal factors and levels of influence of different factors on mycelial biomass

水平 Levels	因素 Factors/(g·L⁻¹)
水平 Levels	A葡萄糖 A Gucose	B小麦粉 B Wheat flour	C酵母粉 C Yeast powder	D 起始pH D Initial pH
1	10	20	0.75	5
2	15	25	1.00	6
3	20	30	1.25	7

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表 2 培养基优化的正交试验结果及极差分析

Table 2. Results of the orthogonal design experiments and range analysis for medium optimization

试验号 Run	因素 Factors				菌丝体生物量 Mycelial biomass/ ( g/100 mL)
试验号 Run	A葡萄糖 A Gucose	B小麦粉 B Wheat flour	C酵母粉 C Yeast powder	D起始pH D Initial pH	菌丝体生物量 Mycelial biomass/ ( g/100 mL)
1	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
K_1j	3.64	3.68	4.23	4.17
K_2j	3.96	4.02	3.91	4.03
K_3j	4.43	4.33	3.89	3.84
k_1j	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 level	3	3	1	1

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表 3 正交试验方差分析

Table 3. Variance analysis on the results of orthogonal experiment

方差来源 Source	平方和 Sum of squares	自由度 Degree of freedom	均方 Mean square	F值 Fvalue	显著性 Significance
A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
表示差异显者（P＜0.01）。 means significant differences (P＜0.01).

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表 4 不同碳氮比的菌包培养基配方

Table 4. Culture medium with different 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 ratio
1	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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参考文献(27)

[1]	贺新生. 现代菌物分类系统[M]. 北京: 科学出版社, 2015: 238.
[2]	黄年来. 大球盖菇的分类地位和特征特性 [J]. 食用菌, 1995, 17(6):11. HUANG N L. Taxonomic status and characteristics of Pleurotus ostreatus [J]. Edible Fungi, 1995, 17(6): 11. (in Chinese)
[3]	姚春馨, 王小艳, 王小蓉, 等. 大球盖菇栽培研究现状与绿色发展前景 [J]. 中国食用菌, 2023, 42(6):90−97. YAO C X, WANG X Y, WANG X R, et al. Research progress on cultivation and green development of Stropharia rugosoannulata [J]. Edible Fungi of China, 2023, 42(6): 90−97. (in Chinese)
[4]	边银丙. 大球盖菇栽培技术及其创新发展方向 [J]. 食药用菌, 2023, 31(06):370−377. BIAN Y B. Cultivation technology and its innovative development direction of Stropharia rugosoannulata [J]. Edible and Medicinal Mushrooms, 2023, 31(06): 370−377. (in Chinese)
[5]	杨晓波, 蔡为明, 金群力, 等. 大球盖菇大棚设施栽培试验 [J]. 食药用菌, 2020, 28(5):328−331. YANG X B, CAI W M, JIN Q L, et al. Cultivation experiment of Stropharia rugoso-annulata in greenhouse [J]. Edible and Medicinal Mushrooms, 2020, 28(5): 328−331. (in Chinese)
[6]	霍捷, 王卫平, 滑帆, 等. 大球盖菇栽培模式研究进展与发展方向探讨 [J]. 中国食用菌, 2020, 39(2):35−38. HUO J, WANG W P, HUA F, et al. Research progress and development direction of the cultivation model of Stropharia rugosoannulata [J]. Edible Fungi of China, 2020, 39(2): 35−38. (in Chinese)
[7]	LIU Y T, SUN J, LUO Z Y, et al. Chemical composition of five wild edible mushrooms collected from Southwest China and their antihyperglycemic and antioxidant activity [J]. Food and Chemical Toxicology: an International Journal Published for the British Industrial Biological Research Association, 2012, 50(5): 1238−1244. doi: 10.1016/j.fct.2012.01.023
[8]	LI X P, CUI W J, CUI Y F, et al. Stropharia rugoso-annulata acetylated polysaccharides alleviate NAFLD via Nrf2/JNK1/AMPK signaling pathways [J]. International Journal of Biological Macromolecules, 2022, 215: 560−570. doi: 10.1016/j.ijbiomac.2022.06.156
[9]	JIN M Z, ZHANG W Q, ZHANG X M, et al. Characterization, chemical modification and bioactivities of a polysaccharide from Stropharia rugosoannulata [J]. Process Biochemistry, 2023, 128: 30−39. doi: 10.1016/j.procbio.2023.02.008
[10]	WU J, SUZUKI T, CHOI J H, et al. An unusual sterol from the mushroom Stropharia rugosoannulata [J]. Tetrahedron Letters, 2013, 54(36): 4900−4902. doi: 10.1016/j.tetlet.2013.06.142
[11]	李正鹏, 吴萍, 陆晓民. 大球盖菇液体菌种培养条件的研究 [J]. 中国林副特产, 2006, (4):12−14. doi: 10.3969/j.issn.1001-6902.2006.04.005 LI Z P, WU P, LU X M. Study on the training condition of liquid culture Stropharia rugoso-annulata [J]. Forest by-Product and Speciality in China, 2006(4): 12−14. (in Chinese) doi: 10.3969/j.issn.1001-6902.2006.04.005
[12]	曾健勇, 张国财, 赵博, 等. 磁化水培养对大球盖菇菌丝生长特性的影响 [J]. 食品工业科技, 2017, 38(20):110−114. ZENG J Y, ZHANG G C, ZHAO B, et al. Effect of magnetized water liquid cultivation on growth characteristics of Stropharia rugoso-annulata mycelium [J]. Science and Technology of Food Industry, 2017, 38(20): 110−114. (in Chinese)
[13]	付婧璇, 耿丹萌, 高华, 等. 香蒲资源化开发中大球盖菇液体菌种的制备与应用 [J]. 河北大学学报(自然科学版), 2019, 39(1):76−79. FU J X, GENG D M, GAO H, et al. Preparation and application of liquid strains of Stropharia rugoso-annulata in the development of Typha resources [J]. Journal of Hebei University (Natural Science Edition), 2019, 39(1): 76−79. (in Chinese)
[14]	刘克芳, 于国荣, 王华丽, 等. 大球盖菇液体菌种培养基的优化 [J]. 中国食用菌, 2021, 40(8):24−28. LIU K F, YU G R, WANG H L, et al. Optimization of liquid spawn formula of Stropharia rugosoannulata [J]. Edible Fungi of China, 2021, 40(8): 24−28. (in Chinese)
[15]	杜瑞垚. 大球盖菇菌丝液体培养及液体菌种固化技术研究[D]. 武汉: 华中农业大学, 2023. DU R Y. Study on the Technology of Mycelia Liquid Culture and Liquid Spawn Solidification in Stropharia Rugosoannulata[D]. Wuhan: Huazhong Agricultural University, 2023. (in Chinese)
[16]	曾志恒, 曾辉, 程翊, 等. 双孢蘑菇发酵液还原糖和总糖的含量测定 [J]. 中国食用菌, 2018, 37(6):40−43,49. ZENG Z H, ZENG H, CHENG Y, et al. Determination of reducing sugar and total sugar content in fermentation liquid of Agaricus bisporus [J]. Edible Fungi of China, 2018, 37(6): 40−43,49. (in Chinese)
[17]	曾志恒, 曾辉, 程翊, 等. 双孢蘑菇发酵液氨基氮含量测定方法的研究 [J]. 食用菌, 2019, 41(5):77−80. doi: 10.3969/j.issn.1000-8357.2019.05.026 ZENG Z H, ZENG H, CHENG Y, et al. Study on determination method of amino nitrogen content in fermentation broth of Agaricus bisporus [J]. Edible Fungi, 2019, 41(5): 77−80. (in Chinese) doi: 10.3969/j.issn.1000-8357.2019.05.026
[18]	戴建清. 双孢蘑菇W192液体菌种摇瓶培养过程中的生理生化分析 [J]. 福建农业学报, 2021, 36(2):182−187. DAI J Q. Physiological and biochemical properties of Agaricus bisporus in shaking flask culture [J]. Fujian Journal of Agricultural Sciences, 2021, 36(2): 182−187. (in Chinese)
[19]	田华. 现代菌物分类系统[M]. 北京: 化学工业出版社, 2019.
[20]	刘金力, 唐琳, 陈思等. 大球盖菇液体菌种培养基碳氮源及培养条件的优化[J]. 黑龙江农业科学, 2019, (04): 94-99. [20] 刘金力, 唐琳, 陈思, 等. 大球盖菇液体菌种培养基碳氮源及培养条件的优化[J]. 黑龙江农业科学, 2019(4): 94-99. LIU J L, TANG L, CHEN S, et al. Optimization of carbon and nitrogen sources and culture conditions for liquid culture medium of Stropharia rugosoannulata [J]. Heilongjiang Agricultural Sciences, 2019, (04): 94-99. (in Chinese) LIU J L, TANG L, CHEN S, et al. Optimization of carbon and nitrogen sources and culture conditions of liquid culture medium of Stropharia rugosoannulata[J]. Heilongjiang Agricultural Sciences, 2019(4): 94-99. (in Chinese)
[21]	周韬. 香菇液体菌种培养基优化及其菌种质量评价体系的建立[D]. 武汉: 华中农业大学, 2017. ZHOU T. Research on medium optimization and quality evaluation system of Lentinula edodes liquid spawn[D]. Wuhan: Huazhong Agricultural University, 2017. (in Chinese)
[22]	曾志恒, 程翊, 曾辉, 等. 双孢蘑菇W192菌株液体发酵培养的研究 [J]. 福建农业学报, 2013, 28(8):763−769. doi: 10.3969/j.issn.1008-0384.2013.08.008 ZENG Z H, CHENG Y, ZENG H, et al. Study on fermentation of Agaricus bisporus strain W192 [J]. Fujian Journal of Agricultural Sciences, 2013, 28(8): 763−769. (in Chinese) doi: 10.3969/j.issn.1008-0384.2013.08.008
[23]	孙萌. 大球盖菇菌丝培养及胞外酶活性变化规律研究[D]. 延吉: 延边大学, 2013. SUN M. Mycelium culture and the variation regularity of extracellular enzyme activity of stropharia rugosoannulata Farlow[D]. Yanji: Yanbian University, 2013. (in Chinese)
[24]	戴建成. 大球盖菇培养基质的优化及工厂化生产的初步探究[D]. 南京: 南京农业大学, 2021. DAI J C. Optimization of Medium Quality and Preliminary Study on Industrial Production of Stropharia Rugosoannulata[D]. Nanjing: Nanjing Agricultural University, 2021. (in Chinese)
[25]	王升厚, 李玉双. 大球盖菇液体母种制备培养基碳氮源的优化 [J]. 北方园艺, 2008, (5):219−221. WANG S H, LI Y S. The optimization of carbonaceous compounds and nitrogenous substances for liquid culture media of Stropharia rugoso annulata [J]. Northern Horticulture, 2008(5): 219−221. (in Chinese)
[26]	黄桂英, 杨林, 左天兴. 提高白僵菌培养基透气性的试验[J]. 云南林业科技, 1996, 25(2): 71-73. [26] 黄桂英, 杨林, 左天兴. 提高白僵菌培养基透气性的试验[J]. 云南林业科技, 1996(2): 71-73. HUANG G Y, YANG L, ZUO T X. A Test on Raising BreathbiIity to Beauveria bassiana medium[J]. Journal of West China Forestry Science, 1996, 25(2): 71-73. (in Chinese) HUANG G Y, YANG L, ZUO T X. A Test on Raising BreathbiIity to Beauveria bassiana medium[J]. Yunnan Forestry Science and Technology, 1996(2): 71-73. (in Chinese)
[27]	曾志恒, 曾辉, 蔡志欣, 等. 双孢蘑菇新型颗粒原种配方的优化 [J]. 生物技术通报, 2021, 37(11):134−141. ZENG Z H, ZENG H, CAI Z X, et al. Formula optimization of the novel particle pre-culture spawn of Agaricus bisporus [J]. Biotechnology Bulletin, 2021, 37(11): 134−141. (in Chinese)