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

doi:10.19303/j.issn.1008-0384.2024.06.007

大球盖菇液体菌种繁育工艺研究

doi: 10.19303/j.issn.1008-0384.2024.06.007

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: 福建省科技计划公益类专项（2021R1035002）；宁夏回族自治区重点研发计划项目（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
计量
- 文章访问数: 190
- HTML全文浏览量: 74
- PDF下载量: 40
- 被引次数: 0
出版历程
- 收稿日期: 2024-04-12
- 修回日期: 2024-05-14
- 网络出版日期: 2024-07-10
- 刊出日期: 2024-06-28

Propagating Stropharia rugosoannulata in Liquid Medium

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

摘要

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。
- 大球盖菇 /
- 液体菌种 /
- 培养基 /
- 优化 /
- 应用
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.
- Stropharia rugosoannulata /
- liquid culture /
- medium /
- optimization /
- application

HTML全文

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.

下载: 全尺寸图片幻灯片

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

下载: 全尺寸图片幻灯片

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

下载: 全尺寸图片幻灯片

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

下载: 全尺寸图片幻灯片

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

下载: 全尺寸图片幻灯片

图 6 不同培养时间的菌丝生物量

Figure 6. Changes on mycelial biomass at different culture time

下载: 全尺寸图片幻灯片

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

下载: 全尺寸图片幻灯片

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

下载: 全尺寸图片幻灯片

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

下载: 全尺寸图片幻灯片

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

下载: 全尺寸图片幻灯片

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

下载: 全尺寸图片幻灯片

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

下载: 全尺寸图片幻灯片

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)
水平 Levels	A葡萄糖 A Glucose	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

下载: 导出CSV

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 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

下载: 导出CSV

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)
试验号 No.	A葡萄糖 A Glucose	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·hmL^-1)
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

下载: 导出CSV

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 square	F值 F value	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
表示差异显著（P＜0.01）。 means significant differences at P＜0.01.

下载: 导出CSV

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(4): 94-99. 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. 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)