Study on liquid spawn reproductive technology of Stropharia rugosoannulata
-
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·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 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了基础。 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. -
Key words:
- Stropharia rugosoannulata /
- liquid spawn /
- medium /
- optimization /
- application
-
表 1 正交试验因素和水平
Table 1. Orthogonal factors and levels
水平
Levels因素 Factors/(g·L−1) A葡萄糖
A GucoseB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 表 2 培养基优化的正交试验结果
Table 2. Results of the orthogonal design experiments and range analysis for medium optimization
试验号
No.因素 Factors 菌丝体
生物量
Mycelial
biomass/
( g·h mL-1)A葡萄糖
A GucoseB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 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 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 **表示差异显者(P<0.01)。
**means significant differences (P<0.01).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
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 -
[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.1001-6902.2006.04.005LI 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.1000-8357.2019.05.026ZENG 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.1008-0384.2013.08.008ZENG 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]. 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) -