金耳类酵母型菌株分离与高产胞外多糖培养基优化

doi:10.3969/j.issn.1004-1524.2023.05.19

浙江农业学报 ›› 2023, Vol. 35 ›› Issue (5): 1154-1160.DOI: 10.3969/j.issn.1004-1524.2023.05.19

金耳类酵母型菌株分离与高产胞外多糖培养基优化

张璐¹^,²(), 李翘楚¹^,², 王增利², 丁强¹, 王鸿磊¹^,^*()

1.中国农业大学烟台研究院,山东烟台 264670
2.中国农业大学食品科学与营养工程学院,北京 100083

收稿日期:2022-05-27 出版日期:2023-05-25 发布日期:2023-06-01
作者简介:张璐(1997—),女,山西吕梁人,硕士研究生,从事生物与医药专业相关研究。E-mail:729133479@qq.com
通讯作者: *王鸿磊,E-mail: whl197749@163.com
基金资助:
山东省现代农业产业技术体系食用菌产业体系烟台综合试验站(SDAIT-07-14)

Isolation of yeast form strain from Tremella aurantialba and optimization of medium for its exopolysaccharide production

ZHANG Lu¹^,²(), LI Qiaochu¹^,², WANG Zengli², DING Qiang¹, WANG Honglei¹^,^*()

1. Yantai Institute of China Agricultural University, Yantai 264670, Shandong, China
2. College of Food Science & Nutrition Engineering, China Agricultural University, Beijing 100083, China

Received:2022-05-27 Online:2023-05-25 Published:2023-06-01

摘要/Abstract

摘要：

为获得纯金耳多糖,采用担孢子弹射法分离获得金耳类酵母型菌株(EF145),通过ITS序列测定对分离的菌株进行鉴定,测定金耳多糖的抗氧化性,通过正交试验优化高产胞外多糖培养基配方。结果显示:EF145菌株的菌体呈卵圆形,大小约为(2.5~4.0)μm×(2.0~4.0)μm,出芽增殖;菌落乳白色、表面凸起、光滑、湿润;其ITS序列与金耳子实体小条带一致,经Blast比对确定该菌株为金耳(Tremella aurantialba);该菌株多糖的羟自由基清除率为57.67%,超氧阴离子清除率为51.73%,DPPH自由基清除率为17.43%。EF145菌株培养的最佳碳源、氮源和无机盐分别是蔗糖、酒石酸铵和磷酸二氮钾,EF-145菌株产胞外多糖最佳培养基配方为蔗糖40 g·L^-1、酒石酸铵7.5 g·L^-1、磷酸二氢钾4 g·L^-1、土豆浸粉3 g·L^-1、维生素B₁ 0.2 g·L^-1。

关键词: 金耳, 类酵母型菌株, 胞外多糖, 培养基优化

Abstract:

In order to obtain pure exopolysaccharide from Tremella aurantialba through liquid fermentation, the yeast form strain EF145 of T. aurantialba was isolated by basidiospore ejection method and identified by ITS method, the antioxidant activity of its exopolysaccharide was studied. Orthogonal test was employed to optimize the medium formula for high yield exopolysaccharide. The result showed that the shape of EF145 was oval with a size of (2.5-4.0) μm×(2.0-4.0) μm, which proliferated by budding and its colonies were milky white, convex, smooth and moist. ITS sequence was consistent with the small band of T. aurantialba fruiting body, which was identified as T. aurantialba by BLAST analysis. The scavenging rates of hydroxyl radical, superoxide anion and DPPH radical of polysaccharide of T. aurantialba were 57.67%, 51.73% and 17.43%, respectively. The best carbon, nitrogen and inorganic salts of EF145 were sucrose, ammonium tartrate and potassium dihydrogen phosphate, respectively. The optimal medium formula for exopolysaccharide production was sucrose 40 g·L^-1, ammonium tartrate 7.5 g·L^-1, potassium dihydrogen phosphate 4 g·L^-1, potato powder 3 g·L^-1 and vitamin B₁ 0.2 g·L^-1.

Key words: Tremella aurantialba, yeast form strain, exopolysaccharide, medium optimization

中图分类号:

S646

张璐, 李翘楚, 王增利, 丁强, 王鸿磊. 金耳类酵母型菌株分离与高产胞外多糖培养基优化[J]. 浙江农业学报, 2023, 35(5): 1154-1160.

ZHANG Lu, LI Qiaochu, WANG Zengli, DING Qiang, WANG Honglei. Isolation of yeast form strain from Tremella aurantialba and optimization of medium for its exopolysaccharide production[J]. Acta Agriculturae Zhejiangensis, 2023, 35(5): 1154-1160.

图/表 10

表1 正交试验因素与水平设置表

Table 1 The factor and level of orthogonal experiment g·L-1

水平 Level	因素 Factors
水平 Level	A蔗糖质量浓度 Sucrose concentration	B酒石酸铵质量浓度 Ammonium tartrate concentration	C磷酸二氢钾质量浓度 Monopotassium phosphate concentration
1	30	7.5	2
2	40	10.0	4
3	50	12.5	6

图1 EF145菌株的分离培养 A,担孢子弹射法;B,EF145菌株平板培养物;C,EF145菌株发酵液;D,EF145菌株菌体显微观察(400×)。

Fig.1 Isolation and culture of strain EF145 A, Basidiospore ejection; B, Plate culture of strain EF145; C, Fermentation broth of strain EF145; D, Micrograph of strain EF145 (400×).

图2 PCR产物电泳图 M,DL2000 marker;1,金耳子实体;2,EF145 菌株。

Fig.2 Electrophorogram of PCR products M, DL2000 marker; 1, Fruiting body of Tremella aurantialba; 2. Strain EF145.

图3 EF145菌株系统发育树

Fig.3 Phylogenetic tree of strain EF145

图4 金耳胞外多糖抗氧化能力测定

Fig.4 Determination of antioxidant capacity of exopolysaccharide from T.aurantialba EF145

图5 碳源、氮源和无机盐对胞外多糖产量的影响柱上无相同小写字母表示差异显著(P<0.05)。下同。

Fig.5 Effect of carbon sources, nitrogen sources and inorganic salts on exopolysaccharide production Data marked without the same lowercase letter indicated significant differences at P<0.05.The same as below.

图6 蔗糖(A)、酒石酸铵(B)、磷酸二氢钾(C)质量浓度对胞外多糖产量的影响

Fig.6 Effect of concentration of sucrose (A), ammonium tartrateon (B) and monopotassium phosphate (C) on exopolysaccharide production

表2 正交试验结果

Table 2 Results of orthogonal experiment

试验号 Number	因素 Factors			胞外多糖含量 Exopolysaccharide production/(g·L^-1)
试验号 Number	A	B	C	胞外多糖含量 Exopolysaccharide production/(g·L^-1)
1	1	1	1	2.850±0.515
2	1	2	2	3.320±0.436
3	1	3	3	2.573±0.722
4	2	1	2	5.163±0.447
5	2	2	3	5.073±0.812
6	2	3	1	3.620±0.353
7	3	1	3	5.003±0.885
8	3	2	1	3.466±0.387
9	3	3	2	4.710±0.252

表3 极差分析结果

Table 3 Result of range analysis

因素 Factor	胞外多糖含量Exopolysaccharide production
因素 Factor	k₁/(g·L^-1)	k₂/(g·L^-1)	k₃/(g·L^-1)	R/(g·L^-1)	最优水平Optimal level
A蔗糖Sucrose concentration	2.914	4.619	4.393	1.705	2
B酒石酸铵Ammonium tartrate concentration	4.339	3.953	3.634	0.705	1
C磷酸二氢钾Monopotassium phosphate concentration	3.312	4.398	4.216	1.086	2

表4 正交试验方差分析结果

Table 4 The variance analysis results of orthogonal experiment

方差来源 Sources of variance	离差平方和 Sum of squares of deviations	自由度 Degree of freedom	均方 Mean square	F值 F value	P值 P value
A蔗糖质量浓度 Sucrose concentration	15.429	2	7.715	58.716	<0.001
B酒石酸铵质量浓度 Ammonium tartrate concentration	2.240	2	1.120	8.523	0.002
C磷酸二氢钾质量浓度 Monopotassium phosphate concentration	6.088	2	3.044	23.167	<0.001
误差Error	2.628	20	0.131

参考文献 21

[1]	LIU X Z, WANG Q M, THEELEN B, et al. Phylogeny of tremellomycetous yeasts and related dimorphic and filamentous basidiomycetes reconstructed from multiple gene sequence analyses[J]. Studies in Mycology, 2015, 81: 1-26. DOI PMID
[2]	LIU X Z, WANG Q M, GÖKER M, et al. Towards an integrated phylogenetic classification of the Tremellomycetes[J]. Studies in Mycology, 2015, 81: 85-147. DOI PMID
[3]	刘正南, 郑淑芳. 金耳的经济价值和开发利用状况[J]. 中国食用菌, 1995, 14(1): 23-24.
	LIU Z N, ZHENG S F. Economic value and development and utilization of Tremella aurantialba[J]. Edible Fungi of China, 1995, 14(1): 23-24. (in Chinese)
[4]	DENG C, SUN Y Y, FU H T, et al. Antioxidant and immunostimulatory activities of polysaccharides extracted from Tremella aurantialba mycelia[J]. Molecular Medicine Reports, 2016, 14(5): 4857-4864. DOI URL
[5]	DU X J, WANG X D, CHEN Y, et al. Antioxidant activity and oxidative injury rehabilitation of chemically modified polysaccharide (TAPA1) from Tremella aurantialba[J]. Macromolecular Research, 2018, 26(6): 479-483. DOI
[6]	DU X J, ZHANG J S, LV Z W, et al. Chemical modification of an acidic polysaccharide (TAPA1) from Tremella aurantialba and potential biological activities[J]. Food Chemistry, 2014, 143: 336-340. DOI URL
[7]	邓云霞. 金耳胞外多糖的结构分析及其生物学活性的探讨[D]. 上海: 华东师范大学, 2004.
	DENG Y X. Study on structural analysis and biological activities of polysaccharides from fermentation filtrate of Tremella aurantialba[D]. Shanghai: East China Normal University, 2004. (in Chinese with English abstract)
[8]	苑小林, 张培德, 陈石根. 金耳水溶性多糖的部分化学性质及抑瘤试验[J]. 复旦学报(自然科学版), 1996, 35(6): 703-706.
	YUAN X L, ZHANG P D, CHEN S G. Preliminary study on chemical properties of a soluble polysaccharide from the mushnoom Tremella aurantia, and its oncosuppressive activity[J]. Journal of Fudan University(Natural Science), 1996, 35(6): 703-706. (in Chinese with English abstract)
[9]	张雯, 赵旌旌, 王捷思, 等. 金耳菌丝体多糖对实验性2型糖尿病大鼠的降血糖作用研究[J]. 天然产物研究与开发, 2010, 22(1): 49-53.
	ZHANG W, ZHAO J J, WANG J S, et al. Hypoglycemic activity of polysaccharides extracted from Tremella aurantialba mycelia in experimental type 2 diabetic rats[J]. Natural Product Research and Development, 2010, 22(1): 49-53. (in Chinese with English abstract)
[10]	魏涛, 张静, 高兆兰. 金耳菌丝体多糖对高血糖小鼠血糖作用的研究[J]. 安徽农业科学, 2012, 40(12): 7034-7035.
	WEI T, ZHANG J, GAO Z L. Hypoglycemic activity of polysaccharides extracted from Tremella aurantialba Mycelium in experimental diabetic mice[J]. Journal of Anhui Agricultural Sciences, 2012, 40(12): 7034-7035. (in Chinese with English abstract)
[11]	蒋剑平, 熊耀康, 俞冰, 等. 金耳多糖对豚鼠哮喘模型肺功能的影响[J]. 中华中医药杂志, 2013, 28(1): 206-209.
	JIANG J P, XIONG Y K, YU B, et al. Effect of Tremella mesenterica polysacchrides on pulmonary function in asthmatic model of Guinea pigs[J]. China Journal of Traditional Chinese Medicine and Pharmacy, 2013, 28(1): 206-209. (in Chinese with English abstract)
[12]	刘欣, 刘虹, 赵照林, 等. 金耳栽培条件的初步研究[J]. 中国食用菌, 2019, 38(5): 15-17.
	LIU X, LIU H, ZHAO Z L, et al. Preliminary study on cultivation condition of Tremella aurantialba[J]. Edible Fungi of China, 2019, 38(5): 15-17. (in Chinese with English abstract)
[13]	刘春卉, 谢红, 刘芰华, 等. 金耳子实体和发酵菌丝体多糖的分离、纯化与结构的比较研究[J]. 菌物系统, 1998, 17(3): 246-250.
	LIU C H, XIE H, LIU J H, et al. Isolation, purification and structural comparison investigation of polysaccharides of artificial cultivated fruit body and fermented mycelia of Tremella aurantialba[J]. Mycosystema, 1998, 17(3): 246-250. (in Chinese with English abstract)
[14]	DAI C X, HUANG X Y, HUANG D M, et al. Detection of submerged fermentation of Tremella aurantialba using data fusion of electronic nose and tongue[J]. Food Process Engineering, 2019, 42: e1300.
[15]	叶彬. 金耳菌种的分子鉴定及出菇实验[D]. 福州: 福建农林大学, 2012.
	YE B. Identification and cultivation of golden Tremella(Tremella aurantialba)[D]. Fuzhou: Fujian Agriculture and Forestry University, 2012. (in Chinese with English abstract)
[16]	刘春卉. 金耳生药学和发酵产物活性的研究[D]. 上海: 华东师范大学, 2007.
	LIU C H. Studies on pharmacognosy of Tremella aurantialba and bioactivities of its fermented products[D]. Shanghai: East China Normal University, 2007. (in Chinese with English abstract)
[17]	姜浩, 孙涛, 姚皓昱, 等. 食用真菌多糖的研究进展[J]. 食品工业科技, 2022, 43(12): 447-456.
	JIANG H, SUN T, YAO H Y, et al. Research progress of edible fungal polysaccharides[J]. Science and Technology of Food Industry, 2022, 43(12): 447-456. (in Chinese with English abstract)
[18]	李娜, 唐传红, 刘艳芳, 等. 深层发酵用高产灵芝胞内多糖菌株的筛选[J]. 食用菌学报, 2021, 28(3): 63-71.
	LI N, TANG C H, LIU Y F, et al. Screening of Ganoderma lucidum strains for high intracellular polysaccharide yield in submerged fermentation[J]. Acta Edulis Fungi, 2021, 28(3): 63-71. (in Chinese with English abstract)
[19]	刘正南, 郑淑芳. 金耳的生理特性及有效优良菌种的制备原理(续)[J]. 中国食用菌, 1995, 14(6):10-11.
	LIU Z N, ZHENG S F. Physiological characteristics of Tremella aurantialba and preparation principle of effective and excellent strains (continued)[J]. Edible Fungi of China, 1995, 14(6):10-11. (in Chinese)
[20]	杨林雷, 李荣春, 曹瑶, 等. 金耳的学名及分类地位考证[J]. 食药用菌, 2020, 28(4): 252-255.
	YANG L L, LI R C, CAO Y, et al. Textual research on the scientific Name and classification status of Auricularia auricula[J]. Edible and Medicinal Mushrooms, 2020, 28(4): 252-255. (in Chinese)
[21]	柴红梅, 赵永昌, 李树红, 等. 金耳酵母状孢子发酵产多糖研究[J]. 中国食用菌, 2008, 27(3): 39-40.
	CHAI H M, ZHAO Y C, LI S H, et al. Study on the polysaccharide production from yeast-like conidia of Tremella aurantiabla[J]. Edible Fungi of China, 2008, 27(3): 39-40. (in Chinese with English abstract)

金耳类酵母型菌株分离与高产胞外多糖培养基优化

Isolation of yeast form strain from Tremella aurantialba and optimization of medium for its exopolysaccharide production

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