一株野生肺形侧耳的鉴定及其液体发酵培养基的优化

doi:10.3969/j.issn.1004-1524.20231282

浙江农业学报 ›› 2024, Vol. 36 ›› Issue (11): 2535-2545.DOI: 10.3969/j.issn.1004-1524.20231282

一株野生肺形侧耳的鉴定及其液体发酵培养基的优化

龚娜(), 刘国丽, 陈珣, 马晓颖, 肇莹, 肖军()

辽宁省农业科学院食用菌研究所,辽宁沈阳 110161

收稿日期:2023-11-13 出版日期:2024-11-25 发布日期:2024-11-27
作者简介:龚娜(1982—),女,辽宁朝阳人,硕士,副研究员,主要从事食药用菌育种与栽培生理等研究工作。E-mail:doll52133@163.com
通讯作者: *肖军,E-mail:xiaojun20002@163.com
基金资助:
辽宁省应用基础研究计划(2022JH2/101300160);辽宁省农业科学院院长基金资助项目(2022QN2303);揭榜挂帅科技攻关专项资助项目(2021JH1/10400035);辽宁省农村科技特派行动计划(2022JH5/10400057);国家食用菌产业技术体系资助项目(CARS-20)

Identification of a wild strain of Pleurotus pulmonarius and optimization of its liquid fermentation culture medium

GONG Na(), LIU Guoli, CHEN Xun, MA Xiaoying, ZHAO Ying, XIAO Jun()

Institute of Edible Fungi, Liaoning Academy of Agricultural Sciences, Shenyang 110161, China

Received:2023-11-13 Online:2024-11-25 Published:2024-11-27

摘要/Abstract

摘要：

为优化肺形侧耳液体发酵的培养基配方,以采集到的野生侧耳菌株为试验材料,通过分子生物学方法确定该菌株为肺形侧耳,利用单因素试验筛选该野生肺形侧耳液体发酵培养基的最适碳源、氮源、无机盐及其质量浓度;根据单因素试验结果,选择玉米粉、豆粕粉、MgSO₄·7H₂O为考察因素,以肺形侧耳菌丝生物量为指标,采用Box-Benhnken设计优化肺形侧耳的液体发酵培养基。单因素试验结果表明,野生肺形侧耳液体发酵培养基的最适碳源、氮源、无机盐分别是玉米粉、豆粕粉和MgSO₄·7H₂O。Box-Benhnken设计试验结果显示:响应面回归模型拟合系数可达0.968 4,肺形侧耳的最佳液体发酵培养基配方为玉米粉24.36 g·L^-1、豆粕粉9.98 g·L^-1、MgSO₄·7H₂O 2.66 g·L^-1;在最佳培养基中,肺形侧耳菌丝体的理论生物量为11.15 mg·mL^-1,实测生物量为11.09 mg·mL^-1。优化后的液体发酵培养基比PDA培养基发酵时间缩短4 d,产量提高1.21 mg·L^-1。

关键词: 肺形侧耳, 发酵, 碳源, 氮源, 无机盐, 培养基, 生物量

Abstract:

In order to optimize the liquid fermentation medium formula of Pleurotus pulmonarius, the collected wild P. pulmonarius strain was used as experimental material, and the strain was determined as P. pulmonarius by molecular biology methods. The optimum carbon source, nitrogen source, inorganic salt and their mass concentrations in the liquid fermentation medium of the wild P. pulmonarius were screened by single factor test. Then, according to the results of single factor test, corn flour, soybean meal and MgSO₄·7H₂O were selected as the investigation factors, and the mycelial biomass of P. pulmonarius was used as the index. The liquid fermentation medium of P. pulmonarius was optimized by Box-Benhnken design. The results of single factor experiments showed that the optimum carbon source, nitrogen source and inorganic salt for liquid fermentation medium of wild P. pulmonarius were corn flour, soybean meal and MgSO₄·7H₂O, respectively. The results of Box-Benhnken design test showed that the fitting coefficient of response surface regression model could reach 0.968 4, and the optimal liquid fermentation medium formula of P. pulmonarius was corn flour 24.36 g·L^-1, soybean meal 9.98 g·L^-1, MgSO₄·7H₂O 2.66 g·L^-1. In the optimal medium, the theoretical biomass of P. pulmonarius mycelium was 11.15 mg·mL^-1, and the measured biomass was 11.09 mg·mL^-1.

Key words: Pleurotus pulmonarius, fermentation, carbon source, nitrogen source, inorganic salt, culture medium, biomass

中图分类号:

S646.14

龚娜, 刘国丽, 陈珣, 马晓颖, 肇莹, 肖军. 一株野生肺形侧耳的鉴定及其液体发酵培养基的优化[J]. 浙江农业学报, 2024, 36(11): 2535-2545.

GONG Na, LIU Guoli, CHEN Xun, MA Xiaoying, ZHAO Ying, XIAO Jun. Identification of a wild strain of Pleurotus pulmonarius and optimization of its liquid fermentation culture medium[J]. Acta Agriculturae Zhejiangensis, 2024, 36(11): 2535-2545.

图/表 12

表1 Box-Behnken实验设计的因素与水平

Table 1 Factors and levels of Box-Behnken design g·L-1

水平 Level	因素Factor
水平 Level	A玉米粉 Corn flour	B豆粕粉 Soybean meal	C MgSO₄·7H₂O
-1	10	6	1
0	20	9	2.5
1	30	12	4

图1 基于ITS序列构建的野生肺形侧耳的系统发育树

Fig.1 Phylogenetic tree of wild Pleurotus pulmonarius based on ITS sequence

图2 不同碳源培养基中野生肺形侧耳菌丝的长势、形态和生物量图A中1、2、3、4、5和6分别是以可溶性淀粉、蔗糖、木屑粉、葡萄糖、果糖和玉米粉为碳源的培养基。图B中柱上无相同小写字母表示差异显著(P<0.05)。

Fig.2 Growth, morphology and biomass of wild P. pulmonarius mycelium under different carbon sources 1, 2, 3, 4, 5 and 6 in figure A were media with soluble starch, sucrose, sawdust, glucose, fructose and corn flour as carbon source, respectively. The bars in figure B marked without the same lowercase letter indicated significant differences at P<0.05.

图3 不同质量浓度玉米粉培养基中肺形侧耳菌丝的生物量和菌球数量

Fig.3 The mycelium biomass and globule number of wild Pleurotus pulmonarius under different mass concentrations of corn meal

图4 不同氮源培养基中野生肺形侧耳菌丝的长势、形态和生物量图A中1、2、3、4、5和6分别是以蛋白胨、酵母膏、麸皮、牛肉膏、麦芽浸粉、豆粕粉为氮源的培养基。图B中柱上无相同小写字母表示差异显著(P<0.05)。

Fig.4 Growth, morphology and biomass of wild P. pulmonarius mycelium under different nitrogen sources 1, 2, 3, 4, 5 and 6 in figure A were media with peptone, yeast, bran, beef extract, malt extract and soybean meal as nitrogen source, respectively. The bars in figure B marked without the same lowercase letter indicated significant differences at P<0.05.

图5 不同质量浓度豆粕培养基中肺形侧耳的菌丝生物量和菌球数量

Fig.5 The mycelium biomass and globule number under different mass concentrations of soybean meal

图6 不同无机盐对肺形侧耳菌丝生长和生物量的影响 Ⅰ,KH2PO4;Ⅱ,MgSO4·7H2O;Ⅲ,NaCl;Ⅳ,CoCl2;Ⅴ,FeSO4;Ⅵ,ZnSO4。柱上无相同小写字母表示差异显著(P<0.05)。

Fig.6 Effect of different inorganic salts on the growth and biomass of P. pulmonarius mycelium Ⅰ, KH2PO4; Ⅱ, MgSO4·7H2O; Ⅲ, NaCl; Ⅳ, CoCl2; Ⅴ, FeSO4; Ⅵ, ZnSO4. The bars marked without the same lowercase letter indicated significant differences at P<0.05.

图7 不同质量浓度MgSO4·7H2O培养基中肺形侧耳的菌丝生物量和菌球数量

Fig.7 The mycelium biomass and globule number under different mass concentrations of MgSO4·7H2O

表2 响应面法试验设计和结果

Table 2 Experimental design and results of response surface method

序号 No.	因素Factor			菌丝生物量 Mycelium biomass/ (mg·mL^-1)
序号 No.	A玉米粉 Corn meal	B豆粕粉 Soybean meal	C MgSO₄·7H₂O	菌丝生物量 Mycelium biomass/ (mg·mL^-1)
1	-1	-1	0	3.65
2	1	-1	0	5.10
3	-1	1	0	5.08
4	1	1	0	9.50
5	-1	0	-1	4.63
6	1	0	-1	8.17
7	-1	0	1	4.43
8	1	0	1	8.97
9	0	-1	-1	5.78
10	0	1	-1	6.92
11	0	-1	1	5.15
12	0	1	1	7.90
13	0	0	0	10.15
14	0	0	0	11.47
15	0	0	0	10.67
16	0	0	0	11.03
17	0	0	0	9.50

表3 回归模型方差分析

Table 3 Variance analysis of regression model

方差来源 Source of variation	平方和 Sum of square	自由度 Degree of freedom	均方 Mean square	F值 F value	P
模型Model	4.220 0	9	0.468 5	23.85	0.000 2
A	0.970 7	1	0.970 7	49.42	0.000 2
B	0.472 1	1	0.472 1	24.03	0.001 7
C	0.004 5	1	0.004 5	0.23	0.646 3
AB	0.088 0	1	0.088 0	4.48	0.072 1
AC	0.010 0	1	0.010 0	0.51	0.498 6
BC	0.026 1	1	0.026 1	1.33	0.286 5
A²	0.897 7	1	0.897 7	45.71	0.000 3
B²	0.987 3	1	0.987 3	50.27	0.000 2
C²	0.489 4	1	0.489 4	24.91	0.001 6
残差Residual	0.137 5	7	0.019 6
失拟项Lack of fit	0.043 5	3	0.014 5	0.62	0.639 3
纯误差Pure error	0.094 0	4	0.023 5
总离差Total dispersion	4.350 0	16

图8 玉米粉、豆粕和MgSO4·7H2O交互作用对肺形侧耳菌丝生物量的影响

Fig.8 Effect of interaction among corn meal, soybean meal and MgSO4·7H2O on biomass of P. pulmonarius mycelium

图9 不同液体发酵培养基对肺形侧耳菌株生长曲线的影响

Fig.9 Effect of different liquid fermentation media on growth curve of P. pulmonarius strain

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一株野生肺形侧耳的鉴定及其液体发酵培养基的优化

Identification of a wild strain of Pleurotus pulmonarius and optimization of its liquid fermentation culture medium

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