不同栽培基质诱导对香菇液体发酵产漆酶活性的影响

doi:10.13560/j.cnki.biotech.bull.1985.2021-0844

摘要/Abstract

摘要：

为探索贵州不同香菇菌株产漆酶能力,得到高产漆酶的栽培基质,从而为提高和改善香菇的产量和品质奠定理论基础,助推贵州香菇产业振兴。试验以贵州香菇主栽品种香菇808、庆科R20以及贵州特有马桑香菇为研究对象,通过拮抗反应、系统发育树及温度试验,对香菇菌株进行了区别性鉴定,并以香菇的主要栽培基质为诱导物,研究3种不同香菇菌株液体发酵产漆酶活性连续12 d的变化规律。马桑香菇、香菇808和庆科R20虽均同为香菇属的下属分支,但菌株间是有区别的,存在一定种源差异。香菇液体产漆酶活性受菌株遗传差异和4种不同诱导培养基的影响均为极显著（P<0.001）,且各处理下漆酶均呈现先升后降的趋势。在培养初期,所有处理下香菇产漆酶活性均较低,在培养中后期漆酶活性较高,峰值也主要集中在第8-11天内。相对菌株庆科R20和马桑香菇,香菇808在含玉米芯、马桑木屑及青杠木屑的诱导培养基中液体产漆酶能力均为最强,最大酶活分别为464.06、238.64、288.94 U/L。而麦麸对庆科R20的诱导力更强,最大酶活达265.07 U/L;麦麸对诱导马桑香菇产漆酶的时间最为集中且反应迅速,最大酶活达161.83 U/L,但高水平诱导力更持久的是马桑木屑,最大酶活达108.61 U/L。香菇808、庆科R20和马桑香菇3种菌株间是有区别的,存在一定种源差异,且从产漆酶水平上看,香菇808、庆科R20和马桑香菇对4种基质木质素降解吸收能力差异较大,最佳基质分别为玉米芯、麦麸和马桑木屑。

关键词: 香菇, 区别性, 栽培基质, 液体发酵, 漆酶

Abstract:

The aims are to explore the laccase-producing capacities of different Lentinula edodes strains in Guizhou and obtain a high-yield laccase cultivation substrate,and therefore lay a theoretical foundation for increasing and improving the yield and quality of L. edodes,for achieving the purpose of revitalizing the mushroom industry in Guizhou. Taking Guizhou L. edodes 808,Qingke R20 and Guizhou’s unique Masang L. edodes as the research objects,the discriminative identification of the three species of L. edodes strains was preliminarily studied through antagonistic reaction,phylogenetic tree and temperature tests. And the main cultivation substrates of L. edodes were used as an inducer,the changing pattern on the laccase production activities of three different L. edodes strains in liquid fermentation for 12 consecutive days were studied. Although Masang L. edodes,L. edodes 808 and Qingke R20 were all subordinate branches of organic L. edodes,and there were differences among the strains,meaning there were certain provenance differences. The liquid laccase activity of L. edodes was significantly affected by the genetic differences of the strains and the 4 different induction media（P<0.001）,and the laccase activity showed a trend of increasing first and then decreasing under each treatment. The laccase activity of L. edodes was lower under all treatments in the early stage of culture,higher in the middle and late stages of culture,and the peak value was mainly concentrated in the 8-11 d. Compared with the strain Qingke R20 and Masang L. edodes,L. edodes 808 had the strongest liquid laccase-producing capacity in the induction medium containing corn cob,Coriariane palensis sawdust and Quercus acutissima sawdust,and their maximum enzyme activity was 464.06,238.64 and 288.94 U/L,respectively. However,wheat bran was more inducible to Qingke R20,with the maximum enzyme activity reaching 265.07 U/L. In addition,wheat bran presented the most concentrated and rapid response time for inducing laccase production from Masang L. edodes,with the maximum enzyme activity reaching 161.83 U/L. However,the high level of inducibility was more durable in C. palensis sawdust,with the maximum enzyme activity reaching 108.61 U/L. In conclusion,there are differences among the three strains of Masang L. edodes,L. edodes 808 and Qingke R20,meaning that there were certain provenance differences. From the perspective of laccase production level,L. edodes 808,Qingke R20 and Masang L. edodes had great differences in lignin degradation and absorption capacity of 4 substrates,and their best substrates were corn cob,wheat bran and C. palensis sawdust,respectively.

Key words: Lentinula edodes, distinctiveness, cultivation substrate, liquid fermentation, laccase activity

熊雪, 李鹏, 张贵合, 向准, 陶文广, 周光燕, 和耀威. 不同栽培基质诱导对香菇液体发酵产漆酶活性的影响[J]. 生物技术通报, 2021, 37(12): 50-59.

XIONG Xue, LI Peng, ZHANG Gui-he, XIANG Zhun, TAO Wen-Guang, ZHOU Guang-yan, HE Yao-wei. Effects of Different Cultivation Substrates on the Laccase Activities of Lentinula edodes During Liquid Fermentation[J]. Biotechnology Bulletin, 2021, 37(12): 50-59.

图/表 9

表1 3种香菇拮抗试验对比

Table 1 Antagonistic reaction of 3 kinds of L. edodes strain

菌株Strain	马桑香菇 Masang L. edodes	香菇808 L. edodes 808	庆科R20 Qingke R20
马桑香菇 Masang L. edodes	-
香菇808 L. edodes 808	+	-
庆科R20 Qingke R20	+	-	-

图1 3种香菇拮抗反应 A:马桑香菇×马桑香菇;B:香菇808×香菇808;C:庆科R20×庆科R20;D:庆科R20（左）×马桑香菇（下）×香菇808（右）

Fig. 1 Antagonistic reaction of 3 kinds of L. edodes strain A:Masang L. edodes×Masang L. edodes. B:L. edodes 808×L. edodes 808. C:Qingke R20×Qingke R20. D:Qingke R20（left）×Masang L. edodes（underside）×L. edodes 808(right)

图2 3种香菇菌株rDNA ITS区段的PCR产物

Fig. 2 PCR products of the rDNA ITS segments of the 3 kinds of L. edodes strain

图3 基于ITS序列构建的香菇系统发育树

Fig. 3 Phylogenetic tree of L. edodes constructed based on ITS sequence

表2 不同温度条件对3种香菇菌株生长的影响

Table 2 Effects of different temperature conditions on the growths of 3 kinds of L. edodes strain

菌株 Strain	温度 Temperature/℃	萌发时间Germination time/h	菌落长势Colony growth	菌落描述 Colony description	日均生长速度 Average daily growth rate/（cm·d^-1）	差异显著性 Significant difference （P<0.05）
马桑香菇 Masang L. edodes	10	48	++	绒毛状白色圆形菌落,边缘整齐	0.11	e
	15	48	+++	绒毛状白色圆形菌落,边缘整齐	0.34	d
	20	48	++++	绒毛状白色圆形菌落,边缘整齐	0.49	b
	25	48	++++	绒毛状白色圆形菌落,边缘整齐	0.89	a
	30	48	+++	绒毛状白色圆形菌落,边缘整齐	0.41	c
香菇808 L. edodes 808	10	48	+	绒毛状白色圆形菌落,边缘整齐	0.15	d
	15	48	++	绒毛状白色圆形菌落,边缘整齐	0.28	c
	20	48	+++	绒毛状白色圆形菌落,边缘整齐	0.44	b
	25	48	++++	绒毛状白色圆形菌落,边缘整齐	0.85	a
	30	48	++++	绒毛状白色圆形菌落,边缘整齐	0.31	c
庆科R20 Qingke R20	10	48	++	绒毛状白色圆形菌落,边缘整齐	0.10	e
	15	48	+++	绒毛状白色圆形菌落,边缘整齐	0.29	c
	20	48	+++	绒毛状白色圆形菌落,边缘整齐	0.50	b
	25	48	++++	绒毛状白色圆形菌落,边缘整齐	0.89	a
	30	48	++	绒毛状白色圆形菌落,边缘较整齐	0.25	d

图4 不同温度条件下香菇菌丝日均生长速度

Fig. 4 Average daily growth rate of L. edodes under differ-ent temperature conditions

表3 4种栽培基质对不同香菇漆酶活性的影响

Table 3 Effects of 4 cultivation substrates on the laccase activities of different L. edodes

培养时间 Incubation period/d	漆酶活性Laccase activity/（U·L^-1）
培养时间 Incubation period/d	菌株 Strain	培养基 Substrate	菌株×培养基 Strain × Substrate
2	65.718^***	23.380^***	6.887^***
3	38.515^***	26.703^***	5.890^***
4	23.114^***	30.396^***	21.908^***
5	40.804^***	31.882^***	31.989^***
6	59.004^***	24.854^***	41.526^***
7	212.19^***	79.513^***	80.377^***
8	353.451^***	133.537^***	111.936^***
9	302.412^***	96.631^***	67.755^***
10	537.164^***	143.962^***	115.817^***
11	371.454^***	164.037^***	91.993^***
12	93.704^***	45.079^***	34.453^***

图5 3种香菇菌株在不同诱导培养基上液体发酵产漆酶活性对比 A:BM+MS:含马桑木屑的基础培养基;B:BM+QG:含青杠木屑基础培养基;C:BM+MF:含麦麸的基础培养基;D:BM+YMX:含玉米芯的基础培养基

Fig.5 Comparison of the laccase production activities of 3 kinds of L. edodes strains in liquid fermentation on different induction media A:BM+MS:Basic medium with C. palensis sawdust. B:BM+QG:Basic medium with Q. acutissima sawdust. C:BM+YMX:Basic medium with corncob. D:BM+YMX:Basic medium with wheat bran

图6 不同栽培基质对香菇液体发酵产漆酶活性的影响 A:马桑香菇;B:香菇808;C:庆科R20

Fig.6 Effects of different cultivation substrates on laccase production activities of different L. edodes strains A:Marsang L. edodes. B:L. edodes 808. C:Qingke R20

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