微白黄链霉菌G-1发酵液抗真菌特性研究和发酵条件优化

doi:10.13560/j.cnki.biotech.bull.1985.2020-0419

摘要/Abstract

摘要：

为明确微白黄链霉菌（Streptomyces albidoflavus）G-1发酵液抗真菌特性和较优发酵条件,本研究采用平板对峙法、生长速率法、单因素试验、RDA分析和响应面CCD设计分别对其抗真菌特性、产抗菌物质关键限制因子和发酵条件较优组合进行了探究。结果表明：S. albidoflavus G-1发酵液对马铃薯早疫病菌、西瓜枯萎病菌、小麦赤霉病菌、辣椒枯萎病菌、黄瓜疫霉病菌和桃褐腐病菌具有较好抑菌效果,抑菌率均可达70%;发酵液在≤80℃、紫外照射24 h、pH 3-13和稀释100倍条件下仍可保持70%以上抑菌率;产抗菌物质关键限制因子为初始pH值、温度、麦芽糖和马铃薯;较优发酵条件为8%麦芽糖、马铃薯16.5%、初始pH 6.5、温度30℃、转速160 r/min、发酵5 d,且在该条件下可使抑菌率提高14.46%。说明菌株G-1发酵液具有良好的抑真菌稳定性,且优化后的发酵条件可使菌株G-1在较低成本和较短时间内产生大量抗菌物质,研究结果为其后期田间应用和规模化发酵奠定了理论基础。

关键词: 微白黄链霉菌G-1, 抗真菌谱, 稳定性, 发酵条件

Abstract:

In order to understand the antifungal properties of the fermentation broth of Streptomyces albidoflavus G-1 and its optimal fermentation conditions,the methods of flat panel confrontation and growth rate were used to study the antifungal properties,and the single factor experiment,RDA analysis and response surface CCD design were applied to study the key limiting factors for the production of antifungal substances and the optimal combination of fermentation conditions. The results showed that fermentation broth of S. albidoflavus G-1 not only had more than 70% of inhibitory rate against Alternaria solani,Fusarium oxysporum f. sp. niveum,Fusarium graminerum,Fusarium oxysporum f. sp. capsicum,Phytophthora melonis and Monilinia fructicola,but also still maintained > 70% inhibitory rate at ≤80℃,UV irradiation 24 h,pH 3-13,and diluted 100 times. In addition,based on RDA analysis,it was found that initial pH value,temperature,maltose and potato were the key limiting factors for producing antifungal substances from strain G-1. The optimal culture condition was 8% maltose and 16.5% potato,initial pH value 6.5,temperature 30° C,speed 160 r / min,and fermentation time 5 d. Moreover,the antifungal rate increased by 14.46% at optimized fermentation condition. Therefore it is concluded that the fermentation broth of strain G-1 not only has fine antifungal stability,but also may largely produce antifungal compaounds with a lower cost and short time at optimized fermentation conditions,which may provide theoretical foundation for the field of large-scale fermentation and field application.

Key words: Streptomyces albidoflavus G-1, antifungal spectrum, stability, fermentation conditions

高振峰, 赵佳. 微白黄链霉菌G-1发酵液抗真菌特性研究和发酵条件优化[J]. 生物技术通报, 2021, 37(3): 53-64.

GAO Zhen-feng, ZHAO Jia. Study on Antifungal Properties of Fermentation Broth from Streptomyces albidoflavus G-1 and Optimization of Its Fermentation Condition[J]. Biotechnology Bulletin, 2021, 37(3): 53-64.

图/表 10

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植物病原真菌	菌株G-1抑菌带/ mm	菌株G-1发酵液抑菌率/%
植物病原真菌	菌株G-1抑菌带/ mm	对照菌落直径/mm	处理菌落直径/ mm	抑菌率/%
马铃薯早疫病菌	14.92±1.62a	59.44±2.96	18.11±2.47	75.86±4.77b
西瓜枯萎病菌	7.83±1.11c	85.33±0.71	26.67±1.58	73.03±1.90c
小麦赤霉病菌	14.08±1.24ab	85.56±0.53	24.78±1.48	75.45±1.86b
辣椒枯萎病菌	13.75±0.97b	82.78±0.97	28.44±0.88	69.86±0.90d
棉花立枯病菌	4.33±1.07d	85.22±0.67	59.00±3.46	32.68±4.44g
桃褐腐病菌	14.42±1.88a	82.22±1.92	21.22±1.39	78.96±2.15a
菜豆菌核病菌	3.42±1.08e	85.11±0.78	52.56±3.21	40.62±4.20f
菜豆炭疽病菌	7.58±1.24c	66.00±2.55	35.11±3.30	50.58±5.63e
番茄灰霉病菌	2.92±0.90f	85.22±0.67	72.89±2.62	15.36±3.52h
黄瓜疫霉菌	14.17±1.27ab	67.33±3.00	20.11±2.76	75.66±4.83b

植物病原真菌	菌株G-1抑菌带/ mm	菌株G-1发酵液抑菌率/%
植物病原真菌	菌株G-1抑菌带/ mm	对照菌落直径/mm	处理菌落直径/ mm	抑菌率/%
马铃薯早疫病菌	14.92±1.62a	59.44±2.96	18.11±2.47	75.86±4.77b
西瓜枯萎病菌	7.83±1.11c	85.33±0.71	26.67±1.58	73.03±1.90c
小麦赤霉病菌	14.08±1.24ab	85.56±0.53	24.78±1.48	75.45±1.86b
辣椒枯萎病菌	13.75±0.97b	82.78±0.97	28.44±0.88	69.86±0.90d
棉花立枯病菌	4.33±1.07d	85.22±0.67	59.00±3.46	32.68±4.44g
桃褐腐病菌	14.42±1.88a	82.22±1.92	21.22±1.39	78.96±2.15a
菜豆菌核病菌	3.42±1.08e	85.11±0.78	52.56±3.21	40.62±4.20f
菜豆炭疽病菌	7.58±1.24c	66.00±2.55	35.11±3.30	50.58±5.63e
番茄灰霉病菌	2.92±0.90f	85.22±0.67	72.89±2.62	15.36±3.52h
黄瓜疫霉菌	14.17±1.27ab	67.33±3.00	20.11±2.76	75.66±4.83b

处理		对照菌落直径/ mm	处理菌落直径/ mm	抑菌率/%
温度 /℃	室温	52.56±1.13	15.44±1.94	78.98±2.90a
	30	52.56±1.13	15.44±1.59	78.01±4.16a
	40	52.56±1.13	14.56±1.13	78.06±3.17a
	50	52.56±1.13	14.78±1.92	79.89±2.49a
	60	52.56±1.13	15.00±2.55	79.46±3.91a
	70	52.56±1.13	15.00±1.41	78.94±5.52a
	80	52.56±1.13	17.22±1.39	78.93±3.24a
	90	52.56±1.13	21.44±1.51	74.27±3.18b
	100	52.56±1.13	15.00±1.41	65.38±3.50c
初始pH值	3	52.56±1.13	15.44±1.51	78.03±3.19a
	5	52.56±1.13	15.22±1.39	78.52±2.81a
	7	52.56±1.13	15.22±0.97	78.52±1.79a
	9	52.56±1.13	15.00±1.22	78.93±2.91a
	11	52.56±1.13	15.11±0.78	78.72±1.78a
	13	52.56±1.13	15.00±1.22	78.95±2.77a
紫外照射时间/ h	0	52.56±1.13	15.33±1.00	78.25±2.29a
	4	52.56±1.13	15.11±1.90	78.70±4.16a
	6	52.56±1.13	15.11±1.45	78.71±3.21a
	8	52.56±1.13	15.00±0.87	78.96±1.93a
	10	52.56±1.13	15.11±1.27	78.73±2.68a
	12	52.56±1.13	15.00±1.32	78.95±2.93a
	24	52.56±1.13	15.00±1.22	78.95±2.72a
发酵液不同稀释倍数	0	54.33±2.18	16.22±0.97	77.25±1.67a
	10	54.33±2.18	16.33±1.12	77.01±1.59a
	50	54.33±2.18	17.00±1.12	75.66±1.98b
	100	54.33±2.18	18.89±1.76	71.80±3.39c
	200	54.33±2.18	26.33±1.58	56.61±4.89d
	300	54.33±2.18	34.67±2.50	39.74±6.27e
	500	54.33±2.18	46.00±2.06	16.65±5.41f

处理		对照菌落直径/ mm	处理菌落直径/ mm	抑菌率/%
温度 /℃	室温	52.56±1.13	15.44±1.94	78.98±2.90a
	30	52.56±1.13	15.44±1.59	78.01±4.16a
	40	52.56±1.13	14.56±1.13	78.06±3.17a
	50	52.56±1.13	14.78±1.92	79.89±2.49a
	60	52.56±1.13	15.00±2.55	79.46±3.91a
	70	52.56±1.13	15.00±1.41	78.94±5.52a
	80	52.56±1.13	17.22±1.39	78.93±3.24a
	90	52.56±1.13	21.44±1.51	74.27±3.18b
	100	52.56±1.13	15.00±1.41	65.38±3.50c
初始pH值	3	52.56±1.13	15.44±1.51	78.03±3.19a
	5	52.56±1.13	15.22±1.39	78.52±2.81a
	7	52.56±1.13	15.22±0.97	78.52±1.79a
	9	52.56±1.13	15.00±1.22	78.93±2.91a
	11	52.56±1.13	15.11±0.78	78.72±1.78a
	13	52.56±1.13	15.00±1.22	78.95±2.77a
紫外照射时间/ h	0	52.56±1.13	15.33±1.00	78.25±2.29a
	4	52.56±1.13	15.11±1.90	78.70±4.16a
	6	52.56±1.13	15.11±1.45	78.71±3.21a
	8	52.56±1.13	15.00±0.87	78.96±1.93a
	10	52.56±1.13	15.11±1.27	78.73±2.68a
	12	52.56±1.13	15.00±1.32	78.95±2.93a
	24	52.56±1.13	15.00±1.22	78.95±2.72a
发酵液不同稀释倍数	0	54.33±2.18	16.22±0.97	77.25±1.67a
	10	54.33±2.18	16.33±1.12	77.01±1.59a
	50	54.33±2.18	17.00±1.12	75.66±1.98b
	100	54.33±2.18	18.89±1.76	71.80±3.39c
	200	54.33±2.18	26.33±1.58	56.61±4.89d
	300	54.33±2.18	34.67±2.50	39.74±6.27e
	500	54.33±2.18	46.00±2.06	16.65±5.41f

来源	平方和	F值	P值	显著性（P<0.05）
模型	18 325.23	42.65	< 0.0001	显著
A	1 791.02	58.36	< 0.0001	显著
B	248.08	8.08	0.0123	显著
C	802.25	26.14	0.0001	显著
D	1 351.36	44.03	< 0.0001	显著
AB	8.61	0.28	0.6042
AC	9.41	0.31	0.5879
AD	82.22	2.68	0.1225
BC	14.03	0.46	0.5092
BD	1.27	0.041	0.8414
CD	111.47	3.63	0.0760
A²	10 703.87	348.79	< 0.0001	显著
B²	4 906.32	159.87	< 0.0001	显著
C²	261.39	8.52	0.0106	显著
D²	196.74	6.41	0.0230	显著
失拟项	401.33	3.40	0.0945	不显著