深海真菌101#的分子鉴定、次生代谢产物及其生物活性研究*

doi:10.11978/2022242

Abstract

Abstract:

In order to provide reference basis for development and application of deep-sea microbial resources, the molecular identification, secondary metabolites and biological activity of a fungal strain (named as 101#) isolated from deep-sea sediment of the South China Sea was studied. The fungal strain was analyzed based on rDNA-ITS gene sequence. The changes in the content of its fermentation products were tracked by high performance liquid chromatography (HPLC). The secondary metabolites were isolated by silica gel column chromatography, Sephadex LH-20 and HPLC. The structures of compounds were identified by nuclear magnetic resonance (NMR), mass spectrometry (MS), and literature analysis. The biological activities were detected by filter paper-agar diffusion and brine shrimp lethal methods, respectively. The results show that the fungal strain 101# was identified as Aspergillus flavus. The optimal fermentation period was 28 d. In addition, the crude extract had certain inhibitory activities against 15 indicator bacteria. The minimum inhibitory concentration (MIC) against 10 Gram-positive and negative bacteria and 3 plant pathogenic bacteria were 0.781 ~ 6.25 μg·mL^-1 and 50 ~ 200 μg·mL^-1, respectively, and the MIC against marine biofouling bacterium (Alteromonas macleodii) and pathogenic fungus (Candida albicans) were 3.125 μg·mL^-1 and 1.563 μg·mL^-1, respectively. The crude extract had lethal activity against brine shrimp, and their LD₅₀ at 1, 12, 24 and 48 h were 74.597, 24.322, 13.797 and 8.559 mg·mL^-1, respectively. Finally, 4 monomer compounds were isolated from the ethyl acetate extraction site of the strain and their structures were identified as 5-chloro-2-hydroxyphenylacetic acid (1), aspergamide A (2), WF-3681 methyl Ester (3) and (E)-But-2-enedioic acid monomethyl Ester (4). The four compounds showed certain inhibitory activities against B. subtilis, C. albicans, B. thuringiensis, Shewanella and M. luteus, which MIC value were 6.25 ~ 200 μg·mL^-1. Furthermore, compounds 1 ~ 4 showed strong brine shrimp lethal activity with LD₅₀ values of 7.40, 11.95, 17.69, 23.35 μg·mL^-1, respectively.

Key words: deep-sea-fungus, molecular identification, secondary metabolite, antibacterial activity, biological toxicity

ZENG Buyan, LIANG Zhifeng, LUO Qinqin, ZENG Ling, YANG Changgeng, WANG Liyun, SUN Yulin. Molecular identification, secondary metabolites and biological activities of a deep-sea-derived fungus 101#^*[J].Journal of Tropical Oceanography, 2023, 42(5): 104-114.

Figures/Tables 8

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Tab 3.

Tab. 4

References 31

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指示菌	阳性对照	粗提物浓度/(μg·mL^-1)						MIC/(μg·mL^-1)
指示菌	阳性对照	12.5	6.25	3.125	1.563	0.781	0.391	MIC/(μg·mL^-1)
枯草芽孢杆菌 (Bacillus subtilis)	＋＋＋＋	＋＋	＋＋	＋	＋	＋	－	0.781
藤黄微球菌 (Micrococcus luteus)	＋＋＋＋	＋＋	＋＋	＋	＋	－	－	1.563
苏云金芽孢杆菌 (Bacillus thuringiensis)	＋＋＋＋	＋＋	＋	＋	＋	－	－	1.563
耐甲氧西林金黄色葡萄球菌 (Methicillin-resistant staphylococcus aureus)	＋＋＋＋	＋＋	＋	＋	－	－	－	3.125
金黄色葡萄球菌 (Staphylococcus aureus)	＋＋＋＋	＋＋	＋	＋	－	－	－	3.125
无乳链球菌 (Streptococcus agalactiae)	＋＋＋＋	＋＋	＋	－	－	－	－	6.250
稀瓦氏菌 (Shewanella)	＋＋＋＋	＋＋	＋	＋	＋	－	－	1.563
大肠杆菌 (Escherichia coli)	＋＋＋＋	＋＋	＋	＋	－	－	－	3.125
福氏志贺菌 (Shigella Flexneri)	＋＋＋＋	＋＋	＋	＋	－	－	－	3.125
肺炎克雷伯菌 (Klebsiella pneumoniae)	＋＋＋＋	＋	＋	－	－	－	－	6.250
麦氏交替单胞菌 (Alteromonas macleodii) SCAU-003	＋＋＋＋	＋＋＋	＋＋	＋	－	－	－	3.125
多耐白色念珠菌 (Candida albicans)	＋＋＋＋	＋＋＋	＋＋	＋	＋	－	－	1.563

指示菌	阳性对照	粗提物浓度/(μg·mL^-1)					MIC/(μg·mL^-1)
指示菌	阳性对照	200	100	50	25	12.5	MIC/(μg·mL^-1)
禾谷镰刀菌 (Fusarium graminearum)	＋＋	＋＋	＋	＋	－	－	50
意大利青霉 (Penicillium italicum)	＋＋＋＋	＋	－	－	－	－	200
尖孢镰刀菌 (Fusarium oxysporum)	＋	＋	－	－	－	－	200
层出镰刀菌 (Fusarium proliferatum)	＋	－	－	－	－	－	>200
盘长孢状刺盘孢 (Colletotrichum gloeosprioides)	＋	－	－	－	－	－	>200
茄镰孢菌 (Fusarium solani)	＋＋	－	－	－	－	－	>200

时间/h	粗提物浓度/( mg·mL^-1)					LD₅₀/(mg·mL^-1)	95%置信区间
时间/h	100	50	25	12.5	6.25	LD₅₀/(mg·mL^-1)	下限	上限
1	64.7	38.9	25.0	10.0	0.0	74.597	54.900	118.483
12	100.0	83.3	56.3	30.0	12.5	24.322	17.238	34.071
24	100.0	100.0	87.5	35.5	18.8	13.797	9.739	19.393
48	100.0	100.0	93.8	65.0	37.5	8.559	4.888	12.455

化合物	枯草芽孢杆菌	多耐白色念珠菌	苏云金芽孢杆菌	希瓦氏菌	藤黄微球菌
1	25	12.5	50	200	>200
2	>200	6.25	25	>200	100
3	25	12.5	12.5	>200	>200
4	12.5	6.25	50	200	12.5
硫酸庆大霉素	12.5	6.25	1.56	12.5	12.5

Molecular identification, secondary metabolites and biological activities of a deep-sea-derived fungus 101#^*

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Molecular identification, secondary metabolites and biological activities of a deep-sea-derived fungus 101#*

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Molecular identification, secondary metabolites and biological activities of a deep-sea-derived fungus 101#^*