深海真菌101#的分子鉴定、次生代谢产物及其生物活性研究*
曾步燕(2000—), 女, 学士, 研究方向为海洋生物活性物质挖掘。email: |
收稿日期: 2022-11-11
修回日期: 2022-12-26
网络出版日期: 2022-12-28
基金资助
国家自然科学基金项目(31902373)
广东省自然科学基金项目(2021A1515011398)
广东省教育厅项目(2021ZDJS034)
广东省教育厅项目(2021KCXTD039)
广东省教育厅项目(2022-K08)
南海海洋生物医药资源研发公共服务平台项目(2017C8B2)
岭南师范学院燕岭优秀青年教师培养计划项目(YL20200213)
岭南师范学院红树林研究院开放课题项目(PYXM05)
Molecular identification, secondary metabolites and biological activities of a deep-sea-derived fungus 101#*
Received date: 2022-11-11
Revised date: 2022-12-26
Online published: 2022-12-28
Supported by
National Natural Science Foundation of China(31902373)
Natural Science Foundation of Guangdong Province, China(2021A1515011398)
Project of Education Department of Guangdong Province, China(2021ZDJS034)
Project of Education Department of Guangdong Province, China(2021KCXTD039)
Project of Education Department of Guangdong Province, China(2022-K08)
Public Service Platform of Biomedical Resources Research and Development of South China Sea(2017C8B2)
Yanling Outstanding Young Teacher Training Program of Lingnan Normal University(YL20200213)
Open Project of Mangrove Research Institute, Lingnan Normal University(PYXM05)
文章对分离自南海深海沉积物的1株真菌101#进行分子鉴定、次生代谢产物及其生物活性研究。基于rDNA-ITS基因序列分析法对菌株进行鉴定, 运用高效液相色谱(high performance liquid chromatography, HPLC)跟踪其发酵产物含量的变化, 采用硅胶柱层析、Sephadex LH-20层析、HPLC等多种方法进行代谢产物分离纯化; 利用核磁共振、质谱等波谱学方法并结合参考文献报道对分离的单体化合物进行结构鉴定。采用滤纸片扩散法和卤虫致死法分别测定抗菌和卤虫致死性活性。结果表明, 该菌株鉴定为黄曲霉(Aspergillus flavus), 第28天的发酵产物含量最丰富。此外, 该粗提物对15种指示菌均具有一定的抑制活性, 其中对10种革兰氏阳性菌和阴性菌的最小抑菌浓度(minimum inhibitory concentration, MIC)范围为0.781~6.25μg·mL-1, 对3种植物病原菌MIC范围为50~200μg·mL-1, 对海洋生物污损菌(麦氏交替单胞菌)和致病真菌(多耐白色念珠菌) MIC值分别为3.125μg·mL-1和1.563μg·mL-1, 对卤虫1、12、24和48 h的半数致死浓度(Median Lethal Dose 50, LD50)分别为74.597、24.322、13.797和8.559mg·mL-1。最后, 从乙酸乙酯萃取部位中共得到4个单体化合物, 分别鉴定为5-chloro-2-hydroxyphenylacetic acid (1)、aspergamide A (2)、WF-3681 methyl Ester (3)和(E)-But-2-enedioic acid monomethyl Ester (4), 这4个化合物对枯草芽孢杆菌、多耐白色念珠菌、苏云金芽孢杆菌、希瓦氏菌和藤黄微球菌显示出一定的抑制活性, 其MIC值范围为6.25~200μg·mL-1, 此外, 化合物 1~4均显示出较强的卤虫致死活性, 其LD50值分别为7.40、11.95、17.69和23.35μg·mL-1。
曾步燕 , 梁志凤 , 罗琴琴 , 曾玲 , 杨长庚 , 王锂韫 , 孙玉林 . 深海真菌101#的分子鉴定、次生代谢产物及其生物活性研究*[J]. 热带海洋学报, 2023 , 42(5) : 104 -114 . DOI: 10.11978/2022242
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.
表1 菌株101#粗提物对12种指示菌的抑制活性及最小抑制浓度(MIC值)(n=3)Tab. 1 Minimum inhibitory concentration (MIC) of crude extract of strain 101# against 12 indicator bacteria (n=3) |
指示菌 | 阳性对照 | 粗提物浓度/(μg·mL-1) | MIC/(μg·mL-1) | |||||
---|---|---|---|---|---|---|---|---|
12.5 | 6.25 | 3.125 | 1.563 | 0.781 | 0.391 | |||
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 |
注: -: 无抑制作用; +: 抑菌圈为6~9mm; + +: 抑菌圈为9~12mm; + + +: 抑菌圈为12~15mm; + + + +: 抑菌圈≥15mm。阳性对照组为硫酸庆大霉素 |
表2 菌株101#粗提物对6种植物病原菌的抑制活性及最小抑制浓度(MIC值)(n=3)Tab. 2 Minimum inhibitory concentration (MIC) of crude extract of strain 101# against 6 plant pathogens (n=3) |
指示菌 | 阳性对照 | 粗提物浓度/(μg·mL-1) | MIC/(μg·mL-1) | ||||
---|---|---|---|---|---|---|---|
200 | 100 | 50 | 25 | 12.5 | |||
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 |
注: -: 无抑制作用; +: 抑菌圈为6~9mm; + +: 抑菌圈为9~12mm; + + +: 抑菌圈为12~15mm; + + + +: 抑菌圈≥15mm。对照组为两性霉素B |
表3 菌株101#粗提物对卤虫致死活性的测试结果Tab 3. Test results of lethal activity of strain 101# crude extract against brine shrimp |
时间/h | 粗提物浓度/( mg·mL-1) | LD50/(mg·mL-1) | 95%置信区间 | |||||
---|---|---|---|---|---|---|---|---|
100 | 50 | 25 | 12.5 | 6.25 | 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 |
表4 化合物1~4对5株指示菌的MIC值(单位: μg·mL-1)Tab. 4 Minimum inhibitory concentration (MIC) of compounds 1 ~ 4 against 5 indicator bacteria |
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 |
*藤黄微球菌、无乳链球菌、福氏志贺菌、麦氏交替单胞菌以及6种植物病原菌均由华南农业大学张晓勇课题组惠赠, 在此表示衷心的感谢!
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