绒泡菌目几种黏菌的培养、代谢产物及其生物活性的研究
发布时间：2019-06-14 18:38
【摘要】：近年,对黏菌的研究已越来越受到部分研究学者的广泛关注。从孢子到孢子的个体发育过程已成为热点研究,其个体发育过程主要包括:多核非细胞结构的原质团阶段和单核的子实体阶段,不同种间存在着差异,并且不同种黏菌原质团对营养物质的需要量不同,因而会表现出在生长状态上的差异。由于从黏菌中分离得到的某些单体化合物显示出抗菌、抗肿瘤、细胞毒性等作用,因此黏菌代谢产物的研究也受到了广泛的关注。本文对绒泡菌目2种黏菌的生活史进行了研究;同时对绒泡菌目3种黏菌在不同培养基上的生长状态进行对比,筛选出相对适宜的培养基;分析了绒泡菌目6种黏菌脂溶性成分;并对大孢钙皮菌代谢产物进行了提取分离,同时对不同极性提取物的生物活性进行了研究,为发掘更好的天然产物资源提供了依据。主要结果如下:采用湿室培养、悬滴培养和有饲培养相结合的方法,对绒泡菌目(Physarales)绒泡菌属(Physarum)的刚丝绒泡菌(Physarum rigidum)和钙皮菌属(Didymium)的鳞钙皮菌(Didymium squamulosum)的生活史进行了观察,记录了其不同时期的形态变化过程,同时对各时期存在的差异进行了比较。两种黏菌间存在的差异主要表现为:刚丝绒泡菌孢子萌发所需时间较短,而鳞钙皮菌形成可见原质团的时间较短,形成子实体时不需要光照,并且子实体成熟所需时间较短。对3种黏菌鳞钙皮菌(D.squamulosum)、大孢钙皮菌(D.megalosporum)和光皮煤绒菌(Fuligo leviderma)在6种不同谷物(高粱米、大黄米、燕麦、荞麦、玉米、薏米)培养基上的生长状态进行对比分析,记录了3种黏菌在培养基上生长10d后的状态。从扇面面积上看,鳞钙皮菌表现为:大黄米高粱米燕麦薏米玉米荞麦;大孢钙皮菌表现为:燕麦玉米薏米大黄米高粱米荞麦。光皮煤绒菌表现为:大黄米燕麦玉米高粱米薏米荞麦。而从扇面前缘浓密程度上看,鳞钙皮菌表现为:大黄米高粱米燕麦薏米玉米荞麦。光皮煤绒菌表现为:大黄米燕麦玉米高粱米薏米荞麦。大孢钙皮菌表现为:燕麦玉米薏米大黄米高粱米荞麦。因此说明在以后对原质团的培养过程中,当想要获得大量原质团,培养鳞钙皮菌和光皮煤绒菌可采用大黄米固体培养基,而培养大孢钙皮菌可采用燕麦固体培养基。采用GC-MS法对煤绒菌(Fuligo septica)、光皮煤绒菌(F.leviderma)、大孢钙皮菌(D.megalosporum)、鳞钙皮菌(D.squamulosum)、刚丝绒泡菌(P.rigidum)、鳞皮菌(Lepidoderma tigrinum)6种黏菌脂溶性代谢产物进行分析。从煤绒菌石油醚提取物中鉴定得出23个脂溶性代谢产物,从光皮煤绒菌石油醚提取物中鉴定得出16个脂溶性代谢产物,从大孢钙皮菌石油醚提取物中鉴定得出42个脂溶性代谢产物,从鳞钙皮菌石油醚提取物中鉴定得出15个脂溶性代谢产物,从刚丝绒泡菌石油醚提取物中鉴定得出12个脂溶性代谢产物,从鳞皮菌石油醚提取物中鉴定得出12个脂溶性代谢产物。不同种黏菌具有产生相同类型脂溶性代谢产物的能力,同时根据亲缘关系的远近进行分析,相近种具有合成相同代谢产物的能力。利用超声波提取法、索氏提取法和重结晶法对大孢钙皮菌原质团干品的代谢产物进行了提取分离,并通过1H-NMR、13C-NMR对两种化合物结构进行分析。从大孢钙皮菌(D.megalosporum)原质团中分离出两种代谢产物,其中化合物1为豆甾醇,化合物2为乳酸。两种代谢产物均为从该种黏菌中首次分离得到。采用CCK-8试剂盒检测法,对大孢钙皮菌甲醇和石油醚提取物进行了对人前列腺癌细胞(PC-3M)、肝癌细胞(HepG2)、人胃癌细胞(SGC)、人乳腺癌细胞(MCF-7)4种肿瘤细胞的抗肿瘤活性研究,实验结果表明,大孢钙皮菌甲醇和石油醚提取物对4种肿瘤细胞增值均具有抑制作用。并且无论对哪种肿瘤细胞,甲醇提取物的抑制作用均高于石油醚提取物。采用滤纸片扩散法,对大孢钙皮菌甲醇和石油醚提取物进行了抗菌活性的研究,4种细菌分别为大肠杆菌(Escherichia coli)、鼠伤寒沙门氏菌(Salmonella typhimurium)、金黄色葡萄球菌(Staphylococcus aureus)、枯草芽孢杆菌(Bacillus subtilis),结果表明,甲醇提取物和石油醚提取物对4种细菌均没有显示出明显的抑制作用,其中仅对金黄色葡萄球菌和枯草芽孢杆菌具有微弱的抑制作用。
[Abstract]:In recent years, the research of the slime has been widely concerned by some research scholars. The individual development of the spores from the spores to the spores has become a hot spot, and the individual development process mainly includes the original mass stage of the polynuclear non-cellular structure and the sub-entity stage of the mononuclear, the difference of the different species, and the different requirements of the different species of the protoplasm to the nutrient substances, And thus exhibit a difference in the growth state. Since some of the monomer compounds isolated from the slime show antibacterial, anti-tumor and cytotoxicity, the study of the metabolites of the slime is also of great concern. In this paper, the life history of 2 kinds of slime molds of the genus Angora is studied, and the growth state of three kinds of slime molds on different medium is compared, and a relatively suitable culture medium is selected. And the biological activity of the different polar extracts is researched, and the basis for finding a better natural product resource is provided. The main results are as follows: using the combination of wet chamber culture, suspension drop culture and feeding culture, the life history of the Physarum velvum and Deiymium squamulosa is observed. The morphological changes in different periods were recorded, and the differences in the different periods were compared. The difference between the two strains is mainly as follows: the time required for the spore germination of the velour is short, and the time for forming the visible protoplasm is short, and the time for forming the sub-solid does not need illumination, and the time required for the sub-entity to mature is shorter. A comparative analysis was made on the growth state of three kinds of slime, such as D. squamusum, D. meegaltosum and Fuligo leviderma, on the medium of six different grains (high rice, rhubarb, oats, buckwheat, corn and coix). The state of the growth of three kinds of slime on the medium was recorded. From the area of the fan surface, the scale-calcium-bearing bacteria are: the barley and the barley, the barley, the coix seed and the buckwheat, and the large-spore-calcium-bearing bacteria are: the oat-corn coix seed and the high-speed rice buckwheat. The preparation method comprises the following steps of: On the other hand, from the front edge of the fan surface, the scale-calcium-bearing bacteria showed that the barley-rice-sorghum coix seed and the barley-corn buckwheat. The preparation method comprises the following steps of: The results of this study were as follows: the oat-corn coix seed, the barley-sorghum rice, and the buckwheat. Therefore, in the course of the culture of the original mass, when it is desired to obtain a large amount of the primary mass, the cultivation of the scale calico and the corduroy can be made by the rhubarb-rice solid culture medium, and the culture of the large-spore calcium-producing bacteria can be made of the oat solid culture medium. Six kinds of liposoluble metabolites of Fulgo septica, F. leviderma, D. megalosporum, D. squamusum, P. rigidum and Lepidoderma tirinum were analyzed by GC-MS. Twenty-three fat-soluble metabolites were identified from the petroleum ether extract of the corduroy, and 16 fat-soluble metabolites were identified from the petroleum ether extract of the photocoaceum, and the 42 liposoluble metabolites were identified from the petroleum ether extract of the genus Daisporium. A total of 15 fat-soluble metabolites were identified from the petroleum ether extract of the Pegasus scale, and 12 liposoluble metabolites were obtained from the petroleum ether extract of the fleas, and 12 fat-soluble metabolites were obtained from the petroleum ether extract of the scale. The different types of slime have the ability to produce the same type of fat-soluble metabolites, and at the same time, according to the near-far of the relationship, the similar species have the ability to synthesize the same metabolic products. In this paper, the metabolites of the dry matter of the original mass of the genus Daisporium are extracted and separated by means of the ultrasonic extraction method, the Soxhlet extraction method and the recrystallization method, and the structure of the two compounds is analyzed by 1H-NMR and 13C-NMR. Two metabolites were isolated from the original mass of D. medegaltosum, wherein the compound 1 is a bean-derived alcohol, and the compound 2 is lactic acid. Both metabolites were obtained for the first time in this kind of slime. The anti-tumor activity of human prostate cancer cells (PC-3M), liver cancer cells (HepG2), human gastric cancer cells (SGC) and human breast cancer cells (MCF-7) in human prostate cancer cells (PC-3M), liver cancer cells (HepG2), human gastric cancer cells (SGC) and human breast cancer cells (MCF-7) were studied by using the CCK-8 kit detection method. The results showed that the addition of methanol and petroleum ether extract to 4 kinds of tumor cells has an inhibitory effect on the value-added of 4 kinds of tumor cells. And the inhibition effect of the methanol extract is higher than that of the petroleum ether. The antibacterial activity of the extracts of the methanol and the petroleum ether was studied by the filter paper diffusion method. The four kinds of bacteria were Escherichia coli, Salmonella typhi, Staphylococcus and Bacillus subtilis, and the results showed that, The methanol extract and the petroleum ether extract have no obvious inhibition effect on the four bacteria, and only the staphylococcus aureus and the bacillus subtilis have weak inhibition effect.
【学位授予单位】：吉林农业大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R915
本文编号：2499593
[Abstract]:In recent years, the research of the slime has been widely concerned by some research scholars. The individual development of the spores from the spores to the spores has become a hot spot, and the individual development process mainly includes the original mass stage of the polynuclear non-cellular structure and the sub-entity stage of the mononuclear, the difference of the different species, and the different requirements of the different species of the protoplasm to the nutrient substances, And thus exhibit a difference in the growth state. Since some of the monomer compounds isolated from the slime show antibacterial, anti-tumor and cytotoxicity, the study of the metabolites of the slime is also of great concern. In this paper, the life history of 2 kinds of slime molds of the genus Angora is studied, and the growth state of three kinds of slime molds on different medium is compared, and a relatively suitable culture medium is selected. And the biological activity of the different polar extracts is researched, and the basis for finding a better natural product resource is provided. The main results are as follows: using the combination of wet chamber culture, suspension drop culture and feeding culture, the life history of the Physarum velvum and Deiymium squamulosa is observed. The morphological changes in different periods were recorded, and the differences in the different periods were compared. The difference between the two strains is mainly as follows: the time required for the spore germination of the velour is short, and the time for forming the visible protoplasm is short, and the time for forming the sub-solid does not need illumination, and the time required for the sub-entity to mature is shorter. A comparative analysis was made on the growth state of three kinds of slime, such as D. squamusum, D. meegaltosum and Fuligo leviderma, on the medium of six different grains (high rice, rhubarb, oats, buckwheat, corn and coix). The state of the growth of three kinds of slime on the medium was recorded. From the area of the fan surface, the scale-calcium-bearing bacteria are: the barley and the barley, the barley, the coix seed and the buckwheat, and the large-spore-calcium-bearing bacteria are: the oat-corn coix seed and the high-speed rice buckwheat. The preparation method comprises the following steps of: On the other hand, from the front edge of the fan surface, the scale-calcium-bearing bacteria showed that the barley-rice-sorghum coix seed and the barley-corn buckwheat. The preparation method comprises the following steps of: The results of this study were as follows: the oat-corn coix seed, the barley-sorghum rice, and the buckwheat. Therefore, in the course of the culture of the original mass, when it is desired to obtain a large amount of the primary mass, the cultivation of the scale calico and the corduroy can be made by the rhubarb-rice solid culture medium, and the culture of the large-spore calcium-producing bacteria can be made of the oat solid culture medium. Six kinds of liposoluble metabolites of Fulgo septica, F. leviderma, D. megalosporum, D. squamusum, P. rigidum and Lepidoderma tirinum were analyzed by GC-MS. Twenty-three fat-soluble metabolites were identified from the petroleum ether extract of the corduroy, and 16 fat-soluble metabolites were identified from the petroleum ether extract of the photocoaceum, and the 42 liposoluble metabolites were identified from the petroleum ether extract of the genus Daisporium. A total of 15 fat-soluble metabolites were identified from the petroleum ether extract of the Pegasus scale, and 12 liposoluble metabolites were obtained from the petroleum ether extract of the fleas, and 12 fat-soluble metabolites were obtained from the petroleum ether extract of the scale. The different types of slime have the ability to produce the same type of fat-soluble metabolites, and at the same time, according to the near-far of the relationship, the similar species have the ability to synthesize the same metabolic products. In this paper, the metabolites of the dry matter of the original mass of the genus Daisporium are extracted and separated by means of the ultrasonic extraction method, the Soxhlet extraction method and the recrystallization method, and the structure of the two compounds is analyzed by 1H-NMR and 13C-NMR. Two metabolites were isolated from the original mass of D. medegaltosum, wherein the compound 1 is a bean-derived alcohol, and the compound 2 is lactic acid. Both metabolites were obtained for the first time in this kind of slime. The anti-tumor activity of human prostate cancer cells (PC-3M), liver cancer cells (HepG2), human gastric cancer cells (SGC) and human breast cancer cells (MCF-7) in human prostate cancer cells (PC-3M), liver cancer cells (HepG2), human gastric cancer cells (SGC) and human breast cancer cells (MCF-7) were studied by using the CCK-8 kit detection method. The results showed that the addition of methanol and petroleum ether extract to 4 kinds of tumor cells has an inhibitory effect on the value-added of 4 kinds of tumor cells. And the inhibition effect of the methanol extract is higher than that of the petroleum ether. The antibacterial activity of the extracts of the methanol and the petroleum ether was studied by the filter paper diffusion method. The four kinds of bacteria were Escherichia coli, Salmonella typhi, Staphylococcus and Bacillus subtilis, and the results showed that, The methanol extract and the petroleum ether extract have no obvious inhibition effect on the four bacteria, and only the staphylococcus aureus and the bacillus subtilis have weak inhibition effect.
【学位授予单位】：吉林农业大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R915
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