絨泡菌目幾種黏菌的培養(yǎng)、代謝產(chǎn)物及其生物活性的研究
發(fā)布時間：2019-06-14 18:38
【摘要】：近年,對黏菌的研究已越來越受到部分研究學者的廣泛關注。從孢子到孢子的個體發(fā)育過程已成為熱點研究,其個體發(fā)育過程主要包括:多核非細胞結構的原質團階段和單核的子實體階段,不同種間存在著差異,并且不同種黏菌原質團對營養(yǎng)物質的需要量不同,因而會表現(xiàn)出在生長狀態(tài)上的差異。由于從黏菌中分離得到的某些單體化合物顯示出抗菌、抗腫瘤、細胞毒性等作用,因此黏菌代謝產(chǎn)物的研究也受到了廣泛的關注。本文對絨泡菌目2種黏菌的生活史進行了研究;同時對絨泡菌目3種黏菌在不同培養(yǎng)基上的生長狀態(tài)進行對比,篩選出相對適宜的培養(yǎng)基;分析了絨泡菌目6種黏菌脂溶性成分;并對大孢鈣皮菌代謝產(chǎn)物進行了提取分離,同時對不同極性提取物的生物活性進行了研究,為發(fā)掘更好的天然產(chǎn)物資源提供了依據(jù)。主要結果如下:采用濕室培養(yǎng)、懸滴培養(yǎng)和有飼培養(yǎng)相結合的方法,對絨泡菌目(Physarales)絨泡菌屬(Physarum)的剛絲絨泡菌(Physarum rigidum)和鈣皮菌屬(Didymium)的鱗鈣皮菌(Didymium squamulosum)的生活史進行了觀察,記錄了其不同時期的形態(tài)變化過程,同時對各時期存在的差異進行了比較。兩種黏菌間存在的差異主要表現(xiàn)為:剛絲絨泡菌孢子萌發(fā)所需時間較短,而鱗鈣皮菌形成可見原質團的時間較短,形成子實體時不需要光照,并且子實體成熟所需時間較短。對3種黏菌鱗鈣皮菌(D.squamulosum)、大孢鈣皮菌(D.megalosporum)和光皮煤絨菌(Fuligo leviderma)在6種不同谷物(高粱米、大黃米、燕麥、蕎麥、玉米、薏米)培養(yǎng)基上的生長狀態(tài)進行對比分析,記錄了3種黏菌在培養(yǎng)基上生長10d后的狀態(tài)。從扇面面積上看,鱗鈣皮菌表現(xiàn)為:大黃米高粱米燕麥薏米玉米蕎麥;大孢鈣皮菌表現(xiàn)為:燕麥玉米薏米大黃米高粱米蕎麥。光皮煤絨菌表現(xiàn)為:大黃米燕麥玉米高粱米薏米蕎麥。而從扇面前緣濃密程度上看,鱗鈣皮菌表現(xiàn)為:大黃米高粱米燕麥薏米玉米蕎麥。光皮煤絨菌表現(xiàn)為:大黃米燕麥玉米高粱米薏米蕎麥。大孢鈣皮菌表現(xiàn)為:燕麥玉米薏米大黃米高粱米蕎麥。因此說明在以后對原質團的培養(yǎng)過程中,當想要獲得大量原質團,培養(yǎng)鱗鈣皮菌和光皮煤絨菌可采用大黃米固體培養(yǎng)基,而培養(yǎng)大孢鈣皮菌可采用燕麥固體培養(yǎng)基。采用GC-MS法對煤絨菌(Fuligo septica)、光皮煤絨菌(F.leviderma)、大孢鈣皮菌(D.megalosporum)、鱗鈣皮菌(D.squamulosum)、剛絲絨泡菌(P.rigidum)、鱗皮菌(Lepidoderma tigrinum)6種黏菌脂溶性代謝產(chǎn)物進行分析。從煤絨菌石油醚提取物中鑒定得出23個脂溶性代謝產(chǎn)物,從光皮煤絨菌石油醚提取物中鑒定得出16個脂溶性代謝產(chǎn)物,從大孢鈣皮菌石油醚提取物中鑒定得出42個脂溶性代謝產(chǎn)物,從鱗鈣皮菌石油醚提取物中鑒定得出15個脂溶性代謝產(chǎn)物,從剛絲絨泡菌石油醚提取物中鑒定得出12個脂溶性代謝產(chǎn)物,從鱗皮菌石油醚提取物中鑒定得出12個脂溶性代謝產(chǎn)物。不同種黏菌具有產(chǎn)生相同類型脂溶性代謝產(chǎn)物的能力,同時根據(jù)親緣關系的遠近進行分析,相近種具有合成相同代謝產(chǎn)物的能力。利用超聲波提取法、索氏提取法和重結晶法對大孢鈣皮菌原質團干品的代謝產(chǎn)物進行了提取分離,并通過1H-NMR、13C-NMR對兩種化合物結構進行分析。從大孢鈣皮菌(D.megalosporum)原質團中分離出兩種代謝產(chǎn)物,其中化合物1為豆甾醇,化合物2為乳酸。兩種代謝產(chǎn)物均為從該種黏菌中首次分離得到。采用CCK-8試劑盒檢測法,對大孢鈣皮菌甲醇和石油醚提取物進行了對人前列腺癌細胞(PC-3M)、肝癌細胞(HepG2)、人胃癌細胞(SGC)、人乳腺癌細胞(MCF-7)4種腫瘤細胞的抗腫瘤活性研究,實驗結果表明,大孢鈣皮菌甲醇和石油醚提取物對4種腫瘤細胞增值均具有抑制作用。并且無論對哪種腫瘤細胞,甲醇提取物的抑制作用均高于石油醚提取物。采用濾紙片擴散法,對大孢鈣皮菌甲醇和石油醚提取物進行了抗菌活性的研究,4種細菌分別為大腸桿菌(Escherichia coli)、鼠傷寒沙門氏菌(Salmonella typhimurium)、金黃色葡萄球菌(Staphylococcus aureus)、枯草芽孢桿菌(Bacillus subtilis),結果表明,甲醇提取物和石油醚提取物對4種細菌均沒有顯示出明顯的抑制作用,其中僅對金黃色葡萄球菌和枯草芽孢桿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.
【學位授予單位】：吉林農(nóng)業(yè)大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：R915
本文編號：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.
【學位授予單位】：吉林農(nóng)業(yè)大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：R915
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