褐多孔菌的发酵培养基优化及其化学成分初探
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摘要
我国大约有真菌18万种,90%以上还没有被人认识,是很重要的国家战略性生物资源。褐多孔菌(Polyporus picipes)是担子菌纲、多孔菌科、多孔菌属真菌,大多寄生于倒木或腐朽木上,具有多种功效,具有抗癌作用,可治疗乳腺癌、前列腺癌,可软化血管、提高免疫能力、预防和延缓心脏的老化、调节大脑神经、抗衰老、抑菌等作用。多孔菌发酵代谢能产生多种产物,如生物碱、多糖、多肽、甾类、三萜等,因此多孔菌将会成为一类具有非常大应用前景的生物资源,在医疗卫生和农业上发挥重要的作用。
鉴于此,本实验利用色谱技术对褐多孔菌菌丝体乙酸乙酯提取物的化学成分进行了分离纯化并通过响应面法对褐多孔菌发酵培养基进行优化用以提高褐多孔菌的抗菌活性,为该菌株杀菌活性成分的提取分离及生物农药的开发应用奠定基础。研究结果如下:通过化学的分离手段对褐多孔菌(Polyporus pecipes)菌丝体进行了化学成分研究,从褐多孔菌菌丝体乙酸乙酯部分中分离到了8个化合物。综合应用EI-MS、1HNMR、13CNMR、DEPT及(–)ESI-MS等现代波谱学方法,并与标准品、文献值以及图谱库对照,鉴定出其中的4个,化合物I为2,4-二羟基-5-甲基-苯乙酮,化合物Ⅱ为麦角甾醇,化合物Ⅲ为麦角甾醇过氧化物,化合物Ⅴ为4,6,8 (14),22 (23)-四烯-3-酮-麦角甾烷,其它化合物未做图谱鉴定。
采用改良的PDA液体培养基(葡萄糖马铃薯液体培养基+酵母粉5.37g/L+葡萄糖6.31g/L),褐多孔菌在初始自然PH,培养液装液量60.74mL/250mL,经28℃,150rpm旋转振荡培养68.32h,次生产代谢物对小麦赤酶的抑制作用最强。褐多孔菌在优化的发酵培养基中的抗菌活性显著提高,表明响应面法在培养基优化中十分有效,相对简单,且节省时间和材料。
There are about 180 thousand fungus in our country, 90% have not yet been recognized, which is a very important national strategic biological resources. Polyporus pecipes, a wood rooting fungus of Polyporaceace family, is found on the trunks of living or dead trees. Pharmacologically, it is traditionally used to treat cancer such as breast cancer and prostatic cancer, and it can be eaten during the early time of its life. It has functions of enhancing immunity, softening blood vessels, preventing ageing of heart and regulating the nerve of brain. Some research results have showed that Polyporus pecipes can produce many secondary metabolites, such as alkaloid、polysaccharides、polypeptide、steroid、Triterpenoids and so on. So it is highly possible that the Polyporus pecipes would become a kind of bio-resource that have exploitation potential and prospect of application. It would play an important role in the health and agriculture area.
For this purpose, used modern chromatographic techniques and isolation methods to purify the chemical constituents of ethyl acetate extract from mycelia of Polyporus pecipes systematically.
In order to distill and separate the antibacterial active of Polyporus picipesand lay the foundation of application and development of biochemical pesticides, the fermentation medium of Polyporus picipes was optimized by response surface methodology which enhance the activity of antibiotics. The following are the results of the study:
Eight compounds were isolated from the ethyl acetate extract of mycelia of Polyporus pecipes, of which compoundⅠwas with spectroscopy identified as 1-(2,4-dihydroxy-5-methylphenyl)ethanpone, compoundⅡidentified as ergosterol,Ⅲwas ergosterol peroxide andⅤwas 4,6,8(14),22(23)-tetraen-3-one-ergostane. other compounds have not been assessed.
The strongest antifungul activity of metabolites from Polyporus picipes was obtained when it was cultured in the improved liquid medium based on PDA (liquid medium based on PDA, glucose6.31g/L and yeast extract5.37g/L) with initial pH and the size of 60.74mL/250ml, Erlenmeyer flask at 150rpm and 28 for 68.32h.The results were showed that the chosen method of optimization of medium composition was efficient, relatively simple as well as time and material saving.
鉴于此,本实验利用色谱技术对褐多孔菌菌丝体乙酸乙酯提取物的化学成分进行了分离纯化并通过响应面法对褐多孔菌发酵培养基进行优化用以提高褐多孔菌的抗菌活性,为该菌株杀菌活性成分的提取分离及生物农药的开发应用奠定基础。研究结果如下:通过化学的分离手段对褐多孔菌(Polyporus pecipes)菌丝体进行了化学成分研究,从褐多孔菌菌丝体乙酸乙酯部分中分离到了8个化合物。综合应用EI-MS、1HNMR、13CNMR、DEPT及(–)ESI-MS等现代波谱学方法,并与标准品、文献值以及图谱库对照,鉴定出其中的4个,化合物I为2,4-二羟基-5-甲基-苯乙酮,化合物Ⅱ为麦角甾醇,化合物Ⅲ为麦角甾醇过氧化物,化合物Ⅴ为4,6,8 (14),22 (23)-四烯-3-酮-麦角甾烷,其它化合物未做图谱鉴定。
采用改良的PDA液体培养基(葡萄糖马铃薯液体培养基+酵母粉5.37g/L+葡萄糖6.31g/L),褐多孔菌在初始自然PH,培养液装液量60.74mL/250mL,经28℃,150rpm旋转振荡培养68.32h,次生产代谢物对小麦赤酶的抑制作用最强。褐多孔菌在优化的发酵培养基中的抗菌活性显著提高,表明响应面法在培养基优化中十分有效,相对简单,且节省时间和材料。
There are about 180 thousand fungus in our country, 90% have not yet been recognized, which is a very important national strategic biological resources. Polyporus pecipes, a wood rooting fungus of Polyporaceace family, is found on the trunks of living or dead trees. Pharmacologically, it is traditionally used to treat cancer such as breast cancer and prostatic cancer, and it can be eaten during the early time of its life. It has functions of enhancing immunity, softening blood vessels, preventing ageing of heart and regulating the nerve of brain. Some research results have showed that Polyporus pecipes can produce many secondary metabolites, such as alkaloid、polysaccharides、polypeptide、steroid、Triterpenoids and so on. So it is highly possible that the Polyporus pecipes would become a kind of bio-resource that have exploitation potential and prospect of application. It would play an important role in the health and agriculture area.
For this purpose, used modern chromatographic techniques and isolation methods to purify the chemical constituents of ethyl acetate extract from mycelia of Polyporus pecipes systematically.
In order to distill and separate the antibacterial active of Polyporus picipesand lay the foundation of application and development of biochemical pesticides, the fermentation medium of Polyporus picipes was optimized by response surface methodology which enhance the activity of antibiotics. The following are the results of the study:
Eight compounds were isolated from the ethyl acetate extract of mycelia of Polyporus pecipes, of which compoundⅠwas with spectroscopy identified as 1-(2,4-dihydroxy-5-methylphenyl)ethanpone, compoundⅡidentified as ergosterol,Ⅲwas ergosterol peroxide andⅤwas 4,6,8(14),22(23)-tetraen-3-one-ergostane. other compounds have not been assessed.
The strongest antifungul activity of metabolites from Polyporus picipes was obtained when it was cultured in the improved liquid medium based on PDA (liquid medium based on PDA, glucose6.31g/L and yeast extract5.37g/L) with initial pH and the size of 60.74mL/250ml, Erlenmeyer flask at 150rpm and 28 for 68.32h.The results were showed that the chosen method of optimization of medium composition was efficient, relatively simple as well as time and material saving.
引文
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丁雪,黄丽丽,康振生,姜云. 2008.拮抗淡紫灰链霉菌xjy的发酵条件优化西北农业学报, 17(6): 197-201
董艳红,张鞍灵,马慧妮. 2007.褐多孔菌发酵代谢物的抑菌活性初步研究.西北林学院学报, 22 (2): 105-108
杜肖娜,王润田,刘殿武. 2001.中药猪苓多糖抑瘤作用机理的初探.中华微生物学和免疫学杂志,, (50): 296-297
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