海带内生真菌的分离鉴定及其抑制乙酰胆碱酯酶活性的发酵工艺优化
Isolation, identification and fermentation process optimization of acetylcholinesterase inhibitory activity of endophytic fungus from Laminaria japonica
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摘要: 本文以青岛海域的海带为原料,对其进行内生真菌的分离纯化,共得到7株内生真菌,采用改良Ellman法对其乙酰胆碱酯酶（ACh E）抑制活性进行筛选,并对其中一株活性最高内生真菌ML-X进行形态学和18S rDNA分子生物学鉴定,通过单因素实验和正交实验筛选出最适合该菌进行液体生长发酵的培养条件。结果表明,分离获得的菌株ML-X为白地霉属,序列号为KY977411,与遗传距离最近的Galactomyces geotrichum strain LMA-21株（JQ668739）同源性达99%。最佳发酵条件为:PS培养基,培养基的初始p H6.0,接种量5%,装瓶量150 m L/500 m L,培养温度31℃,发酵8 d,当发酵液粗浸膏为5 mg/m L时,对乙酰胆碱酯酶的抑制率可达28.91%±0.25%。Abstract: In this study, Laminaria japonica from Qingdao sea area was used to isolate and purify endophytic fungus.A total of seven strains of endophytic fungi were isolated. Using modified Ellman method to determine the inhibitory rate of ACh E and active strains were identified.Strain ML-X showed the highest AChE inhibitory activity and was identified, by physiologicalbiochemical characteristics and molecular biological techniques of 18 S rDNA sequencing.Based on single factor and orthogonal test, the culture conditions of liquid fermentation of the endophytic fungi were screened out. The results showed that the strain ML-X was confirmed as Geotrichum candidum, and the optimal fermentation conditions were as follows: PS medium was the suitable medium for the strain at pH6.0, incubated with 5% inoculum and 150 m L/500 m L culture volume at 31 ℃ for 8 days.Under these conditions, the strain ML-X exhibited a higher ACh E inhibitory activity, the inhibition ratio of Ach E was 28.91%± 0.25% when the concentration of crude extract from fermentation broth was 5 mg/m L.
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