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Batch Culture Fermentation of Endophytic Fungi and Extraction of Their Metabolites
内生真菌的分批培养发酵及其代谢物的提取   

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PLOS ONE
Feb 2013

 

Abstract

Antibiosis is one of the possible modes of action shown by endophytic fungi having antifungal activity. To test if antifungal activity in endophytic fungi is due to antibiosis, assay of the metabolites of endophytic fungi was needed. To obtain metabolites for bioassay batch culture fermentation and extraction of metabolites was done. Fungus was multiplied on wickerham media at incubation temperature of 25 ± 2 °C for 4 weeks and then extracted with solvents of different polarity. All the solvent extracts were dried under vacuum rotary evaporator to get dried crude fungal extract, which was subjected to further fractionation and bioassay.

Keywords: Fermentation (发酵), Endophytic fungi (内生真菌), Batch culture (批文化), Metabolites of endophytic fungi (内生真菌metabolites of), Solvent extraction (溶剂萃取)

Materials and Reagents

  1. Ethyl acetate (Rankem)
  2. Butanol (Qualigens)
  3. Methanol (Qualigens)
  4. Hexane (Qualigens)
  5. Fungus culture
  6. Measuring cylinder
  7. Whatman filter paper
  8. Detergent
  9. Glass jars of 5 L capacity
  10. Malt extract (HiMedia Laboratories)
  11. Yeast extract (HiMedia Laboratories)
  12. Peptone (HiMedia Laboratories)
  13. Glucose (Qualigens)
  14. Needle
  15. Spirit lamp
  16. Commercial disinfectant (70% ethanol)
  17. Vacuum pump
  18. Malt extract
  19. Yeast extract
  20. Wickerham medium (see Recipes)

Equipment

  1. Inoculation flasks
  2. Conical flasks of 1 L capacity
  3. Vacuum rotary evaporator (Heidolph Instruments GmbH)
  4. pH meter (Eutech Instruments pH tutor)
  5. Autoclave (Nat steel)
  6. BOD incubator (Toshiba)
  7. Laminar air flow hood (Toshiba)
  8. Filteration assembly 
  9. Hand blender (inalsaappliances.com)
  10. Fume hood
  11. Microbalances (Sartorious, model: RC210P )

Procedure

  1. 5 discs of 5 mm diameter of endophytic fungus from petridish were inoculated in Wickerham medium.
  2. Flasks with inoculated media were incubated at 24 °C for 24 days under static culture condition without light.
  3. One flask of medium without any inoculam served as a control.
  4. After 24 days of incubation, 250 ml of ethyl acetate was added to each flask, mixed, and left overnight.
  5. Ethyl acetate immersed fungus culture was blended with a hand blender for 15 min and filtered by whatman filter paper under vacuum (Wicklow et al., 1998).
  6. The filtrate was collected and residual aqueous phase was partitioned thrice times with equal volumes of ethyl acetate in a separator funnel (Figure 1).


    Figure 1. Partitioning by separator funnel

  7. Aqueous phase obtained after ethyl acetate extraction was further partitioned three times with equal volumes of saturated butanol.
  8. Aqueous phase obtained after butanol extraction was discarded after immersing in detergent.
  9. The ethyl acetate and butanol extracts were dried with vacuum rotary evaporator.
  10. Dried ethyl acetate extract resuspended in 90% methanol and extracted with n-hexane.
  11. After drying the hexane, butanol, and methanol extracts with vacuum rotary evaporator they were subjected to further experimentation. Schematic diagram for the extraction of the metabolite has been given in Figure 2.


    Figure 2. Schematic diagram of extraction procedure for obtaining crude fungal extracts

Recipes

  1. Wickerham medium
    Malt extract 3 g/L
    Yeast extract 3 g/L
    Peptone 5 g/L
    Glucose 10 g/L
    All the media chemicals were weighed and dissolved in distilled water and pH was measured. After adjusting the pH in range of 7.2-7.4, media was distributed in conical flasks (300 ml in 1 L conical flask). These flasks were subjected to autoclaving at 121 °C temperature and 15 psi pressure for 20 min.

Acknowledgments

This protocol was adopted from Kumar and Kaushik (2013). Authors are grateful to their host institution, The Energy and Resources Institute (TERI), New Delhi, India for funding the research. Susheel Kumar is grateful to University Grant Commission, New Delhi for a research fellowship.

References

  1. Kumar, S. and Kaushik, N. (2013). Endophytic fungi isolated from oil-seed crop Jatropha curcas produces oil and exhibit antifungal activity. PLoS One 8(2): e56202.
  2. Wicklow, D. T., Joshi, B. K., Gamble, W. R., Gloer, J. B. and Dowd, P. F. (1998). Antifungal metabolites (monorden, monocillin IV, and cerebrosides) from Humicola fuscoatra traaen NRRL 22980, a mycoparasite of Aspergillus flavus sclerotia. Appl Environ Microbiol 64(11): 4482-4484.

简介

抗生素是具有抗真菌活性的内生真菌所显示的可能的作用模式之一。 为了测试内生真菌中的抗真菌活性是否是由于抗生素,需要测定内生真菌的代谢物。 为了获得用于生物测定的代谢物,进行批次培养发酵和代谢物的提取。 将真菌在wickerham培养基上在25±2℃的孵育温度下繁殖4周,然后用不同极性的溶剂萃取。 所有溶剂提取物在真空旋转蒸发器下干燥,得到干燥的粗真菌提取物,对其进行进一步分馏和生物测定。

关键字:发酵, 内生真菌, 批文化, 内生真菌metabolites of, 溶剂萃取

材料和试剂

  1. 乙酸乙酯(Rankem)
  2. 丁醇(Qualigens)
  3. 甲醇(Qualigens)
  4. 己烷(Qualigens)
  5. 真菌文化
  6. 测量缸
  7. Whatman过滤纸
  8. 洗涤剂
  9. 5升容量的玻璃瓶
  10. 麦芽提取物(HiMedia Laboratories)
  11. 酵母提取物(HiMedia Laboratories)
  12. 蛋白胨(HiMedia Laboratories)
  13. 葡萄糖(Qualigens)

  14. 精神灯
  15. 商业消毒剂(70%乙醇)
  16. 真空泵
  17. 麦芽提取物
  18. 酵母提取物
  19. Wickerham medium(见配方)

设备

  1. 接种瓶
  2. 1升容量的锥形瓶
  3. 真空旋转蒸发器(Heidolph Instruments GmbH)
  4. pH计(Eutech Instruments pH tutor)
  5. 高压釜(Nat steel)
  6. BOD孵化器(东芝)
  7. 层流风罩(东芝)
  8. 过滤组件
  9. 手动搅拌机(inalsaappliances.com)
  10. 通风橱
  11. 微型天平(Sartorious,型号:RC210P)

程序

  1. 将来自petridish的5个直径为5mm内生真菌的圆盘接种在Wickerham培养基中
  2. 将具有接种培养基的烧瓶在无光的静态培养条件下在24℃下孵育24天。
  3. 一个没有任何接种体的培养基作为对照
  4. 孵育24天后,向每个烧瓶中加入250ml乙酸乙酯,混合,并放置过夜。
  5. 将乙酸乙酯浸渍的真菌培养物用手动搅拌器混合15分钟,并在真空下通过whatman滤纸过滤(Wicklow等人,1998)。
  6. 收集滤液,在分液漏斗中用等体积的乙酸乙酯将残留的水相分离三次(图1)。


    图1.按分隔符漏斗分区

  7. 在乙酸乙酯萃取后获得的水相进一步用等体积的饱和丁醇分配三次。
  8. 在丁醇萃取后获得的水相在浸入洗涤剂之后被丢弃
  9. 用真空旋转蒸发器干燥乙酸乙酯和丁醇提取物。
  10. 将干乙酸乙酯萃取物重悬浮于90%甲醇中,并用正己烷萃取。
  11. 用真空旋转蒸发器干燥己烷,丁醇和甲醇萃取物后,进行进一步实验。 图2中给出了代谢物提取的示意图

    图2.获得粗真菌提取物的提取程序示意图

食谱

  1. Wickerham培养基
    麦芽提取物3 g/L
    酵母提取物3 g/L
    蛋白胨5 g/L
    葡萄糖10g/L
    称量所有介质化学品并溶解在蒸馏水中,并测量pH。 在将pH调节在7.2-7.4的范围内后,将培养基分布在锥形瓶(300ml,在1L锥形瓶中)中。 将这些烧瓶在121℃的温度和15psi的压力下进行高压灭菌20分钟

致谢

该协议从Kumar和Kaushik(2013)获得。 作者感谢他们的主办机构,能源和资源研究所(TERI),新德里,印度资助研究。 Susheel Kumar感谢新德里大学奖学金委员会的研究奖学金。

参考文献

  1. Kumar,S.和Kaushik,N。(2013)。 从油籽作物中分离的内生真菌<麻木麻疯树产生油和展览 抗真菌活性。 PLoS One 8(2):e56202
  2. Wicklow,D.T.,Joshi,B.K.,Gamble,W.R.,Gloer,J.B。和Dowd,P.F。(1998)。 来自Humicola fuscoatra的抗真菌代谢物(monorden,monocillin IV和cerebrosides) traaen NRRL 22980,黄色曲霉菌的霉菌寄生虫。 Appl Environ Microbiol 64(11):4482-4484。
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Copyright: © 2013 The Authors; exclusive licensee Bio-protocol LLC.
引用:Kumar, S. and Kaushik, N. (2013). Batch Culture Fermentation of Endophytic Fungi and Extraction of Their Metabolites. Bio-protocol 3(19): e926. DOI: 10.21769/BioProtoc.926.
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