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Marine-derived Aspergillus terreus M7 with antibacterial effect, and separation and application of secondary metabolite of Aspergillus terreus M7

A technology of Aspergillus terreus and Aspergillus genus, applied in the field of microorganisms, can solve the problems of increasing antibiotic resistance and achieve the effect of simple operation and low cost

Active Publication Date: 2021-12-14
CHINA PHARM UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The frequent use of clinical antibiotics directly leads to the continuous increase of resistance to existing antibiotics

Method used

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  • Marine-derived Aspergillus terreus M7 with antibacterial effect, and separation and application of secondary metabolite of Aspergillus terreus M7
  • Marine-derived Aspergillus terreus M7 with antibacterial effect, and separation and application of secondary metabolite of Aspergillus terreus M7
  • Marine-derived Aspergillus terreus M7 with antibacterial effect, and separation and application of secondary metabolite of Aspergillus terreus M7

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] Embodiment 1: Bacterial strain morphological identification and molecular biological identification

[0036] (1) Seed liquid preparation: Inoculate the glycerol tube of the strain M7 isolated from the sponge sample from the South China Sea stored in a -80°C ultra-low temperature refrigerator on a PDA plate, and culture at 28°C for 3 days. PDA plate (fungus medium: 5.0g potato powder; 20.0g glucose; 20.0g agar; sea crystal 33.0g; H 2 (1L; adjust the pH to 6.0) after the obvious hyphae grow out, add an appropriate amount of sterile water, wash the mycelium with a sterile inoculation loop, and inoculate the spore suspension into 200ml PDB liquid medium (fungus Medium: 10.0g potato powder; 20.0g glucose; 33.0g sea crystal; H 2 (0 1L; adjust pH to 6.0), 28°C, 200rpm, 3 days.

[0037] Morphological identification: Inoculate the seed liquid on the MEA plate with 2ul per point by the three-point method (20.0g maltose extract; 1.0g peptone; 20.0g glucose; 0.005g CuSO 4 .5H 2...

Embodiment 2

[0046] Embodiment 2: strain fermentation

[0047] (1) seed solution preparation: preparation method is the same as embodiment 1 (1)

[0048] (2) Small-batch fermentation: inoculate the seed liquid into rice solid medium, corn solid medium, and soybean solid medium according to the ratio of 1ml / 10g, and the culture time is 30 days; the culture conditions are light and dark respectively; the culture temperature Both are at 28°C. Set up two parallels for each group.

[0049] For small batches of fermentation products, soak overnight in 3 times the volume of ethanol, ultrasonically extract for 30 minutes the next day, and filter under reduced pressure to obtain the filtrate. Repeat this step three times. The obtained filtrates were combined, and the fermented extract was obtained by rotary steaming under reduced pressure, and 3 ml of methanol was added to dissolve the extract to obtain the fermented crude extract, which was used for antibacterial experiments.

[0050] The indi...

Embodiment 3

[0053] Embodiment 3: Separation and purification of target compound

[0054] (1) Dissolve the fermented extract in methanol, mix the sample with silica gel G (100-200 mesh), and then separate it with a silica gel G chromatography column, and gradually elute with dichloromethane-methanol, and the elution gradient is CH 2 Cl 2 、CH 2 Cl 2 -MeOH(80:1),CH 2 Cl 2 -MeOH(60:1),CH 2 Cl 2 -MeOH(40:1),CH 2 Cl 2 -MeOH(20:1),CH 2 Cl 2 -MeOH (10:1). Active components were screened with Acinetobacter baumannii as indicator bacteria. See 2(2) for active ingredient screening. The active fractions were combined for the next step of separation.

[0055] (2) The combined active components were dissolved in methanol and then introduced into a Sephadex LH-20 gel column for further purification. The sample after passing through the column was screened for active components with Acinetobacter baumannii as indicator bacteria. See 2(2) for active ingredient screening. The active fractio...

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Abstract

The invention belongs to the technical field of microorganisms, and relates to marine-derived Aspergillus terreus M7 with an antibacterial effect, and separation and application of a secondary metabolite of the Aspergillus terreus M7. The strain is classified and named as Aspergillus terreus M7, and is preserved in China Center for Type Culture Collection (CCTCC) on June 7, 2021, with the preservation number of CCTCC NO: M 2021679. The strain is fermented by a soybean solid culture medium, and is subjected to methanol extraction, silica gel G chromatography, Sephadex LH-20 gel column chromatography, silica gel H chromatography and preparative liquid phase separation and purification to obtain a compound terramide A. The terramide A has obvious antibacterial activity on acinetobacter baumannii ATCC 19606, and has the potential of being developed into an antibacterial agent.

Description

technical field [0001] The invention belongs to the technical field of microorganisms, and relates to an extraction and separation method and application of a secondary metabolite of Aspergillus terreus which can be used as an antibacterial agent. Background technique [0002] Many clinically used antibiotics are mostly derived from microbial secondary metabolites or their derivatives. However, with the continuous development of natural microbial resources, it has become more and more difficult to discover new species of microorganisms to develop new antibiotics. The frequent use of clinical antibiotics directly leads to the increasing resistance to existing antibiotics. Compared with the terrestrial environment, the marine environment has the characteristics of no light, low nutrition, high pressure, high salt, and low temperature. Due to the harsh living conditions in the ocean, the microorganisms living in it, especially fungi, have evolved a more complex metabolic syst...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/14C12P17/12A61P31/04C12R1/66
CPCC12N1/14C12P17/12A61P31/04Y02A50/30
Inventor 王颖金国虔祁江峰
Owner CHINA PHARM UNIV
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