Extraction and crystallization process of 5-oxomilbemycins fermentation liquor

A technology of ketomilbemycin and fermented liquid, which is applied in the field of extraction and crystallization process of 5-ketomilbemycin fermented liquid, can solve the problem of no relevant reports on extraction and separation process and crystallization process, low purity of crude extract, Complicated operation process and other problems to achieve the effect of overcoming the extreme instability of intermediates, high yield and reducing impurity content

Active Publication Date: 2018-09-28
ZHEJIANG HISUN PHARMA CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

This method uses a mixed solvent to extract twice, the operation process is complicated, and because the extracted purified product is not further crystallized, the crude extract has low purity and poor stability
[0007] The laboratory used genetic engineering technology to construct the genetically engineered strain F2-18 that can directly ferment and produce 5-ketomilbemycin (literature Huang Jun, Lin Jiatan, Zhou Min et al. Streptomyces hygroscopicus HS023milF gene knockout construction 5-ketomilbemycin genetically engineered bacteria. "Acta Microbiology", 2015,55(1):107-113), the oxidation reaction step is omitted, but the extraction and separation of 5-ketomilbemycin fermentation broth technology and crystallization process but no related reports

Method used

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  • Extraction and crystallization process of 5-oxomilbemycins fermentation liquor
  • Extraction and crystallization process of 5-oxomilbemycins fermentation liquor
  • Extraction and crystallization process of 5-oxomilbemycins fermentation liquor

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0040] Embodiment 1: Preparation of 5-ketomilbemycin fermented liquid

[0041] 5-ketomilbemycin-producing bacteria Streptomyces milbemycinicus F2-18 is a genetically engineered bacterium obtained by Streptomyces milbemycinicus (Streptomyces milbemycinicus, deposit number: CGMCC No. 7677) through genetic recombination technology, references Huang Jun, Lin Jiatan, Zhou Min et al. Knockout of Streptomyces hygroscopicus HS023milF gene to construct genetically engineered bacteria producing 5-ketomilbemycin. "Acta Microbiology", 2015,55(1):107-113. The method of using the engineering bacteria to prepare 5-ketomilbemycin fermentation broth refers to the invention patent CN201310541829.8.

[0042] 1. Take Streptomyces F2-18 spore suspension from the cryopreservation tube and inoculate it on a plate of solid MS medium (mannitol 20g / L, soybean powder 20g / L, agar 20g / L), and culture at 28°C for 7 days;

[0043] 2. Inoculate the cultured fungal spores into a 15L seed tank with 10L of see...

Embodiment 2

[0050] Embodiment 2: the ethanol extraction of 5-ketomilbemycin fermented liquid

[0051] 1) After getting 12.5L of 5-ketomilbemycin fermented liquid among the embodiment 1 and passing through the frame filter, collect and obtain mycelium 5.2Kg;

[0052] 2) 5.2Kg of mycelium was soaked in 26L of 98% ethanol, and 25.6L of 5-ketomilbemycin extract was obtained after plate and frame filtration, concentrated at a negative pressure of 0.09Mpa until the volume concentration of ethanol was 50%, and 5-ketomilbemycin was obtained. Ketomilbemycin ethanol concentrate 12.7L;

[0053] 3) Take 12.7 L of the 5-ketomilbemycin ethanol concentrate and extract and layer with 12.7 L of n-heptane to obtain 12.0 L of n-heptane phase. Detection by HPLC (see figure 1 ), the measured concentration of 5-ketomilbemycin A3 is 520mg / L, HPLC purity 19.3%, the concentration of A4 is 2002mg / L, HPLC purity 59.2%.

Embodiment 3

[0054] Example 3: The methanol extraction of 5-ketomilbemycin fermentation broth

[0055] 1) After 12.5 L of 5-ketomilbemycin fermentation broth in Example 1 was filtered through a plate frame, 5.2 Kg of mycelium was collected;

[0056] 2) 5.2Kg of mycelium was soaked in 26L of 98% methanol, and 25.6L of 5-ketomilbemycin extract was obtained after plate and frame filtration, concentrated at a negative pressure of 0.09Mpa until the volume concentration of methanol was 50%, and 5-ketomilbemycin was obtained. Ketomilbemycin Methanol Concentrate 12.7L;

[0057] 3) Take 12.7 L of 5-ketomilbemycin methanol concentrate and extract and separate with 12.7 L of n-heptane to obtain 12.2 L of n-heptane phase. Adopt HPLC detection, record the concentration of 5-ketomilbemycin A3 to be 510mg / L, the concentration of A4 is 1970mg / L.

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Abstract

The invention discloses an extraction and crystallization process of a 5-oxomilbemycins fermentation liquor. The extraction and crystallization process comprises pretreatment of the fermentation liquor: mycelium immersion, extraction, filtration and concentration; n-heptane extraction, concentration, stirring and crystallization; n-heptane leaching drying; and the like. A 5-oxomilbemycins crystalproduced by the process disclosed by the invention has the purity up to 99 percent which is more than the purity of a 5-oxomilbemycins coarse product by 25 to 35 percent; furthermore, the process is simple, high in yield and high in stability; no silicagel column chromatographic purification process is involved in a production process, so that the cost is reduced, and the burden for environmentalprotection is greatly reduced.

Description

technical field [0001] The invention relates to the technical field of compound separation and purification, more specifically, to a process for extracting and crystallizing a 5-ketomilbemycin fermentation broth. Background technique [0002] [0003] 5-ketomilbemycin A3: R=CH 3 ;A4: R=CH 2 CH 3 [0004] 5-ketomilbemycins (5-oxomilbemycins) is a sixteen-membered ring macrocyclic lactone compound, which is the precursor of milbemycin, and its A3 and A4 components can be obtained by oxomilbemycin A3 and A4. Milbemycinoxime (milbemycinoxime) is an anthelmintic drug, which was launched in 1986 by Sankyo Co., Ltd. of Japan. Milbexime has a broad-spectrum anti-parasitic effect, and has a good repellent effect on internal and external parasites, especially nematodes and arthropods, and has a good effect on the control and prevention of most common parasitic diseases. It is usually used to prevent heartworm disease, control dog and cat diseases caused by nematodes and hookw...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07D493/22
CPCC07D493/22
Inventor 沈敢锋余贞滕云叶小形阮海宁郑玲辉
Owner ZHEJIANG HISUN PHARMA CO LTD
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