阅读说明：本技术 一种利用泡沫提取和大孔树脂纯化分离原人参二醇的方法 (Method for separating protopanoxadiol by using foam extraction and macroporous resin purification ) 是由 卢文玉 赵方龙 南伟华 张传波 于 2019-09-19 设计创作，主要内容包括：本发明公开了一种利用泡沫提取和大孔树脂纯化分离原人参二醇的方法,步骤：(1)将能合成原人参二醇的酿酒酵母菌株置于发酵罐A中,通空气,发酵,有泡沫产生；(2)将泡沫转移至无菌容器B中,泡破裂,原人参二醇附着在无菌容器B的器壁上,而泡沫携带的发酵液和菌体聚集在无菌容器B的底部,再移到发酵罐A中,继续发酵；每间隔4-8h,停止发酵液和菌体的转移,向无菌容器B内通入乙醇水溶液,将无菌容器B器壁上附着的原人参二醇溶解得原人参二醇溶液；(3)将原人参二醇溶液经大孔树脂提纯。本发明的方法能高效的将原人参二醇从泡沫中分离出来,实现原人参二醇的提取。提取效率高,纯度高。(The invention discloses a method for separating protopanoxadiol by utilizing foam extraction and macroporous resin purification, which comprises the following steps: (1) putting a saccharomyces cerevisiae strain capable of synthesizing protopanaxadiol into a fermentation tank A, introducing air, and fermenting to generate foam; (2) transferring the foam into a sterile container B, breaking the foam, attaching protopanaxadiol on the wall of the sterile container B, collecting the fermentation liquid and thalli carried by the foam at the bottom of the sterile container B, transferring the fermentation liquid and thalli into a fermentation tank A, and continuing to ferment; stopping transferring the fermentation liquor and thallus every 4-8h, introducing ethanol water solution into the sterile container B, and dissolving the protopanaxadiol attached to the wall of the sterile container B to obtain protopanaxadiol solution; (3) purifying the protopanaxadiol solution with macroporous resin. The method can efficiently separate the protopanoxadiol from the foam, and realizes the extraction of the protopanoxadiol. High extraction efficiency and high purity.)

1. A method for purifying and separating protopanoxadiol by using foam extraction and macroporous resin is characterized by comprising the following steps:

(1) placing a saccharomyces cerevisiae strain capable of synthesizing protopanaxadiol in a fermentation tank A, introducing air, and fermenting to generate foams containing the protopanaxadiol;

(2) transferring the foam into a sterile container B through an exhaust hole on a fermentation tank A, wherein when bubbles of the foam are broken, protopanoxadiol is attached to the wall of the sterile container B, and fermentation liquor and thalli carried by the foam are accumulated at the bottom of the sterile container B and then transferred into the fermentation tank A for continuous fermentation; stopping transferring the bottom fermentation liquid and thallus of the sterile container B every 4-8h, introducing ethanol water solution into the sterile container B, and dissolving protopanaxadiol attached to the wall of the sterile container B to obtain protopanaxadiol solution;

(3) passing the protopanoxadiol solution through macroporous resin column C, eluting with ethanol or ethanol water solution, collecting eluate, and evaporating to obtain protopanaxadiol.

2. The method as claimed in claim 1, wherein the step (1) is: putting a saccharomyces cerevisiae strain capable of synthesizing protopanaxadiol into a fermentation tank A; introducing air at 2L/min. L for 0-12 hr, and fermenting; introducing air at 2-4L/min & L for 12-24 hr, and fermenting; introducing air at 2-4L/min & L for 24-48h, and fermenting; introducing air at 3-6L/min & L for 48-60 hr, and fermenting; introducing air at a rate of 3-6L/min & L for 60-108h, and fermenting; introducing air at 2-6L/min & L for fermentation at 108-120 h; introducing air at 2L/min & L for fermentation at 120-144 h; there is a foam production containing protopanaxadiol.

3. The method as set forth in claim 1, characterized in that the concentration by volume of the ethanol aqueous solution in the step (2) is 40% -80%.

4. The method of claim 1, wherein the macroporous resin is Diaion HP-20, HDP-100, or X-5.

5. The method as set forth in claim 1, wherein the volume concentration of the ethanol aqueous solution in the step (3) is 70-99%.

Technical Field

The invention belongs to the technical field of bioengineering, and particularly relates to a method for separating protopanoxadiol by foam extraction and macroporous resin purification.

Background

Protopanaxadiol is one of triterpene sapogenins in Ginseng radix, and is precursor of dammarane type saponin. The molecule is polymerized from six isoprene units, and has a relative molecular mass of 460.743. Modern medical research shows that the triterpenoid sapogenin, especially protopanaxadiol (PPD), has high anticancer activity and can kill tumor cell effectively. In addition, it has no toxic side effect on human body, can also enhance the immunity of the organism, and can effectively enhance the anticancer curative effect when being mixed with other anticancer drugs. Besides, PPD has very excellent curative effect and effect on coronary heart disease, depression treatment, learning ability and the like. At present, various companies at home and abroad are actively developing medicines taking PPD as a main component, and various new products enter the market or clinical test stage. An anti-tumor drug 'Yisheng' capsule (the main components are PPD and ginsenoside Rh2) developed by Hainan Asia pharmacy is used as a national class of anti-cancer auxiliary drugs, and the clinical phase-3 test is completed at home at present; the anti-depression national class 1 new medicine "Youxiniding" capsule developed by Shanghai Chinese medicine innovation research center is undergoing phase 3 clinical research, and the medicine contains more than 93% of PPD component.

The traditional plant extraction method of the triterpenoid sapogenin is limited by factors such as high raw material cost, pesticide residue, environmental protection and the like, and the production efficiency is low. In recent years, the production of triterpene sapogenin by microbial fermentation by adopting a synthetic biology method has been advanced to a certain extent. For example, Zhang Zhi et al integrate the synthesis pathway of protopanaxadiol into Saccharomyces cerevisiae cells, and optimize the metabolic engineering and fermentation to make the protopanaxadiol yield reach 1.2 g/L.

The subject group improves the cell activity by optimizing key enzymes in the way and engineering the cell stress resistance characteristic, so that the yield of the protopanaxadiol is improved to 4.2 g/L. The above studies make it possible to produce protopanaxadiol by microbial fermentation, however, no studies on extraction of protopanaxadiol after fermentation have been reported yet. During PPD fermentation, more than 98% of PPD is secreted extracellularly, forming a large amount of foam, which makes the fermentation process difficult to control. In addition, these foams adsorb to the tank walls, forming a large amount of solid matter, and also increasing the difficulty of post-extraction of the product. At present, no report is available on the research on the separation process after the protopanaxadiol is produced by a fermentation method.

Disclosure of Invention

The invention aims to overcome the defects of the prior art and provides a method for separating protopanoxadiol by using foam extraction and macroporous resin purification.

The technical scheme of the invention is summarized as follows:

a method for purifying and separating protopanoxadiol by using foam extraction and macroporous resin comprises the following steps:

(1) placing a saccharomyces cerevisiae strain capable of synthesizing protopanaxadiol in a fermentation tank A, introducing air, and fermenting to generate foams containing the protopanaxadiol;

(2) transferring the foam into a sterile container B through an exhaust hole on a fermentation tank A, wherein when bubbles of the foam are broken, protopanoxadiol is attached to the wall of the sterile container B, and fermentation liquor and thalli carried by the foam are accumulated at the bottom of the sterile container B and then transferred into the fermentation tank A for continuous fermentation; stopping transferring the bottom fermentation liquid and thallus of the sterile container B every 4-8h, introducing ethanol water solution into the sterile container B, and dissolving protopanaxadiol attached to the wall of the sterile container B to obtain protopanaxadiol solution;

(3) passing the protopanoxadiol solution through macroporous resin column C, eluting with ethanol or ethanol water solution, collecting eluate, and evaporating to obtain protopanaxadiol.

Preferably, step (1) is: putting a saccharomyces cerevisiae strain capable of synthesizing protopanaxadiol into a fermentation tank A; introducing air at 2L/min. L for 0-12 hr, and fermenting; introducing air at 2-4L/min & L for 12-24 hr, and fermenting; introducing air at 2-4L/min & L for 24-48h, and fermenting; introducing air at 3-6L/min & L for 48-60 hr, and fermenting; introducing air at a rate of 3-6L/min & L for 60-108h, and fermenting; introducing air at 2-6L/min & L for fermentation at 108-120 h; introducing air at 2L/min & L for fermentation at 120-144 h; there is a foam production containing protopanaxadiol.

The volume concentration of the ethanol water solution in the step (2) is 40-80%.

The macroporous resin is Diaion HP-20, HDP-100 or X-5.

The volume concentration of the ethanol water solution in the step (3) is 70-99%.

The invention has the advantages that:

experiments prove that the method can efficiently separate the protopanoxadiol belonging to the triterpene sapogenin from the foam of the fermentation liquor, the transfer efficiency of the protopanaxadiol reaches 74.8 percent or more, and the extraction of the protopanaxadiol is realized. Purifying with macroporous resin to obtain protopanaxadiol with purity of 74.4% or above.

Drawings

FIG. 1 is a schematic diagram of the process of the present invention.

Detailed Description

The present invention will be further illustrated by the following specific examples.

The experimental procedures used in the following examples are all conventional ones unless otherwise specified.

Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.

The saccharomyces cerevisiae strain capable of synthesizing protopanaxadiol is the protopanaxadiol saccharomyces cerevisiae engineering bacterium W3a-ssPy, but the invention is not limited, and other saccharomyces cerevisiae strains capable of synthesizing protopanaxadiol can also be used in the invention.