CN110396120B - Method for extracting and separating S-adenosylmethionine from S-adenosylmethionine fermentation liquor - Google Patents
Method for extracting and separating S-adenosylmethionine from S-adenosylmethionine fermentation liquor Download PDFInfo
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- CN110396120B CN110396120B CN201910112234.8A CN201910112234A CN110396120B CN 110396120 B CN110396120 B CN 110396120B CN 201910112234 A CN201910112234 A CN 201910112234A CN 110396120 B CN110396120 B CN 110396120B
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Abstract
The invention provides a method for extracting and separating S-adenosylmethionine from S-adenosylmethionine fermentation liquor, which enables the total yield of the S-adenosylmethionine to reach more than 70 percent and is carried out according to the following steps: a. wall breaking: collecting yeast by the fermentation liquor through a disc centrifuge, adding pure water with the volume of 30-100% of the fermentation liquor to wash the bacteria, and quickly cooling the obtained bacteria liquid after instantaneous high-temperature wall breaking by adopting a starch jet liquefying device to ensure that the cell breakage rate reaches more than 99%; b. crude extraction: b, passing the wall breaking liquid obtained in the step a through a microfiltration membrane with the aperture of 50-200 mu m, collecting filtrate, controlling the rejection rate of the microfiltration membrane on S-adenosylmethionine to be less than or equal to 5%, passing the dialysate through an ultrafiltration membrane (the material is PVDF, and the rejection molecular weight is 3000-10000D), removing macromolecular substances such as protein and the like, and obtaining a crude S-adenosylmethionine extracting solution; c. refining: purifying the crude extract obtained in the step b by using weak acid type cation exchange resin, and decoloring the eluent by using decoloring resin to obtain refined S-adenosylmethionine solution; d. salifying: and c, concentrating the refined liquid obtained in the step c by 5-15 times through a nanofiltration membrane (the material is PVDF, the molecular weight cut-off is 100-500D), and synthesizing double salts with sulfuric acid and p-toluenesulfonic acid in a certain molar ratio to obtain the ademetionine p-toluenesulfonic acid sulfate.
Description
Technical Field
The invention relates to the technical field of bioengineering, in particular to a method for extracting yeast after wall breaking fermentation by using a traditional starch jet liquefaction device.
Background
Ademetionine, also known as S-Adenosyl-methionine (SAM), is a metabolite formed by combining sulfur-containing amino acid-methionine (methionine) and adenosine triphosphate (ATP-adenosin ethephosphate) which is a main energy substance of a human body, is a physiologically active substance existing in all tissues and body fluids of the human body, participates in the transmethylation action, polyamine synthesis and transsulfuration action of the human body, and has better curative effects on arthritis, depression, liver dysfunction, pancreatitis and the like. In 1999, FDA approval of S-adenosylmethionine as a health product in the united states was rapidly becoming one of the best-marketed nutraceuticals in the united states. With the improvement of the quality of life of people and the update of health concepts, the demand of S-adenosylmethionine is increased.
At present, the industrial production of S-adenosylmethionine mostly adopts a yeast fermentation method (intracellular expression method). However, the separation and purification of S-adenosylmethionine from the fermentation broth has been a technical problem. S-adenosylmethionine is an intracellular product, the mature yeast has thick cell wall and is not easy to break, and the S-adenosylmethionine is sensitive to temperature and pH value and is easy to be transformed (from active S-S form to inactive R-S isomer) and decomposed (into S-adenosyl-homocysteine, adenine, methionine and the like). There are many methods for disrupting yeast cells, and the conventional methods include: extrusion cell disruption, ultrasonic methods, and chemical reagent methods. The extrusion cell disruption method needs more nitrogen, has high cost and is only suitable for laboratories; when the ultrasonic method is used for crushing, heat is generated, the stability of S-adenosylmethionine is influenced, and the ultrasonic method is not suitable for industrial production; the chemical reagent method has high corrosivity, brings inorganic salt ions, influences subsequent purification, is generally used for detecting the fermentation efficiency in a laboratory, and is not suitable for industrialization. Therefore, a method for breaking the cell wall, which can break the yeast cells with high efficiency, does not bring other impurities into the yeast cells and can effectively maintain the activity of S-adenosylmethionine, needs to be found.
In conclusion, the traditional method for separating and purifying S-adenosylmethionine has high cost, long separation process, low product yield and higher cost, and the S-adenosylmethionine with poor stability is easy to transform and degrade in the long-time separation process, thereby easily causing the excessive high content of product impurities and ash. Research and development of a new extraction and refining technology for separating and purifying S-adenosylmethionine from biological fermentation liquid with high efficiency and low cost are problems to be solved urgently in the current production.
Disclosure of Invention
The invention aims to provide a novel separation and purification technology for separating and purifying S-adenosylmethionine from biological fermentation liquor with high efficiency and low cost. In the process, a starch jet liquefaction device is adopted for instantly breaking the walls at high temperature, and a circulating freezing process is adopted for rapidly cooling, so that the wall breaking rate of the saccharomycetes can reach more than 99%, and the stability of the S-adenosylmethionine can be ensured to the maximum extent. In the extraction process, advanced membrane equipment is adopted for separation and concentration, and a microfiltration membrane is used for removing broken yeast cells and collecting wall-broken liquid; the ultrafiltration membrane can effectively remove macromolecular substances such as small cell fragments, foreign proteins, pigments and the like in the feed liquid; the nanofiltration membrane is used for concentrating the decolored solution and removing small molecular impurities. In addition, the purification process of S-adenosylmethionine also adopts the process of connecting weak acid type cation exchange resin and decolorizing resin in series.
Specifically, the object of the present invention can be achieved by the following technical measures:
the method comprises the following steps:
wall breaking: the fermentation liquor passes through a disc centrifuge at the rotation speed of 1000-. Adjusting the concentration of the bacteria to 10-50%, feeding into a starch spraying liquefying device, instantaneously breaking the wall at a high temperature of 95-120 ℃, cooling the feed liquid to 15-45 ℃ within 30S by adopting a circulating chilled water process after spraying, wherein the cell breakage rate reaches more than 99%, and the yield of S-adenosylmethionine is more than 95%;
crude extraction: and c, passing the wall breaking liquid obtained in the step a through a microfiltration membrane with the aperture of 50-500 mu m, collecting filtrate, adding pure water with the volume of 30-100% of that of the wall breaking liquid, and cleaning, wherein the retention rate of the microfiltration membrane on S-adenosylmethionine is controlled to be less than 5%. Passing the dialysate through an ultrafiltration membrane (the material is PVDF, the cut-off molecular weight is 3000-10000D), adding pure water with the volume of 30-100% of the material liquid to clean the ultrafiltration membrane, controlling the cut-off rate of the ultrafiltration membrane on S-adenosylmethionine to be less than 5%, and performing ultrafiltration on the dialysate, namely the S-adenosylmethionine crude extract, wherein the concentration of the S-adenosylmethionine is 3-7 g/L;
refining: adjusting pH of the crude extractive solution obtained in step b to 3-10, and passing through weak acid type cation exchange resin at flow rate of 0.5-5 BV. Eluting the eluent by using 0.05-5mol/L sulfuric acid solution, connecting the eluent with decolorizing resin in series, controlling the flow rate to be 0.5-5BV, and controlling the light transmittance of the decolorizing solution to be more than 95 percent to obtain S-adenosylmethionine refined solution;
salifying: and c, passing the refined liquid obtained in the step c through a nanofiltration membrane (the material is PVDF, the molecular weight cutoff is 100-500D), adding pure water with the volume of 30-100% of that of the liquid to clean the nanofiltration membrane, and controlling the concentration multiple to be 5-15 times and the loss rate of S-adenosylmethionine to be less than 5%. The concentrated solution is mixed with sulfuric acid and p-toluenesulfonic acid in a certain molar ratio to synthesize double salt, and the finished product of the ademetionine p-toluenesulfonic acid sulfate is obtained.
The extraction method provided by the invention can be used for obtaining the finished product of the ademetionine p-toluenesulfonic acid sulfate, the product purity is more than 99%, the total yield is more than 70%, and the S-S isomer is more than 65%. The other indexes of the ademetionine p-toluenesulfonic acid sulfate product all meet the export standards of Europe and America.
The invention has the beneficial effects that:
(1) the method creatively adopts the traditional starch injection liquefying device for saccharomycete cell disruption, and adopts the high-temperature instantaneous wall-breaking and rapid cooling method to ensure that the wall-breaking rate of the saccharomycete reaches 99 percent and the product yield is more than 95 percent. The process belongs to physical wall breaking, no other substances are introduced in the operation process, the wall breaking rate is high, and the product loss rate is low.
(2) The separation and concentration of S-adenosylmethionine are carried out by adopting a membrane separation mode, the process is simple and reliable to operate, strong in pollution resistance and easy to clean, the production cost is greatly reduced, and industrial amplification is easy to realize.
(3) The purification process of S-adenosylmethionine adopts the technique of weak acid type cation exchange resin elution and decolorizing resin series connection, thereby shortening the extraction period, improving the efficiency and saving the cost.
(4) The total yield of the S-adenosylmethionine provided by the extraction method is more than 70 percent and is far higher than that of other post-extraction methods.
Description of the drawings:
FIG. 1 is a schematic diagram of the technical scheme of the present invention.
The specific implementation mode is as follows:
the following examples further illustrate the present invention but are not to be construed as limiting the invention. Modifications or substitutions to methods, procedures, or conditions of the invention may be made without departing from the spirit and scope of the invention. Referring to fig. 1, the present invention provides the following embodiments:
example 1:
the fermentation liquor passes through a disc centrifuge at the rotating speed of 3000rpm, yeast is collected, and pure water with the volume of 30 percent of the fermentation liquor is added to wash the thalli. Adjusting the concentration of the bacteria to 10%, feeding the bacteria into a starch spraying liquefaction device, instantaneously breaking the wall at a high temperature, wherein the spraying temperature is 105 ℃, cooling the feed liquid to 40 ℃ 30S after spraying, the cell breakage rate is 99.2%, and the yield of S-adenosylmethionine is 98.5%; passing the wall-broken liquid through a microfiltration membrane with the aperture of 500 mu m, collecting filtrate, and adding 30% pure water for cleaning. Passing the microfiltration dialysate through an ultrafiltration membrane (made of PVDF and having a molecular weight cutoff of 6000D), adding pure water with a volume of 30% of the feed liquid, and cleaning the ultrafiltration membrane, wherein the concentration of S-adenosylmethionine in the ultrafiltration dialysate is 4 g/L; the pH of the ultrafiltration dialysate was adjusted to 5.0, and the dialysate was passed through a weak acid type cation exchange resin at a flow rate of 2 BV. Eluting the eluent by using 0.2mol/L sulfuric acid solution, connecting the eluent in series with decolorizing resin, and controlling the flow rate to be 2BV to obtain S-adenosylmethionine refined solution; and (3) passing the refined liquid through a nanofiltration membrane (the material is PVDF, the molecular weight cut-off is 300D), adding pure water with the volume of 30% of the liquid to clean the nanofiltration membrane, controlling the concentration multiple to be 15 times, and synthesizing the concentrated liquid, sulfuric acid and p-toluenesulfonic acid in a certain molar ratio into double salt to obtain the finished product of the ademetionine p-toluenesulfonic acid sulfate.
The obtained ademetionine p-toluenesulfonic acid sulfate is checked, the product purity is 99.2%, the total yield is 71.3%, the S-S isomer is 67.4%, and the product meets European and American export standards.
Example 2:
the fermentation liquor passes through a disc centrifuge, the rotating speed is 5000rpm, yeast is collected, and pure water with the volume of 50 percent of the fermentation liquor is added to wash the thalli. Adjusting the concentration of the bacteria to 50%, feeding the bacteria into a starch spraying liquefaction device, instantaneously breaking the wall at a high temperature, wherein the spraying temperature is 110 ℃, cooling the feed liquid to 45 ℃ 30S after spraying, the cell breakage rate is 99.6%, and the yield of S-adenosylmethionine is 96.5%; passing the wall-broken liquid through a microfiltration membrane with the aperture of 200 mu m, collecting filtrate, and adding 50% volume of pure water for cleaning. Passing the microfiltration dialysate through ultrafiltration membrane (made of PVDF with molecular weight cutoff of 8000D), adding pure water 30% of the volume of the filtrate to clean the ultrafiltration membrane, wherein the concentration of S-adenosylmethionine in the ultrafiltration dialysate is 6.5 g/L; the ultrafiltration dialysate was adjusted to pH5.0 and passed through a weak acid type cation exchange resin at a flow rate of 1.5 BV. Eluting the eluent by using 0.2mol/L sulfuric acid solution, connecting the eluent in series with decolorizing resin, and controlling the flow rate to be 2.5BV to obtain S-adenosylmethionine refined solution; and (3) passing the refined liquid through a nanofiltration membrane (the material is PVDF, the molecular weight cut-off is 300D), adding pure water with the volume of 50% of the liquid volume to clean the nanofiltration membrane, controlling the concentration multiple to be 8 times, and synthesizing the concentrated liquid, sulfuric acid and p-toluenesulfonic acid in a certain molar ratio into double salt to obtain the finished product of the ademetionine p-toluenesulfonic acid sulfate.
The obtained ademetionine p-toluenesulfonic acid sulfate is checked, the product purity is 99.3%, the total yield is 73.4%, the S-S isomer is 63.2%, and the product meets European and American export standards.
Example 3:
comparison of the wall-breaking methods of various yeasts
The fermentation liquor passes through a disc centrifuge at the rotating speed of 6000rpm, yeast is collected, and pure water accounting for 50% of the volume of the fermentation liquor is added to wash the thalli. Adjusting the concentration of the bacteria to 30 percent. The breaking of yeast cells is carried out in quadruplicate by a high-temperature instantaneous wall breaking method, an ultrasonic method, a hot water wall breaking method and a dilute sulfuric acid wall breaking method respectively. Comparing the wall-breaking rate, the yield of S-adenosylmethionine and the ratio of S-S isomer. The results are given in the following table:
Claims (3)
1. a method for extracting and separating S-adenosylmethionine from S-adenosylmethionine fermentation broth is characterized by comprising the following steps: a, breaking the wall: collecting yeast by the fermentation liquor through a disc centrifuge, adding pure water with the volume of 30-100% of the fermentation liquor to wash the bacteria, and quickly cooling the obtained bacteria liquid after instantaneous high-temperature wall breaking by adopting a starch jet liquefying device to ensure that the cell breakage rate reaches more than 99%; b, crude extraction: b, passing the wall breaking liquid obtained in the step a through a microfiltration membrane with the aperture of 50-200 mu m, removing broken bacteria residues, controlling the retention rate of the microfiltration membrane on S-adenosylmethionine to be less than or equal to 5%, passing the dialysate through an ultrafiltration membrane, wherein the material is PVDF, the retention molecular weight is 3000-10000D, and removing macromolecular substances such as protein and the like to obtain a crude S-adenosylmethionine extract; c, refining: purifying the crude extract obtained in the step b by using weak acid type cation exchange resin, and decoloring the eluent by using decoloring resin to obtain refined S-adenosylmethionine solution; d, salifying: c, passing the refined liquid obtained in the step c through a nanofiltration membrane, wherein the material is PVDF, the molecular weight cutoff is 100-500D, concentrating the solution by 5-15 times, and synthesizing double salts with sulfuric acid and p-toluenesulfonic acid in a certain molar ratio to obtain adenosylmethionine p-toluenesulfonic acid sulfate;
instantly breaking the wall at high temperature by adopting a starch jetting and liquefying device in the step a, wherein the jetting temperature is 95-120 ℃;
and a, quickly cooling the feed liquid to 15-45 ℃ within 30 seconds by adopting a circulating chilled water process.
2. The method for extracting and separating S-adenosylmethionine from S-adenosylmethionine fermentation broth according to claim 1, wherein said weak acid cation exchange resin treatment of step c has a loading concentration of 1-10g/L, a loading pH of 3-10, and the eluent is 0.05-5mol/L sulfuric acid solution.
3. The method for extracting and separating S-adenosylmethionine from S-adenosylmethionine fermentation broth according to claim 1, wherein the weak acid type cation exchange resin purification and decolorization in step c are performed by a series process at a flow rate of 0.5-5 BV.
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