A method for extracting S-adenosylmethionine from a S-adenosylmethionine fermentation broth

Abstract

The present application relates to a kind of methods for extracting S-adenosyl methionine from S-adenosyl methionine fermentation liquor, S-adenosyl methionine fermentation liquor is acid hydrolysis, emulsification is broken, microfiltration, two water phase extraction, macroporous resin is removed PEG, and then by solvated crystallization, anion resin is decolorized, recrystallization, vacuum drying, and S-adenosyl methionine sulfate finished product is obtained.The present application adopts two water phase extraction macroporous resin separation method and unique solvent recrystallization purification method, with high extraction efficiency, mild operating conditions, strong selectivity, green environmental protection and other unique advantages, especially can maintain (S, S) -SAM biological activity, improve product quality.

Description

Technical Field

[0001] This invention belongs to the field of bio-fermentation extraction technology, and in particular relates to a method for extracting S-adenosylmethionine from S-adenosylmethionine fermentation broth. Background Technology

[0002] S-Adenosylmethionine (SAM) is a common co-substrate involved in methyl transfer. It is synthesized from adenosine triphosphate (ATP) and methionine by methionine adenosyltransferase. All biological cellular processes, including transmethylation, transsulfuration, and aminopropylation, utilize the SAM metabolic pathway. SAM exists in two isomers: (R,S)-SAM and (S,S)-SAM, of which only (S,S)-SAM is biologically active. SAM is unstable and can spontaneously racemize to form the biologically inactive (R,S)-SAM. It is generally prepared as SAM sulfate, p-toluenesulfonic acid-SAM salt, or stored at low temperatures to improve its stability.

[0003] SAM molecular formula C 15 H 22 N6O5S, molecular weight 399.49

[0004] Structural formula of S-adenosylmethionine:

[0005]

[0006] SAM is a crystalline or crystalline white powder, readily soluble in water, slightly soluble in methanol and ethanol, and almost insoluble in diethyl ether and butanone. SAM is relatively stable at pH < 3; otherwise, it is prone to hydrolysis.

[0007] SAM is an important and physiologically active substance in the human body, primarily used clinically for the treatment of liver disease, arthritis, and depression. Furthermore, SAM is an important chemopreventive drug for cancer, increasingly attracting attention. SAM also shows good therapeutic effects on hyperlipidemia, arteriosclerosis, fibromyalgia, migraines, and Alzheimer's disease.

[0008] Currently, the production methods for S-adenosylmethionine both domestically and internationally include chemical synthesis, in vitro enzyme-catalyzed synthesis, and microbial fermentation, with microbial fermentation being the primary method.

[0009] There are two main routes for the chemical synthesis of SAM: one is to synthesize racemic SAM using 5'-methylthioadenosine and DL-2-amino-4-bromobutylic acid, which has only 50% biological activity; the other is to synthesize SAM through S-adenosine-L-homocysteine ​​methylation, which produces a product containing 20-30% (R, S) isomers. Due to the large number of isomers in the chemically synthesized product, the difficulty in separation and purification, and the unstable nature of the SAM molecule, the industrialization prospects of SAM production by chemical synthesis are limited.

[0010] In vitro enzymatic synthesis utilizes SAM synthase to catalyze the reaction of Met (methionine) and ATP to produce SAM in vitro. Genetic engineering technology has facilitated the rapid development of this method. These genetic engineering techniques not only enhance enzyme activity but also achieve higher substrate conversion rates, facilitating purification. In vitro enzymatic synthesis has the potential for industrial application.

[0011] Microbial fermentation has become the main method for industrial production of SAM in recent years. By fermenting certain microorganisms, especially by adding a certain amount of Met (methionine) to the culture medium, SAM can be accumulated in large quantities within the cells, and then extracted and purified to obtain SAM products.

[0012] Methods for extracting SAM from SAM fermentation broth generally begin with physical or chemical methods to disrupt the cell walls. This involves altering the permeability of yeast cells in the fermentation broth or causing cell rupture, releasing SAM. Physical disruption methods include cell disruption by compression, ultrasonic methods, and liquid nitrogen grinding. Chemical disruption methods primarily involve using reagents such as acetic acid, ethyl acetate, toluene, sulfuric acid, and perchloric acid to release SAM from the cell walls. Then, SAM is extracted by binding with precipitating reagents (such as picric acid or picric acid); alternatively, SAM can be adsorbed onto a cation exchange resin and then eluted; or it can be extracted using ultrafiltration or gel column chromatography.

[0013] The above production process has at least the following technical problems:

[0014] 1. The method of extracting SAM by binding it with precipitating reagents (such as picric acid, picric acid, etc.) is complicated. It requires the addition of a variety of organic solvents for extraction. The salt-forming reagent itself introduces pigments, which increases the difficulty of extraction and production costs.

[0015] In addition to SAM, the fermentation broth also contains a large amount of protein. Protein is an amphoteric compound, and when SAM is adsorbed by cation exchange, some protein will also be adsorbed. The resulting SAM eluent also contains a certain amount of protein, which crystallizes directly and has poor quality. Summary of the Invention

[0016] The purpose of this invention is to overcome the shortcomings of the prior art and provide a method for extracting S-adenosylmethionine from S-adenosylmethionine fermentation broth. By providing a method including aqueous two-phase extraction with macroporous resin separation and acetone recrystallization purification, the yield and purity of S-adenosylmethionine are improved while maintaining its stability.

[0017] The technical solution adopted to achieve the above objectives is as follows:

[0018] A method for extracting S-adenosylmethionine from S-adenosylmethionine fermentation broth, characterized by the following process steps: acid hydrolysis, emulsification and crushing of the S-adenosylmethionine fermentation broth, microfiltration, aqueous two-phase extraction, PEG removal with macroporous resin, followed by dissolution and crystallization, decolorization with anion exchange resin, recrystallization, and vacuum drying to obtain S-adenosylmethionine sulfate product, wherein the aqueous two-phase is polyethylene glycol / ammonium phosphate with a volume ratio of 1:1.

[0019] The polyethylene glycol is PEG400 or PEG600, preferably PEG600.

[0020] The concentration of polyethylene glycol is 20-25% (w / v).

[0021] The concentration of ammonium phosphate is 20-25% (w / v).

[0022] The recrystallization solvent is icy acetone.

[0023] The S-adenosylmethionine fermentation broth was acid-hydrolyzed, emulsified, and broken down. A 10% (w / w) sulfuric acid solution was heated to 65–80°C, and then the 10% (w / w) sulfuric acid was passed through a pore size... Spray and atomize the mixture with the fermentation broth. The mixing ratio is: fermentation broth (V): 10% (w / w) sulfuric acid (V) = 1: 0.35~0.0.45. The mixing time is 8~10 minutes, and the hydrolysis temperature is controlled at 45~65℃. After the acid hydrolysis is completed, the mixture is rapidly cooled to 5~8℃ and then passed through a high-speed emulsifying shearing machine to break up the mycelium, thus obtaining an emulsified acidified hydrolysate.

[0024] The microfiltration refers to passing an emulsified and acidified hydrolysate of S-adenosylmethionine into a microfiltration membrane (0.1 μm pore size), washing it with water, collecting the dialysate, and obtaining an S-adenosylmethionine dialysate. The concentration factor of the microfiltration membrane is 3.0 to 5.0.

[0025] The aqueous two-phase extraction involves adding PEG / ammonium phosphate at a volume ratio of 1:1, with PEG concentration of 20-25% (w / v) and ammonium phosphate concentration of 20-25% (w / v). The aqueous two-phase extraction time is 0.5-2 hours, during which the mixture is stirred every 20 minutes. After settling, the mixture is allowed to separate into layers. The lower ammonium phosphate phase waste liquid is treated as wastewater, and the upper PEG phase (containing S-adenosylmethionine) is collected. The upper PEG phase (containing S-adenosylmethionine) is fed into a macroporous resin at a feed rate of 1-3 BV / h. After feeding, the mixture is eluted in stages with anhydrous methanol and water at a rate of 0.5-0.8 BV / h. The methanol eluent is concentrated to remove methanol, and the PEG is recovered for reuse in the aqueous two-phase cycle. The water eluent is transferred to the next step.

[0026] It should be noted that the PEG refers to PEG400 or PEG600, with PEG600 being the most effective.

[0027] The process of dissolution and crystallization involves adding 5 to 8 times the volume of anhydrous methanol to the above-washed liquid to precipitate S-adenosylmethionine sulfate, followed by filtration to obtain crude S-adenosylmethionine sulfate.

[0028] The anion exchange resin decolorization process involves dissolving the crude S-adenosylmethionine sulfate in water, controlling the concentration at 22-25% (w / v), decolorizing with anion exchange resin, and filtering to obtain a purified S-adenosylmethionine sulfate solution.

[0029] The recrystallization process involves adding 4 to 6 times the volume of icy acetone to the above purified S-adenosylmethionine sulfate solution to precipitate the S-adenosylmethionine sulfate, followed by filtration and vacuum drying to obtain the finished S-adenosylmethionine sulfate product.

[0030] Compared with traditional methods for extracting S-adenosylmethionine from fermentation broth, the technical advantages of this invention are as follows:

[0031] This patent employs a two-phase aqueous extraction technology, which has unique advantages such as high extraction efficiency, mild operating conditions, strong selectivity, and environmental friendliness. In particular, it can maintain the bioactivity of (S,S)-SAM and improve product quality.

[0032] This patent uses macroporous resin to separate PEG and SAM, enabling PEG to be recycled and reducing production costs.

[0033] The three patents respectively use ice-cold methanol for dissolution and ice-cold acetone for crystallization of SAM, resulting in high-quality products with high SAM activity. Detailed Implementation

[0034] The invention is illustrated below with examples. It should be understood that these examples are for illustrative purposes only and not for limiting the invention. The scope and core content of the invention are defined by the claims.

[0035] 800L of S-adenosylmethionine fermentation broth (fermentation titer 32g / L) was obtained by fermenting yeast and subjected to the following experiments:

[0036] Example 1

[0037] Take 100L of S-adenosylmethionine fermentation broth (fermentation potency 32g / L), heat a 10% (w / w) sulfuric acid solution to 65-80℃, and then pass the 10% (w / w) sulfuric acid through a pore size... Spray and atomize the mixture with the fermentation broth. The mixing ratio is: fermentation broth (V): 10% (w / w) sulfuric acid (V) = 1: 0.35~0.0.45. The mixing time is 8 minutes, and the hydrolysis temperature is controlled at 45~65℃. After the acid hydrolysis is completed, the mixture is rapidly cooled to 5~8℃ and then passed through a high-speed emulsifying shearing machine to break up the mycelium, thus obtaining an emulsified acidified hydrolysate.

[0038] The above S-adenosylmethionine emulsified acidified hydrolysate was fed into a microfiltration membrane (pore size 0.1 μm), washed with water, and the dialysate was collected to obtain S-adenosylmethionine dialysate. The concentration factor of the microfiltration membrane was 3.0.

[0039] Add PEG400 and ammonium phosphate at a volume ratio of 1:1, with PEG400 and ammonium phosphate concentrations both at 20% (w / v). Perform aqueous two-phase extraction for 0.5–2 hours, stirring every 20 minutes. Allow the mixture to settle and separate into layers. The lower ammonium phosphate phase waste liquid is treated as wastewater, while the upper PEG400 phase (containing S-adenosylmethionine) is collected. Feed the upper PEG400 phase (containing S-adenosylmethionine) into a macroporous resin at a feed rate of 1 BV / h. After feeding, elute separately with anhydrous methanol and water at a rate of 0.5 BV / h. Concentrate the methanol-eluted solution to remove methanol and recover PEG400 for reuse in the aqueous two-phase cycle. Transfer the water-eluted solution to the next step.

[0040] Add 5 times the volume of ice-cold anhydrous methanol to the above water washing section liquid to precipitate S-adenosylmethionine sulfate. Filter the solution to obtain crude S-adenosylmethionine sulfate.

[0041] The crude S-adenosylmethionine sulfate was dissolved in water to control the concentration at 22% (w / v), decolorized by anion exchange resin, and filtered to obtain a purified S-adenosylmethionine sulfate solution.

[0042] Add 4 times the volume of ice-cold acetone to the above purified S-adenosylmethionine sulfate solution to precipitate S-adenosylmethionine sulfate. Filter and dry under vacuum to obtain the finished S-adenosylmethionine sulfate product.

[0043] Example 2

[0044] Take 100L of S-adenosylmethionine fermentation broth (fermentation potency 32g / L), heat a 10% (w / w) sulfuric acid solution to 65-80℃, and then pass the 10% (w / w) sulfuric acid through a pore size... Spray and atomize the mixture with the fermentation broth. The mixing ratio is: fermentation broth (V): 10% (w / w) sulfuric acid (V) = 1: 0.35~0.0.45. The mixing time is 9 minutes, and the hydrolysis temperature is controlled at 45~65℃. After the acid hydrolysis is completed, the mixture is rapidly cooled to 5~8℃ and then passed through a high-speed emulsifying shearing machine to break up the mycelium, thus obtaining an emulsified acidified hydrolysate.

[0045] The above S-adenosylmethionine emulsified acidified hydrolysate was fed into a microfiltration membrane (pore size 0.1 μm), washed with water, and the dialysate was collected to obtain S-adenosylmethionine dialysate. The concentration factor of the microfiltration membrane was 4.0.

[0046] Add PEG600 and ammonium phosphate at a volume ratio of 1:1, with both PEG600 and ammonium phosphate concentrations at 22% (w / v). Perform aqueous two-phase extraction for 0.5–2 hours, stirring every 20 minutes. Allow the mixture to settle and separate into layers. The lower ammonium phosphate phase waste liquid is treated as wastewater, while the upper PEG600 phase (containing S-adenosylmethionine) is collected. Feed the upper PEG600 phase (containing S-adenosylmethionine) into a macroporous resin at a feed rate of 2 BV / h. After feeding, elute separately with anhydrous methanol and water at a rate of 0.6 BV / h. Concentrate the methanol-eluted solution to remove methanol and recover PEG600 for reuse in the aqueous two-phase cycle. Transfer the water-eluted solution to the next step.

[0047] Add 6 times the volume of ice-cold anhydrous methanol to the above water washing section liquid to precipitate S-adenosylmethionine sulfate. Filter the solution to obtain crude S-adenosylmethionine sulfate.

[0048] The crude S-adenosylmethionine sulfate was dissolved in water to control the concentration at 23% (w / v), decolorized by anion exchange resin, and filtered to obtain a purified S-adenosylmethionine sulfate solution.

[0049] Add 5 times the volume of ice-cold acetone to the above purified S-adenosylmethionine sulfate solution to precipitate S-adenosylmethionine sulfate. Filter and dry under vacuum to obtain the finished S-adenosylmethionine sulfate product.

[0050] Example 3

[0051] Take 100L of S-adenosylmethionine fermentation broth (fermentation potency 32g / L), heat a 10% (w / w) sulfuric acid solution to 65-80℃, and then pass the 10% (w / w) sulfuric acid through a pore size... Spray and atomize the mixture with the fermentation broth. The mixing ratio is: fermentation broth (V): 10% (w / w) sulfuric acid (V) = 1: 0.35~0.0.45. The mixing time is 10 minutes, and the hydrolysis temperature is controlled at 45~65℃. After the acid hydrolysis is completed, the mixture is rapidly cooled to 5~8℃ and then passed through a high-speed emulsifying shearing machine to break up the mycelium, thus obtaining an emulsified acidified hydrolysate.

[0052] The above S-adenosylmethionine emulsified acidified hydrolysate was fed into a microfiltration membrane (pore size 0.1 μm), washed with water, and the dialysate was collected to obtain S-adenosylmethionine dialysate. The concentration factor of the microfiltration membrane was 5.0.

[0053] Add PEG400 and ammonium phosphate at a volume ratio of 1:1, with PEG400 and ammonium phosphate concentrations both at 25% (w / v). Perform aqueous two-phase extraction for 0.5–2 hours, stirring every 20 minutes during this time. Allow the mixture to settle and separate into layers. The lower ammonium phosphate phase waste liquid is treated as wastewater, while the upper PEG400 phase (containing S-adenosylmethionine) is collected. Feed the upper PEG400 phase (containing S-adenosylmethionine) into a macroporous resin at a feed rate of 3 BV / h. After feeding, elute separately with anhydrous methanol and water at a rate of 0.8 BV / h. Concentrate the methanol-eluted solution to remove methanol and recover PEG400 for reuse in the aqueous two-phase cycle. Transfer the water-eluted solution to the next step.

[0054] Add 7 times the volume of ice-cold anhydrous methanol to the above water washing section liquid to precipitate S-adenosylmethionine sulfate. Filter the solution to obtain crude S-adenosylmethionine sulfate.

[0055] The crude S-adenosylmethionine sulfate was dissolved in water to control the concentration at 24% (w / v), decolorized by anion exchange resin, and filtered to obtain a purified S-adenosylmethionine sulfate solution.

[0056] Add 6 times the volume of ice-cold acetone to the above purified S-adenosylmethionine sulfate solution to precipitate S-adenosylmethionine sulfate. Filter and dry under vacuum to obtain the finished S-adenosylmethionine sulfate product.

[0057] Example 4

[0058] Take 100L of S-adenosylmethionine fermentation broth (fermentation potency 32g / L), heat a 10% (w / w) sulfuric acid solution to 65-80℃, and then pass the 10% (w / w) sulfuric acid through a pore size... Spray and atomize the mixture with the fermentation broth. The mixing ratio is: fermentation broth (V): 10% (w / w) sulfuric acid (V) = 1: 0.35~0.0.45. The mixing time is 9 minutes, and the hydrolysis temperature is controlled at 45~65℃. After the acid hydrolysis is completed, the mixture is rapidly cooled to 5~8℃ and then passed through a high-speed emulsifying shearing machine to break up the mycelium, thus obtaining an emulsified acidified hydrolysate.

[0059] The above S-adenosylmethionine emulsified acidified hydrolysate was fed into a microfiltration membrane (pore size 0.1 μm), washed with water, and the dialysate was collected to obtain S-adenosylmethionine dialysate. The concentration factor of the microfiltration membrane was 4.0.

[0060] Add PEG600 and ammonium phosphate at a volume ratio of 1:1, with both PEG600 and ammonium phosphate concentrations at 22% (w / v). Perform aqueous two-phase extraction for 0.5–2 hours, stirring every 20 minutes. Allow the mixture to settle and separate into layers. The lower ammonium phosphate phase waste liquid is treated as wastewater, while the upper PEG600 phase (containing S-adenosylmethionine) is collected. Feed the upper PEG600 phase (containing S-adenosylmethionine) into a macroporous resin at a feed rate of 2 BV / h. After feeding, elute separately with anhydrous methanol and water at a rate of 0.7 BV / h. Concentrate the methanol-eluted solution to remove methanol and recover PEG600 for reuse in the aqueous two-phase cycle. Transfer the water-eluted solution to the next step.

[0061] Add 7 times the volume of ice-cold anhydrous methanol to the above water washing section liquid to precipitate S-adenosylmethionine sulfate. Filter the solution to obtain crude S-adenosylmethionine sulfate.

[0062] The crude S-adenosylmethionine sulfate was dissolved in water to control the concentration at 25% (w / v), decolorized by anion exchange resin, and filtered to obtain a purified S-adenosylmethionine sulfate solution.

[0063] Add 5 times the volume of ice-cold acetone to the above purified S-adenosylmethionine sulfate solution to precipitate S-adenosylmethionine sulfate. Filter and dry under vacuum to obtain the finished S-adenosylmethionine sulfate product.

[0064] Example 5

[0065] Take 100L of S-adenosylmethionine fermentation broth (fermentation potency 32g / L), heat a 10% (w / w) sulfuric acid solution to 65-80℃, and then pass the 10% (w / w) sulfuric acid through a pore size... Spray and atomize the mixture with the fermentation broth. The mixing ratio is: fermentation broth (V): 10% (w / w) sulfuric acid (V) = 1: 0.35~0.0.45. The mixing time is 9 minutes, and the hydrolysis temperature is controlled at 45~65℃. After the acid hydrolysis is completed, the mixture is rapidly cooled to 5~8℃ and then passed through a high-speed emulsifying shearing machine to break up the mycelium, thus obtaining an emulsified acidified hydrolysate.

[0066] The above S-adenosylmethionine emulsified acidified hydrolysate was fed into a microfiltration membrane (pore size 0.1 μm), washed with water, and the dialysate was collected to obtain S-adenosylmethionine dialysate. The concentration factor of the microfiltration membrane was 4.0.

[0067] Add PEG600 and ammonium phosphate at a volume ratio of 1:1, with PEG600 and ammonium phosphate concentrations both at 25% (w / v). Perform aqueous two-phase extraction for 0.5–2 hours, stirring every 20 minutes. Allow the mixture to settle and separate into layers. The lower ammonium phosphate phase waste liquid is treated as wastewater, while the upper PEG600 phase (containing S-adenosylmethionine) is collected. Feed the upper PEG600 phase (containing S-adenosylmethionine) into a macroporous resin at a feed rate of 2 BV / h. After feeding, elute separately with anhydrous methanol and water at a rate of 0.7 BV / h. Concentrate the methanol-eluted solution to remove methanol and recover PEG600 for reuse in the aqueous two-phase cycle. Transfer the water-eluted solution to the next step.

[0068] Add 7 times the volume of ice-cold anhydrous methanol to the above water washing section liquid to precipitate S-adenosylmethionine sulfate. Filter the solution to obtain crude S-adenosylmethionine sulfate.

[0069] The crude S-adenosylmethionine sulfate was dissolved in water to control the concentration at 23% (w / v), decolorized by anion exchange resin, and filtered to obtain a purified S-adenosylmethionine sulfate solution.

[0070] Add 5 times the volume of ice-cold acetone to the above purified S-adenosylmethionine sulfate solution to precipitate S-adenosylmethionine sulfate. Filter and dry under vacuum to obtain the finished S-adenosylmethionine sulfate product.

[0071] Comparison Case 1

[0072] Take 100L of S-adenosylmethionine fermentation broth (fermentation potency 32g / L), heat a 10% (w / w) sulfuric acid solution to 65-80℃, and then pass the 10% (w / w) sulfuric acid through a pore size... Spray and atomize the mixture with the fermentation broth. The mixing ratio is: fermentation broth (V): 10% (w / w) sulfuric acid (V) = 1: 0.35~0.0.45. The mixing time is 8 minutes, and the hydrolysis temperature is controlled at 45~65℃. After the acid hydrolysis is completed, the mixture is rapidly cooled to 5~8℃ and then passed through a high-speed emulsifying shearing machine to break up the mycelium, thus obtaining an emulsified acidified hydrolysate.

[0073] The above S-adenosylmethionine emulsified acidified hydrolysate was fed into a microfiltration membrane (pore size 0.1 μm), washed with water, and the dialysate was collected to obtain S-adenosylmethionine dialysate. The concentration factor of the microfiltration membrane was 3.0.

[0074] Add PEG400 and ammonium phosphate at a volume ratio of 1:1, with PEG400 and ammonium phosphate concentrations both at 20% (w / v). Perform aqueous two-phase extraction for 0.5–2 hours, stirring every 20 minutes. Allow the mixture to settle and separate into layers. The lower ammonium phosphate phase waste liquid is treated as wastewater, while the upper PEG400 phase (containing S-adenosylmethionine) is collected. Feed the upper PEG400 phase (containing S-adenosylmethionine) into a macroporous resin at a feed rate of 1 BV / h. After feeding, elute separately with anhydrous methanol and water at a rate of 0.5 BV / h. Concentrate the methanol-eluted solution to remove methanol and recover PEG400 for reuse in the aqueous two-phase cycle. Transfer the water-eluted solution to the next step.

[0075] Add 5 times the volume of ice-cold anhydrous methanol to the above water washing section liquid to precipitate S-adenosylmethionine sulfate. Filter the solution to obtain crude S-adenosylmethionine sulfate.

[0076] The crude S-adenosylmethionine sulfate was dissolved in water to control the concentration at 22% (w / v), decolorized by anion exchange resin, and filtered to obtain a purified S-adenosylmethionine sulfate solution.

[0077] The above purified S-adenosylmethionine sulfate solution was mixed with 4 times its volume of ice-cold methanol to precipitate S-adenosylmethionine sulfate. The solution was then filtered and dried under vacuum to obtain the final product of S-adenosylmethionine sulfate.

[0078] Comparison Case 2

[0079] Take 100L of S-adenosylmethionine fermentation broth (fermentation potency 32g / L), heat a 10% (w / w) sulfuric acid solution to 65-80℃, and then pass the 10% (w / w) sulfuric acid through a pore size... Spray and atomize the mixture with the fermentation broth. The mixing ratio is: fermentation broth (V): 10% (w / w) sulfuric acid (V) = 1: 0.35~0.0.45. The mixing time is 8 minutes, and the hydrolysis temperature is controlled at 45~65℃. After the acid hydrolysis is completed, the mixture is rapidly cooled to 5~8℃ and then passed through a high-speed emulsifying shearing machine to break up the mycelium, thus obtaining an emulsified acidified hydrolysate.

[0080] The above S-adenosylmethionine emulsified acidified hydrolysate was fed into a microfiltration membrane (pore size 0.1 μm), washed with water, and the dialysate was collected to obtain S-adenosylmethionine dialysate. The concentration factor of the microfiltration membrane was 3.0.

[0081] Prepare a 20% (w / v) PEG400 aqueous solution. Add the above S-adenosylmethionine dialysate and stir every 20 minutes. Allow the mixture to stand and separate into layers. Separate the lower waste liquid and send it for wastewater treatment. Collect the upper PEG400 phase (containing S-adenosylmethionine). Feed the upper PEG400 phase (containing S-adenosylmethionine) into a macroporous resin at a feed rate of 1 BV / h. After feeding, elute separately with anhydrous methanol and water at a rate of 0.5 BV / h. Concentrate the methanol eluent to remove methanol and recover PEG400 for reuse in the aqueous two-phase cycle. Transfer the water eluent to the next step.

[0082] Add 5 times the volume of ice-cold anhydrous methanol to the above water washing section liquid to precipitate S-adenosylmethionine sulfate. Filter the solution to obtain crude S-adenosylmethionine sulfate.

[0083] The crude S-adenosylmethionine sulfate was dissolved in water to control the concentration at 22% (w / v), decolorized by anion exchange resin, and filtered to obtain a purified S-adenosylmethionine sulfate solution.

[0084] The above purified S-adenosylmethionine sulfate solution was mixed with 4 times its volume of ice-cold methanol to precipitate S-adenosylmethionine sulfate. The solution was then filtered and dried under vacuum to obtain the final product of S-adenosylmethionine sulfate.

[0085] Comparison Case 3

[0086] Take 100L of S-adenosylmethionine fermentation broth (fermentation potency 32g / L), heat a 10% (w / w) sulfuric acid solution to 65-80℃, and then pass the 10% (w / w) sulfuric acid through a pore size... Spray and atomize the mixture with the fermentation broth. The mixing ratio is: fermentation broth (V): 10% (w / w) sulfuric acid (V) = 1: 0.35~0.0.45. The mixing time is 8 minutes, and the hydrolysis temperature is controlled at 45~65℃. After the acid hydrolysis is completed, the mixture is rapidly cooled to 5~8℃ and then passed through a high-speed emulsifying shearing machine to break up the mycelium, thus obtaining an emulsified acidified hydrolysate.

[0087] The above S-adenosylmethionine emulsified acidified hydrolysate was fed into a microfiltration membrane (pore size 0.1 μm), washed with water, and the dialysate was collected to obtain S-adenosylmethionine dialysate. The concentration factor of the microfiltration membrane was 3.0.

[0088] Prepare a 20% (w / v) ammonium phosphate aqueous solution, add the above S-adenosylmethionine dialysate, stir once every 20 minutes, let stand and separate the layers, separate the lower layer wastewater and send it to wastewater treatment, collect the upper aqueous phase (containing S-adenosylmethionine); feed the upper aqueous phase (containing S-adenosylmethionine) into a macroporous resin at a feed rate of 1 BV / h. After feeding, elute separately with anhydrous methanol and water at a rate of 0.5 BV / h. Concentrate the methanol eluent to remove methanol, combine it with the water eluent, and proceed to the next step.

[0089] Add 5 times the volume of ice-cold anhydrous methanol to the above water washing section liquid to precipitate S-adenosylmethionine sulfate. Filter the solution to obtain crude S-adenosylmethionine sulfate.

[0090] The crude S-adenosylmethionine sulfate was dissolved in water to control the concentration at 22% (w / v), decolorized by anion exchange resin, and filtered to obtain a purified S-adenosylmethionine sulfate solution.

[0091] The above purified S-adenosylmethionine sulfate solution was mixed with 4 times its volume of ice-cold methanol to precipitate S-adenosylmethionine sulfate. The solution was then filtered and dried under vacuum to obtain the final product of S-adenosylmethionine sulfate.

[0092] Comparison of experimental results

[0093] The yield, purity, and SS-type isomers of S-adenosylmethionine sulfate obtained in Examples 1-5 and Comparative Examples 1-3 were determined. The results are shown in Table 1 below.

[0094] Table 1 Comparison of S-adenosylmethionine sulfate results between examples and comparative examples

[0095] Experiment No. Yield % purity% % of SS-type isomers Experiment 1 80.2 99.6 67.8 Experiment 2 81.6 99.2 67.5 Experiment 3 80.5 99.3 67.9 Experiment 4 81.8 99.1 67.6 Experiment 5 81.5 98.9 67.6 Comparison-1 76.7 96.6 62.8 Comparison-2 68.3 97.5 64.4 Comparison-3 69.8 96.8 62.2

[0096] As can be seen from the test data in Table 1 above, the purification and extraction of S-adenosylmethionine using the method disclosed in this invention in Examples 1-5 can significantly improve the yield and purity of S-adenosylmethionine, with a yield higher than 80%, a purity higher than 98.5%, and SS-type isomers higher than 67%.

Claims

1. A method for extracting S-adenosylmethionine sulfate from S-adenosylmethionine fermentation broth, characterized in that... The process steps are as follows: acid hydrolysis, emulsification and crushing of S-adenosylmethionine fermentation broth, microfiltration, aqueous two-phase extraction, PEG removal with macroporous resin, followed by dissolution and crystallization, decolorization with anion exchange resin, recrystallization, and vacuum drying to obtain the S-adenosylmethionine sulfate product. The acid hydrolysis and emulsification of the S-adenosylmethionine fermentation broth involves heating a 10% w / w sulfuric acid solution to 65–80°C, then spraying the 10% w / w sulfuric acid through an orifice with a diameter of 2–5 mm to atomize and mix it with the fermentation broth. The mixing ratio is: fermentation broth: 10% w / w sulfuric acid = 1:0.35–0.0.45, and the mixing time is 8–10 minutes, with the hydrolysis temperature controlled at 45–65°C. After acid hydrolysis, the solution is rapidly cooled to 5–8°C and then passed through a high-speed emulsification shearing machine to break up the mycelium, yielding an emulsified acid hydrolysate. The aqueous two-phase extraction and macroporous resin PEG removal process involves adding PEG / ammonium phosphate at a volume ratio of 1:1, with both PEG and ammonium phosphate concentrations at 20-25%. The aqueous two-phase extraction time is 0.5-2 hours, during which the mixture is stirred every 20 minutes. After settling, the mixture is allowed to separate into layers. The lower ammonium phosphate phase waste liquid is treated as wastewater, and the upper PEG phase is collected. The upper PEG phase is fed into the macroporous resin at a feed rate of 1-3 BV / h. After feeding, the resin is eluted in stages with anhydrous methanol and water at a rate of 0.5-0.8 BV / h. The methanol-eluted liquid is concentrated to remove methanol and recover PEG for reuse in the aqueous two-phase cycle. The water-eluted liquid is then transferred to the next step. The solubilization and crystallization process involves adding 5 to 8 times the volume of anhydrous methanol to the above-mentioned water washing section solution to precipitate S-adenosylmethionine sulfate, followed by filtration to obtain crude S-adenosylmethionine sulfate. The anion exchange resin decolorization process involves dissolving the crude S-adenosylmethionine sulfate in water, controlling the concentration at 22-25%, decolorizing with anion exchange resin, and filtering to obtain a refined S-adenosylmethionine sulfate solution. The recrystallization and vacuum drying process involves adding 4 to 6 times the volume of icy acetone to the above purified S-adenosylmethionine sulfate solution to precipitate the S-adenosylmethionine sulfate, followed by filtration and vacuum drying to obtain the finished S-adenosylmethionine sulfate product.

2. The method for extracting S-adenosylmethionine sulfate from S-adenosylmethionine fermentation broth according to claim 1, characterized in that... The PEG is PEG400 or PEG600.