A method for separating and extracting spermidine from fermentation broth

Abstract

The present invention discloses a method for separating and extracting spermidine from a fermentation broth, belonging to the field of biochemical engineering. The method comprises: S1, fermentation broth pretreatment: the spermidine fermentation broth is sequentially passed through an inorganic ceramic membrane to obtain a clarified liquid; S2, ultrafiltration and concentration: the clarified liquid obtained in step S1 is passed through an ultrafiltration membrane, and the permeate is concentrated through a reverse osmosis membrane; S3, extraction: the concentrated liquid obtained in step S2 is extracted with an organic solvent to obtain an extract phase; S4, organic solvent evaporation; S5, two-step molecular distillation: the spermidine material after solvent evaporation in step S4 is separated by two-step molecular distillation. The first step of molecular distillation removes low-boiling-point free amines and putrescine, and the second step of molecular distillation re-extracts the heavy components from the first step to obtain pure spermidine. The purification method of the present invention is simple and has high spermidine purification efficiency. The resulting product has a purity of >98% and a yield of >80%.

Description

Technical Field

[0001] The invention relates to a method for separating and extracting spermidine from fermentation liquid, and belongs to the field of biochemical engineering. Background Art

[0002] Spermidine (Spd), also known as spermidine, has the chemical name N-(3-aminopropyl)-1,4-butanediamine. It is a polyamine biosynthesized from putrescine and adenosylmethionine.

[0003] Spermidine intake has cardiovascular protective effects. Previous studies have reported that oral administration of spermidine to mice significantly increases spermidine levels in whole blood, serum, and tissues, prolongs lifespan, reduces cardiac hypertrophy, and exerts cardioprotective effects. In a high-fat diet-induced obese mouse model, spermidine inhibits lipid accumulation in atherosclerotic plaques and reduces the formation of a necrotic core by stimulating cholesterol efflux from vascular smooth muscle cells. In a study by Ma et al., spermidine alleviated high-fat diet-induced hepatic steatosis in mice by regulating lipid metabolism and enhancing antioxidant capacity. Furthermore, it reduced adipose tissue inflammation by inhibiting the expression of inflammatory cytokines and chemokines. Furthermore, exogenous spermidine supplementation can induce enhanced autophagy, extending lifespan in yeast, fruit flies, and mice, and reducing oxidative stress in yeast and mice. Short-term spermidine supplementation in aged mice can restore basal levels of autophagy, alleviate mitochondrial dysfunction and oxidative stress, reverse the regenerative capacity of aged muscle satellite cells, and prevent aging. In the nervous system, oral supplementation of spermidine to the experimental autoimmune encephalomyelitis (EVE) mouse model can slow down the progression of EVE and alleviate the typical demyelinating lesions and inflammatory responses of EVE mice.

[0004] Spermidine is a high-boiling substance that is prone to decomposition or oxidation at high temperatures. Currently, industrial production mainly uses distillation to purify spermidine. However, due to the long heating time, spermidine is prone to oxidation reactions, resulting in low product purity and high production costs.

[0005] The main method for preparing spermidine is chemical synthesis. CN109096122B discloses a method for synthesizing spermidine. This invention uses aminopropanol and butyrolactone as raw materials, and then undergoes reduction, amino protection, and other key process steps to produce spermidine with a purity exceeding 98% and an overall yield exceeding 35%. CN113735716B reacts 2-pyrrolidone with di-tert-butyl dicarbonate. The resulting product is added to a 1,3-propylenediamine solution. After the reaction is completed and post-processed, tert-butyl [3(3-aminoalanylcarbamoyl)propyl]carbamate is obtained. This is then reduced, deprotected, and further processed to obtain spermidine.

[0006] In addition to chemical synthesis, plant extraction and biological fermentation are also common methods for obtaining spermidine. CN112544888A discloses a method for preparing a natto extract rich in spermidine, wherein spermidine is extracted from freeze-dried natto powder using a supercritical carbon dioxide fluid containing aqueous ethanol as a carrier. The spermidine content in the resulting extract ranges from 1.0 mg / g to 2.5 mg / g. CN111019960A discloses an enzymatic method for preparing spermidine, comprising steps such as plasmid construction, induced expression of strains, and bacterial cell crushing. The supernatant of the crushed cells is used as a crude enzyme solution, and S-adenosylmethionine is used as a reaction substrate to obtain spermidine at a concentration of 1 g / L.

[0007] Plant extraction requires large quantities of plants, has a long growth cycle, and is difficult to cultivate, especially for rare and valuable species, which poses a serious threat to the survival of some plants. Furthermore, the chemically active nature of most plant components requires demanding storage environments, resulting in high storage costs. Compared to plant extraction, biosynthetic fermentation offers better efficiency and lower raw material costs. Compared to chemical synthesis, biofermentation is more environmentally friendly and natural, making it more readily accepted by consumers. Therefore, biofermentation technology is poised to become the mainstream method for industrial mass production of spermidine.

[0008] Spermidine has active chemical properties and a low boiling point, which makes it difficult to separate and purify spermidine. Summary of the Invention

[0009] The present invention aims to provide a method for separating and purifying spermidine with low energy consumption, low pollution, a simple process, and high purity and yield. The method utilizes molecular distillation technology, which utilizes differences in the mean free paths of molecules to separate and purify different substances. This thermal separation process operates at a pressure of 0.001 mbar. Its low evaporation temperature makes it well-suited for heat-sensitive, high-boiling-point materials.

[0010] Compared with traditional distillation, molecular distillation has the following technical advantages:

[0011] (1) Short time, which can reduce the chance of thermal decomposition; (2) Low distillation temperature, and the evaporation temperature is much lower than the boiling point of the material, which can effectively protect the separated material and is suitable for the separation of high-boiling-point and heat-sensitive materials; (3) High vacuum degree and low pressure can inhibit oxidation damage of the material; (4) The distillation liquid film is thin, and the heat transfer efficiency is high and uniform; (5) High degree of separation: no boiling and bubbling phenomenon, the target product is selectively evaporated, and other impurities are removed.

[0012] To achieve the above object, the present invention adopts the following technical solutions:

[0013] The present invention provides a method for separating and purifying spermidine from a fermentation broth, the method comprising the following steps:

[0014] (1) Fermentation broth pretreatment: The microbial fermentation broth containing spermidine is passed through an inorganic ceramic membrane to obtain a clarified liquid;

[0015] (2) Ultrafiltration and concentration: the clarified liquid obtained in step (1) is passed through an ultrafiltration membrane, the ultrafiltration permeate is collected, the permeate is concentrated through a reverse osmosis membrane, and the concentrate is collected;

[0016] (3) Extraction: extracting the concentrated solution obtained in step (2) with an organic solvent extract, allowing it to stand for stratification, discarding the aqueous layer, and collecting the organic solvent extract layer;

[0017] (4) solvent evaporation: the organic extract phase obtained in step (3) is subjected to reduced pressure distillation to remove the organic solvent in the system;

[0018] (5) Two-stage molecular distillation: The molecular distillation system is evacuated and maintained in a high vacuum state, the spermidine mixture obtained in step (4) is subjected to a first-stage molecular distillation, and the heavy component is collected. The heavy component is subjected to a second-stage molecular distillation to collect the light component, which is spermidine.

[0019] In one embodiment of the present invention, in step (1), the pore size of the ceramic membrane is 50 nm-200 nm.

[0020] In one embodiment of the present invention, in step (2), the molecular weight cutoff of the ultrafiltration membrane is 1000-5000 Daltons, and the pressure is 0.5 mPa. The purpose of the ultrafiltration membrane filtration is to remove impurities such as residual protein and residual sugar with smaller molecular weight in the fermentation broth.

[0021] In one embodiment of the present invention, in step (2), the molecular weight cut-off of the reverse osmosis membrane is 100 Daltons and the pressure is 2 mPa. The reverse osmosis membrane can remove most of the water in the feed liquid and concentrate the ultrafiltrate.

[0022] In one embodiment of the present invention, in step (3), the organic solvent is a lower alcohol or ether that can dissolve spermidine and is immiscible with water. The purpose of using an organic solvent for extraction is to transfer spermidine from the aqueous phase to the organic phase after reverse osmosis concentration in the fermentation broth, thereby achieving the purpose of preliminary separation and purification.

[0023] In one embodiment of the present invention, the organic solvent includes but is not limited to diethyl ether, petroleum ether, n-butanol, 2-butanol, ethyl acetate, methyl isobutyl ketone, etc.

[0024] In one embodiment of the present invention, the volume ratio of the organic solvent is (v / v): 10% to 200%.

[0025] In one embodiment of the present invention, the addition amount of the organic solvent is 20% to 200%.

[0026] In one embodiment of the present invention, 30-80 mL of organic solvent is used per 1 g of spermidine.

[0027] In one embodiment of the present invention, 40-50 mL of organic solvent is used per 1 g of spermidine.

[0028] In one embodiment of the present invention, in step (3), the organic solvent extraction is performed in 3 to 4 times, and the difference in the amount of organic solvent used in each extraction does not exceed 30%;

[0029] In one embodiment of the present invention, the amount of organic solvent used in each extraction is decreased.

[0030] In one embodiment of the present invention, the purpose of distilling off the solvent in the organic phase in step (4) is to completely remove the light component solvent in the system to facilitate the next separation.

[0031] In one embodiment of the present invention, in step (4), the purpose of removing the organic solvent by reduced pressure distillation is to avoid oxidative degradation of spermidine under high temperature and aerobic conditions.

[0032] In one embodiment of the present invention, in step (5), the primary molecular distillation is intended to separate low-boiling point organic impurities such as putrescine from the fermentation broth. During the primary molecular distillation, the pressure is set to 0.1-10 mbar and the distillation temperature is set to 20-40°C.

[0033] In one embodiment of the present invention, in step (5), the secondary molecular distillation is intended to separate and purify spermidine in the heavy component of the primary molecular distillation. During the secondary molecular distillation, the pressure is set to 0.01-0.1 mbar and the distillation temperature is set to 20-40°C.

[0034] In one embodiment of the present invention, in step (4), the solvent is evaporated by reduced pressure distillation with a vacuum degree of 0.1 to 500 mbar.

[0035] In one embodiment of the present invention, the distillation temperature is 20-60°C.

[0036] The present invention also provides spermidine prepared by the method.

[0037] Beneficial effects

[0038] Due to the structural characteristics of spermidine, it cannot be crystallized from solution. Therefore, it is mostly obtained in the form of spermidine trihydrochloride on the market, making it difficult to directly extract spermidine monomer. Compared with the existing technology, the spermidine purification method provided by the present invention can directly purify high-quality spermidine monomer with a yield of over 80% and a purity of over 98.5%. In addition, this method does not use toxic or harmful organic solvents, is environmentally friendly, has a simple process and is easy to operate, and has low production costs, making the purified product highly competitive in the market. This method is applicable to the purification of spermidine produced by either biocatalysis or fermentation methods. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] Figure 1 : Example 2 product liquid phase spectrogram.

[0040] Figure 2 : Example 3 product liquid phase spectrum.

[0041] Figure 3 : Example 4 product liquid phase spectrogram.

[0042] Figure 4 : Liquid phase spectrum of comparative example 1 product.

[0043] Figure 5 : Liquid phase spectrum of comparative example 2 product.

[0044] Figure 6 : Liquid phase spectrum of comparative example 3 product. DETAILED DESCRIPTION

[0045] The present invention will be described in more detail below with reference to specific embodiments. The E. coli involved in the following examples is E. coli BWΔGΔD-DEFK-A, the construction method of which is described in the Chinese invention patent application publication number CN114874962A.

[0046] Unless otherwise specified, the raw materials or chemical reagents used in the examples of the present invention were obtained through conventional commercial channels. In the examples of the present invention, the content of spermidine was detected by high performance liquid chromatography (HPLC) external standard method.

[0047] It should be noted that the experimental method of the present invention is effective for the purification of spermidine in spermidine fermentation broth, and is not only effective for the purification of spermidine in spermidine fermentation broth obtained by the method of the present invention. The following is merely an example to better illustrate the technical effects of the present invention.

[0048] The detection methods involved in the following embodiments are:

[0049] Detection method of spermidine and putrescine content

[0050] HPLC detection pre-column derivatization method:

[0051] Add 750 μL of 0.4 mol / L perchloric acid to 250 μL of fermentation broth. After 15 minutes, shake and centrifuge for 1 minute. Take 250 μL of supernatant and add 100 μL of internal standard solution (100 mg / L 1,7-diaminoheptane), 75 μL of saturated NaHCO3, 25 μL of 2 mol / L NaOH, and 500 μL of 5 g / L dansyl chloride in sequence. Derivatize at 50°C in the dark for 45 minutes. After derivatization, add 25 μL of concentrated ammonia water to terminate the reaction. React at 50°C in the dark for 15 minutes. After the reaction, 500 μL of acetonitrile was added and mixed, and the mixture was centrifuged for 1 min. 500 μL of the supernatant was mixed with 500 μL of acetonitrile and centrifuged for 3 min. The supernatant was filtered through a 0.22 μm organic filter membrane, and the spermidine and putrescine contents in the sample were detected by high performance liquid chromatography. The putrescine peak time was about 3.5 min, and the spermidine peak time was about 12.4 min.

[0052] Example 1: Preparation of spermidine fermentation broth

[0053] The spermidine fermentation broth is obtained by biosynthesis of Escherichia coli BWΔGΔD-DEFK-A, wherein the content of spermidine in the spermidine fermentation broth is 2.08 g / L;

[0054] The specific method for the preparation of spermidine fermentation liquid is as follows:

[0055] (1) Preparation of fermentation medium

[0056] Yeast extract 2.8g / L, peptone 7g / L, diammonium phosphate 5g / L, potassium dihydrogen phosphate 15g / L, citric acid 1.9g / L, trace elements 100x 10ml / L;

[0057] Trace element solution 100x: FeSO4·7H2O 10g / L, ZnSO4·7H2O 2.25g / L, CuSO4·5H2O 1g / L, MnSO4·5H2O 0.5g / L, Na2B4O7·10H2O 0.23g / L, CaCl2·2H2O 2g / L and (NH4)6Mo7O240.1g / L.

[0058] (2) Preparation of seed solution

[0059] Escherichia coli BWΔGΔD-DEFK-A was added to the seed culture medium and cultured in a shake flask at 37°C for 24 hours to prepare a seed solution;

[0060] Seed culture medium composition (1 L): tryptone 1%, yeast extract 0.5%, sodium chloride 10g, trace element solution 100x

[0061] 10 mL, dilute to 1 L with purified water, pH 6.8, and sterilize at 116°C for 30 minutes.

[0062] (3) Preparation of spermidine fermentation broth

[0063] Add fermentation medium and 0.2 ml / L of 50% defoamer to the fermentor, adjust the pH to about 6.8, adjust the speed to 300 rpm, inoculate the seed liquid obtained in step (2) into the above fermentation medium, the inoculation amount is 10% (v / v), the temperature of the bacterial culture stage is set to 37°C, and 50% ammonia water is added to control the pH value to 7.0. During this period, glucose (concentration below 1%) is added until the yield no longer increases. The total reaction time is 32 h.

[0064] Finally, a spermidine fermentation broth with a spermidine content of 2.08 g / L was obtained.

[0065] Example 2: Purification of spermidine

[0066] The specific steps are as follows:

[0067] (1) Fermentation broth pretreatment

[0068] Pour 25 L of spermidine fermentation broth prepared in Example 1 into a ceramic membrane circulation tank, control the membrane pressure at about 0.2 mPa, and the temperature at 35°C ± 5°C. Remove insoluble matter such as bacterial residue from the fermentation broth through the interception effect of the ceramic membrane with a pore size of 50 nm to obtain a ceramic membrane clarified liquid.

[0069] (2) Ultrafiltration and concentration

[0070] The ceramic membrane clarified liquid obtained in step (1) is passed through an ultrafiltration membrane with a molecular weight cut-off of 3000 Daltons at a pressure of 0.6 mPa, and the permeate is collected to obtain an ultrafiltrate;

[0071] The ultrafiltrate was pumped into the upstream tank of the reverse osmosis membrane (molecular weight cut-off of 100 Daltons), and the reverse osmosis membrane equipment was started. The membrane pressure was controlled at about 1.5 mPa and the temperature was controlled at 30°C ± 5°C. Most of the water was removed to obtain the reverse osmosis membrane concentrate.

[0072] (3) Extraction

[0073] The content of spermidine in the reverse osmosis membrane concentrate was detected to be about 53.6 g; 3.22 L of ethyl acetate was added to the reverse osmosis membrane concentrate obtained in step (2) for extraction according to the addition amount of 60 mL of ethyl acetate per 1 g of spermidine; the mixture was allowed to stand for 30 minutes; the aqueous layer and the organic layer were fully separated, the organic solvent layer was collected, and a total of 3 extractions were performed, and the organic solvent layers obtained each time were combined to obtain an ethyl acetate extract phase.

[0074] (4) Evaporation of solvent

[0075] The ethyl acetate extract of the organic solvent layer collected in step (3) was transferred to a vacuum distillation apparatus and subjected to vacuum distillation at 38° C. and 100 mbar to evaporate the ethyl acetate and collect the remaining spermidine mixture in the evaporator.

[0076] (5) Two-stage molecular distillation

[0077] First-stage molecular distillation: The spermidine mixture obtained in step (4) is transferred to a molecular distillation preheating system, the molecular distillation system is evacuated and maintained in a high vacuum state, and the spermidine mixture in the preheater is molecularly distilled at a molecular distillation pressure of 0.9 mbar and a distillation temperature of 38° C.; and the heavy components are collected.

[0078] Secondary molecular distillation: The heavy components obtained after the primary molecular distillation are collected and transferred back to the molecular distillation preheating system. The molecular distillation system is evacuated and maintained in a high vacuum state. The spermidine mixture in the preheater is molecularly distilled at a molecular distillation pressure of 0.1 mbar and a distillation temperature of 38°C. The light components, namely spermidine, are collected.

[0079] The results show:

[0080] HPLC external standard method ( Figure 1 ), the purity of spermidine in the spermidine obtained in the embodiment of the present invention is 93.56%, and the total recovery rate of spermidine relative to the fermentation broth is 72.88%.

[0081] Example 3: Purification of spermidine

[0082] (1) Fermentation broth pretreatment

[0083] Pour 25 L of spermidine fermentation broth prepared in Example 1 into a ceramic membrane circulation tank, control the membrane pressure at about 0.2 mPa, and the temperature at 35°C ± 5°C. Remove insoluble matter such as bacterial residue from the fermentation broth through the interception effect of the ceramic membrane with a pore size of 50 nm to obtain a ceramic membrane clarified liquid.

[0084] (2) Ultrafiltration and concentration

[0085] The ceramic membrane clarified liquid obtained in step (1) is passed through an ultrafiltration membrane with a molecular weight cut-off of 3000 Daltons at a pressure of 0.6 mPa, and the permeate is collected to obtain an ultrafiltrate;

[0086] The ultrafiltrate was pumped into the upstream tank of the reverse osmosis membrane (molecular weight cut-off of 100 Daltons), and the reverse osmosis membrane equipment was started. The membrane pressure was controlled at about 1.5 mPa and the temperature was controlled at 30°C ± 5°C. Most of the water was removed to obtain the reverse osmosis membrane concentrate.

[0087] (3) Extraction

[0088] The content of spermidine in the reverse osmosis membrane concentrate was detected to be approximately 49.5 g; 3.96 L of ether was added to the reverse osmosis membrane concentrate obtained in step (2) for extraction, using 80 mL of ether for every 1 g of spermidine; the mixture was allowed to stand for 30 min; the aqueous layer and the organic layer were fully separated, the organic solvent layer was collected, and extraction was performed three times in total. The organic solvent layers obtained each time were combined to obtain an ether extract phase.

[0089] (4) Evaporation of solvent

[0090] The ether extract phase of the organic solvent layer collected in step (3) was transferred to a vacuum distillation apparatus and subjected to vacuum distillation at 35° C. and normal pressure to evaporate the ether and collect the remaining spermidine mixture in the evaporator.

[0091] (5) Molecular distillation

[0092] First-stage molecular distillation: The spermidine mixture obtained in step (4) is transferred to a molecular distillation preheating system, the molecular distillation system is evacuated and maintained in a high vacuum state, and the spermidine mixture in the preheater is molecularly distilled at a molecular distillation pressure of 0.9 mbar and a distillation temperature of 38° C.; and the heavy components are collected.

[0093] Secondary molecular distillation: The heavy components obtained after the primary molecular distillation are collected and transferred back to the molecular distillation preheating system. The molecular distillation system is evacuated and maintained in a high vacuum state. The spermidine mixture in the preheater is molecularly distilled at a molecular distillation pressure of 0.1 mbar and a distillation temperature of 38°C. The light components, namely spermidine, are collected.

[0094] The results show:

[0095] HPLC external standard method ( Figure 2 ), the purity of spermidine in the spermidine obtained in the embodiment of the present invention is 97.67%, and the total recovery rate of spermidine relative to the fermentation broth is 80.74%.

[0096] Example 4: Purification of spermidine

[0097] (1) Fermentation broth pretreatment

[0098] Pour 25 L of spermidine fermentation broth prepared in Example 1 into a ceramic membrane circulation tank, control the membrane pressure at about 0.2 mPa, and the temperature at 35°C ± 5°C. Remove insoluble matter such as bacterial residue from the fermentation broth through the interception effect of the ceramic membrane with a pore size of 50 nm to obtain a ceramic membrane clarified liquid.

[0099] (2) Ultrafiltration and concentration

[0100] The ceramic membrane clarified liquid obtained in step (1) is passed through an ultrafiltration membrane with a molecular weight cut-off of 3000 Daltons at a pressure of 0.6 mPa, and the permeate is collected to obtain an ultrafiltrate;

[0101] The ultrafiltrate was pumped into the upstream tank of the reverse osmosis membrane (molecular weight cut-off of 100 Daltons), and the reverse osmosis membrane equipment was started. The membrane pressure was controlled at about 1.5 mPa and the temperature was controlled at 30°C ± 5°C. Most of the water was removed to obtain the reverse osmosis membrane concentrate.

[0102] (3) Extraction

[0103] The content of spermidine in the reverse osmosis membrane concentrate was detected to be approximately 51.5 g; 4.12 L of ether was added to the reverse osmosis membrane concentrate obtained in step (2) for extraction, using 80 mL of ether for every 1 g of spermidine; the mixture was allowed to stand for 30 min; the aqueous layer and the organic layer were fully separated, the organic solvent layer was collected, and extraction was performed three times in total. The organic solvent layers obtained each time were combined to obtain an ether extract phase.

[0104] (4) Evaporation of solvent

[0105] The ether extract phase of the organic solvent layer collected in step (3) was transferred to a vacuum distillation apparatus and subjected to vacuum distillation at 30° C. and 300 mbar to quickly distill off the ether and collect the remaining spermidine mixture in the evaporator.

[0106] (5) Molecular distillation

[0107] First-stage molecular distillation: The spermidine mixture obtained in step (4) is transferred to a molecular distillation preheating system, the molecular distillation system is evacuated and maintained in a high vacuum state, and the spermidine mixture in the preheater is molecularly distilled at a molecular distillation pressure of 0.9 mbar and a distillation temperature of 38° C.; and the heavy components are collected.

[0108] Secondary molecular distillation: The heavy components obtained after the primary molecular distillation are collected and transferred back to the molecular distillation preheating system. The molecular distillation system is evacuated and maintained in a high vacuum state. The spermidine mixture in the preheater is molecularly distilled at a molecular distillation pressure of 0.02 mbar and a distillation temperature of 30°C. The light components, namely spermidine, are collected.

[0109] The results show:

[0110] HPLC external standard method ( Figure 3 ), the purity of spermidine in the spermidine obtained in the embodiment of the present invention is 98.67%, and the total recovery rate of spermidine relative to the fermentation broth is 83.24%.

[0111] Comparative Example 1

[0112] The specific implementation method is the same as that of Example 2, except that the molecular distillation in step (5) is changed to vacuum distillation. The conditions of the vacuum distillation are: vacuum degree 0.5 mbar, number of plates 15, glass spring packing, reflux ratio 3, kettle temperature 80°C, and the second fraction is collected at a temperature of about 67°C.

[0113] The results showed that: the HPLC external standard method ( Figure 4 ), the content of spermidine in the spermidine obtained in the comparative example of the present invention is 85.63%, and the total recovery rate of spermidine relative to the fermentation broth is 31.24%.

[0114] Compared with molecular distillation, vacuum distillation has very high requirements for evaporation rate. High evaporation rate will increase the requirement for system temperature. However, spermidine is unstable at high temperature, resulting in low overall yield and quality.

[0115] Comparative Example 2

[0116] The specific implementation method is the same as that of Example 2, except that the organic solvent in step (3) is adjusted to n-butanol.

[0117] Specifically, the extraction agent in step (3) of Example 2 is replaced by n-butanol from ethyl acetate, and the amount of the extraction agent after replacement also needs to be adjusted accordingly to ensure that the extraction yield is equivalent. Step (3) is specifically as follows:

[0118] The content of spermidine in the reverse osmosis membrane concentrate was detected to be approximately 53.2 g; 3.46 L of n-butanol was added to the reverse osmosis membrane concentrate obtained in step (2) for extraction, using 65 mL of n-butanol per 1 g of spermidine; the mixture was allowed to stand for 30 min; the aqueous layer and the organic layer were fully separated, the organic solvent layer was collected, and extraction was performed three times in total. The organic solvent layers obtained each time were combined to obtain the n-butanol extract phase.

[0119] In addition, due to the solvent adjustment, the evaporation conditions are also adjusted accordingly according to the different properties of the solvent. Specifically, step (4) of evaporating the solvent is as follows: the n-butanol extraction phase of the organic solvent layer collected in step (3) is transferred to a vacuum distillation apparatus, and vacuum distilled at 50° C. and 40 mbar to evaporate the n-butanol, and the remaining spermidine mixture in the evaporator is collected.

[0120] The results showed that, when other conditions remained unchanged, the final HPLC external standard method ( Figure 5 ), the content of spermidine in the spermidine obtained in the comparative example of the present invention is 96.3%, and the total recovery rate of spermidine relative to the fermentation broth is 63.24%.

[0121] This indicates that after changing to a solvent with a high boiling point, the evaporation temperature will be higher and the evaporation time will be prolonged. Under high-temperature and long-term evaporation conditions, spermidine will be carried away by the solvent and lost on the one hand, and on the other hand, high temperature will also cause spermidine to degrade, which will affect the overall extraction yield.

[0122] Comparative Example 3

[0123] The specific implementation method is the same as that of Example 4, except that the molecular distillation conditions of step (5) are changed from two-step molecular distillation to one-step molecular distillation; step (5) is specifically as follows:

[0124] One-step molecular distillation: the spermidine mixture obtained in step (4) is transferred to a molecular distillation preheating system, the molecular distillation system is evacuated and maintained in a high vacuum state, and the spermidine mixture in the preheater is molecularly distilled at a molecular distillation pressure of 0.02 mbar and a distillation temperature of 30° C.; and the light fraction is collected.

[0125] The results showed that: when other conditions remained unchanged, the final HPLC external standard method was used to detect ( Figure 6 ), the content of spermidine in the spermidine obtained in the comparative example of the present invention is 58.5%, and the total recovery rate of spermidine relative to the fermentation broth is 87.24%.

[0126] This shows that although the conditions of single-step molecular distillation are simpler and the yield is higher, the light component impurities are not removed and the purity is very low.

[0127] Although the present invention has been disclosed above in terms of preferred embodiments, it is not intended to limit the present invention. Anyone familiar with this technology can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be based on the definition of the claims.

Claims

1. A method for separating and purifying spermidine from a fermentation broth, characterized in that: The method comprises the following steps: (1) Fermentation broth pretreatment: The microbial fermentation broth containing spermidine is filtered using an inorganic ceramic membrane to obtain a clarified liquid; (2) Ultrafiltration and concentration: The clarified liquid obtained in step (1) is filtered using an ultrafiltration membrane, the permeate is collected, and the permeate is concentrated through a reverse osmosis membrane to obtain a concentrated liquid; (3) Extraction: The concentrated solution obtained in step (2) is extracted with an organic solvent, allowed to stand for stratification, the aqueous layer is discarded, and the organic solvent extraction layer is collected; (4) Distilling the solvent: subjecting the organic extract phase obtained in step (3) to low-temperature, reduced-pressure distillation to remove the organic solvent in the system; the organic solvent is ether or ethyl acetate; the distillation temperature is 20-60°C; (5) Molecular distillation: First-stage molecular distillation: the material after solvent removal obtained in step (4) is transferred to a molecular distillation preheating container, and the molecular distillation system is evacuated and maintained in a high vacuum state, the material in the preheating container is molecularly distilled and the heavy components are collected; the pressure of the first-stage molecular distillation is set to 0.1~10 mbar, and the distillation temperature is set to 20~40℃; Secondary molecular distillation: The heavy components obtained after the primary molecular distillation are collected and transferred back to the molecular distillation preheating container. The molecular distillation system is evacuated and maintained in a high vacuum state. The spermidine mixture in the preheating container is molecularly distilled. The pressure of the secondary molecular distillation is set to 0.01~0.1 mbar and the distillation temperature is set to 20~40℃; and the light component, namely spermidine, is collected.

2. The method for separating and purifying spermidine from a fermentation broth according to claim 1, wherein: In step (1), the pore size of the inorganic ceramic membrane is 50 nm-200 nm.

3. The method for separating and purifying spermidine from a fermentation broth according to claim 1, wherein: In step (2), the molecular weight cut-off of the ultrafiltration membrane is 1000-5000 Daltons.

4. The method for separating and purifying spermidine from a fermentation broth according to claim 1, wherein: Use 30-80 mL of organic solvent for every 1 g of spermidine.

5. The method for separating and purifying spermidine from a fermentation broth according to claim 1, wherein: In step (4), the solvent is evaporated by reduced pressure distillation with a vacuum degree of 0.1-500 mbar.

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