A method for extracting high-purity sphingomyelin from egg yolk powder

Abstract

The present invention relates to the field of natural drug extraction and separation, and discloses a method for extracting high-purity sphingomyelin from egg yolk powder, comprising the following steps: A, de-oiled egg yolk powder is subjected to alcohol extraction, filter residue is filtered, filtrate is combined, and de-oiled egg yolk powder alcohol extract is obtained by concentration and drying; B, the de-oiled egg yolk powder alcohol extract is completely dissolved, silica gel column chromatography is performed, and a post-separation chromatography column is left; C, the post-separation chromatography column is eluted using an alkane-short-chain alcohol system, eluent is combined, and concentrated and dried to obtain a sphingomyelin crude product; D, the sphingomyelin crude product is completely dissolved, secondary silica gel column chromatography is performed, eluted using an alkane-short-chain alcohol-water system, eluent is collected, concentrated and freeze-dried, and a sphingomyelin product is obtained. The present invention realizes that while being able to obtain high-purity sphingomyelin, egg yolk lecithin can be obtained, the utilization rate of raw materials is improved, and the production cost of sphingomyelin is greatly reduced.

Description

Technical Field

[0001] The invention relates to the field of natural drug extraction and separation, and in particular to a method for extracting high-purity sphingomyelin from egg yolk powder. Background Art

[0002] Sphingomyelin (SM) is a sphingolipid composed of phosphocholine (or phosphoethanolamine) connected to the C-1 hydroxyl group of ceramide. It is one of the bioactive lipids in human milk and is composed of sphingosine, fatty acids, phosphoric acid and nitrogenous bases. Sphingomyelin is the most abundant phospholipid in human milk, accounting for 29.7%-45.5% of the total phospholipids. It has a high proportion in the milk fat globule membrane and has many benefits for human health. It is a very important component of the phospholipid part of milk. It is reported that supplementing sphingomyelin can prevent diseases and maintain good health. Phospholipids and sphingomyelin play an important role in infant nutrition. As a nutritional carrier of long-chain polyunsaturated fatty acids and sphingomyelin, phospholipids have a profound impact on the improvement of cognitive function. Some studies have shown that sphingomyelin has a potential anti-cancer effect, and its two major metabolites, ceramide and sphingosine, are involved in physiological activities such as cell growth, differentiation, and apoptosis in the body. Sphingomyelin has the above-mentioned benefits for human health, which makes sphingomyelin have a good application prospect.

[0003] SM from natural sources mostly comes from animal brains, eggs and other tissues, and its content in animal tissues is relatively low. Therefore, the cost of separating and refining SM from natural sources alone is high, and the yield is too low. In addition, there are many impurities in the phospholipids from animal sources. In the process of extracting egg yolk lecithin from natural sources, for example, sphingomyelin (SM), lysophosphatidylcholine (LPC), lysophosphatidylethanolamine (LPE), phosphatidylinositol (PI) and other components have similar polarity and are difficult to separate. Therefore, from searching the published technical reports, it can be found that there is no report on the extraction process of high-purity sphingomyelin, only reports on enriching SM-rich components to increase the SM content, and no technical reports on the separation and preparation of pure SM from the perspective of pharmaceutical excipients and impurity separation. Therefore, in order to obtain SM that can meet the requirements of pharmaceutical use, the technical invention scheme starts from egg yolk powder rich in SM, and obtains SM with a purity of more than 98% through a series of technical means.

[0004] SM obtained by extraction from egg yolk powder and SM obtained by enzymatic hydrolysis can better guarantee the natural fatty acid state of SM. Due to the uncertainty of hydrolysis, enzymatic hydrolysis will inevitably change the natural state of SM. It is precisely because the content of SM from natural sources is very low that the SM used in the impurity inspection of medicinal phospholipid products is currently obtained by enzymatic hydrolysis. However, it is unreasonable to use SM obtained by enzymatic hydrolysis as a reference for SM detection in natural products, and there are often a series of problems such as large fluctuations in test results and inaccurate content determination. The best detection method is to use SM from natural sources as a reference, but the content of SM from natural sources is very low, and it is difficult to extract and separate. There has long been a lack of a practical acquisition method. If there is no complete set of core technology, simply extracting SM will inevitably make its extraction cost high and cause adverse effects on the environment due to the large consumption of raw materials. Summary of the invention

[0005] The purpose of the present invention is to solve the problem that the prior art lacks a mature method for obtaining natural sphingomyelin.

[0006] In order to solve the above technical problems, the present invention adopts the following technical solutions:

[0007] A method for extracting high-purity sphingomyelin from egg yolk powder comprises the following steps:

[0008] A. extracting the de-oiled egg yolk powder with alcohol, filtering the residue, combining the filtrate, concentrating and drying to obtain the de-oiled egg yolk powder alcohol extract;

[0009] B. Completely dissolving the de-oiled egg yolk powder alcohol extract, performing silica gel column chromatography, eluting and separating with a chloroform-ethanol system, and leaving a chromatography column after separation;

[0010] C. eluting the separated chromatography column with an alkane-short-chain alcohol system, combining the eluates, concentrating and drying to obtain a crude sphingomyelin;

[0011] D. Completely dissolving the crude sphingomyelin product, performing secondary silica gel column chromatography, using an alkane-short-chain alcohol-water system for elution, collecting the eluate, concentrating and freeze-drying to obtain a sphingomyelin product.

[0012] Preferably, in step D, the alkane-short-chain alcohol-water system comprises: alkane, short-chain alcohol, and water are mixed in a volume ratio of 3:5.85-5.95:0.05-0.15.

[0013] Preferably, in step D, the crude sphingomyelin is completely dissolved in chloroform, the crude sphingomyelin and silica gel are filled in a mass ratio of 1:1-5, and the column pressure is 0.5-3.0 mPa.

[0014] Preferably, in step C, the alkane-short-chain alcohol system comprises: alkanes and short-chain alcohols mixed in a volume ratio of 3:1-5.

[0015] Preferably, in step B, the de-oiled egg yolk powder alcohol extract is completely dissolved with alkane, and the de-oiled egg yolk powder alcohol extract is mixed with silica gel in a mass ratio of 1:1 to 5, and the silica gel column chromatography is performed with a diameter-to-height ratio of 1:1 to 1:20, and elution is performed with an alkane-short-chain alcohol system, and the alkane-short-chain alcohol system includes: alkanes and short-chain alcohols are mixed in a volume ratio of 3:0.1 to 1.5, the column pressure is -0.1 to 3.0 mPa, and the column temperature is 10 to 60°C.

[0016] In this step, PC is eluted and separated by silica gel column chromatography. In the process of silica gel column chromatography, the elution effect of pure alkane alone is not good. The polarity is increased by adding a proper proportion of short-chain alcohol, but SM and LPC must not be eluted. Therefore, the eluent of this step needs to control the volume ratio of alkane to short-chain alcohol between 3:0.5 and 3:1.5. Preferably, 3:1 is the best condition for eluting PC.

[0017] Preferably, in step B, the de-oiled egg yolk powder alcohol extract is adsorbed by alumina before being subjected to the silica gel column chromatography separation, specifically: the de-oiled egg yolk powder alcohol extract is completely dissolved with a short-chain alcohol, the de-oiled egg yolk powder alcohol extract and the alumina are stirred at a mass ratio of 1:0.2-1, adsorbed for 1-10 hours, filtered, concentrated, dried, and then dissolved again, and subjected to the silica gel column chromatography separation; the chromatographic liquid obtained by the silica gel column chromatography separation is collected, concentrated, decolorized with activated carbon, filtered, concentrated, refined, and freeze-dried to obtain an egg yolk lecithin product; the egg yolk lecithin content in the egg yolk lecithin product is greater than 70%.

[0018] In this step, a large amount of phosphatidylethanolamine (PE) is removed by adding alumina and stirring and adsorbing, while sphingomyelin (SM) and egg yolk phosphatidylcholine (PC) are enriched, ensuring that a higher purity egg yolk phosphatidylcholine product is obtained.

[0019] Preferably, the alkane includes one or more of chloroform, dichloromethane, n-heptane, and n-octane, and the short-chain alcohol includes one or more of methanol, ethanol, n-butanol, and isopropanol.

[0020] In the alkane-short-chain alcohol-water system or alkane-short-chain alcohol system used for elution, the longer the carbon chain of the alcohol, the smaller the polarity, and the smaller the selectivity of the eluent, which can easily make the target sample unable to be eluted or incompletely eluted. At the same time, alcohols with too long carbon chains will be accompanied by strong pungent odors and are not suitable for use. The length of short-chain alcohols is generally controlled within 5 carbons.

[0021] Preferably, in step A, the alcohol used for the alcohol extraction includes one or more of methanol, ethanol and their aqueous solutions; the mass ratio of the de-oiled egg yolk powder to the alcohol is 1:5-15, the number of extractions is 2-6 times, the extraction time is 1-8 hours, and the extraction temperature is 15-50°C.

[0022] Preferably, in step A, the deoiled egg yolk powder is obtained by deoiling the egg yolk powder, and the deoiling includes one of an acetone deoiling method and a supercritical method; when the acetone deoiling method is applied, the mass ratio of the egg yolk powder to acetone is 1:1-10, the deoiling times are 1-5 times, and the deoiling temperature is 20-45°C. After deoiling, the acetone insoluble matter is combined and dried to obtain the deoiled egg yolk powder; when the supercritical method is applied, the egg yolk powder is placed in an extraction kettle for continuous extraction, the temperature is maintained at 25-45°C, the CO2 flow rate is controlled at 50-120kg / h, the extraction kettle pressure is 10-25Mpa, the separation kettle 1 pressure is 5-20Mpa, and the separation kettle 2 pressure is 5-15Mpa, which is continued for 4-10h to obtain the deoiled egg yolk powder.

[0023] A sphingomyelin product obtained by the method for extracting high-purity sphingomyelin from egg yolk powder as described above, wherein the purity of the sphingomyelin product is higher than 98%.

[0024] A sphingomyelin (SM) and egg yolk phosphatidylcholine (PC) product obtained by the method for extracting high-purity sphingomyelin from egg yolk powder as described above.

[0025] Compared with the prior art, the present invention has the following beneficial effects:

[0026] Therefore, the technical solution of the present invention uses egg yolk powder rich in SM as the starting material. Through the technical solution of the present invention, not only high-purity SM with a purity higher than 98% can be obtained, but also egg yolk lecithin with a PC content greater than 70% can be obtained, which greatly reduces the extraction cost of SM. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 The process route diagram of sphingomyelin extraction of the technical solution of the present invention;

[0028] Figure 2 This is a comparison chart of the effect of the second-order eluent on the purity of SM crude product and SM finished product. DETAILED DESCRIPTION

[0029] In order to make the purpose, technical scheme and advantages of the present invention clearer, the present invention will be further described in detail below in conjunction with the accompanying drawings so that those skilled in the art can better understand the present invention and implement it, but the examples given are not intended to limit the present invention. Without departing from the spirit and essence of the present invention, modifications or replacements made to the method, steps or conditions of the present invention all belong to the scope of the present invention. Unless otherwise specified, the technical means used in the embodiments are conventional means well known to those skilled in the art.

[0030] Example 1

[0031] like Figure 1 As shown, 1 kg of egg yolk powder was taken, 10 L of acetone was added for deoiling, and deoiling was performed once at 20 ° C, filtered, and dried to obtain 732 g of deoiled egg yolk powder. Then 10 L of 95% ethanol was used for alcohol extraction, the extraction temperature was 15 ° C, the extraction time was 1 h, the filter residue was filtered, the extraction was repeated 2 times, the filtrate was combined, and the deoiled egg yolk powder alcohol extract 484 g was obtained. Ethanol was added to dissolve to prepare a raw material solution, and 96.8 g of aluminum oxide was added for stirring and adsorption for 1 h. Then, after filtration, concentration and drying, the product was dissolved again in chloroform and filled with 2.42 kg of silica gel for column chromatography (diameter-to-height ratio of 1:1). The column temperature was 10°C, the first eluent was chloroform-ethanol (3:0.1), and the column pressure during the elution process was 1.0 mPa until the egg yolk phosphatidylcholine (PC) was completely eluted. The second eluent was then replaced with chloroform-ethanol (3:2) with a column pressure of 1.1 mPa. Ten column volumes were eluted, the eluates were combined, concentrated and dried to obtain 6.16 g of crude sphingomyelin with a sphingomyelin purity of 95.1%. The crude sphingomyelin was dissolved in chloroform to prepare a raw material solution, and 20g of silica gel was filled for column chromatography (diameter-to-height ratio of 1:2), and the eluent was chloroform-ethanol-water (3:1.85:0.15). The column pressure during the elution process was 0.5mPa, until the sphingomyelin was completely eluted, and the eluent containing sphingomyelin was combined, concentrated and dried to obtain 5.62g of sphingomyelin with a purity of 99.8%. 0.3% (based on solid content) of activated carbon was added to the PC eluent for decolorization and adsorption. After complete elution, it was filtered, concentrated, refined, and freeze-dried to obtain refined egg yolk lecithin with a PC content of 74.6%.

[0032] Example 2

[0033] like Figure 1As shown, 250g of egg yolk powder is taken, 1500ml of acetone is added for deoiling, and the oil is repeatedly degreased 3 times at 30°C, filtered, and dried to obtain 162.85g of deoiled egg yolk powder. Then 1950ml of 95% ethanol is used for alcohol extraction, the extraction temperature is 25°C, the extraction time is 4h, the filter residue is filtered, the extraction is repeated 3 times, the filtrate is combined, concentrated and dried, and 123.95g of deoiled egg yolk powder alcohol extract is obtained. Methanol is added to dissolve and prepare the raw material liquid, and 62g of aluminum oxide is added for stirring and adsorption for 3h. Then filter, concentrate and dry, and dissolve it again with dichloromethane to obtain a concentrated solution. Fill 375g of silica gel for column chromatography (diameter-to-height ratio of 1:10), the column temperature is 25°C, the first eluent is dichloromethane-methanol (3:0.5), the column pressure during the elution process is 3.0mPa, until the egg yolk phosphatidylcholine is completely eluted, then replace the second eluent with dichloromethane-methanol (3:3), the column pressure during the elution process is 2.9mPa, elute for 10 column volumes, combine the eluates, concentrate and dry to obtain 2.01g of crude sphingomyelin with a sphingomyelin purity of 88.4%. The crude sphingomyelin was dissolved in chloroform to prepare a raw material solution, and 8g of silica gel was filled for column chromatography (diameter-to-height ratio of 1:5), and the eluent was dichloromethane-ethanol-water (3:1.95:0.05). The column pressure during the elution process was 1.5mPa, until the sphingomyelin was completely eluted, and the eluent containing sphingomyelin was combined, concentrated and dried to obtain 1.74g of sphingomyelin with a purity of 99.5%. 0.1% (based on solid content) of activated carbon was added to the PC eluent for decolorization and adsorption. After complete elution, it was filtered, concentrated, refined, and freeze-dried to obtain refined egg yolk lecithin with a PC content of 76.1%.

[0034] Example 3

[0035] like Figure 1As shown, 500g of egg yolk powder was taken, 500ml of acetone was added for deoiling, and the deoiling was repeated 5 times at 45°C, filtered, and dried to obtain 371g of deoiled egg yolk powder. Then 1855ml of 95% ethanol was used for alcohol extraction, the extraction temperature was 45°C, the extraction time was 8h, the filter residue was filtered, the extraction was repeated 6 times, the filtrate was combined, concentrated and dried, and 292g of deoiled egg yolk powder alcohol extract was obtained. Isopropanol was added to dissolve and prepare a raw material solution, and 233.6g of aluminum oxide was added for stirring and adsorption for 5h. Then, the product was filtered, concentrated, dried, and dissolved again in n-heptane to obtain a concentrate, which was loaded with 292 g of silica gel for column chromatography (diameter-to-height ratio of 1:15). The column temperature was 35°C, and the first eluent was n-heptane-isopropanol (3:1). The column pressure during the elution process was 1.6 mPa until the egg yolk phosphatidylcholine was completely eluted. The second eluent was n-heptane-isopropanol (3:4). The column pressure during the elution process was 1.8 mPa. 10 column volumes were eluted, and the eluates were combined, concentrated, and dried to obtain 4.81 g of crude sphingomyelin with a sphingomyelin purity of 79.6%. The crude sphingomyelin was dissolved in chloroform to prepare a raw material solution, and 25g of silica gel was filled for column chromatography (diameter-to-height ratio of 1:3), and the eluent was chloroform-ethanol-water (3:1.90:0.10). The column pressure during the elution process was 0.9mPa until the sphingomyelin was completely eluted, and the eluent containing sphingomyelin was combined, concentrated and dried to obtain 3.66g of sphingomyelin with a purity of 99.1%. 0.5% (based on solid content) of activated carbon was added to the PC eluent for decolorization and adsorption. After complete elution, it was filtered, concentrated, refined, and freeze-dried to obtain refined egg yolk lecithin with a PC content of 72.8%.

[0036] Example 4

[0037] like Figure 1As shown, 750g of egg yolk powder is taken and added into a supercritical extraction kettle for continuous extraction. The extraction kettle temperature is 45°C, CO2 flow rate is 120kg / h, extraction kettle pressure is 25MPa, separation kettle 1 pressure is 20MPa, and separation kettle 2 pressure is 15MPa. After 4h, 355.5g of de-oiled egg yolk powder is obtained. 5330ml of 95% ethanol is used for alcohol extraction. The extraction temperature is 50°C, the extraction time is 8h, and the extraction is repeated 4 times. The filter residue is filtered and concentrated and dried to obtain 201.7g of de-oiled egg yolk powder alcohol extract. N-butyl alcohol is added to dissolve and made into a raw material solution, and 201.7g of aluminum oxide is added for stirring and adsorption for 8h. Then, after filtration, concentration and drying, the product was dissolved again in n-octane and filled with 605 g of neutral alumina for column chromatography (diameter-to-height ratio of 1:20). The column temperature was 50°C and the first eluent was n-octane-n-butanol (3:1.2). The column pressure during the elution process was 1.2 mPa until the egg yolk phosphatidylcholine was completely eluted. The second eluent was n-octane-n-butanol (3:5). The column pressure during the elution process was 1.8 mPa. 10 column volumes were eluted, the eluates were combined, concentrated and dried to obtain 7.64 g of crude sphingomyelin with a sphingomyelin purity of 74.5%. The crude sphingomyelin was dissolved in chloroform to prepare a raw material solution, and 7.64 g of silica gel was filled for column chromatography (diameter-to-height ratio of 1:2), and the eluent was chloroform-ethanol-water (3:1.85:0.15). The column pressure during the elution process was 2.0 mPa until the sphingomyelin was completely eluted, and the eluent containing sphingomyelin was combined, concentrated and dried to obtain 5.62 g of sphingomyelin with a purity of 98.5%. 0.7% (based on solid content) of activated carbon was added to the PC eluent for decolorization and adsorption. After complete elution, it was filtered, concentrated, refined, and freeze-dried to obtain refined egg yolk lecithin with a PC content of 71.4%.

[0038] Example 5

[0039] like Figure 1As shown, 1kg of egg yolk powder is taken and added into a supercritical extraction kettle for continuous extraction. The extraction kettle temperature is 25°C, CO2 flow rate is 50kg / h, the extraction kettle pressure is 10MPa, the separation kettle 1 pressure is 5MPa, and the separation kettle 2 pressure is 5MPa. After 10 hours, 676g of de-oiled egg yolk powder is obtained. 6760ml of 95% ethanol is used for alcohol extraction. The extraction temperature is 20°C, and the extraction time is 2h. The filter residue is filtered, and the extraction is repeated 3 times. The filtrate is combined and concentrated and dried to obtain 473g of de-oiled egg yolk powder alcohol extract. N-butyl alcohol is added to dissolve and become a raw material solution, and 330g of aluminum oxide is added for stirring and adsorption for 10h. Then, after filtration, concentration and drying, the product was dissolved again in chloroform and filled with 2365g of neutral alumina for column chromatography (diameter-to-height ratio of 1:8). The column temperature was 60°C, and the first eluent was chloroform-n-butanol (3:1.5). The column pressure during the elution process was 2.0mPa until the egg yolk phosphatidylcholine was completely eluted. The second eluent was replaced with chloroform-n-butanol (3:5). The column pressure during the elution process was 1.8mPa. 10 column volumes were eluted, the eluates were combined, concentrated and dried to obtain 11.26g of crude sphingomyelin with a sphingomyelin purity of 69.6%. The crude sphingomyelin was dissolved in chloroform to prepare a raw material solution, and 66g of silica gel was filled for column chromatography (diameter-to-height ratio of 1:8), and the eluent was chloroform-ethanol-water (3:1.90:0.10). The column pressure during the elution process was 3.0mPa, until the sphingomyelin was completely eluted, and the eluent containing sphingomyelin was combined, concentrated and dried to obtain 7.81g of sphingomyelin with a purity of 98.2%. 0.7% (based on solid content) of activated carbon was added to the PC eluent for decolorization and adsorption. After complete elution, it was filtered, concentrated, refined, and freeze-dried to obtain refined egg yolk lecithin with a PC content of 70.4%.

[0040] Comparative Example 1

[0041] Take 2kg of egg yolk powder and add it to a supercritical extraction kettle for continuous extraction. The extraction kettle temperature is 35°C, the CO2 flow rate is 80kg / h, the extraction kettle pressure is 15MPa, the separation kettle 1 pressure is 10MPa, and the separation kettle 2 pressure is 8MPa. Continue for 6h to obtain 1.2kg of de-oiled egg yolk powder. Use 5.5L 95% ethanol for alcohol extraction. The extraction temperature is 35°C and the extraction time is 4h. Filter the filter residue, extract repeatedly for 2 times, combine the filtrate, concentrate and dry, and obtain 848g of de-oiled egg yolk powder alcohol extract. Add ethanol to dissolve and prepare a raw material solution, add 169.6g of aluminum oxide and stir and adsorb for 1h. The product was then filtered, concentrated and dried, and then dissolved in chloroform for the second time. 4.8 kg of neutral alumina was loaded for column chromatography (diameter-to-height ratio of 1:5). The eluent was chloroform-ethanol (3:1). The column pressure during the elution process was 1.8 mPa until the egg yolk phosphatidylcholine was completely eluted. The second eluent was replaced with chloroform-ethanol (3:6). The column pressure during the elution process was 2.4 mPa. 10 column volumes were eluted, the eluates were combined, concentrated and dried to obtain 26.49 g of crude sphingomyelin with a sphingomyelin purity of 63.9%. The crude sphingomyelin was dissolved in chloroform to prepare a raw material solution, and 210g of silica gel was filled for column chromatography (diameter-to-height ratio of 1:15). The column pressure during the elution process was 2.9mPa, and the eluent was chloroform-ethanol-water (3:1.95:0.05) until the sphingomyelin was completely eluted. The eluent containing sphingomyelin was combined, concentrated and dried to obtain 16.86g of sphingomyelin with a purity of 96.7%. 1.5% (based on solid content) of activated carbon was added to the PC eluent for decolorization and adsorption. After complete elution, it was filtered, concentrated, refined, and freeze-dried to obtain refined egg yolk lecithin with a PC content of 66.5%.

[0042] Comparative Example 2

[0043] In order to fully evaluate the effect of the technology of the present invention, the key steps of eluting sphingomyelin were investigated. In the process of extracting sphingomyelin using egg yolk powder as the starting material, the first-stage elution and the second-stage elution are the key steps to ensure that the final purity of sphingomyelin can reach more than 98%. When the de-oiled egg yolk powder alcohol extract is subjected to alumina column chromatography, most of the SM and LPC are adsorbed in the alumina, and at the same time, the alumina will also adsorb a large amount of PC. In other words, after the de-oiled egg yolk powder alcohol extract is subjected to alumina column chromatography, there are mainly SM and LPC and a large amount of PC in the alumina chromatography column. Since the polarity of PC and SM is very similar, the first-stage elution needs to wash away most of the PC, and the completeness of the removal of the first-stage PC directly affects the purity of the subsequent second column chromatography. Therefore, in order to ensure the purity of the SM in the second-stage elution, chloroform-ethanol (3:1) is first used to elute all PC, and then the ethanol ratio is increased to elute SM. As the polarity increases, LPC will be eluted together with SM. Therefore, the total mass of SM and LPC, the percentage of SM, and the purity of SM after the secondary column chromatography are used as evaluation indicators to investigate the second-stage elution ratio. The implementation plan is carried out according to the technical scheme of Example 1, and the second-stage elution ratio is used as a single factor for investigation. The effects of chloroform and ethanol ratios of 3:1, 3:2, 3:3, 3:4, 3:5, 3:6, and 3:7 on the final SM purity were investigated respectively. The research results show that with the increase of polarity, LPC is gradually eluted and the proportion of SM gradually decreases. When the first-stage SM percentage is less than 68%, the secondary column chromatography cannot obtain sphingomyelin with a purity of more than 98%. This is mainly because the polarity of LPC and SM is very similar, and it is difficult to obtain high-purity SM. The results are as follows. Figure 2 shown.

[0044] The above disclosure is only the preferred embodiment of the present invention, which certainly cannot be used to limit the scope of the present invention. Therefore, equivalent changes made according to the claims of the present invention are still within the scope of the present invention.

Claims

1. A method for extracting sphingomyelin from egg yolk powder, characterized in that: The steps include: A. extracting the de-oiled egg yolk powder with alcohol, filtering the residue, combining the filtrate, concentrating and drying to obtain the de-oiled egg yolk powder alcohol extract; B. Dissolving the de-oiled egg yolk powder alcohol extract completely with an alkane, performing silica gel column chromatography separation, eluting with an n-heptane-isopropanol system or a chloroform-ethanol system or a dichloromethane-methanol system, and leaving a chromatography column after separation; the de-oiled egg yolk powder alcohol extract is subjected to alumina adsorption before the silica gel column chromatography separation; the de-oiled egg yolk powder alcohol extract is subjected to alumina adsorption before the silica gel column chromatography separation, specifically: dissolving the de-oiled egg yolk powder alcohol extract completely with a short-chain alcohol, stirring at a mass ratio of the de-oiled egg yolk powder alcohol extract to the alumina of 1:0.2-1, adsorbing for 1-10 hours, filtering, concentrating, drying, dissolving for a second time, and performing the silica gel column chromatography separation; C. eluting the separated chromatographic column with a n-heptane-isopropanol system, a chloroform-ethanol system, or a dichloromethane-methanol system, combining the eluates, concentrating and drying to obtain a crude sphingomyelin; D. completely dissolving the crude sphingomyelin product, performing secondary silica gel column chromatography, using a chloroform-ethanol-water system or a dichloromethane-ethanol-water system for elution, collecting the eluate, concentrating and freeze-drying to obtain a sphingomyelin product; the purity of the sphingomyelin product is higher than 98%; When the elution system of step B is chloroform and ethanol, they are mixed in a volume ratio of 3:0.1, the elution system of step C is chloroform and ethanol mixed in a volume ratio of 3:2, and the elution system of step D is chloroform, ethanol, and water mixed in a volume ratio of 3:1.85:0.15; When the elution system of step B is dichloromethane and methanol, they are mixed in a volume ratio of 3:0.5, the elution system of step C is a mixture of dichloromethane and methanol in a volume ratio of 3:3, and the elution system of step D is a mixture of dichloromethane, ethanol, and water in a volume ratio of 3:1.95:0.05; When the elution system of step B is n-heptane and isopropanol, they are mixed in a volume ratio of 3:1, the elution system of step C is a mixture of n-heptane and isopropanol in a volume ratio of 3:4, and the elution system of step D is a mixture of chloroform, ethanol and water in a volume ratio of 3:1.90:0.

10.

2. The method for extracting sphingomyelin from egg yolk powder as claimed in claim 1, characterized in that: In step D, the crude sphingomyelin is completely dissolved in chloroform, the crude sphingomyelin and silica gel are filled in a mass ratio of 1:1-5, and the column pressure is 0.5-3.0 mPa.

3. The method for extracting sphingomyelin from egg yolk powder as claimed in claim 1, characterized in that: In step B, the de-oiled egg yolk powder alcohol extract and silica gel are separated by silica gel column chromatography in a mass ratio of 1:1-5, with a diameter-to-height ratio of 1:1-1:20, a column pressure of -0.1-3.0 mPa, and a column temperature of 10-60°C.

4. The method for extracting sphingomyelin from egg yolk powder as claimed in claim 1, characterized in that: In step B, the chromatographic liquid obtained by the silica gel column chromatography separation is collected, concentrated, decolorized with activated carbon, filtered, concentrated, refined, and freeze-dried to obtain an egg yolk lecithin product; the egg yolk lecithin content of the egg yolk lecithin product is greater than 70%.

5. The method for extracting sphingomyelin from egg yolk powder according to any one of claims 1, 3 and 4, characterized in that: The alkane includes one or more of n-heptane and n-octane, and the short-chain alcohol includes one or more of methanol, ethanol, n-butanol, and isopropanol.

6. The method for extracting sphingomyelin from egg yolk powder as claimed in claim 1, characterized in that: In step A, the alcohol used for the alcohol extraction includes one or more of methanol, ethanol and their aqueous solutions; the mass ratio of the de-oiled egg yolk powder to the alcohol is 1:5-15, the number of extractions is 2-6 times, the extraction time is 1-8 hours, and the extraction temperature is 15-50°C.

7. The method for extracting sphingomyelin from egg yolk powder as claimed in claim 1, characterized in that: In step A, the deoiled egg yolk powder is obtained by deoiling the egg yolk powder, and the deoiling includes one of an acetone deoiling method and a supercritical method; when the acetone deoiling method is used, the mass ratio of the egg yolk powder to acetone is 1:1-10, the deoiling times are 1-5 times, and the deoiling temperature is 20-45°C. After deoiling, the acetone insoluble matter is combined and dried to obtain the deoiled egg yolk powder; when the supercritical method is used, the egg yolk powder is placed in an extraction kettle for continuous extraction, the temperature is maintained at 25-45°C, the CO2 flow rate is controlled at 50-120kg / h, the extraction kettle pressure is 10-25Mpa, the separation kettle 1 pressure is 5-20Mpa, and the separation kettle 2 pressure is 5-15Mpa, which is continued for 4-10h to obtain the deoiled egg yolk powder.

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