Krill oil extract as well as preparation method and application thereof

Abstract

The invention discloses a krill oil extract as well as a preparation method and application thereof, and particularly relates to the technical field of separation and preparation of natural products. The extraction method comprises the five steps of enzymolysis, dehydration, purification, free fatty acid removal and concentration and drying, in the krill oil extract obtained through the preparation method, a raw material rich in LPC-DHA is obtained for the first time, and the content of LPC-DHA and sn-1-LPC-DHA in the raw material prepared through the preparation method is higher; a molecular distillation method is mostly adopted for removing fatty acid industrially, the method is high in cost, an alkaline solution is added for salifying to remove free fatty acid, the production cost is reduced, and meanwhile, the good yield is kept.

Description

Technical Field

[0001] The present invention relates to the technical field of separation and preparation of natural products, and particularly relates to a krill oil extract, a preparation method thereof, and an application thereof. Background Art

[0002] Docosahexaenoic acid (DHA) is a crucial essential fatty acid in the human body and plays a vital role in maintaining the normal functions of the brain, retina, heart, liver, and other important tissues. DHA is an important component of brain cell membranes and can promote the growth and development of nerve cells; it is rich in the retina, and appropriate intake of DHA helps protect eyesight and prevent eye diseases. In addition, DHA also has the effects of reducing blood viscosity and inhibiting platelet aggregation, which is also beneficial for reducing the risk of cardiovascular and cerebrovascular diseases; DHA also has certain anti-inflammatory and immunomodulatory effects, which helps maintain the normal function of the human immune system.

[0003] DHA is mainly derived from deep-sea fish, shrimps, and algae, and in addition, it also exists in eggs. Currently, the main structural types of DHA are triglyceride type and ethyl ester type, and a small amount of DHA sources are phospholipid type. For example, DHA in Antarctic krill oil and egg yolk has a phospholipid type structure.

[0004] Triglyceride-type DHA and ethyl ester-type DHA are hydrolyzed by pancreatic lipase in the small intestine to release free DHA or monoacylglycerol, and are absorbed as triglycerides (TAG) in chylomicrons, preferentially incorporated into adipose tissue and the heart, but not into the brain and eyes. The liver then absorbs the remaining residues and incorporates DHA into cell membrane lipids or synthesizes low-density lipoproteins for transport to other tissues. For phospholipid-type DHA, most of it is hydrolyzed by phospholipase A2 into free DHA and LPC-DHA; the released free DHA undergoes the same metabolic pathway as the free DHA released from triglyceride-type DHA; and for the released LPC-DHA, sn-1-LPC-DHA can directly enter the blood, and a part of it is also reshaped by DHA absorbed by the liver in the form of phosphatidylcholine (PC) and secreted into the blood in the form of sn-2DHA LPC. Both sn-1 and sn-2 LPC-DHA can enter the brain and eyes through the Mfsd2a pathway to increase the DHA content in the brain and eyes. However, currently, naturally occurring phospholipid-type DHA is basically present at the sn-2 position, and is basically hydrolyzed by phospholipase A2 into free DHA, and only a very small part will be hydrolyzed into sn-1DHA-LPC and enter the brain and eyes.

[0005] DHA is crucial for the functions of the human brain and eyes. However, among the currently naturally occurring DHA, very little can be truly absorbed by the brain and eyes. Triglyceride DHA and ethyl ester DHA are hydrolyzed into free DHA and basically only enter the blood circulation. Due to their site relationship, phospholipid DHA is also basically hydrolyzed into free DHA and cannot enter the brain and eyes.

[0006] sn-1-LPC-DHA can directly enter the brain to supplement the DHA content in the human brain. In addition, LPC has good emulsifying properties. It can promote the digestion and absorption of some fat-soluble nutrients, and at the same time can increase the excretion of fat substances such as cholesterol and triglycerides in the blood, reduce blood lipid content, and has good benefits for human cardiovascular and cerebrovascular diseases. Summary of the Invention

[0007] The object of the present invention is to provide a krill oil extract containing DHA that can enter the brain and lysophosphatidylcholine (sn-1-LPC-DHA) rich in DHA, and its preparation method and application, aiming at the deficiencies of the existing technology.

[0008] In the first aspect, the present invention provides a krill oil extract, wherein the LPC content is 0-72%, and the sn-1-LPC-DHA content is 0-20%;

[0009] In some embodiments, the LPC content is 18-60%, preferably 35.8-48.4%; the sn-1-LPC-DHA content is 0-15%, preferably 0-10.3%.

[0010] In the second aspect, the present invention also provides a preparation method of a krill oil extract, comprising the following steps:

[0011] 1) Enzymatic hydrolysis: Mix krill oil and water to a uniform emulsion state. After preheating, add lipase and stir. After stirring, raise the temperature to inactivate the enzyme to obtain a mixed solution;

[0012] 2) Dehydration: Add an organic solvent to the mixed solution in step 1) for demulsification, and after demulsification, concentrate and dehydrate under vacuum to obtain a dehydrated product;

[0013] 3) Purification: Add acetone to the dehydrated product in step 2), stir and let stand to remove the supernatant; add acetone again, stir and let stand to remove the supernatant, and concentrate under vacuum to obtain a crude product;

[0014] 4) Removal of free fatty acids: The crude product in step 3) is dissolved in an organic solvent, and the pH is adjusted to 8 by dropping a sodium hydroxide ethanol solution and reacting. After the reaction, the pH of the solution is adjusted to 8 by dropping a sodium hydroxide ethanol solution again. After the reaction, filter to obtain a filtrate;

[0015] 5) Concentration and drying: The filtrate was concentrated under vacuum to obtain a dark red paste product.

[0016] In some embodiments, in step 1), the mass ratio of the krill oil to water is 1:1 - 45, preferably 1:1 - 30, and more preferably 1:1 - 15; the preheating temperature is 24 - 180°C, preferably 32 - 120°C, and more preferably 40 - 60°C; the mass of the added lipase accounts for 1.8 - 27% of the mass of the krill oil, preferably 2.4 - 18%, and more preferably 3 - 9%.

[0017] In some embodiments, in step 2), the organic solvent is one or a combination of methanol, ethanol, acetone, or ethyl acetate, preferably methanol, ethanol, or acetone, and more preferably ethanol; the volume of the added organic solvent is 0.6 - 18 times the mass of water in step (1), preferably 0.8 - 6 times, and more preferably 1 - 2 times.

[0018] In some embodiments, in step 3), the mass of acetone added to the dehydrated product is 3 - 30 times the mass of the krill oil, preferably 4 - 20 times, and more preferably 5 - 10 times; the mass of acetone added again is 1.2 - 45 times the mass of the krill oil, preferably 1.6 - 15 times, and more preferably 2 - 5 times.

[0019] In some embodiments, in step 4), the organic solvent is one or a combination of methanol, ethanol, acetone, or ethyl acetate, preferably methanol, ethanol, or acetone, and more preferably ethanol; the mass of the organic solvent is 0.6 - 3 times the mass of the crude product, preferably 0.8 - 2 times, and more preferably 1 time; the mass fraction of sodium hydroxide in the sodium hydroxide ethanol solution is 0 - 6%, preferably 0 - 4%, and more preferably 0 - 2%.

[0020] In the third aspect, the present invention provides a product comprising a krill oil extract or a krill oil extract prepared by the preparation method and a pharmaceutically acceptable excipient.

[0021] In some embodiments, the product can be formulated into tablets, capsules, oral liquids, or gummies.

[0022] In the fourth aspect, the present invention provides an application of a krill oil extract, a krill oil extract prepared by the preparation method of the krill oil extract, or the product in the preparation of drugs, health foods, dietary supplements, and / or foods for eye protection, anti - inflammation, and anti - cardio - cerebrovascular diseases.

[0023] Advantages of the present invention:

[0024] 1. The present invention obtains, for the first time, a raw material enriched with LPC - DHA, providing a raw material basis for the subsequent development of products.

[0025] 2. The raw materials prepared by the preparation method of the present invention have higher contents of LPC-DHA and sn-1-LPC-DHA.

[0026] 3. In industry, the molecular distillation method is mostly used to remove fatty acids. This method has a high cost. The present invention uses an alkaline solution to form salts to remove free fatty acids, which not only reduces the production cost but also maintains a good yield. Detailed implementation manners

[0027] For the convenience of understanding by those skilled in the art, the present invention will be further described below in conjunction with embodiments. The content mentioned in the implementation manners does not limit the present invention.

[0028] Example 1

[0029] (1) Mix 100 g of krill oil with 100 g of water, stir until a uniform emulsion state is obtained, preheat to 50 °C, then add lipase accounting for 3% of the mass of krill oil, react for 60 min, and after the reaction ends, raise the temperature to 90 °C to inactivate the enzyme to obtain a mixed solution;

[0030] (2) Add 95% ethanol with a volume 1-2 times that of the water in step (1) to the mixed solution in step (1) for demulsification. After demulsification, vacuum concentrate to an anhydrous state to obtain a dehydrated product;

[0031] (3) Add acetone 5 times the mass of krill oil to the dehydrated product in step (2), stir for 60 min, then cool to below 0 °C, stand for 8 h, and remove the supernatant. Then add acetone 3 times the mass of krill oil, stir for 60 min, cool to below 0 °C, stand for 3 h, remove the supernatant, and vacuum concentrate to remove the remaining acetone to obtain a crude product;

[0032] (4) Dissolve the crude product obtained in step (3) in 95% ethanol with a mass 1 time that of it, dropwise add a 1% sodium hydroxide ethanol solution until the pH of the solution reaches 6, then react for 60 min. After the reaction ends, dropwise add a 1% sodium hydroxide ethanol solution until the pH of the solution is 8. After the reaction ends, filter to remove the precipitated fatty acid salts to obtain a filtered solution;

[0033] (5) Vacuum concentrate the filtered solution in step (4) to remove ethanol, control the moisture content of the material below 2%, and the residual amounts of acetone and ethanol below 5000 ppm. Finally, 40 g of a brownish-red paste-like product is obtained, with a yield of 40%. The content of sn-1-LPC-DHA detected in the product is 6%, and the content of LPC is 35.8%.

[0034] Example 2

[0035] (1) Mix 100 g of krill oil with 500 g of water, stir until it becomes a homogeneous emulsion state, preheat to 50 °C, then add lipase accounting for 3% of the mass of krill oil, react for 60 min, after the reaction ends, raise the temperature to 90 °C to inactivate the enzyme, and obtain a mixed solution;

[0036] (2) Add 95% ethanol with a volume 1 - 2 times that of the water in step (1) to the mixed solution in step (1) for demulsification, after demulsification, vacuum concentrate to an anhydrous state to obtain a dehydrated product;

[0037] (3) Add acetone 5 times the mass of krill oil to the dehydrated product in step (2), stir for 60 min, then cool to below 0 °C, let it stand for 8 h, and remove the supernatant. Then add acetone 3 times the mass of krill oil, stir for 60 min, cool to below 0 °C, let it stand for 4 h, remove the supernatant, and vacuum concentrate to remove the remaining acetone to obtain a crude product;

[0038] (4) Dissolve the crude product obtained in step (3) in 95% ethanol with a mass 1 time that of it, dropwise add a 1% sodium hydroxide ethanol solution until the pH of the solution reaches 6, then react for 60 min. After the reaction ends, dropwise add a 1% sodium hydroxide ethanol solution until the pH of the solution is 8. After the reaction ends, filter to remove the precipitated fatty acid salts to obtain a filtered solution;

[0039] (5) Vacuum concentrate the filtered solution in step (4) to remove ethanol, control the moisture content of the material below 2%, and the residual amounts of acetone and ethanol below 5000 ppm. Finally, obtain 40 g of a brown - red paste - like product, with a yield of 40%. The content of sn - 1 - LPC - DHA in the product is detected to be 10.3%, and the content of LPC is 48.4%.

[0040] Example 3

[0041] (1) Mix 100 g of krill oil with 1000 g of water, stir until it becomes a homogeneous emulsion state, preheat to 50 °C, then add lipase accounting for 3% of the mass of krill oil, react for 60 min, after the reaction ends, raise the temperature to 90 °C to inactivate the enzyme, and obtain a mixed solution;

[0042] (2) Add 95% ethanol with a volume 1 - 2 times that of the water in step (1) to the mixed solution in step (1) for demulsification, after demulsification, vacuum concentrate to an anhydrous state to obtain a dehydrated product;

[0043] (3) Add acetone 10 times the mass of krill oil to the dehydrated product in step (2), stir for 60 min, then cool to below 0 °C, let it stand for 16 h, and remove the supernatant. Then add acetone 5 times the mass of krill oil, stir for 60 min, cool to below 0 °C, let it stand for 8 h, remove the supernatant, and vacuum concentrate to remove the remaining acetone to obtain a crude product;

[0044] (4) The crude product obtained in step (3) is dissolved in ethanol with a mass equal to its own mass, and a 1% sodium hydroxide ethanol solution is added dropwise until the pH of the solution reaches 6, and the reaction proceeds for 60 min. After the reaction, a 1% sodium hydroxide ethanol solution is added dropwise again until the pH of the solution reaches 8. After the reaction, the precipitated fatty acid salts are filtered off to obtain a filtered solution;

[0045] (5) The filtered solution in step (4) is concentrated under vacuum to remove ethanol, and the moisture content of the material is controlled below 2%, and the residual amounts of acetone and ethanol are below 5000 ppm. Finally, 50 g of a brownish-red paste product is obtained, with a yield of 50%. The content of sn-1-LPC-DHA detected in the product is 8.5%, and the LPC content is 45.3%.

[0046] Example 4

[0047] (1) 100 g of krill oil is mixed with 1500 g of water, stirred until a homogeneous emulsion state is achieved, preheated to 50 °C, and then 3% of the mass of the krill oil of lipase is added, and the reaction proceeds for 60 min. After the reaction, the temperature is raised to 90 °C to inactivate the enzyme; a mixed solution is obtained;

[0048] (2) 95% ethanol with a volume 1 - 2 times that of the water in step (1) is added to the mixed solution in step (1) for demulsification. After demulsification, it is concentrated under vacuum to an anhydrous state to obtain a dehydrated product;

[0049] (3) Acetone with a mass 10 times that of the krill oil is added to the dehydrated product in step (2), stirred for 60 min, then cooled to below 0 °C, and left standing for 16 h. The supernatant is removed. Then, acetone with a mass 5 times that of the krill oil is added, stirred for 60 min, cooled to below 0 °C, and left standing for 8 h. The supernatant is removed, and the remaining acetone is removed by vacuum concentration to obtain a crude product;

[0050] (4) The crude product obtained in step (3) is dissolved in ethanol with a mass equal to its own mass, and a 1% sodium hydroxide ethanol solution is added dropwise until the pH of the solution reaches 6, and the reaction proceeds for 60 min. After the reaction, a 1% sodium hydroxide ethanol solution is added dropwise again until the pH of the solution reaches 8. After the reaction, the precipitated fatty acid salts are filtered off to obtain a filtered solution;

[0051] (5) The filtered solution in step (4) is concentrated under vacuum to remove ethanol, and the moisture content of the material is controlled below 2%, and the residual amounts of acetone and ethanol are below 5000 ppm. Finally, 50 g of a brownish-red paste product is obtained, with a yield of 50%. The content of sn-1-LPC-DHA detected in the product is 8.7%, and the LPC content is 44.6%.

[0052] Example 5

[0053] (1) Mix 100 g of krill oil with 500 g of water, stir until it becomes a homogeneous emulsion state, preheat to 50 °C, then add lipase accounting for 6% of the mass of krill oil, react for 60 min, after the reaction ends, raise the temperature to 90 °C to inactivate the enzyme, and obtain a mixed solution;

[0054] (2) Add 95% ethanol with a volume 1 - 2 times that of the water in step (1) to the mixed solution in step (1) for demulsification, and after demulsification, vacuum concentrate to an anhydrous state to obtain a dehydrated product;

[0055] (3) Add acetone with a mass 5 times that of krill oil to the dehydrated product in step (2), stir for 60 min, then cool to below 0 °C, let it stand for 8 h, and remove the supernatant. Then add acetone with a mass 2 times that of krill oil, stir for 60 min, cool to below 0 °C, let it stand for 4 h, remove the supernatant, and vacuum concentrate to remove the remaining acetone to obtain a crude product;

[0056] (4) Dissolve the crude product obtained in step (3) in ethanol with a mass 1 time that of it, dropwise add a 1% sodium hydroxide ethanol solution until the pH of the solution reaches 6, then react for 60 min. After the reaction ends, dropwise add a 1% sodium hydroxide ethanol solution until the pH of the solution is 8. After the reaction ends, filter to remove the precipitated fatty acid salts to obtain a filtered solution;

[0057] (5) Vacuum concentrate the filtered solution in step (4) to remove ethanol, control the moisture content of the material below 2%, and the residual amounts of acetone and ethanol below 5000 ppm. Finally, obtain 46 g of a brown - red paste - like product, with a yield of 46%. The content of sn - 1 - LPC - DHA in the product is detected to be 7.6%, and the LPC content is 42.8%.

[0058] Example 6

[0059] (1) Mix 100 g of krill oil with 500 g of water, stir until it becomes a homogeneous emulsion state, preheat to 50 °C, then add lipase accounting for 9% of the mass of krill oil, react for 60 min, after the reaction ends, raise the temperature to 90 °C to inactivate the enzyme, and obtain a mixed solution;

[0060] (2) Add 95% ethanol with a volume 1 - 2 times that of the water in step (1) to the mixed solution in step (1) for demulsification, and after demulsification, vacuum concentrate to an anhydrous state to obtain a dehydrated product;

[0061] (3) Add acetone with a mass 10 times that of krill oil to the dehydrated product in step (2), stir for 60 min, then cool to below 0 °C, let it stand for 8 h, and remove the supernatant. Then add acetone with a mass 5 times that of krill oil, stir for 60 min, cool to below 0 °C, let it stand for 4 h, remove the supernatant, and vacuum concentrate to remove the remaining acetone to obtain a crude product;

[0062] (4) The crude product obtained in step (3) is dissolved in 95% ethanol with a mass equal to its own mass, and a 1% sodium hydroxide ethanol solution is added dropwise until the pH of the solution reaches 6, followed by reacting for 60 min. After the reaction, a 1% sodium hydroxide ethanol solution is added dropwise again until the pH of the solution reaches 8. After the reaction, the precipitated fatty acid salt is filtered off to obtain a filtered solution;

[0063] (5) The filtered solution in step (4) is concentrated under vacuum to remove ethanol, and the moisture content of the material is controlled below 2%, and the residual amounts of acetone and ethanol are below 5000 ppm. Finally, 43.7 g of a brownish-red paste product is obtained, with a yield of 43.7%. The content of sn-1-LPC-DHA in the product is detected to be 9%, and the content of LPC is 43.2%.

[0064] Example 7

[0065] (1) 100 g of krill oil is mixed with 500 g of water, stirred until a homogeneous emulsion state is achieved, preheated to 50 °C, and then 9% of lipase based on the mass of krill oil is added. After reacting for 30 min, the temperature is raised to 90 °C to inactivate the enzyme, obtaining a mixed solution;

[0066] (2) 95% ethanol with a volume 1 - 2 times that of the water in step (1) is added to the mixed solution in step (1) for demulsification. After demulsification, it is concentrated under vacuum to an anhydrous state to obtain a dehydrated product;

[0067] (3) Acetone with a mass 10 times that of the krill oil is added to the dehydrated product in step (2), stirred for 60 min, then cooled to below 0 °C, and left standing for 8 h. The supernatant is removed. Then, acetone with a mass 5 times that of the krill oil is added, stirred for 60 min, cooled to below 0 °C, and left standing for 4 h. The supernatant is removed, and the remaining acetone is removed by vacuum concentration to obtain a crude product;

[0068] (4) The crude product obtained in step (3) is dissolved in ethanol with a mass equal to its own mass, and a 1% sodium hydroxide ethanol solution is added dropwise until the pH of the solution reaches 6, followed by reacting for 60 min. After the reaction, a 1% sodium hydroxide ethanol solution is added dropwise again until the pH of the solution reaches 8. After the reaction, the precipitated fatty acid salt is filtered off to obtain a filtered solution;

[0069] (5) The filtered solution in step (4) is concentrated under vacuum to remove ethanol, and the moisture content of the material is controlled below 2%, and the residual amounts of acetone and ethanol are below 5000 ppm. Finally, 46 g of a brownish-red paste product is obtained, with a yield of 46%. The content of sn-1-LPC-DHA in the product is detected to be 7.6%, and the content of LPC is 42.3%.

[0070] Example 8

[0071] (1) Mix 100 g of krill oil with 500 g of water, stir until it becomes a homogeneous emulsion state, preheat to 50 °C, then add lipase accounting for 9% of the mass of krill oil, react for 120 min, after the reaction, raise the temperature to 90 °C to inactivate the enzyme, and obtain a mixed solution;

[0072] (2) Add 95% ethanol with a volume 1 - 2 times that of the water in step (1) to the mixed solution in step (1) for demulsification. After demulsification, vacuum concentrate to an anhydrous state to obtain a dehydrated product;

[0073] (3) Add acetone with a mass 10 times that of krill oil to the dehydrated product in step (2), stir for 60 min, then cool to below 0 °C, stand for 8 h, and remove the supernatant. Then add acetone with a mass 5 times that of krill oil, stir for 60 min, cool to below 0 °C, stand for 4 h, remove the supernatant, and vacuum concentrate to remove the remaining acetone to obtain a crude product;

[0074] (4) Dissolve the crude product obtained in step (3) in 95% ethanol with a mass 1 times that of it, dropwise add a 1% sodium hydroxide ethanol solution until the pH of the solution reaches 6, then react for 60 min. After the reaction, dropwise add a 1% sodium hydroxide ethanol solution until the pH of the solution is 8. After the reaction, filter to remove the precipitated fatty acid salts to obtain a filtered solution;

[0075] (5) Vacuum concentrate the filtered solution in step (4) to remove ethanol, control the moisture content of the material below 2%, and the residual amounts of acetone and ethanol below 5000 ppm. Finally, obtain 46 g of a brown - red paste - like product, with a yield of 46%. The content of sn - 1 - LPC - DHA detected in the product is 6.8%, and the content of LPC is 37.6%.

[0076] Example 9

[0077] (1) Mix 100 g of krill oil with 500 g of water, stir until it becomes a homogeneous emulsion state, preheat to 40 °C, then add lipase accounting for 9% of the mass of krill oil, react for 120 min, after the reaction, raise the temperature to 90 °C to inactivate the enzyme, and obtain a mixed solution;

[0078] (2) Add 95% ethanol with a volume 1 - 2 times that of the water in step (1) to the mixed solution in step (1) for demulsification. After demulsification, vacuum concentrate to an anhydrous state to obtain a dehydrated product;

[0079] (3) Add acetone with a mass 10 times that of krill oil to the dehydrated product in step (2), stir for 60 min, then cool to below 0 °C, stand for 8 h, and remove the supernatant. Then add acetone with a mass 5 times that of krill oil, stir for 60 min, cool to below 0 °C, stand for 4 h, remove the supernatant, and vacuum concentrate to remove the remaining acetone to obtain a crude product;

[0080] (4) The crude product obtained in step (3) is dissolved in 95% ethanol with a mass 1 time that of the crude product, and a 1% sodium hydroxide ethanol solution is added dropwise until the pH of the solution reaches 6, followed by reacting for 60 min. After the reaction ends, a 1% sodium hydroxide ethanol solution is added dropwise again until the pH of the solution is 8. After the reaction ends, the precipitated fatty acid salt is filtered off to obtain the filtered solution;

[0081] (5) The filtered solution in step (4) is concentrated under vacuum to remove ethanol, and the moisture content of the material is controlled below 2%, and the residual amounts of acetone and ethanol are below 5000 ppm. Finally, 46 g of a brownish-red paste product is obtained, with a yield of 46%. The content of sn-1-LPC-DHA detected in the product is 6.5%, and the content of LPC is 37.8%.

[0082] Example 10

[0083] (1) 100 g of krill oil is mixed with 500 g of water, stirred until a homogeneous emulsion state is achieved, preheated to 60 °C, and then 9% of lipase based on the mass of krill oil is added, followed by reacting for 120 min. After the reaction ends, the temperature is raised to 90 °C to inactivate the enzyme, obtaining a mixed solution;

[0084] (2) 95% ethanol with a volume 1 - 2 times that of the water in step (1) is added to the mixed solution in step (1) for demulsification. After demulsification, it is concentrated under vacuum to an anhydrous state to obtain the dehydrated product;

[0085] (3) Acetone with a mass 10 times that of krill oil is added to the dehydrated product in step (2), stirred for 60 min, then cooled to below 0 °C, and left standing for 8 h. The supernatant is removed. Then, acetone with a mass 5 times that of krill oil is added, stirred for 60 min, cooled to below 0 °C, and left standing for 4 h. The supernatant is removed, and the remaining acetone is removed by vacuum concentration to obtain the crude product;

[0086] (4) The crude product obtained in step (3) is dissolved in 95% ethanol with a mass 1 time that of the crude product, and a 1% sodium hydroxide ethanol solution is added dropwise until the pH of the solution reaches 6, followed by reacting for 60 min. After the reaction ends, a 1% sodium hydroxide ethanol solution is added dropwise again until the pH of the solution is 8. After the reaction ends, the precipitated fatty acid salt is filtered off to obtain the filtered solution;

[0087] (5) The filtered solution in step (4) is concentrated under vacuum to remove ethanol, and the moisture content of the material is controlled below 2%, and the residual amounts of acetone and ethanol are below 5000 ppm. Finally, 46 g of a brownish-red paste product is obtained, with a yield of 46%. The content of sn-1-LPC-DHA detected in the product is 7.1%, and the content of LPC is 40.1%.

[0088] Comparative Example 1

[0089] (1) Mix 100 g of krill oil with 500 g of 80% ethanol solution, stir until it is in a dissolved state, preheat to 50 °C, then add lipase at 3% of the mass of krill oil, react for 60 min, and after the reaction, raise the temperature to 90 °C to inactivate the enzyme to obtain a mixed solution;

[0090] (2) Vacuum concentrate the mixed solution in step (1) at 60 °C to an anhydrous state to obtain a dehydrated product;

[0091] (3) Add acetone 10 times the mass of krill oil to the dehydrated product in step (2), stir for 60 min, then cool to below 0 °C, let stand for 8 h, and remove the supernatant. Then add acetone 5 times the mass of krill oil, stir for 60 min, cool to below 0 °C, let stand for 4 h, remove the supernatant, and vacuum concentrate to remove the remaining acetone to obtain a crude product;

[0092] (4) Dissolve the crude product obtained in step (3) in ethanol of 1 times its mass, add a 1% sodium hydroxide ethanol solution dropwise until the pH of the solution reaches 6, then react for 60 min. After the reaction, add a 1% sodium hydroxide ethanol solution dropwise until the pH of the solution is 8. After the reaction, filter to remove the precipitated fatty acid salts to obtain a filtered solution;

[0093] (5) Vacuum concentrate the filtered solution in step (4) to remove ethanol, control the moisture content of the material below 2%, and the residual amounts of acetone and ethanol below 5000 ppm. Finally, obtain 52 g of a brownish-red paste product with a yield of 52%. The content of sn-1-LPC-DHA in the product is detected to be 0.023%, and the content of LPC is 5.8%.

[0094] Comparative Example 2

[0095] (1) Mix 100 g of krill oil with 500 g of 60% ethanol solution, stir until it is in a dissolved state, preheat to 50 °C, then add lipase at 3% of the mass of krill oil, react for 60 min, and after the reaction, raise the temperature to 90 °C to inactivate the enzyme to obtain a mixed solution;

[0096] (2) Vacuum concentrate the mixed solution in step (1) at 60 °C to an anhydrous state to obtain a dehydrated product;

[0097] (3) Add acetone 10 times the mass of krill oil to the dehydrated product in step (2), stir for 60 min, then cool to below 0 °C, let stand for 8 h, and remove the supernatant. Then add acetone 5 times the mass of krill oil, stir for 60 min, cool to below 0 °C, let stand for 4 h, remove the supernatant, and vacuum concentrate to remove the remaining acetone to obtain a crude product;

[0098] (4) The crude product obtained in step (3) is dissolved in ethanol with a mass 1 time that of the crude product, and a 1% sodium hydroxide ethanol solution is added dropwise until the pH of the solution reaches 6, followed by reacting for 60 min. After the reaction, a 1% sodium hydroxide ethanol solution is added dropwise again until the pH of the solution is 8. After the reaction, the precipitated fatty acid salt is filtered off to obtain the filtered solution;

[0099] (5) The filtered solution in step (4) is concentrated under vacuum to remove ethanol, and the moisture content of the material is controlled below 2%, and the residual amounts of acetone and ethanol are below 5000 ppm. Finally, 52 g of a brownish-red paste product is obtained, with a yield of 52%. The content of sn-1-LPC-DHA in the product is detected to be 1.02%, and the content of LPC is 9.18%.

[0100] Comparative Example 3

[0101] (1) 100 g of krill oil is mixed with 500 g of water, stirred until a homogeneous emulsion state is obtained, the pH is adjusted to 9.5, and after preheating to 60 °C, a lipase accounting for 3% of the mass of the krill oil is added, and the reaction is carried out for 120 min. After the reaction, the temperature is raised to 90 °C to inactivate the enzyme, obtaining a mixed solution;

[0102] (2) 95% ethanol with a volume 1 - 2 times that of the water in step (1) is added to the mixed solution in step (1) for demulsification, and after demulsification, it is concentrated under vacuum to an anhydrous state to obtain the dehydrated product;

[0103] (3) Acetone with a mass 10 times that of the krill oil is added to the dehydrated product in step (2), stirred for 60 min, then cooled to below 0 °C, and left standing for 8 h, and the supernatant is removed. Then, acetone with a mass 5 times that of the krill oil is added, stirred for 60 min, cooled to below 0 °C, left standing for 4 h, and the supernatant is removed, and the remaining acetone is removed by vacuum concentration to obtain the crude product;

[0104] (4) The crude product obtained in step (3) is dissolved in 95% ethanol with a mass 1 time that of the crude product, and a 1% sodium hydroxide ethanol solution is added dropwise until the pH of the solution reaches 6, followed by reacting for 60 min. After the reaction, a 1% sodium hydroxide ethanol solution is added dropwise again until the pH of the solution is 8. After the reaction, the precipitated fatty acid salt is filtered off to obtain the filtered solution;

[0105] (5) The filtered solution in step (4) is concentrated under vacuum to remove ethanol, and the moisture content of the material is controlled below 2%, and the residual amounts of acetone and ethanol are below 5000 ppm. Finally, 51 g of a brownish-red paste product is obtained, with a yield of 51%. The content of sn-1-LPC-DHA in the product is detected to be 2.18%, and the content of LPC is 12.16%.

[0106] Comparative Example 4

[0107] Mix 100 g of krill oil with 500 g of water, stir until a homogeneous emulsion state is achieved, preheat to 50 °C, then add lipase at 3% of the mass of the krill oil, react for 60 min, and after the reaction, raise the temperature to 90 °C to inactivate the enzyme to obtain a mixed solution;

[0108] (2) Add 95% ethanol with a volume 1 - 2 times that of the water in step (1) to the mixed solution in step (1) for demulsification. After demulsification, vacuum concentrate to an anhydrous state to obtain a dehydrated product;

[0109] (3) Add acetone 5 times the mass of the krill oil to the dehydrated product in step (2), stir for 60 min, then cool to below 0 °C, let stand for 8 h, and remove the supernatant. Then add acetone 3 times the mass of the krill oil, stir for 60 min, cool to below 0 °C, let stand for 4 h, remove the supernatant, and vacuum concentrate to remove the remaining acetone to obtain a crude product;

[0110] (4) Dissolve the crude product obtained in step (3) in 95% ethanol with a mass 1 time that of the crude product, add a 2% aqueous sodium hydroxide solution dropwise until the pH of the solution reaches 6, then react for 60 min. After the reaction, add a 2% aqueous sodium hydroxide solution dropwise until the pH of the solution is 7. After the reaction, filter to remove the precipitated fatty acid salts to obtain a filtered solution; no salt precipitates under these conditions

[0111] Comparative Example 5

[0112] (1) Mix 100 g of krill oil with 500 g of 2.6% aqueous sodium hydroxide solution, stir until a homogeneous emulsion state is achieved, preheat to 65 °C, then react for 60 min, and rotary evaporate to remove ethanol to obtain a mixed solution;

[0113] (2) Add 1000 mL of water to the mixed solution in step (1), stir evenly, adjust the pH to 3 with hydrochloric acid, separate and collect the upper oil phase, and rotary evaporate and dry to obtain a crude product;

[0114] (3) Add acetone 5 times the mass of the krill oil to the dehydrated crude product in step (2), stir for 60 min, then cool to below 0 °C, let stand for 8 h, and remove the supernatant. Then add acetone 3 times the mass of the krill oil, stir for 60 min, cool to below 0 °C, let stand for 4 h, remove the supernatant, and vacuum concentrate to remove the remaining acetone to obtain a crude product;

[0115] (4) Dissolve the crude product obtained in step (3) in 95% ethanol with a mass 1 time that of the crude product, add a 1% sodium hydroxide ethanol solution dropwise until the pH of the solution reaches 6, then react for 60 min. After the reaction, add a 1% sodium hydroxide ethanol solution dropwise until the pH of the solution is 8. After the reaction, filter to remove the precipitated fatty acid salts to obtain a filtered solution;

[0116] (5) Concentrate the filtered solution in step (4) under vacuum to remove ethanol, control the moisture content of the material below 2%, and the residual amounts of acetone and ethanol below 5000 ppm. Finally, 55 g of a brownish-red paste product is obtained, with a yield of 55%. The content of sn-1-LPC-DHA in the product is below the detection limit, and the content of LPC is below the detection limit.

[0117] Comparative Example 6

[0118] (1) Mix 100 g of krill oil with 500 g of 1.6% sodium hydroxide aqueous solution, stir until a homogeneous emulsion state is obtained, preheat to 65 °C, react for 60 min, and then remove ethanol by rotary evaporation to obtain a mixed solution;

[0119] (2) Add 1000 mL of water to the mixed solution in step (1), stir evenly, adjust the pH to 3 with hydrochloric acid, separate and collect the upper oil phase, and dry by rotary evaporation to obtain a crude product;

[0120] (3) Add acetone 5 times the mass of krill oil to the crude product dehydrated in step (2), stir for 60 min, cool to below 0 °C, stand for 8 h, and remove the supernatant. Then add acetone 3 times the mass of krill oil, stir for 60 min, cool to below 0 °C, stand for 4 h, remove the supernatant, and concentrate under vacuum to remove the remaining acetone to obtain a crude product;

[0121] (4) Dissolve the crude product obtained in step (3) in 95% ethanol with a mass 1 time that of the crude product, dropwise add 1% sodium hydroxide ethanol solution until the pH of the solution reaches 6, react for 60 min, and then dropwise add 1% sodium hydroxide ethanol solution until the pH of the solution is 8. After the reaction is completed, filter to remove the precipitated fatty acid salts to obtain a filtered solution;

[0122] (5) Concentrate the filtered solution in step (4) under vacuum to remove ethanol, control the moisture content of the material below 2%, and the residual amounts of acetone and ethanol below 5000 ppm. Finally, 55 g of a brownish-red paste product is obtained, with a yield of 55%. The content of sn-1-LPC-DHA in the product is below the detection limit, and the content of LPC is below the detection limit.

[0123] Among them, in the above examples, the moisture content of the material refers to the determination method of moisture in foods in GB5009.3-2016; the residual amounts of acetone and ethanol refer to the second method in General Principles 0861, Volume IV of the Chinese Pharmacopoeia 2020 Edition;

[0124] The contents of sn-1-LPC-DHA and LPC in the brownish-red paste product are determined by HPLC.

[0125] It should be understood that the detailed description of the technical solutions of the present invention by means of the preferred embodiments above is illustrative rather than restrictive. Those of ordinary skill in the art can modify the technical solutions described in each embodiment on the basis of reading the specification of the present invention, or perform equivalent replacements for some of the technical features; and these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of each embodiment of the present invention.

Claims

1. A krill oil extract, characterized in that, Wherein the LPC content is 0 - 72%, and the sn-1-LPC-DHA content is 0 - 20%.

2. The krill oil extract according to claim 1, wherein Wherein the LPC content is 18 - 60%, preferably 35.8 - 48.4%; wherein the sn-1-LPC-DHA content is 0 - 15%, preferably 0 - 10.3%.

3. The preparation method of the krill oil extract according to any one of claims 1-2, characterized in that, Comprising the following steps: 1) Enzymatic hydrolysis: Mix krill oil and water to a uniform emulsion state. After preheating, add lipase and stir. After stirring, raise the temperature to inactivate the enzyme to obtain a mixed solution; 2) Dehydration: Add an organic solvent to the mixed solution in step 1) for demulsification. After demulsification, concentrate and dehydrate under vacuum to obtain a dehydrated product; 3) Purification: Add acetone to the dehydrated product in step 2), stir and let stand to remove the supernatant; add acetone again, stir and let stand to remove the supernatant, and concentrate under vacuum to obtain a crude product; 4) Removal of free fatty acids: Dissolve the crude product in step 3) in an organic solvent, add sodium hydroxide ethanol solution to adjust the pH to 8 and react. After the reaction, add sodium hydroxide ethanol solution again to adjust the pH of the solution to 8. After the reaction, filter to obtain a filtrate; 5) Concentration and drying: Concentrate the filtrate under vacuum to obtain a brownish-red paste product.

4. The preparation method according to claim 3, characterized in that, In step 1), the mass ratio of the krill oil to water is 1:1 - 45, preferably 1:1 - 30, more preferably 1:1 - 15; the preheating temperature is 24 - 180°C, preferably 32 - 120°C, more preferably 40 - 60°C; the mass of the added lipase accounts for 1.8 - 27% of the mass of the krill oil, preferably 2.4 - 18%, more preferably 3 - 9%.

5. The preparation method according to claim 3, characterized in that In step 2), the organic solvent is one or a combination of methanol, ethanol, acetone or ethyl acetate, preferably methanol, ethanol or acetone, more preferably ethanol; the volume of the added organic solvent is 0.6 - 18 times the mass of water in step (1), preferably 0.8 - 6 times, more preferably 1 - 2 times.

6. The preparation method according to claim 3, characterized in that, In step 3), the mass of acetone added to the dehydrated product is 3 - 30 times the mass of the krill oil, preferably 4 - 20 times, more preferably 5 - 10 times; the mass of acetone added again is 1.2 - 45 times the mass of the krill oil, preferably 1.6 - 15 times, more preferably 2 - 5 times.

7. The preparation method according to claim 3, characterized in that In step 4), the organic solvent is one or a combination of methanol, ethanol, acetone or ethyl acetate, preferably methanol, ethanol or acetone, more preferably ethanol; the mass of the organic solvent is 0.6 - 3 times the mass of the crude product, preferably 0.8 - 2 times, more preferably 1 time; the mass fraction of sodium hydroxide in the sodium hydroxide ethanol solution is 0 - 6%, preferably 0 - 4%, more preferably 0 - 2%.

8. A product, characterized in that, Comprising the krill oil extract according to any one of claims 1 - 2 or the krill oil extract prepared by the preparation method according to any one of claims 3 - 7 and a pharmaceutically acceptable excipient.

9. The product according to claim 8, wherein, The product can be made into tablets, capsules, oral liquids or gummies.

10. Use of the krill oil extract according to any one of claims 1-2, the krill oil extract prepared by the preparation method of the krill oil extract according to any one of claims 3-7, or the product according to any one of claims 8-9 in the preparation of drugs, health foods, dietary supplements and / or foods for eye protection, anti-inflammatory, and anti-cardiovascular and cerebrovascular diseases.