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 in particular to a krill oil extract and a preparation method and application thereof. Background Art

[0002] Docosahexaenoic acid (DHA) is an 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 neuronal cells. It is abundant in the retina, and proper intake of DHA helps protect vision and prevent eye diseases. In addition, DHA can reduce blood viscosity and inhibit platelet aggregation, and is also beneficial in 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 mainly comes from deep-sea fish, shrimp and algae, and also exists in eggs. At present, the structural types of DHA are mainly triglyceride type and ethyl ester type, and a small amount of DHA from other sources is phospholipid type, such as Antarctic krill oil and DHA in egg yolk, which are phospholipid structures.

[0004] Triglyceride DHA and ethyl ester DHA are hydrolyzed into free DHA or monoacylglycerol by pancreatic lipase in the small intestine and are absorbed as triglycerides (TAG) in chylomicrons, preferentially incorporated into adipose tissue and heart, but not into the brain and eyes. The liver absorbs the remaining residues and integrates DHA into cell membrane lipids or synthesizes low-density lipoproteins for transport to other tissues. Most of the phospholipid DHA 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 DHA; and the sn-1-LPC-DHA in the released LPC-DHA can directly enter the blood, and some of the DHA absorbed by the liver in the form of phosphatidylcholine (PC) is remodeled 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, increasing the DHA content in the brain and eyes. However, the currently naturally existing phospholipid DHA basically exists at the sn-2 position, and is basically hydrolyzed into free DHA by phospholipase A2. Only a very small part will be hydrolyzed into sn-1DHA-LPC and enter the brain and eyes.

[0005] DHA is essential for the brain and eye functions of the human body, but very little of the naturally occurring DHA can actually be absorbed by the brain and eyes. Triglyceride-type DHA and ethyl ester-type DHA are hydrolyzed into free DHA, which basically only enters the blood circulation. Phospholipid-type DHA is also basically hydrolyzed into free DHA due to its site relationship and cannot enter the brain and eyes.

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

[0007] The purpose of the present invention is to address the deficiencies of the prior art and provide a krill oil extract containing DHA that can enter the brain and lysophosphatidylcholine (sn-1-LPC-DHA) rich in DHA, as well as a preparation method and application thereof.

[0008] In a 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%; and the sn-1-LPC-DHA content is 0-15%, preferably 0-10.3%.

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

[0011] 1) Enzymatic hydrolysis: krill oil and water are mixed into a uniform emulsion state, preheated, lipase is added and stirred, and after stirring, the temperature is raised to inactivate the enzyme to obtain a mixed solution;

[0012] 2) Dehydration: adding an organic solvent to the mixed solution in step 1) to demulsify, and after demulsification, vacuum concentrating and dehydrating to obtain a dehydrated product;

[0013] 3) Purification: Add acetone to the product after dehydration in step 2), stir and let stand, and remove the supernatant; add acetone again, stir and let stand, and remove the supernatant, and vacuum concentrate 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 sodium hydroxide ethanol solution is added dropwise to adjust the pH to 8 and then reacted. After the reaction is completed, sodium hydroxide ethanol solution is added dropwise to adjust the solution pH to 8. After the reaction is completed, the solution is filtered to obtain a filtrate;

[0015] 5) Concentration and drying: The filtrate is concentrated in vacuo to obtain a brown-red paste product.

[0016] In some embodiments, in step 1), the mass ratio of 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, preferably 4-20 times, and more preferably 5-10 times, of the krill oil; the mass of acetone added again is 1.2-45 times, preferably 1.6-15 times, and more preferably 2-5 times, of the krill oil.

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

[0021] In some embodiments, the product can be made into tablets, capsules, oral liquids or soft candies.

[0022] In a fourth aspect, the present invention provides a krill oil extract, a method for preparing a krill oil extract, and the use of the krill oil extract or product obtained in the preparation of eye protection, anti-inflammatory, anti-cardiovascular and cerebrovascular disease medicines, health foods, dietary supplements and / or foods.

[0023] Beneficial effects of the present invention:

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

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

[0026] 3. In industry, molecular distillation is mostly used to remove fatty acids, which is costly. The present invention uses alkaline solution to form salts to remove free fatty acids, thereby reducing production costs while maintaining a good yield. DETAILED DESCRIPTION

[0027] In order to facilitate the understanding of those skilled in the art, the present invention is further described below in conjunction with embodiments, and the contents mentioned in the implementation modes are not intended to limit the present invention.

[0028] Example 1

[0029] (1) 100 g of krill oil and 100 g of water were mixed and stirred until a uniform emulsion state was obtained. After preheating to 50° C., lipase in an amount of 3% by mass of the krill oil was added and reacted for 60 min. After the reaction was completed, the mixture was heated to 90° C. to inactivate the enzyme and obtain a mixed solution;

[0030] (2) adding 95% ethanol in an amount of 1 to 2 times the volume of the water in step (1) to the mixed solution in step (1) to break the emulsion, and after breaking the emulsion, vacuum concentrate to an anhydrous state to obtain a dehydrated product;

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

[0032] (4) The crude product obtained in step (3) is dissolved in 95% ethanol in an amount equal to 1% by weight, and a 1% sodium hydroxide ethanol solution is added dropwise until the pH value of the solution reaches 6, and then the reaction is continued for 60 minutes. After the reaction is completed, a 1% sodium hydroxide ethanol solution is added dropwise until the pH value of the solution reaches 8. After the reaction is completed, the precipitated fatty acid salt is filtered out to obtain a filtered solution;

[0033] (5) The filtered solution in step (4) was concentrated under vacuum to remove ethanol, and the moisture content of the material was controlled to be below 2%, and the residual acetone and ethanol content was below 5000 ppm. Finally, 40 g of a brown-red paste product was obtained with a yield of 40%. The sn-1-LPC-DHA content detected in the product was 6%, and the LPC content was 35.8%.

[0034] Example 2

[0035] (1) 100 g of krill oil and 500 g of water were mixed and stirred until a uniform emulsion state was obtained. After preheating to 50° C., lipase in an amount of 3% by mass of the krill oil was added and reacted for 60 min. After the reaction was completed, the temperature was raised to 90° C. to inactivate the enzyme and obtain a mixed solution;

[0036] (2) adding 95% ethanol in an amount of 1 to 2 times the volume of the water in step (1) to the mixed solution in step (1) to break the emulsion, and after breaking the emulsion, vacuum concentrate to an anhydrous state to obtain a dehydrated product;

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

[0038] (4) The crude product obtained in step (3) is dissolved in 1 times the mass of 95% ethanol, and a 1% sodium hydroxide ethanol solution is added dropwise until the pH of the solution reaches 6, and then reacted for 60 minutes. After the reaction, a 1% sodium hydroxide ethanol solution is added dropwise until the pH of the solution reaches 8. After the reaction is completed, the precipitated fatty acid salt is filtered out to obtain a filtered solution;

[0039] (5) The filtered solution in step (4) was concentrated under vacuum to remove ethanol, and the moisture content of the material was controlled to be below 2%, and the residual acetone and ethanol content was below 5000 ppm. Finally, 40 g of a brown-red paste product was obtained with a yield of 40%. The sn-1-LPC-DHA content detected in the product was 10.3%, and the LPC content was 48.4%.

[0040] Example 3

[0041] (1) 100 g of krill oil and 1000 g of water were mixed and stirred until a uniform emulsion state was obtained. After preheating to 50° C., lipase in an amount of 3% by mass of the krill oil was added and reacted for 60 min. After the reaction was completed, the mixture was heated to 90° C. to inactivate the enzyme and obtain a mixed solution;

[0042] (2) adding 95% ethanol in an amount of 1 to 2 times the volume of the water in step (1) to the mixed solution in step (1) to break the emulsion, and after breaking the emulsion, vacuum concentrate to an anhydrous state to obtain a dehydrated product;

[0043] (3) Add acetone 10 times the mass of krill oil to the product after dehydration in step (2), stir for 60 minutes, cool to below 0°C, let stand for 16 hours, and remove the supernatant. Then add acetone 5 times the mass of krill oil, stir for 60 minutes, cool to below 0°C, let stand for 8 hours, 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 1 times the mass of ethanol, and a 1% sodium hydroxide ethanol solution is added dropwise until the pH of the solution reaches 6, and the reaction is continued for 60 minutes. After the reaction is completed, a 1% sodium hydroxide ethanol solution is added dropwise until the pH of the solution reaches 8. After the reaction is completed, the precipitated fatty acid salt is filtered out to obtain a filtered solution;

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

[0046] Example 4

[0047] (1) 100 g of krill oil and 1500 g of water were mixed and stirred until a uniform emulsion state was obtained. After preheating to 50° C., lipase in an amount of 3% by mass of the krill oil was added and reacted for 60 min. After the reaction was completed, the temperature was raised to 90° C. to inactivate the enzyme to obtain a mixed solution.

[0048] (2) adding 95% ethanol in an amount of 1 to 2 times the volume of the water in step (1) to the mixed solution in step (1) to break the emulsion, and after breaking the emulsion, vacuum concentrate to an anhydrous state to obtain a dehydrated product;

[0049] (3) Add acetone 10 times the mass of krill oil to the product after dehydration in step (2), stir for 60 minutes, cool to below 0°C, let stand for 16 hours, and remove the supernatant. Then add acetone 5 times the mass of krill oil, stir for 60 minutes, cool to below 0°C, let stand for 8 hours, remove the supernatant, and vacuum concentrate to remove the remaining acetone to obtain a crude product;

[0050] (4) The crude product obtained in step (3) is dissolved in 1 times the mass of ethanol, and a 1% sodium hydroxide ethanol solution is added dropwise until the pH of the solution reaches 6, and then reacted for 60 minutes. After the reaction, a 1% sodium hydroxide ethanol solution is added dropwise until the pH of the solution reaches 8. After the reaction is completed, the precipitated fatty acid salt is filtered out to obtain a filtered solution;

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

[0052] Example 5

[0053] (1) 100 g of krill oil and 500 g of water were mixed and stirred until a uniform emulsion state was obtained. After preheating to 50° C., lipase in an amount of 6% by mass of the krill oil was added and reacted for 60 min. After the reaction was completed, the temperature was raised to 90° C. to inactivate the enzyme and obtain a mixed solution;

[0054] (2) adding 95% ethanol in an amount of 1 to 2 times the volume of the water in step (1) to the mixed solution in step (1) to break the emulsion, and after breaking the emulsion, vacuum concentrate to an anhydrous state to obtain a dehydrated product;

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

[0056] (4) The crude product obtained in step (3) is dissolved in 1 times the mass of ethanol, and a 1% sodium hydroxide ethanol solution is added dropwise until the pH of the solution reaches 6, and then reacted for 60 minutes. After the reaction, a 1% sodium hydroxide ethanol solution is added dropwise until the pH of the solution reaches 8. After the reaction is completed, the precipitated fatty acid salt is filtered out to obtain a filtered solution;

[0057] (5) The filtered solution in step (4) was concentrated under vacuum to remove ethanol, and the moisture content of the material was controlled to be below 2%, and the residual acetone and ethanol content was below 5000 ppm. Finally, 46 g of a brown-red paste product was obtained with a yield of 46%. The sn-1-LPC-DHA content detected in the product was 7.6%, and the LPC content was 42.8%.

[0058] Example 6

[0059] (1) 100 g of krill oil and 500 g of water were mixed and stirred until a uniform emulsion state was obtained. After preheating to 50° C., lipase in an amount of 9% by mass of the krill oil was added and reacted for 60 min. After the reaction was completed, the temperature was raised to 90° C. to inactivate the enzyme and obtain a mixed solution;

[0060] (2) adding 95% ethanol in an amount of 1 to 2 times the volume of the water in step (1) to the mixed solution in step (1) to break the emulsion, and after breaking the emulsion, vacuum concentrate to an anhydrous state to obtain a dehydrated product;

[0061] (3) Add acetone 10 times the mass of krill oil to the product dehydrated in step (2), stir for 60 minutes, cool to below 0°C, let stand for 8 hours, and remove the supernatant. Then add acetone 5 times the mass of krill oil, stir for 60 minutes, cool to below 0°C, let stand for 4 hours, 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 in an amount equal to 1% by weight, and a 1% sodium hydroxide ethanol solution is added dropwise until the pH value of the solution reaches 6, and then the reaction is continued for 60 minutes. After the reaction is completed, a 1% sodium hydroxide ethanol solution is added dropwise until the pH value of the solution reaches 8. After the reaction is completed, the precipitated fatty acid salt is filtered out to obtain a filtered solution;

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

[0064] Example 7

[0065] (1) 100 g of krill oil and 500 g of water were mixed and stirred until a uniform emulsion state was obtained. After preheating to 50° C., lipase with a mass percentage of 9% of the krill oil was added and reacted for 30 min. After the reaction was completed, the temperature was raised to 90° C. to inactivate the enzyme and obtain a mixed solution;

[0066] (2) adding 95% ethanol in an amount of 1 to 2 times the volume of the water in step (1) to the mixed solution in step (1) to break the emulsion, and after breaking the emulsion, vacuum concentrate to an anhydrous state to obtain a dehydrated product;

[0067] (3) Add acetone 10 times the mass of krill oil to the product dehydrated in step (2), stir for 60 minutes, cool to below 0°C, let stand for 8 hours, and remove the supernatant. Then add acetone 5 times the mass of krill oil, stir for 60 minutes, cool to below 0°C, let stand for 4 hours, remove the supernatant, and vacuum concentrate to remove the remaining acetone to obtain a crude product;

[0068] (4) The crude product obtained in step (3) is dissolved in 1 times the mass of ethanol, and a 1% sodium hydroxide ethanol solution is added dropwise until the pH of the solution reaches 6, and then reacted for 60 minutes. After the reaction, a 1% sodium hydroxide ethanol solution is added dropwise until the pH of the solution reaches 8. After the reaction is completed, the precipitated fatty acid salt is filtered out to obtain a filtered solution;

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

[0070] Example 8

[0071] (1) 100 g of krill oil was mixed with 500 g of water, stirred until a uniform emulsion was formed, preheated to 50° C., and lipase (9% by mass of krill oil) was added, reacted for 120 min, and after the reaction was completed, heated to 90° C. to inactivate the enzyme to obtain a mixed solution;

[0072] (2) adding 95% ethanol in an amount of 1 to 2 times the volume of the water in step (1) to the mixed solution in step (1) to break the emulsion, and after breaking the emulsion, vacuum concentrate to an anhydrous state to obtain a dehydrated product;

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

[0074] (4) The crude product obtained in step (3) is dissolved in 95% ethanol in an amount equal to 1% by weight, and a 1% sodium hydroxide ethanol solution is added dropwise until the pH value of the solution reaches 6, and then the reaction is continued for 60 minutes. After the reaction is completed, a 1% sodium hydroxide ethanol solution is added dropwise until the pH value of the solution reaches 8. After the reaction is completed, the precipitated fatty acid salt is filtered out to obtain a filtered solution;

[0075] (5) The filtered solution in step (4) was concentrated under vacuum to remove ethanol, and the moisture content of the material was controlled to be below 2%, and the residual acetone and ethanol content was below 5000 ppm. Finally, 46 g of a brown-red paste product was obtained with a yield of 46%. The sn-1-LPC-DHA content detected in the product was 6.8%, and the LPC content was 37.6%.

[0076] Example 9

[0077] (1) 100 g of krill oil and 500 g of water were mixed and stirred until a uniform emulsion state was obtained. After preheating to 40° C., lipase with a mass percentage of 9% of the krill oil was added and reacted for 120 min. After the reaction was completed, the temperature was raised to 90° C. to inactivate the enzyme and obtain a mixed solution;

[0078] (2) adding 95% ethanol in an amount of 1 to 2 times the volume of the water in step (1) to the mixed solution in step (1) to break the emulsion, and after breaking the emulsion, vacuum concentrate to an anhydrous state to obtain a dehydrated product;

[0079] (3) Add acetone 10 times the mass of krill oil to the product after dehydration in step (2), stir for 60 minutes, cool to below 0°C, let stand for 8 hours, and remove the supernatant. Then add acetone 5 times the mass of krill oil, stir for 60 minutes, cool to below 0°C, let stand for 4 hours, 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 in an amount equal to 1% by weight, and a 1% sodium hydroxide ethanol solution is added dropwise until the pH value of the solution reaches 6, and then the reaction is continued for 60 minutes. After the reaction is completed, a 1% sodium hydroxide ethanol solution is added dropwise until the pH value of the solution reaches 8. After the reaction is completed, the precipitated fatty acid salt is filtered out to obtain a filtered solution;

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

[0082] Example 10

[0083] (1) 100 g of krill oil and 500 g of water were mixed and stirred until a uniform emulsion state was obtained. After preheating to 60° C., lipase with a mass percentage of 9% of the krill oil was added and reacted for 120 min. After the reaction was completed, the temperature was raised to 90° C. to inactivate the enzyme and obtain a mixed solution;

[0084] (2) adding 95% ethanol in an amount of 1 to 2 times the volume of the water in step (1) to the mixed solution in step (1) to break the emulsion, and after breaking the emulsion, vacuum concentrate to an anhydrous state to obtain a dehydrated product;

[0085] (3) Add acetone 10 times the mass of krill oil to the product dehydrated in step (2), stir for 60 minutes, cool to below 0°C, let stand for 8 hours, and remove the supernatant. Then add acetone 5 times the mass of krill oil, stir for 60 minutes, cool to below 0°C, let stand for 4 hours, remove the supernatant, and vacuum concentrate to remove the remaining acetone to obtain a crude product;

[0086] (4) The crude product obtained in step (3) is dissolved in 95% ethanol in an amount equal to 1% by weight, and a 1% sodium hydroxide ethanol solution is added dropwise until the pH value of the solution reaches 6, and then the reaction is continued for 60 minutes. After the reaction is completed, a 1% sodium hydroxide ethanol solution is added dropwise until the pH value of the solution reaches 8. After the reaction is completed, the precipitated fatty acid salt is filtered out to obtain a filtered solution;

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

[0088] Comparative Example 1

[0089] (1) 100 g of krill oil was mixed with 500 g of 80% ethanol solution, stirred until dissolved, preheated to 50° C., and then lipase (3% by mass of krill oil) was added, reacted for 60 min, and after the reaction was completed, the temperature was raised to 90° C. to inactivate the enzyme, thereby obtaining a mixed solution;

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

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

[0092] (4) The crude product obtained in step (3) is dissolved in 1 times the mass of ethanol, and a 1% sodium hydroxide ethanol solution is added dropwise until the pH of the solution reaches 6, and then reacted for 60 minutes. After the reaction, a 1% sodium hydroxide ethanol solution is added dropwise until the pH of the solution reaches 8. After the reaction is completed, the precipitated fatty acid salt is filtered out to obtain a filtered solution;

[0093] (5) The filtered solution in step (4) was concentrated under vacuum to remove ethanol, and the moisture content of the material was controlled to be below 2%, and the residual acetone and ethanol content was below 5000 ppm. Finally, 52 g of a brown-red paste product was obtained with a yield of 52%. The sn-1-LPC-DHA content detected in the product was 0.023%, and the LPC content was 5.8%.

[0094] Comparative Example 2

[0095] (1) 100 g of krill oil was mixed with 500 g of 60% ethanol solution, stirred until dissolved, preheated to 50° C., and then lipase (3% by mass of krill oil) was added, reacted for 60 min, and after the reaction was completed, the temperature was raised to 90° C. to inactivate the enzyme, thereby obtaining a mixed solution;

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

[0097] (3) Add acetone 10 times the mass of krill oil to the product dehydrated in step (2), stir for 60 minutes, cool to below 0°C, let stand for 8 hours, and remove the supernatant. Then add acetone 5 times the mass of krill oil, stir for 60 minutes, cool to below 0°C, let stand for 4 hours, 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 1 times the mass of ethanol, and a 1% sodium hydroxide ethanol solution is added dropwise until the pH of the solution reaches 6, and then reacted for 60 minutes. After the reaction, a 1% sodium hydroxide ethanol solution is added dropwise until the pH of the solution reaches 8. After the reaction is completed, the precipitated fatty acid salt is filtered out to obtain a filtered solution;

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

[0100] Comparative Example 3

[0101] (1) 100 g of krill oil and 500 g of water were mixed and stirred until a uniform emulsion state was obtained, the pH was adjusted to 9.5, and the mixture was preheated to 60° C., and lipase in an amount of 3% by mass of the krill oil was added, and the mixture was reacted for 120 min. After the reaction was completed, the mixture was heated to 90° C. to inactivate the enzyme, and a mixed solution was obtained;

[0102] (2) adding 95% ethanol in an amount of 1 to 2 times the volume of the water in step (1) to the mixed solution in step (1) to break the emulsion, and after breaking the emulsion, vacuum concentrate to an anhydrous state to obtain a dehydrated product;

[0103] (3) Add acetone 10 times the mass of krill oil to the product dehydrated in step (2), stir for 60 minutes, cool to below 0°C, let stand for 8 hours, and remove the supernatant. Then add acetone 5 times the mass of krill oil, stir for 60 minutes, cool to below 0°C, let stand for 4 hours, remove the supernatant, and vacuum concentrate to remove the remaining acetone to obtain a crude product;

[0104] (4) The crude product obtained in step (3) is dissolved in 95% ethanol in an amount equal to 1% by weight, and a 1% sodium hydroxide ethanol solution is added dropwise until the pH value of the solution reaches 6, and then the reaction is continued for 60 minutes. After the reaction is completed, a 1% sodium hydroxide ethanol solution is added dropwise until the pH value of the solution reaches 8. After the reaction is completed, the precipitated fatty acid salt is filtered out to obtain a filtered solution;

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

[0106] Comparative Example 4

[0107] 100 g of krill oil was mixed with 500 g of water, stirred until a uniform emulsion state was obtained, and then preheated to 50° C., lipase (3% by mass of krill oil) was added, and reacted for 60 min. After the reaction was completed, the temperature was raised to 90° C. to inactivate the enzyme, and a mixed solution was obtained;

[0108] (2) adding 95% ethanol in an amount of 1 to 2 times the volume of the water in step (1) to the mixed solution in step (1) to break the emulsion, and after breaking the emulsion, vacuum concentrate to an anhydrous state to obtain a dehydrated product;

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

[0110] (4) The crude product obtained in step (3) is dissolved in 95% ethanol (1 times its mass), and a 2% aqueous sodium hydroxide solution is added dropwise until the pH of the solution reaches 6, and then the reaction is continued for 60 minutes. After the reaction is completed, a 2% aqueous sodium hydroxide solution is added dropwise until the pH of the solution reaches 7. After the reaction is completed, the precipitated fatty acid salt is filtered out to obtain a filtered solution; under this condition, no salt precipitation occurs.

[0111] Comparative Example 5

[0112] (1) 100 g of krill oil was mixed with 500 g of 2.6% sodium hydroxide aqueous solution, stirred until a uniform emulsion state was obtained, preheated to 65° C., reacted for 60 min, and then ethanol was removed by rotary evaporation to obtain a mixed solution;

[0113] (2) adding 1000 mL of water to the mixed solution in step (1), stirring evenly, adjusting the pH to 3 with hydrochloric acid, separating and collecting the upper oil phase, and rotary evaporating and drying to obtain a crude product;

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

[0115] (4) The crude product obtained in step (3) is dissolved in 95% ethanol in an amount equal to 1% of its mass, and a 1% sodium hydroxide ethanol solution is added dropwise until the pH of the solution reaches 6, and then the reaction is continued for 60 minutes. After the reaction is completed, a 1% sodium hydroxide ethanol solution is added dropwise until the pH of the solution reaches 8. After the reaction is completed, the precipitated fatty acid salt is filtered out to obtain a filtered solution;

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

[0117] Comparative Example 6

[0118] (1) 100 g of krill oil was mixed with 500 g of 1.6% sodium hydroxide aqueous solution, stirred until a uniform emulsion state was obtained, preheated to 65° C., reacted for 60 min, and then ethanol was removed by rotary evaporation to obtain a mixed solution;

[0119] (2) adding 1000 mL of water to the mixed solution in step (1), stirring evenly, adjusting the pH to 3 with hydrochloric acid, separating and collecting the upper oil phase, and rotary evaporating and drying 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 minutes, cool to below 0°C, let stand for 8 hours, and remove the supernatant. Then add acetone 3 times the mass of krill oil, stir for 60 minutes, cool to below 0°C, let stand for 4 hours, remove the supernatant, and vacuum concentrate to remove the remaining acetone to obtain a crude product;

[0121] (4) The crude product obtained in step (3) is dissolved in 95% ethanol in an amount equal to 1% of its mass, and a 1% sodium hydroxide ethanol solution is added dropwise until the pH of the solution reaches 6, and then the reaction is continued for 60 minutes. After the reaction is completed, a 1% sodium hydroxide ethanol solution is added dropwise until the pH of the solution reaches 8. After the reaction is completed, the precipitated fatty acid salt is filtered out to obtain a filtered solution;

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

[0123] Among them, in the above embodiments, the moisture content of the material refers to the determination method of moisture in food in GB5009.3-2016; the acetone and ethanol residues refer to the second method of 0861 of the fourth general rules of the Chinese Pharmacopoeia 2020 edition;

[0124] The contents of sn-1-LPC-DHA and LPC in the brown-red paste product were 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 is illustrative rather than restrictive. A person skilled in the art may modify the technical solutions described in the embodiments, or replace some of the technical features by equivalents, based on reading the specification of the present invention; and these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A krill oil extract, characterized in that 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, characterized in that The LPC content is 18-60%, preferably 35.8-48.4%; the sn-1-LPC-DHA content is 0-15%, preferably 0-10.3%.

3. The method for preparing the krill oil extract according to any one of claims 1-2, characterized in that: The following steps are involved: 1) Enzymatic hydrolysis: krill oil and water are mixed into a uniform emulsion state, preheated, lipase is added and stirred, and after stirring, the temperature is raised to inactivate the enzyme to obtain a mixed solution; 2) Dehydration: adding an organic solvent to the mixed solution in step 1) to demulsify, and after demulsification, vacuum concentrating and dehydrating to obtain a dehydrated product; 3) Purification: Add acetone to the product after dehydration in step 2), stir and let stand, and remove the supernatant; add acetone again, stir and let stand, and remove the supernatant, and vacuum concentrate to obtain a crude product; 4) Removal of free fatty acids: The crude product in step 3) is dissolved in an organic solvent, and sodium hydroxide ethanol solution is added dropwise to adjust the pH to 8 and then reacted. After the reaction is completed, sodium hydroxide ethanol solution is added dropwise to adjust the solution pH to 8. After the reaction is completed, the solution is filtered to obtain a filtrate; 5) Concentration and drying: The filtrate is concentrated in vacuo to obtain a brown-red paste product.

4. The preparation method according to claim 3, characterized in that: In step 1), the mass ratio of 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%.

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, 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.

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, preferably 4-20 times, and more preferably 5-10 times, of the krill oil; the mass of acetone added again is 1.2-45 times, preferably 1.6-15 times, and more preferably 2-5 times, of the krill oil.

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, 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 times; the mass fraction of sodium hydroxide in the sodium hydroxide ethanol solution is 0-6%, preferably 0-4%, and more preferably 0-2%.

8. A product, characterized in that The invention comprises the krill oil extract according to any one of claims 1 to 2 or the krill oil extract prepared by the preparation method according to any one of claims 3 to 7 and pharmaceutically acceptable excipients.

9. The product according to claim 8, characterized in that The product can be made into tablets, capsules, oral liquids or soft candies.

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