Application of hydrolyzed krill oil extract in aspect of preventing thrombosis

Abstract

The invention discloses application of a hydrolyzed krill oil extract to prevention of thrombosis, and particularly relates to the technical field of health care products. The hydrolyzed krill oil extract with high content of LPC and sn-1-LPC-DHA is prepared through the preparation method, the content of LPC is close to 50%, most of LPC-DHA is sn-1-LPC-DHA, the bioavailability of DHA is improved, thrombosis in a zebra fish model can be effectively prevented, and part of evaluation indexes are superior to those of a known antithrombotic drug aspirin.

Description

[0001] This application claims priority to Chinese Application No. 2024110629564, filed on August 5, 2024, entitled “A Krill Oil Extract, Preparation Method and Application Thereof”, the contents of which are incorporated herein by reference in their entirety. Technical Field

[0002] The invention relates to the technical field of health care products, and in particular to use of a hydrolyzed krill oil extract in preventing thrombosis. Background Art

[0003] 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 nerve cells. It is abundant in the retina, and a moderate intake of DHA helps protect vision and prevent eye diseases. In addition, DHA can also reduce blood viscosity and inhibit platelet aggregation, and is also very 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.

[0004] 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.

[0005] 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.

[0006] 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.

[0007] 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.

[0008] Thrombus is a small piece of blood formed on the surface of the peeling or repaired part of the inner surface of the cardiovascular system. It may be caused by damage to the heart and blood vessel lining, changes in blood flow and blood properties, etc. Thrombosis will cause harm to different tissues and organs. For example, when the coronary artery that supplies blood to the heart is blocked by a thrombus, the heart cells will be ischemic and hypoxic. If not treated in time, it may cause infarction, that is, myocardial infarction.

[0009] There are three main factors in thrombosis: damage to the cardiovascular endothelium, changes in blood flow status, and increased vascular coagulability, any of which can promote thrombosis. The vascular intima is located in the innermost layer of the vascular wall and is composed of the endothelium and subendothelial matrix. Its structural integrity and normal function are crucial to the physiological function of blood vessels, and are also a key link in preventing atherosclerosis and related cardiovascular and cerebrovascular diseases. The vascular intima is directly in contact with the blood mainly in the form of a continuous single cell layer of endothelial cells. Therefore, in the process of vascular intima damage, endothelial cells are the first to show functional abnormalities or structural damage. For example, normal vascular endothelium can shield the invasion of low-density lipoprotein or other harmful substances, deposit under the intima, and block the initiation and progression of atherosclerotic lesions. This is particularly important in atherosclerosis with large changes in vascular bifurcation and blood flow shear force. Summary of the invention

[0010] The purpose of the present invention is to address the deficiencies of the prior art and provide a use of a hydrolyzed krill oil extract containing DHA that can enter the brain and lysophosphatidylcholine (sn-1-LPC-DHA) rich in DHA.

[0011] In a first aspect, the present invention provides a use of a hydrolyzed krill oil extract in the preparation of a food, a health product, or a dietary supplement for preventing thrombosis.

[0012] In some embodiments, the LPC content of the hydrolyzed krill oil extract is 0-90%.

[0013] In some embodiments, the LPC content of the hydrolyzed krill oil extract is 10-72%.

[0014] In some embodiments, the LPC content of the hydrolyzed krill oil extract is 20-60%.

[0015] In some embodiments, the LPC content in the hydrolyzed krill oil extract is 30-50%.

[0016] In some embodiments, the sn-1-LPC-DHA content in the hydrolyzed krill oil extract is 0-30%.

[0017] In some embodiments, the sn-1-LPC-DHA content in the hydrolyzed krill oil extract is 0-20%.

[0018] In some embodiments, the sn-1-LPC-DHA content in the hydrolyzed krill oil extract is 0-15%.

[0019] In some embodiments, the sn-1-LPC-DHA content of LPC-DHA in the hydrolyzed krill oil extract is ≥ 50%.

[0020] In some embodiments, the content of sn-1-LPC-DHA in LPC-DHA in the hydrolyzed krill oil extract is ≥70%, further ≥80%, ≥90%, ≥95%, ≥99%.

[0021] In some embodiments, the total content of EPA and DHA in the hydrolyzed krill oil extract is 20-50%.

[0022] In some embodiments, the total content of EPA and DHA in the hydrolyzed krill oil extract is 30-40%.

[0023] In some embodiments, the mass ratio of EPA to DHA in the hydrolyzed krill oil extract is 1:1-5.

[0024] In some embodiments, the mass ratio of EPA to DHA in the hydrolyzed krill oil extract is 1:2-4.

[0025] In some embodiments, the present invention also provides a method for preparing a hydrolyzed krill oil extract, comprising the following steps:

[0026] 1) Enzymatic hydrolysis: krill oil and water are mixed, lipase is added and stirred, and after stirring, the temperature is raised to inactivate the enzyme to obtain a mixed solution;

[0027] 2) Dehydration: adding an organic solvent to the mixed solution in step 1) to demulsify, and concentrating and dehydrating after demulsification to obtain a dehydrated product;

[0028] 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;

[0029] 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 before reaction. After the reaction is completed, the pH of the solution is adjusted to 8. After the reaction is completed, the solution is filtered to obtain a filtrate;

[0030] 5) Concentration and drying: The filtrate is concentrated and dried to obtain a brown-red paste product.

[0031] 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%.

[0032] In some embodiments, in step 2), the organic solvent includes but is not limited to 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.

[0033] 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.

[0034] In some embodiments, in step 4), the organic solvent includes but is not limited to one or more combinations 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%.

[0035] In some embodiments, the hydrolyzed krill oil extract is used to improve platelet aggregation, cardiac output, or blood flow rate in a subject with thrombosis.

[0036] In some embodiments, the food, health product, dietary supplement further comprises a food acceptable additive.

[0037] In some embodiments, the food, health product, dietary supplement can be made into tablets, capsules, soft capsules, gel candies, oral liquids or soft candies.

[0038] Beneficial effects of the present invention:

[0039] The hydrolyzed krill oil extract with high content of LPC and sn-1-LPC-DHA prepared by the preparation method of the present invention can effectively prevent thrombosis in zebrafish models, and some evaluation indicators are even better than the known antithrombotic drug aspirin. The LPC content in the hydrolyzed krill oil extract is nearly 50%, and the strong emulsification performance of LPC plays an emulsifying role in blood vessels, which can emulsify fatty substances such as triacylglycerol to reduce blood flow velocity. At the same time, DHA and EPA in the form of LPC can improve their effects on anti-thrombosis. In addition, the vast majority of LPC-DHA in the hydrolyzed krill oil extract is sn-1-LPC-DHA, which improves the bioavailability of DHA and has the effect of preventing thrombosis. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] Figure 1 Histogram of the total opacity of zebrafish heart area in each experimental group

[0041] Figure 2 Histogram of the total opacity of the zebrafish trunk area in each experimental group

[0042] Figure 3 Typical images of zebrafish platelet aggregation in each experimental group

[0043] Figure 4 Blood flow velocity bar graph of zebrafish in each experimental group

[0044] Figure 5 Histogram of zebrafish cardiac output in each experimental group DETAILED DESCRIPTION

[0045] 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.

[0046] Example 1

[0047] (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;

[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 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;

[0050] (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;

[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, 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%.

[0052] Example 2

[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 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;

[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 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;

[0056] (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;

[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, 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%.

[0058] Example 3

[0059] (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;

[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 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;

[0062] (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;

[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, 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%.

[0064] Example 4

[0065] (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.

[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 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;

[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, 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%.

[0070] Example 5

[0071] (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;

[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 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;

[0074] (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;

[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 7.6%, and the LPC content was 42.8%.

[0076] Example 6

[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 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;

[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 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;

[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, 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%.

[0082] Example 7

[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 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;

[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 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;

[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 7.6%, and the LPC content was 42.3%.

[0088] Example 8

[0089] (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;

[0090] (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;

[0091] (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;

[0092] (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;

[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, 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%.

[0094] Example 9

[0095] (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;

[0096] (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;

[0097] (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;

[0098] (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;

[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, 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%.

[0100] Example 10

[0101] (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;

[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, 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%.

[0106] Comparative Example 1

[0107] (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;

[0108] (2) concentrating the mixed solution in step (1) at 60° C. in a vacuum condition to an anhydrous state to obtain a dehydrated product;

[0109] (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;

[0110] (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;

[0111] (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%.

[0112] Comparative Example 2

[0113] (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;

[0114] (2) concentrating the mixed solution in step (1) at 60° C. in a vacuum condition to an anhydrous state to obtain a dehydrated product;

[0115] (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;

[0116] (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;

[0117] (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%.

[0118] Comparative Example 3

[0119] (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;

[0120] (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;

[0121] (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;

[0122] (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;

[0123] (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%.

[0124] Comparative Example 4

[0125] 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;

[0126] (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;

[0127] (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;

[0128] (4) The crude product obtained in step (3) is dissolved in 95% ethanol in an amount equal to 1% of 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; no salt is precipitated under this condition.

[0129] Comparative Example 5

[0130] (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;

[0131] (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;

[0132] (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;

[0133] (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;

[0134] (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.

[0135] Comparative Example 6

[0136] (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;

[0137] (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;

[0138] (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;

[0139] (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;

[0140] (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.

[0141] 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;

[0142] The contents of sn-1-LPC-DHA and LPC in the brown-red paste product were determined by HPLC. Example 11 Evaluation test of the preventive efficacy of arachidonic acid-induced zebrafish thrombosis

[0143] 1. Experimental Materials

[0144] 1.1. Experimental samples

[0145] Hydrolyzed krill oil extract group (prepared in Example 2); positive control group (aspirin); model control group (arachidonic acid modeling, no medication). Each group of samples was prepared with 0.5% Tween 80 to a specified concentration and used freshly prepared every day.

[0146] Each group of samples was prepared in water, and the sample concentrations as well as the concentrations of DHA and EPA contained therein were tested, as shown in Table 1.

[0147] Table 1

[0148]

[0149] o-Dianisidine (purchased from Merck), arachidonic acid (purchased from Merck), and aspirin (purchased from Beijing Solaibao Technology Co., Ltd.).

[0150] 1.2 Experimental animals

[0151] Zebrafish were raised in fish farming water at 28°C and bred and provided by the fish farming center of Nanjing Northern Lights Quality Inspection Technology Service Co., Ltd.

[0152] 2. Evaluation methods and results

[0153] 2.1 Statistics

[0154] The statistical results were expressed as mean ± SE and analyzed using SPSS 26.0 software. p < 0.05 indicated that the difference was statistically significant.

[0155] 2.2 Evaluation of thrombosis prevention efficacy—heart / trunk red blood cell staining

[0156] The melanin allele mutant Ablino strain zebrafish larvae of 3 dpf (days post fertilization) with normal development and consistent status were randomly selected and placed in a 6-well plate. Thirty zebrafish were treated in each well (experimental group), and the capacity of each well was 3 mL. The experimental group was given water-soluble samples of each group, and the sample concentrations are shown in Table 1. 25 mg / L aspirin was added to the positive drug group. At the same time, a normal control group (no treatment) and a model control group (no medication) were set up. After treatment at 28°C for 2 hours, except for the normal group, all other experimental groups were given water-soluble arachidonic acid to establish a zebrafish thrombosis model. After staining with o-dianisidine, 15 fish were randomly selected from each group and placed under a stereo microscope for photography. The images were analyzed using NIS-Elements D5.41 advanced image processing software, and the staining intensity of heart and trunk red blood cells was calculated (the parameter in the software is the sum of opacities). The statistical analysis results of this indicator were used to evaluate the efficacy of the samples in preventing thrombosis. The results are shown in Table 2. Figure 1 and Figure 2 .

[0157] Heart region opacity improvement rate (%) = (heart region opacity area of ​​model control group - heart region opacity area of ​​drug-treated group) / (heart region opacity area of ​​model control group - heart region opacity area of ​​normal control group) * 100%. Other improvement rates were calculated using similar methods.

[0158] As shown in Table 2, the hydrolyzed krill oil extract group can significantly reduce the opacity of the trunk and increase the opacity of the heart area, and the effect is better than the positive control group aspirin.

[0159] Table 2

[0160]

[0161] 2.3 Evaluation of the efficacy of preventing thrombosis—platelet aggregation

[0162] Normal and consistent 4dpf platelet fluorescence strain zebrafish larvae were randomly selected and placed in a 6-well plate. Thirty zebrafish were treated in each well (experimental group), and the volume of each well was 3mL. The experimental group was given water-soluble samples of each group. The sample concentrations are shown in Table 1. 25mg / L aspirin was added to the positive drug group. At the same time, a normal control group (no treatment) and a model control group (no medication) were set up. After 2h of treatment at 28℃, arachidonic acid was given to all experimental groups except the normal group to establish a zebrafish thrombosis model. Fifteen fish were randomly selected from each group and placed under a fluorescence microscope to observe platelet aggregation and take pictures. The images were analyzed using NIS-Elements D 5.41 advanced image processing software to calculate platelet aggregation (the parameter in the software is the sum of brightness). The statistical analysis results of this index were used to evaluate the efficacy of the sample in preventing thrombosis, as shown in Tables 3 and 4. Figure 3 shown.

[0163] Compared with the control group, the total brightness of the zebrafish trunk in the model group increased, and the platelet fluorescence intensity increased significantly, indicating that more thrombus formation occurred; compared with the model group, the hydrolyzed krill oil extract group could reduce the total brightness of the zebrafish trunk area and reduce thrombus formation.

[0164] Table 3

[0165] Group Total brightness of the trunk area (mean±SE) Normal control group 103321.11±7739.86**** Model control group 189416.69±11624.81 Hydrolyzed Krill Oil Extract 114565.72±13824.53*** Improvement rate of hydrolyzed krill oil extract group 86.9％

[0166] 2.4 Evaluation of the efficacy of preventing thrombosis—blood flow velocity and cardiac output testing

[0167] AB strain zebrafish larvae at 3dpf with normal development and consistent status were randomly selected and placed in a 6-well plate. 30 zebrafish were treated in each well (experimental group), and the capacity of each well was 3mL. The experimental group was given water-soluble samples of each group. The sample concentrations are shown in Table 1. 25mg / L aspirin was added to the positive drug group. At the same time, a normal control group (no treatment) and a model control group (no medication) were set up. After treatment at 28°C for 2h, except for the normal group, all other experimental groups were given water-soluble arachidonic acid to establish a zebrafish thrombosis model. Ten zebrafish were randomly selected from each group and placed in a blood flow analyzer to observe and record the blood flow velocity and cardiac output. The statistical analysis results of this index were used to evaluate the efficacy of the samples in preventing thrombosis, as shown in Table 4. Figure 4 and Figure 5 shown.

[0168] As shown in Table 4, compared with the model group, the positive control group and the hydrolyzed krill oil extract group could significantly increase the blood flow velocity and cardiac output of zebrafish, among which the hydrolyzed krill oil extract group had a better effect.

[0169] In the hydrolyzed krill oil extract group, the blood flow velocity of zebrafish increased significantly, and the improvement rate was 2.5 times that of the positive control group. In addition, the improvement rate of cardiac output reached 125%, which was even better than the blank control group.

[0170] Table 4

[0171]

[0172] 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. Use of a hydrolyzed krill oil extract in the preparation of food, health products, and dietary supplements for preventing, alleviating, or improving thrombosis.

2. The use according to claim 1, characterized in that The LPC content in the hydrolyzed krill oil extract is 0-90%.

3. The use according to claim 2, characterized in that: The sn-1-LPC-DHA content in the hydrolyzed krill oil extract is 0-30%.

4. The use according to claim 2, characterized in that: The content of sn-1-LPC-DHA in LPC-DHA in the hydrolyzed krill oil extract is ≥50%.

5. The use according to claim 2, characterized in that: The total content of EPA and DHA in hydrolyzed krill oil extract is 20-50%.

6. The use according to claim 5, characterized in that In hydrolyzed krill oil extract, the mass ratio of EPA to DHA is 1:1-5.

7. The use according to any one of claims 1 to 6, characterized in that: The hydrolyzed krill oil extract is prepared by a preparation method comprising the following steps: 1) Enzymatic hydrolysis: krill oil and water are mixed, 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 concentrating and dehydrating after demulsification 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 the pH is adjusted to 8 before reaction. After the reaction is completed, the pH of the solution is adjusted to 8. After the reaction is completed, the solution is filtered to obtain a filtrate; 5) Concentration and drying: The filtrate is concentrated and dried to obtain a brown-red paste product.

8. The use according to any one of claims 1 to 7, characterized in that The food, health product, and dietary supplement further include food-acceptable additives.

9. The use according to claim 8, characterized in that The food, health product, and dietary supplement can be made into tablets, capsules, soft capsules, gel candies, oral liquids, or soft candies.

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