Green preparation method of euphausia superba oil with high phospholipid content

Abstract

The invention discloses a green preparation method of euphausia superba oil with high phospholipid content, which comprises the following steps: mixing euphausia superba oil with water added with phytosterol, stirring, centrifuging, layering the mixed system to obtain an upper glyceride layer and a lower phospholipid gelatinous layer; and finally, transferring part of liquid from the obtained glyceride layer, and carrying out reduced pressure evaporation dehydration treatment on the residual mixed system to obtain the euphausia superba oil with high phospholipid content. According to the method, the phospholipid is induced to form micelles in a water phase by utilizing the amphipathy of the phospholipid, so that the efficient phase separation of the phospholipid and the triglyceride is realized. Meanwhile, phytosterol and water are added into the euphausia superba oil, so that the euphausia superba oil is enriched in a water phase to form micelles, interface modification is performed by utilizing the phytosterol, the enrichment effect is improved, the euphausia superba oil with high phospholipid content is expected to be obtained, and the problems of high solvent consumption, high production cost and the like in the prior art are solved.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of deep processing of Antarctic krill oil, and particularly relates to a green preparation method of phospholipid with high phospholipid content in Antarctic krill oil. BACKGROUND

[0002] Antarctic krill (Euphausia superba) Euphausia superba ) mainly concentrates in the continental shelf waters of the Antarctic, has a huge biological storage capacity and high nutritional value, and has great development and utilization potential. Antarctic krill oil is rich in phospholipids, omega-3 long-chain polyunsaturated fatty acids (omega-3 LC-PUFA), astaxanthin, vitamins and other functional ingredients. Among them, omega-3 LC-PUFA such as eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) mainly exist in the form of phospholipids. Studies have shown that phospholipid omega-3 PUFA has better biological activity than triglyceride omega-3 PUFA and ethyl ester omega-3 PUFA, higher tissue absorption rate and bioavailability. In addition, phospholipid omega-3 LC-PUFA is the only structure that can penetrate the blood-brain barrier among the three, and can play a good protective role on the central nervous system, especially the brain. Studies have shown that phospholipid omega-3 PUFA shows better effects of lowering blood lipids, lowering blood sugar, anti-inflammatory and improving memory than triglyceride omega-3 PUFA. Therefore, the content of phospholipids determines the strength of the efficacy of Antarctic krill oil, and in order to improve the quality of Antarctic krill oil products and increase the value of the products, it is urgent to increase the content of phospholipids.

[0003] At present, the methods for purifying and enriching phospholipids from oil mainly include organic solvent extraction method and column chromatography method. The organic solvent extraction method is a relatively traditional method, and the principle of this method is to separate and purify phospholipids according to the different solubilities of phospholipids and other lipid components in different solvents, and the most commonly used method is cold acetone extraction. Although the organic solvent extraction method can be applied to large-scale industrial production, the production conditions are harsh, and there are problems such as environmental unfriendliness of organic reagents, complex solvent recovery process, solvent residues in products, etc. The principle of column chromatography method is to separate and purify phospholipids according to the differences in the interaction ability of lipid components with column fillers, and to achieve the purpose of separation and purification of phospholipids through different elution sequences. Although the column chromatography method can obtain products with high phospholipid content, it has problems such as low processing capacity, long elution time, high toxicity and large consumption of solvents, and high production cost. Therefore, it is urgent to establish a new phospholipid enrichment technology that is green and environmentally friendly, has a simple process flow, low production cost, and can achieve high phospholipid content. SUMMARY

[0004] In view of the above problems, the purpose of the present application is to provide a green method for enriching phospholipids from Antarctic krill oil. By adding phytosterol and water to Antarctic krill oil, the krill oil is enriched in the water phase to form micelles, and the interface is modified with phytosterol to improve the enrichment effect, so as to obtain Antarctic krill oil with high phospholipid content, so as to solve the problems of large solvent consumption and high production cost in the prior art method.

[0005] In order to achieve the above purpose, the present application provides a green preparation method of Antarctic krill oil with high phospholipid content, comprising the following steps: S1, mixing Antarctic krill oil with water to which phytosterol is added to obtain an oil-water mixed system; S2, stirring the oil-water mixed system obtained in step S1; S3, centrifuging the stirred mixed system obtained in step S2 to separate the mixed system into upper glyceride layer and lower phospholipid gum layer; S4, removing part of the liquid from the glyceride layer obtained in step S3, and performing vacuum evaporation dehydration treatment on the remaining mixed system to obtain Antarctic krill oil with high phospholipid content.

[0006] In an embodiment of the present application, in step S1, the concentration of phytosterol in water is 0.2-1.5 g / L, preferably 0.2-1.0 g / L, and further preferably 0.3-0.8 g / L. The water should be soft water such as distilled water, pure water, and preferably pure water. The mass ratio (kg / kg) of water to Antarctic krill oil should be 1:4-1:10, preferably 1:6.

[0007] In an embodiment of the present application, in step S1, the phytosterol is at least one of beta-sitosterol, campesterol, and stigmasterol.

[0008] In an embodiment of the present application, in step S2, the stirring temperature of the oil-water mixed system is 10-40℃; the stirring speed is 400-600 rpm; and the stirring time is 3-12 h, preferably 12 h.

[0009] In an embodiment of the present application, in step S3, the centrifugation conditions of the mixed system are 3000-5000 g, preferably 5000 g; the centrifugation time is 3-5 min, preferably 5 min; and the centrifugation temperature is 4-40℃, preferably 20℃.

[0010] In an embodiment of the present application, in step S4, the mass of the removed liquid is 33-43% of the mass of the Antarctic krill oil. When the ratio of the Antarctic krill oil to water is 1:6, the phospholipid content in the Antarctic krill oil obtained by the reduced pressure evaporation is about 60% when the amount of the upper glyceride taken after centrifugation is 33% of the mass of the Antarctic krill oil, the phospholipid content in the Antarctic krill oil obtained by the reduced pressure evaporation is about 65% when the amount of the upper glyceride taken after centrifugation is 39% of the mass of the Antarctic krill oil, and the phospholipid content in the Antarctic krill oil obtained by the reduced pressure evaporation is about 70% when the amount of the upper glyceride taken after centrifugation is 43% of the mass of the Antarctic krill oil.

[0011] In an embodiment of the present application, the reduced pressure evaporation condition in step S4 is that the vacuum degree is 0.01-0.04 MPa and the temperature is 60-100 ℃.

[0012] The present application also provides the Antarctic krill oil with high phospholipid content prepared by the above method.

[0013] The present application also provides the use of the above Antarctic krill oil in food and medicine.

[0014] Beneficial effects: 1. The present application uses the amphiphilic property of phospholipids to induce the formation of micelles and self-assembly aggregation in the aqueous phase, realizes the efficient phase separation of phospholipids and triglycerides, and increases the phospholipid content to ≥55% and the concentration is controllable; in the process of enriching krill oil, the added phytosterol can modify the oil-water interface to improve the enrichment effect. The present application solves the green processing problem of high phospholipid products, significantly improves the health efficacy and processing applicability of the products, and provides core raw material support for the development of high-value functional food and medical products.

[0015] 2. The present application can realize the effective separation of phospholipids and triglycerides by the method of phospholipid hydration condensation, and can obtain Antarctic krill phospholipid oil with a higher phospholipid content (50-70%) by controlling the process conditions, which can be used as a good raw material for health food, special medical formula food and medical products.

[0016] 3. The present application does not use organic solvents, but uses water as a medium to effectively enrich krill oil, and the preparation method has the significant advantages of green and no pollution, and the prepared krill oil has good antioxidant property, which greatly expands its application field.

[0017] 4. The production process of the present application is simple and the production cost is low. DETAILED DESCRIPTION

[0018] Raw material sources Commercialized Antarctic krill oil (phospholipid content about 40%) was stored in a refrigerator at -18 ℃ in the dark. Phosphatidylcholine standard was from Avanti Polar Lipids (USA). Phosphatidylethanolamine standard was from TCI (Shanghai) Chemical Industry Development Co., Ltd. The mixture of 37 fatty acid methyl ester standards was from Sigma (USA).

[0019] Detection process 1. Phospholipid content detection: 1.1 High performance liquid chromatography-corona charged aerosol detector (CAD) analysis method Liquid separation was performed using a Thermo liquid chromatography system (UltiMate 3000 SD) and an Agilent normal phase silica gel column (Zorbax Rx-SIL; 4.6 mm × 250 mm, 5 µm). The injection volume was 4 μL, and the column oven temperature and the flow rate of the chromatographic column were maintained at 40 ℃ and 1.0 mL / min, respectively. The mobile phase was composed of isopropanol (I), n-hexane (II), and a solution of methanol / water (2:1, v / v) containing 0.05% glacial acetic acid and 0.01% triethylamine (III). The elution gradient conditions are shown in Table 1 below. After each determination run, the chromatographic column was equilibrated with mobile phase II for 10 min. The liquid phase back-end detector was detected using a Thermo CAD detector (Corona Veo). The CAD detector parameters were as follows: atomization temperature 35 ℃, power rate 1.00, sampling frequency 10 Hz, filter constant 5.0 s, and power function value 1.00. The data processing and quantitative analysis were performed using Thermo Chromeleon software (7.2 SR5).

[0020] 1.2 Standard solution and sample preparation Phosphatidylcholine and phosphatidylethanolamine standards were dissolved in chromatographic grade chloroform-methanol (chloroform / methanol, v / v, 2:1) to prepare a phosphatidylcholine standard stock solution with a concentration of 5 mg / mL and a phosphatidylethanolamine standard stock solution with a concentration of 2 mg / mL. Chromatographic grade chloroform was used to dilute the phosphatidylcholine and phosphatidylethanolamine standard stock solutions to obtain standard solutions of different concentrations for calibration, with the concentration gradient of the phosphatidylcholine standard solutions being 5 mg / mL, 2 mg / mL, 1 mg / mL, 0.5 mg / mL, 0.2 mg / mL, and 0.1 mg / mL, respectively, and the concentration gradient of the phosphatidylethanolamine standard solutions being 2 mg / mL, 1 mg / mL, 0.5 mg / mL, 0.2 mg / mL, 0.1 mg / mL, 0.05 mg / mL, 0.02 mg / mL, and 0.01 mg / mL, respectively. All standard solutions were prepared fresh before analysis to prevent lipid decomposition or oxidation. The raw signal current of CAD is related to the number of charges on the particles and is not linearly related to the sample concentration, so a power function curve is constructed by plotting the average peak area against the sample concentration. The calibration curve is fitted according to the CAD response signal of the standard solutions at different concentration points. For actual samples, 500 mg of lipid sample was first weighed and fully dissolved in chloroform to prepare a sample solution with a concentration of 5 mg / mL. The solutions of the standard and sample were filtered through a 0.22 µm microporous organic membrane and then directly injected into the HPLC-CAD system for analysis. In this patent, the standard curve information of phosphatidylcholine and phosphatidylethanolamine is shown in Table 4. The contents of phosphatidylcholine and phosphatidylethanolamine in Antarctic krill oil were calculated by the standard curve method.

[0021] Table 1 Elution gradient conditions for analyzing phospholipids by forward chromatography

[0022] Table 2 Standard curve, retention time, and correlation coefficient of PC and PE

[0023] 2. Fatty acid composition: 2.1 Gas chromatography-hydrogen flame ionization detector (FID) analysis method Gas chromatography separation of fatty acid methyl esters was performed using an FID detector and a DB-FastFAME capillary column (30 m x 0.25 mm i.d., 0.25 μm). The chromatography method was optimized according to the conditions of national standard GB 5009.168-2016. The initial oven temperature was held at 80 °C for 0.5 min, then ramped to 165 °C at 40 °C / min and held for 1 min, followed by ramping to 185 °C at 2 °C / min and held for 1 min, and finally ramped to 230 °C at 4 °C / min and held for 3 min. The injector temperature was set at 250 °C, the injection volume was 1 μL, and the split ratio was 50:1. The constant carrier gas (N2) flow was set at 1.0 mL / min, and the FID temperature was set at 260 °C.

[0024] 2.2 Standard solution and sample preparation A standard stock solution was prepared by dissolving a mixture of 37 fatty acid methyl ester standards in chromatographic grade chloroform. In a round-bottom flask, 5 mg of lipid sample, 200 μL of internal standard solution (1 mg / mL undecanoic triglyceride dissolved in chloroform), and 2 mL of 0.5 mol / L sodium hydroxide-methanol solution were added. After thorough mixing, the round-bottom flask was refluxed in a water bath at 80 °C for 5 min, and then 2 mL of boron trifluoride-methanol solution (14%, w / w) was added through the inlet at the top of the condenser and reacted at 80 °C for 2 min. Subsequently, the round-bottom flask was cooled to room temperature, and 1.5 mL of n-hexane was added to extract the fatty acid methyl esters by thorough shaking. After standing, the upper n-hexane organic layer was collected, and an appropriate amount of anhydrous sodium sulfate was added to remove excess water for 1 h. Before GC analysis, the n-hexane solution containing fatty acid methyl esters was filtered through a 0.22 μm microporous organic membrane. All fatty acids were qualitatively determined by comparing their chromatographic retention times with those of the 37 fatty acid methyl ester standard mixture. The fatty acid content was calculated by internal standard and response factor according to the following formula:

[0025] In the formula, FAi is the content of fatty acid i in the sample, in units of grams per hundred grams (g / 100 g); F is the response factor of fatty acid methyl ester i; A is the peak area of fatty acid methyl ester i in the sample; A is the peak area of the internal standard, undecanoic acid methyl ester, added to the sample; p is the concentration of undecanoic triglyceride, in units of milligrams per milliliter (mg / mL); and V is the injection volume of the internal standard solution. i i i C11 C11 C11 ​​​​​is the volume of triundecanoin added to the sample in milliliters (mL); 1.0067 is the conversion factor for converting triundecanoin to fatty acid methyl esters; m is the mass of the sample in milligrams (mg); F FAME-FAi is the conversion factor for converting fatty acid methyl ester i to fatty acid.

[0026] Response factor F for fatty acid methyl ester i i is calculated according to the following equation:

[0027] F in the equation i is the response factor for fatty acid methyl ester i; p i is the concentration of each fatty acid methyl ester i in the mix standard in milligrams per milliliter (mg / mL); A 11 is the peak area of triundecanoin; A i is the peak area of fatty acid methyl ester i; p 11 is the concentration of triundecanoin in the mix standard in milligrams per milliliter (mg / mL).

[0028] Finally, the content of the individual fatty acid is divided by the total fatty acid content to obtain the relative content of the individual fatty acid.

[0029] 3. Peroxide value determination: Dissolve 0.01 g of Euphausia superba oil in 1.5 mL of a mixture of dichloromethane and 95% ethanol (volume / volume, w / w, 3:2). Then, add 5 mM ferrous ammonium sulfate hexahydrate (100 μί), 1 M methanolic xylenol orange tetrasodium salt (200 μί), and 0.25 M methanolic sulfuric acid (200 μί), and mix well. After 30 min at room temperature in the dark, add pure water (1 mL) to the reaction. Then centrifuge at 4000 x g for 5 min. Take the upper layer of the mixture (200 μί) and measure the absorbance at 560 nm (A 560 ). After drawing a standard curve of cumene hydroperoxide, calculate the POV value (mmol / kg oil).

[0030] 4. Thiobarbituric acid value determination: Mix 0.10 g of Euphausia superba oil with a mixture (2.5 mL) including distilled water (196 mL), concentrated hydrochloric acid solution (4.17 mL), thiobarbituric acid (0.75 g), and trichloroacetic acid (30 g). After heating the mixture in a boiling water bath for 10 min, centrifuge at 3000 x g for 10 min after cooling, and take the upper layer of the mixture (200 μί) and measure the absorbance at 532 nm (A 532). The malondialdehyde concentration was converted to TBARS value according to the following formula.

[0031] TBARS (ppm) = A 532 x 2.77 Example 1 A green preparation method of high phospholipid content Antarctic krill oil, comprising the following steps: S1: A mixture of β-sitosterol and water with a concentration of 0.5 g / L is added to Antarctic krill oil to obtain an oil-water mixed system, and the mass ratio of water to Antarctic krill oil in the mixed system is 1:6.

[0032] S2: The oil-water mixed system obtained in S1 is stirred at 10°C and a rotation speed of 600 rpm for 12 h.

[0033] S3: The oil-water mixed system obtained in S2 is centrifuged at a centrifugal temperature of 22°C, a centrifugal force of 5000 g, and a centrifugal time of 5 min to obtain an upper glyceride layer and a lower phospholipid gum layer.

[0034] S4: A liquid equivalent to 33% of the mass of Antarctic krill oil is removed from the glyceride liquid layer of S3, and the remaining mixed system is dehydrated at a vacuum degree of 0.04 Mpa and a temperature of 100°C for 1 h to obtain Antarctic krill phospholipids with a purity of about 60%.

[0035] Example 2 The difference between Example 2 and Example 1 is that in step S4, the mass of the removed liquid is 39% of the mass of Antarctic krill oil.

[0036] A green preparation method of high phospholipid content Antarctic krill oil, comprising the following steps: S1: A mixture of β-sitosterol and water with a concentration of 0.5 g / L is added to Antarctic krill oil to obtain an oil-water mixed system, and the mass ratio of water to Antarctic krill oil in the mixed system is 1:6.

[0037] S2: The oil-water mixed system obtained in S1 is stirred at 10°C and a rotation speed of 600 rpm for 12 h.

[0038] S3: The oil-water mixed system obtained in S2 is centrifuged at a centrifugal temperature of 22°C, a centrifugal force of 5000 g, and a centrifugal time of 5 min to obtain an upper glyceride layer and a lower phospholipid gum layer.

[0039] S4: A liquid equivalent to 39% of the mass of Antarctic krill oil is removed from the glyceride liquid layer of S3, and the remaining mixed system is dehydrated at a vacuum degree of 0.04 Mpa and a temperature of 100°C for 1 h to obtain Antarctic krill phospholipids with a purity of about 65%.

[0040] Example 3 The difference between Example 3 and Example 1 is that in step S4, the mass of the removed liquid is 43% of the mass of the Antarctic krill oil.

[0041] A green preparation method of Antarctic krill oil with high phospholipid content, comprising the following steps: S1: A mixture of β-sitosterol and water with a concentration of 0.5 g / L is added to Antarctic krill oil to obtain an oil-water mixed system, and the mass ratio of water to Antarctic krill oil in the mixed system is 1:6.

[0042] S2: The oil-water mixed system obtained in S1 is stirred at 10°C and a rotation speed of 600 rpm for 12 h.

[0043] S3: The oil-water mixed system obtained in S2 is centrifuged at a centrifugal temperature of 22°C, a centrifugal force of 5000 g, and a centrifugal time of 5 min to obtain an upper glyceride layer and a lower phospholipid gum layer.

[0044] S4: From the glyceride liquid layer of step S3, a liquid equivalent to 43% of the mass of the Antarctic krill oil is removed, and the remaining mixed system is dehydrated at a vacuum degree of 0.03 Mpa and a temperature of 100°C for 0.5 h to obtain Antarctic krill phospholipids with a purity of about 70%.

[0045] Comparative Example 1 The difference between Comparative Example 1 and Example 1 is that the addition concentration of β-sitosterol is 4.0 g / L.

[0046] S1: A mixture of β-sitosterol and water with a concentration of 4.0 g / L is added to Antarctic krill oil to obtain an oil-water mixed system, and the mass ratio of water to Antarctic krill oil in the mixed system is 1:6.

[0047] S2: The oil-water mixed system obtained in S1 is stirred at 10°C and a rotation speed of 600 rpm for 12 h.

[0048] S3: The oil-water mixed system obtained in S2 is centrifuged at a centrifugal temperature of 22°C, a centrifugal force of 5000 g, and a centrifugal time of 5 min to obtain an upper glyceride layer and a lower phospholipid gum layer.

[0049] S4: From the glyceride liquid layer of step S3, a liquid equivalent to 33% of the mass of the Antarctic krill oil is removed, and the remaining mixed system is dehydrated at a vacuum degree of 0.04 Mpa and a temperature of 100°C for 1 h to obtain Antarctic krill phospholipids with a purity of about 60%.

[0050] Comparative Example 2 The difference between Comparative Example 2 and Example 1 is that the phytosterol is replaced by phytostanol.

[0051] S1: A mixture of β-sitosterol and water with a concentration of 0.5 g / L was added to the Euphausia superba oil to obtain an oil-water mixed system, and the mass ratio of water to Euphausia superba oil in the mixed system was 1:6.

[0052] S2: The oil-water mixed system obtained in S1 was stirred at 10 °C and a rotation speed of 600 rpm for 12 h.

[0053] S3: The oil-water mixed system obtained in S2 was centrifuged at a centrifugal temperature of 22 °C, a centrifugal force of 5000 g and a centrifugal time of 5 min to obtain a glyceride layer on the top and a phospholipid gum layer on the bottom.

[0054] S4: From the glyceride liquid layer in step S3, an amount of liquid equivalent to 33% of the mass of Euphausia superba oil was removed, and the remaining mixed system was dehydrated at a vacuum degree of 0.04 Mpa and a temperature of 100 °C for 1 h to obtain Euphausia superba phospholipids with a purity of about 58%.

[0055] Comparative Example 3 The difference between Comparative Example 3 and Example 1 is that the addition of β-sitosterol is omitted.

[0056] S1: Pure water was added to Euphausia superba oil at a mass ratio of 1:6 (water / Euphausia superba oil, kg / kg).

[0057] S2: The oil-water mixed system obtained in S1 was stirred at 10 °C and a rotation speed of 600 rpm for 12 h.

[0058] S3: The oil-water mixed system obtained in S2 was centrifuged at a centrifugal temperature of 22 °C, a centrifugal force of 5000 g and a centrifugal time of 5 min to obtain a glyceride layer on the top and a phospholipid gum layer on the bottom.

[0059] S4: From the glyceride liquid layer in step S3, an amount of liquid equivalent to 33% of the mass of Euphausia superba oil was removed, and the remaining mixed system was dehydrated at a vacuum degree of 0.04 Mpa and a temperature of 100 °C for 1 h to obtain Euphausia superba phospholipids with a purity of about 58%.

[0060] Comparative Example 4 The difference between Comparative Example 4 and Example 2 is that in step S1, the mass ratio of water to Euphausia superba oil is 1:2.

[0061] S1: A mixture of β-sitosterol and water with a concentration of 0.5 g / L was added to the Euphausia superba oil to obtain an oil-water mixed system, and the mass ratio of water to Euphausia superba oil in the mixed system was 1:2.

[0062] S2: The oil-water mixture system obtained in S1 was stirred at 10 ℃ and a rotation speed of 600 rpm for 12 h.

[0063] S3: The oil-water mixture system obtained in S2 was centrifuged at a centrifugal temperature of 22 ℃, a centrifugal force of 5000 g and a centrifugal time of 5 min to obtain a glyceride layer on the top and a phospholipid gum layer on the bottom.

[0064] S4: From the glyceride liquid layer in step S3, a liquid equivalent to 39% of the mass of the Antarctic krill oil was removed, and the remaining mixture was dehydrated at a vacuum degree of 0.04 Mpa and a temperature of 100 ℃ for 1 h to obtain Antarctic krill phospholipids with a purity of about 58%.

[0065] Comparative Example 5 Comparative Example 5 differs from Comparative Example 3 in that the stirring temperature in step S2 is 60 ℃.

[0066] S1: Pure water was added to the Antarctic krill oil at a mass ratio of 1:6 (water / Antarctic krill oil, kg / kg).

[0067] S2: The oil-water mixture system obtained in S1 was stirred at 60 ℃ and a rotation speed of 600 rpm for 12 h.

[0068] S3: The oil-water mixture system obtained in S2 was centrifuged at a centrifugal temperature of 22 ℃, a centrifugal force of 5000 g and a centrifugal time of 5 min to obtain a glyceride layer on the top and a phospholipid gum layer on the bottom.

[0069] S4: From the glyceride liquid layer in step S3, a liquid equivalent to 33% of the mass of the Antarctic krill oil was removed, and the remaining mixture was dehydrated at a vacuum degree of 0.04 Mpa and a temperature of 100 ℃ for 1 h to obtain Antarctic krill phospholipids with a purity of about 57%.

[0070] Comparative Example 6 Comparative Example 6 differs from Comparative Example 3 in that the stirring temperature in step S2 is 80 ℃.

[0071] S1: Pure water was added to the Antarctic krill oil at a mass ratio of 1:6 (water / Antarctic krill oil, kg / kg).

[0072] S2: The oil-water mixture system obtained in S1 was stirred at 80 ℃ and a rotation speed of 600 rpm for 12 h.

[0073] S3: The oil-water mixture system obtained in S2 was centrifuged at a centrifugal temperature of 22 ℃, a centrifugal force of 5000 g and a centrifugal time of 5 min to obtain a glyceride layer on the top and a phospholipid gum layer on the bottom.

[0074] S4: Remove liquid equivalent to 33% of the mass of Antarctic krill oil from the glycerol ester liquid layer in step S3, and then dehydrate the remaining mixture for 1 h under vacuum of 0.04 MPa and temperature of 100 °C to obtain Antarctic krill phospholipids with a purity of approximately 55%.

[0075] Comparative Example 7 The difference between Comparative Example 7 and Example 1 is that in step S4, the mass of the liquid transferred is 20% of the mass of Antarctic krill oil.

[0076] S1: Add a mixture of β-sitosterol (0.5 g / L) and water to Antarctic krill oil to obtain an oil-water mixture system with a mass ratio of water to Antarctic krill oil of 1:6.

[0077] S2: The oil-water mixture obtained in S1 was stirred at 10 ℃ and 600 rpm for 12 h.

[0078] S3: Centrifuge the oil-water mixture obtained in S2 at a temperature of 22 ℃, a centrifugal force of 5000 g, and a centrifugation time of 5 min to obtain an upper glycerol ester layer and a lower phospholipid colloidal layer.

[0079] S4: Remove liquid equivalent to 20% of the mass of Antarctic krill oil from the glycerol ester liquid layer in step S3, and then dehydrate the remaining mixture for 1 h under vacuum of 0.04 MPa and temperature of 100 °C to obtain Antarctic krill phospholipids with a purity of approximately 50%.

[0080] Comparative Example 8 The difference between Comparative Example 8 and Example 2 is that a 1.5% aqueous solution of citric acid was used instead of the mixture of β-sitosterol and water.

[0081] S1: Add 1.5% citric acid solution to Antarctic krill oil and mix them at a mass ratio of 1:6 (citric acid solution / Antarctic krill oil, kg / kg) to obtain an oil-water mixture system.

[0082] S2: The oil-water mixture obtained in S1 was stirred at 10 ℃ and 600 rpm for 12 h.

[0083] S3: Centrifuge the oil-water mixture obtained in S2 at a temperature of 22 ℃, a centrifugal force of 5000 g, and a centrifugation time of 5 min to obtain an upper glycerol ester layer and a lower phospholipid colloidal layer.

[0084] S4: Remove liquid equivalent to 39% of the mass of Antarctic krill oil from the glycerol ester liquid layer in step S3, and then dehydrate the remaining mixture for 1 h under vacuum of 0.04 MPa and temperature of 100 °C to obtain Antarctic krill phospholipids with a purity of approximately 57%.

[0085] The phospholipid content of Antarctic krill oil feedstock and the high-phospholipid Antarctic krill phospholipids obtained in Examples 1-3 were tested, and the results are shown in Table 3. The Antarctic krill oil feedstock contained 382.43 mg / g of phosphatidylcholine and 6.75 mg / g of phosphatidylethanolamine, totaling 389.18 mg / g. The Antarctic krill oil sample with approximately 60% phospholipid content obtained in Example 1 contained 607.27 mg / g of phosphatidylcholine and 9.76 mg / g of phosphatidylethanolamine, totaling 617.03 mg / g. The Antarctic krill oil sample with approximately 65% ​​phospholipid content obtained in Example 2 contained 641.03 mg / g of phosphatidylcholine and 11.91 mg / g of phosphatidylethanolamine, totaling 652.94 mg / g. The Antarctic krill oil sample obtained in Example 3, with a phospholipid content of approximately 70%, had a phosphatidylcholine content of 694.23 mg / g and a phosphatidylethanolamine content of 11.36 mg / g, totaling 705.59 mg / g. This demonstrates that the present invention, through control of process conditions, can effectively achieve the green preparation of Antarctic krill oil with varying phospholipid contents.

[0086] Table 3. Phospholipid content of commercially available krill oil, Antarctic krill oil obtained in Examples 1-3 and Comparative Examples 1-8.

[0087] Note: Experimental data in the table are expressed as mean ± standard deviation (n=3); where PC represents phosphatidylcholine and PE represents phosphatidylethanolamine. Different lowercase letters in the table indicate that the differences between samples are statistically significant. p <0.05.

[0088] The fatty acid composition of Antarctic krill oil feedstock and the high-phospholipid-content Antarctic krill phospholipids obtained in Examples 1-3 was analyzed, and the results are shown in Table 4. In the Antarctic krill oil feedstock, saturated fatty acids accounted for 44.60%, monounsaturated fatty acids for 30.16%, and polyunsaturated fatty acids for 25.24%, of which EPA+DHA accounted for 23.83%. In the Antarctic krill oil sample with a phospholipid content of approximately 60% obtained in Example 1, saturated fatty acids accounted for 40.86%, monounsaturated fatty acids for 23.90%, and polyunsaturated fatty acids for 35.24%, of which EPA+DHA accounted for 32.83%. In the Antarctic krill oil sample with a phospholipid content of approximately 65% ​​obtained in Example 2, saturated fatty acids accounted for 37.45%, monounsaturated fatty acids for 24.79%, and polyunsaturated fatty acids for 37.76%, of which EPA+DHA accounted for 35.26%. In Example 3, the Antarctic krill oil sample with a phospholipid content of approximately 70% contained 33.78% saturated fatty acids, 25.84% monounsaturated fatty acids, and 40.38% polyunsaturated fatty acids, of which EPA+DHA accounted for 38.56%. It is evident that the high-phospholipid Antarctic krill oil prepared by this invention has a higher content of polyunsaturated fatty acids, particularly EPA and DHA, resulting in better health benefits.

[0089] Table 4. Fatty acid content in commercially available krill oil, Antarctic krill oil obtained in Examples 1-3 and Comparative Examples 1-8

[0090] Note: Experimental data in the table are expressed as mean ± standard deviation (n=3); where SFA represents saturated fatty acids; MUFA represents monounsaturated fatty acids; and PUFA represents polyunsaturated fatty acids. Different lowercase letters in the table indicate that the differences between samples are significant. p <0.05.

[0091] The oxidation index of Antarctic krill oil and the high-phospholipid-content Antarctic krill phospholipids prepared in Examples 1-3 were tested, and the results are shown in Table 5. The peroxide value of Antarctic krill oil was 1.45 mmol / kg, and the thiobarbituric acid value was 0.13 mg MDA / kg. The peroxide value of the Antarctic krill phospholipid prepared in Example 1 was 1.56 mmol / kg, and the thiobarbituric acid value was 0.14 mg MDA / kg. The peroxide value of the Antarctic krill phospholipid prepared in Example 2 was 1.67 mmol / kg, and the thiobarbituric acid value was 0.14 mg MDA / kg. The peroxide value of the Antarctic krill phospholipid prepared in Example 3 was 1.84 mmol / kg, and the thiobarbituric acid value was 0.15 mg MDA / kg. This indicates that the high-phospholipid-content Antarctic krill phospholipids prepared in these examples have a similar degree of oxidation and secondary oxidation product content to the raw oil, and possess good quality.

[0092] Table 5. Peroxide value (POV) and thiobarbituric acid value (TBARS) of commercially available krill oil, Examples 1-3 and Comparative Examples 1-8 Antarctic krill oil.

[0093] Note: Experimental data in the table are expressed as "mean ± standard deviation" (n=3); different lowercase letters in the table indicate that the differences between samples are significant. p <0.05.

[0094] The difference between Comparative Example 1 and Example 1 lies in the amount of phytosterols added. The prepared sample contained 601.83 mg / g of phosphatidylcholine and 9.14 mg / g of phosphatidylethanolamine, totaling 610.97 mg / g. Saturated fatty acids accounted for 41.29%, monounsaturated fatty acids for 23.78%, and polyunsaturated fatty acids for 34.93%, of which EPA+DHA accounted for 31.03%. The peroxide value was 1.65 mmol / kg, and the thiobarbituric acid value was 0.15 mg MDA / kg. The corresponding phospholipid and fatty acid contents of Comparative Example 1 were lower than those of Example 1, indicating that the increased addition of phytosterols had a certain inhibitory effect on the enrichment of phospholipids and fatty acids and the enhancement of antioxidant properties.

[0095] The difference between Comparative Example 2 and Example 1 is that phytosterols were added instead of phytosterols. The phosphatidylcholine content of the prepared sample was 579.36 mg / g, and the phosphatidylethanolamine content was 5.59 mg / g, totaling 584.95 mg / g, which was significantly lower than that of Example 1. Saturated fatty acids accounted for 43.13%, monounsaturated fatty acids accounted for 23.12%, and polyunsaturated fatty acids accounted for 33.75%, of which EPA+DHA accounted for 30.09%. The peroxide value was 1.57 mmol / kg, and the thiobarbituric acid value was 0.15 mgMDA / kg. The corresponding fatty acid content and peroxide value were also lower than those of Example 1, indicating that different phytosterols have different enrichment effects on phospholipids. The phytosterols selected in this invention are more conducive to better enrichment of phospholipids and improve their antioxidant properties.

[0096] The difference between Comparative Example 3 and Example 1 is that no phytosterols were added. The prepared sample contained 571.55 mg / g of phosphatidylcholine and 6.89 mg / g of phosphatidylethanolamine, totaling 578.44 mg / g. Saturated fatty acids accounted for 42.57%, monounsaturated fatty acids for 23.53%, and polyunsaturated fatty acids for 33.90%, of which EPA+DHA accounted for 30.90%. The peroxide value was 1.73 mmol / kg, and the thiobarbituric acid value was 0.16 mg MDA / kg. The values ​​of Comparative Example 3 were significantly lower than those of Example 1, indicating that the addition of phytosterols plays an important role in the effective enrichment of phospholipids.

[0097] The difference between Comparative Example 4 and Example 2 is that the mass ratio of water to Antarctic krill oil was increased. The prepared sample contained 577.78 mg / g of phosphatidylcholine and 6.87 mg / g of phosphatidylethanolamine, totaling 584.65 mg / g. Saturated fatty acids accounted for 42.32%, monounsaturated fatty acids for 23.98%, and polyunsaturated fatty acids for 33.70%, of which EPA+DHA accounted for 30.36%. The peroxide value was 1.79 mmol / kg, and the thiobarbituric acid value was 0.15 mg MDA / kg.

[0098] The difference between Comparative Examples 5 and 6 and Comparative Example 3 is that high-temperature hydration was used instead of low-temperature hydration. Experiments showed that this reaction resulted in a higher phospholipid content compared to low-temperature hydration, but high-temperature hydration led to oil oxidation and quality loss. Comparative Example 5 yielded a phosphatidylcholine content of 565.67 mg / g and a phosphatidylethanolamine content of 6.22 mg / g, totaling 571.89 mg / g. The sample contained 43.87% saturated fatty acids, 24.08% monounsaturated fatty acids, and 32.05% polyunsaturated fatty acids, with EPA+DHA accounting for 29.71%. Comparative Example 6 yielded a phosphatidylcholine content of 541.41 mg / g and a phosphatidylethanolamine content of 5.88 mg / g, totaling 547.29 mg / g. The sample contained 44.23% saturated fatty acids, 23.97% monounsaturated fatty acids, and 31.80% polyunsaturated fatty acids, with EPA+DHA accounting for 28.22%. Comparative Example 5 had a peroxide value of 3.58 mmol / kg and a thiobarbituric acid value of 0.24 mg MDA / kg. Comparative Example 6 had a peroxide value of 3.73 mmol / kg and a thiobarbituric acid value of 0.27 mg MDA / kg. This indicates that its oxidation level was much higher than that of Comparative Example 3, and its quality had deteriorated significantly.

[0099] The difference between Comparative Example 7 and Comparative Example 3 is that only a liquid equivalent to 20% of the mass of Antarctic krill oil was removed from the glycerol ester liquid layer. The resulting sample contained 488.67 mg / g of phosphatidylcholine and 6.70 mg / g of phosphatidylethanolamine, totaling 495.37 mg / g, indicating a significant decrease in phospholipid content. The sample contained 44.36% saturated fatty acids, 25.36% monounsaturated fatty acids, and 30.28% polyunsaturated fatty acids, of which EPA+DHA accounted for 27.68%. Comparative Example 7 had a peroxide value of 1.98 mmol / kg and a thiobarbituric acid value of 0.14 mg MDA / kg.

[0100] Compared to Comparative Example 3, Comparative Example 8 used a 1.5% citric acid solution instead of purified water and phytosterols. Experiments showed that the phospholipid content of this reaction was similar to that of Comparative Example 3, but the use of acidic substances such as citric acid as electrolytes resulted in higher costs. Secondly, the acidification process generates wastewater, causing environmental pollution. Thirdly, the acidic environment corrodes equipment, increasing maintenance costs. The obtained sample contained 565.98 mg / g of phosphatidylcholine and 7.44 mg / g of phosphatidylethanolamine, totaling 573.42 mg / g. The sample contained 42.48% saturated fatty acids, 23.21% monounsaturated fatty acids, and 32.31% polyunsaturated fatty acids, with EPA+DHA accounting for 29.72%. The content of phospholipids and fatty acids decreased compared to the examples. The peroxide value of Comparative Example 8 was 3.02 mmol / kg, and the thiobarbituric acid value was 0.21 mg MDA / kg. This indicates that its oxidation degree was much higher than that of Comparative Example 3, and its quality deteriorated significantly.

[0101] The embodiments provided above are not intended to limit the scope of the invention, nor are the described steps intended to limit the order of execution. Any obvious modifications made to the invention by those skilled in the art based on existing common knowledge also fall within the scope of protection defined by the claims.

Claims

1. A green preparation method for Antarctic krill oil with high phospholipid content, characterized in that, Includes the following steps: S1. Mix Antarctic krill oil with water containing phytosterols to obtain an oil-water mixture system; S2. Stir the oil-water mixture obtained in step S1. S3. Centrifuge the mixed system obtained in step S2 to separate the mixed system into an upper glyceride layer and a lower phospholipid gel layer. S4. Remove a portion of the liquid from the glycerol ester layer obtained in step S3, and perform vacuum evaporation and dehydration treatment on the remaining mixture to obtain Antarctic krill oil with high phospholipid content.

2. The green preparation method according to claim 1, characterized in that, In step S1, the concentration of phytosterols in water is 0.2~1.5 g / L, and the mass ratio of water to Antarctic krill oil is 1:4~1:

10.

3. The green preparation method according to claim 1, characterized in that, In step S1, the phytosterol is at least one of β-sitosterol, campesterol, and stigmasterol.

4. The green preparation method according to claim 1, characterized in that, In step S2, the stirring temperature of the oil-water mixture is 10~40 ℃; the stirring speed is 400~600 rpm; and the stirring time is 3~12 h.

5. The green preparation method according to claim 1, characterized in that, In step S3, the centrifugation speed is 3000~5000 g, the centrifugation time is 3~5 min, and the centrifugation temperature is 4-40℃.

6. The green preparation method according to claim 1, characterized in that, In step S4, the mass of the liquid transferred is 33-43% of the mass of the Antarctic krill oil.

7. The green preparation method according to claim 1, characterized in that, During the reduced pressure evaporation in step S4, the vacuum level is 0.01~0.04 MPa and the temperature is 60~100 ℃.

8. Antarctic krill oil with high phospholipid content prepared by the green preparation method according to any one of claims 1 to 7.

9. The use of Antarctic krill oil as described in claim 8 in food and pharmaceuticals.

Images