A method for preparing antarctic krill oil by food-grade double-concentration ethanol low-temperature standing impurity removal

Abstract

The application discloses a kind of food-grade double-concentration ethanol low-temperature standing impurity removal preparation Euphausia Superba oil method, it relates to aquatic resources deep processing technical field, the method with Euphausia Superba powder as starting raw material, after low-temperature crushing sieving, first with volume fraction 75% ethanol aqueous solution is extracted once, again with volume fraction 90% ethanol aqueous solution to the first filter residue is extracted twice, after the filtrate of two times is combined and is impurity removal under the condition of 8 DEG C light avoidance, again by centrifugation, membrane filtration, reduced pressure concentration and nitrogen charging dissolving treatment processing, finally food-grade Euphausia Superba oil product is prepared, by the gradient extraction of double-concentration ethanol, the step-by-step sufficient extraction of phospholipid type lipid and weak polar lipid is realized, product yield and total phospholipid content are effectively improved, by low-temperature standing impurity removal step, protein colloid, chitinous particle and the like separable insoluble are formed flocculation precipitate and are efficiently removed, and filtration load is significantly reduced.

Description

Technical Field

[0001] This invention relates to the field of deep processing technology of aquatic resources, specifically a method for preparing Antarctic krill oil by low-temperature static purification with food-grade dual-concentration ethanol. Background Technology

[0002] Antarctic krill is a small crustacean planktonic organism with a huge biomass. It is rich in protein, chitin, astaxanthin, and unique phospholipid-type Omega-3 polyunsaturated fatty acids, of which eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) exist mainly in the form of phospholipids. This makes Antarctic krill oil superior to traditional triglyceride-type fish oil in terms of bioavailability.

[0003] Currently, the main methods for preparing Antarctic krill oil include organic solvent extraction, supercritical fluid extraction, and low-carbon alcohol extraction. Organic solvent extraction often uses non-polar or low-polar solvents such as n-hexane, petroleum ether, No. 6 solvent oil, cyclohexane, and ethyl acetate, posing safety risks due to solvent residue. Furthermore, the desolventizing process requires high temperatures, which can easily lead to the oxidative degradation of polyunsaturated fatty acids and astaxanthin. While supercritical carbon dioxide extraction produces pure products with no solvent residue, it requires significant equipment investment and has high operating costs. It also has low extraction efficiency for highly polar phospholipids, limiting its industrial application. Low-carbon alcohol extraction typically uses an aqueous ethanol solution as the extraction solvent. Ethanol is a permitted solvent in food processing, offering advantages such as high safety and mild operating conditions, making it more suitable for preparing food-grade Antarctic krill oil.

[0004] However, existing ethanol extraction technologies, when using a single concentration of ethanol for a single extraction, suffer from insufficient process stability. While lower concentrations of ethanol can extract phospholipids more fully, they also release a large amount of water-soluble colloidal impurities such as proteins and polysaccharides. This leads to increased extract viscosity, difficulties in solid-liquid separation, a significantly increased filtration load, and a higher proportion of separable insoluble matter, affecting product purity and yield. Conversely, using higher concentrations of ethanol reduces the polarity of the extraction system, resulting in insufficient release of amphiphilic phospholipids and a lower total phospholipid content in the product.

[0005] Therefore, there is an urgent need in this field to develop a method that uses food-grade ethanol aqueous solution as the extraction solvent, has clear and simple process steps, mild operating conditions, can effectively reduce the proportion of separable insoluble matter before filtration, and can stably obtain high-quality Antarctic krill oil, so as to meet the needs of industrial production of food-grade Antarctic krill oil. Summary of the Invention

[0006] To address the challenges in existing Antarctic krill oil preparation technologies, such as the difficulty in achieving both yield and product purity when using single-concentration ethanol extraction, the high content of colloidal impurities and separable insoluble matter in the extract leading to heavy subsequent filtration loads, and the risk of solvent residue and cumbersome processes due to the introduction of non-food-grade organic solvents or complex operations like shear emulsification, countercurrent extraction, and salting-out, this invention designs a method for preparing Antarctic krill oil using food-grade dual-concentration ethanol at low temperature and static purification. This method uses Antarctic krill powder as the starting material, which is first extracted with a 75% (v / v) ethanol aqueous solution after low-temperature pulverization and sieving, followed by a second extraction with a 90% (v / v) ethanol aqueous solution. The filter residue undergoes a second extraction. The two extracts are combined and allowed to stand in the dark at 8°C to remove impurities. Then, the mixture is centrifuged, filtered through a membrane, concentrated under reduced pressure, and desolvated with nitrogen to finally produce food-grade Antarctic krill oil. Through gradient extraction with dual concentrations of ethanol, the phospholipids and weakly polar lipids are extracted stepwise and fully, effectively improving the product yield and total phospholipid content. The low-temperature standing impurity removal step allows separable insoluble substances such as protein colloids and shell particles to form flocculated precipitates and be efficiently removed, significantly reducing the filtration load. Through gentle operation and nitrogen protection throughout the process, lipid oxidation is effectively controlled, ensuring product quality and meeting the industrial production requirements of food-grade Antarctic krill oil.

[0007] To solve the above-mentioned technical problems, the present invention provides the following technical solution: a method for preparing Antarctic krill oil by low-temperature static purification with food-grade dual-concentration ethanol, the specific steps of which are as follows: S100. Take Antarctic krill powder, place it in a -5℃ environment for 30 minutes to equilibrate, and then perform low-temperature pulverization. After pulverization, pass it through a 40-mesh standard sieve to obtain krill raw material powder. By equilibrating and pulverizing Antarctic krill powder at low temperature, the krill tissue becomes brittle, which is conducive to cell wall rupture. At the same time, it avoids the oxidative degradation of heat-sensitive components such as phospholipids and astaxanthin caused by friction heating during the pulverization process. S200. Add a 75% (v / v) ethanol aqueous solution to the krill raw material powder. The mass-volume ratio of the krill raw material powder to the ethanol aqueous solution is 1 kg: 5 L. Stir and extract at a constant temperature of 25°C for 2 hours. After extraction, separate by vacuum filtration to obtain a primary extract and a primary filter residue. Utilize the high polarity of the 75% (v / v) ethanol aqueous solution to disrupt the binding between phospholipids and proteins and polysaccharides, allowing phospholipid lipids to fully dissolve into the extract. S300. Add a 90% ethanol aqueous solution to the primary filter residue. The amount of the 90% ethanol aqueous solution added is 1 kg: 5 L based on the initial mass of the krill raw material powder mentioned in S100. Stir and extract for 2 hours under constant temperature of 35°C. After extraction, separate by vacuum filtration to obtain a secondary extract. Utilize the low polarity of the 90% ethanol aqueous solution to effectively penetrate into the residual tissue of the primary filter residue and further extract the weakly polar lipids that have not yet been released, thus achieving secondary recovery of residual lipids after the primary extraction. S400. Transfer all of the primary and secondary extracts to a sealed, light-proof container and mix them thoroughly to obtain a combined extract. Place the combined extract in a constant temperature environment at 8°C and let it stand in the dark for 8 hours. Under low temperature conditions, the protein-polysaccharide complexes, fine chitin particles and other high molecular weight colloidal impurities that were originally in a dissolved or suspended state in the extract gradually form large-particle-size flocculent precipitates, which can be efficiently separated and removed in the subsequent centrifugation process. S500. After standing, the combined extract is transferred to a centrifuge and centrifuged at 4000 r / min for 10 min. After centrifugation, the upper clear supernatant is collected and the lower precipitate is discarded. The supernatant is then filtered under pressure using a food-grade filter membrane with a pore size of 0.45 μm-1.0 μm to obtain a clear filtrate. Through the combination of centrifugation and membrane filtration, large-particle flocculent precipitates and fine suspended particles are removed one after another to ensure that the feed liquid entering the concentration process has a high purity, thereby reducing scaling and heat transfer deterioration problems in the subsequent concentration process. S600. The clarified filtrate is transferred to a rotary evaporator and concentrated under reduced pressure at 42°C and a vacuum of -0.090 MPa in the dark until no distillate is produced. Ethanol and water are removed to obtain crude krill oil. Ethanol and water are efficiently removed under mild conditions by evaporating under reduced pressure at a lower temperature. At the same time, the light-protected conditions protect the photosensitive components of astaxanthin from photodegradation and retain the natural active substances in the oil to the maximum extent. S700. The crude krill oil is transferred to a sealed desolventizing tank, and nitrogen gas with a purity of ≥99.9% is introduced at a temperature of 35℃-45℃ for bubbling desolventizing treatment for 20min-60min. After desolventizing, aseptic filling is carried out under continuous nitrogen purging and light-proof conditions to obtain food-grade Antarctic krill oil product. The high-purity nitrogen bubbling desolventizing further removes residual trace solvents and moisture, while the inert protective atmosphere of nitrogen effectively isolates oxygen, preventing the oxidative rancidity of polyunsaturated fatty acids during filling and storage, and ensuring the oxidative stability of the finished oil.

[0008] Furthermore, in S100, the moisture content of the Antarctic krill powder is not higher than 8.0%, and the temperature of the krill raw material powder is always maintained within the range of -5℃ to 0℃ during the low-temperature pulverization process. By controlling the moisture content of the raw material and the temperature of the pulverization process, the pulverization efficiency and sieving smoothness are ensured, while avoiding powder adhesion due to excessive moisture and accelerated lipid oxidation due to increased temperature.

[0009] Furthermore, in S200 and S300, the vacuum degree of the vacuum filtration is controlled between -0.06MPa and -0.08MPa. By controlling the vacuum degree of filtration, the solid-liquid separation efficiency is ensured while avoiding excessively high vacuum degrees that would cause fine particles to pass through the filter paper and make the filtrate turbid.

[0010] Furthermore, in S500, the food-grade filter membrane is one of a polypropylene membrane, a polytetrafluoroethylene membrane, or a polyethersulfone membrane, and the filtration pressure is controlled at 0.1MPa-0.3MPa.

[0011] Furthermore, in the S600, the rotation speed of the rotary evaporator is controlled at 60 r / min-80 r / min. During the concentration process, the evaporation flask is wrapped with aluminum foil to avoid light. By controlling the rotation speed, the liquid material forms a uniform liquid film on the inner wall of the evaporation flask to increase the evaporation area and improve the concentration efficiency. At the same time, the aluminum foil wrapping effectively shields the photosensitive active ingredients of astaxanthin from external light.

[0012] Furthermore, in the S700, the nitrogen flow rate for nitrogen bubbling is controlled at 0.5L / min-1.0L / min. By controlling the appropriate nitrogen flow rate, nitrogen forms uniform and fine bubbles in the crude krill oil, increasing the gas-liquid contact area to promote the mass transfer and removal of residual solvent and water, while avoiding excessive flow rate that could cause oil splashing or entrainment by nitrogen, resulting in product loss.

[0013] Furthermore, this method does not use hexane, petroleum ether, No. 6 solvent oil, cyclohexane, or ethyl acetate as extraction solvents throughout the entire process, but instead uses food-grade ethanol aqueous solution as the sole extraction solvent system.

[0014] Furthermore, this method does not include shear emulsification, countercurrent extraction, or salting-out purification steps. By eliminating these complex additional processes, the operation is simplified, equipment investment and production costs are reduced, and the problems of excessive oxidation that may be introduced by shear emulsification and product loss and wastewater discharge caused by salting-out are avoided.

[0015] Furthermore, in the S700, the food-grade Antarctic krill oil product contains 40.5%-43.5% total phospholipids, 7.2%-8.2% eicosapentaenoic acid, and 3.6%-4.2% docosahexaenoic acid, and has a peroxide value not exceeding 3.0 meq / kg.

[0016] Compared with existing technologies, this method for preparing Antarctic krill oil by low-temperature static purification with food-grade dual-concentration ethanol has the following advantages: I. This invention employs a gradient extraction process using a dual-concentration ethanol system consisting of a 75% (v / v) and a 90% (v / v) ethanol aqueous solution. First, a single extraction at room temperature is performed with 75% ethanol, followed by a supplementary extraction with 90% ethanol on the filter residue. This allows the extraction advantages of the two ethanol concentrations to complement each other. The 75% ethanol aqueous solution has high polarity, effectively disrupting the binding between phospholipids and proteins / polysaccharides, allowing phospholipid lipids to fully dissolve into the extract. The 90% ethanol aqueous solution, with relatively low polarity, effectively penetrates the residual tissue of the first-stage filter residue, further extracting any remaining weakly polar lipids. This dual-concentration gradient extraction design ensures more thorough and balanced extraction of total phospholipids, EPA, and DHA. It avoids the problems of increased extract viscosity and subsequent filtration difficulties caused by the co-dissolution of large amounts of water-soluble colloidal impurities during single extraction with lower concentration ethanol, while also avoiding the defects of insufficient phospholipid release and low total phospholipid content in the product due to insufficient polarity of the system during single extraction with higher concentration ethanol.

[0017] Second, after extraction with dual-concentration ethanol, this invention adds a low-temperature settling and impurity removal step. The combined extract is placed in a constant temperature environment at 8°C and kept in the dark for 8 hours. This allows the protein-polysaccharide complexes, fine chitin particles, and other high-molecular colloidal impurities that were originally in a dissolved or suspended state in the extract to gradually form large-particle-size flocculent precipitates. This significantly improves the purity of the feed solution entering the membrane filtration process, significantly reduces the solid load on the membrane filtration, extends the service life of the filter membrane, and also reduces the risk of membrane fouling and clogging.

[0018] Other advantages, objectives and features of the invention will be set forth in part in the description which follows, and in part will be apparent to those skilled in the art from the following examination or study, or may be learned from the practice of the invention. Attached Figure Description

[0019] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the accompanying drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are merely some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without any creative effort.

[0020] Figure 1 A flowchart illustrating the steps of a method for preparing Antarctic krill oil using food-grade dual-concentration ethanol through low-temperature static purification. Detailed Implementation

[0021] To further illustrate the technical means and effects of the present invention in achieving its intended purpose, the following detailed description of the specific implementation methods, structures, features, and effects of the present invention, in conjunction with the accompanying drawings and preferred embodiments, is provided below.

[0022] To address the challenges in existing Antarctic krill oil preparation technologies, such as the difficulty in achieving both yield and purity during single-concentration ethanol extraction, the high content of colloidal impurities and separable insoluble matter in the extract leading to heavy subsequent filtration loads, and the risk of solvent residue and cumbersome processes due to the introduction of non-food-grade organic solvents or complex operations, this invention first describes its application scenarios. This invention is primarily applied to the deep processing of Antarctic krill, the preparation of marine functional oils, and the industrial production of food-grade Antarctic krill oil. In these scenarios, the traditional single-stage extraction method using a single concentration of ethanol suffers from drawbacks: at lower ethanol concentrations, a large amount of water-soluble colloidal impurities are co-dissolved, increasing the viscosity of the extract, making solid-liquid separation difficult, and resulting in a high proportion of separable insoluble matter (up to 0.68%) before filtration. Furthermore, at higher ethanol concentrations, insufficient system polarity leads to inadequate phospholipid release, resulting in low total phospholipid content and unsatisfactory yields. This invention utilizes a dual-concentration gradient extraction system consisting of 75% and 90% ethanol aqueous solutions by volume, and adds an 8°C low-temperature static settling step for impurity removal after extraction. Combined with a mild post-treatment process of low-temperature vacuum concentration and nitrogen desolvation, this invention achieves efficient, stable, and high-quality preparation of food-grade Antarctic krill oil.

[0023] Unified Test Instructions All Antarctic krill oil samples prepared in the embodiments and comparative examples of this invention were tested for performance using the following uniform method: Yield determination: The yield is calculated as the percentage of the final product Antarctic krill oil mass to the initial Antarctic krill powder mass, i.e., the ratio of the final product Antarctic krill oil mass to the initial Antarctic krill powder mass.

[0024] Total phospholipid content determination: The determination shall be carried out in accordance with SN / T3851-2014 "Determination of phosphatidylcholine, phosphatidylethanolamine and phosphatidylinositol in exported food by high performance liquid chromatography" or equivalent methods, and shall be expressed as the mass percentage of total phospholipids in the finished Antarctic krill oil.

[0025] Eicosapentaenoic acid (EPA) content determination: The determination was carried out by gas chromatography in accordance with GB5009.168-2016 "National Food Safety Standard for Determination of Fatty Acids in Food", and the EPA content was expressed as the mass percentage of EPA in the finished Antarctic krill oil.

[0026] Determination of docosahexaenoic acid (DHA) content: The determination was carried out by gas chromatography in accordance with GB5009.168-2016 "National Food Safety Standard - Determination of Fatty Acids in Food", and the DHA content was expressed as the mass percentage of DHA in the finished Antarctic krill oil.

[0027] Peroxide value determination: The determination was carried out by titration method in GB5009.227-2016 "National Food Safety Standard for Determination of Peroxide Value in Food", and the unit is meq / kg.

[0028] Determination of the proportion of separable insoluble matter before filtration: Take the combined extract or the first extract and centrifuge under the same conditions as the subsequent centrifugation steps (4000 r / min, centrifugation for 10 min). Collect the separable sediment, dry it, weigh it, and convert it into the percentage of the mass of the sediment to the mass of the starting Antarctic krill powder raw material.

[0029] Example 1 In this embodiment, Antarctic krill powder with a moisture content of 6.5% was equilibrated at -5℃ for 30 minutes, then pulverized and sieved. A first extract was obtained by stirring at 100 rpm for 2 hours with a 75% ethanol aqueous solution at 25℃ for 2 hours. A second extract was obtained by stirring at 100 rpm for 2 hours with a 90% ethanol aqueous solution at 35℃ for 2 hours. The two extracts were combined and allowed to stand at 8℃ in the dark for 8 hours for low-temperature impurity removal. After centrifugation at 4000 rpm for 10 minutes and pressure filtration through a 0.8 μm polypropylene membrane, the extract was concentrated under reduced pressure at 42℃ and -0.090 MPa in the dark. Finally, the extract was desolvated by nitrogen bubbling at 40℃ for 40 minutes and then bottled under nitrogen bubbling in the dark.

[0030] This embodiment provides a method for preparing Antarctic krill oil by low-temperature static purification with food-grade dual-concentration ethanol, such as... Figure 1 As shown, the specific steps of this method are as follows: S100. Take 100 kg of Antarctic krill powder with a moisture content of 6.5%, place it in a -5℃ environment for 30 min to equilibrate, and then perform low-temperature pulverization. During the pulverization process, control the temperature of the krill raw material powder to be kept within the range of -5℃ to 0℃. After pulverization, pass it through a 40-mesh standard sieve to obtain krill raw material powder. S200. Add 500L of 75% ethanol aqueous solution to the krill raw material powder. The mass-volume ratio of the krill raw material powder to the ethanol aqueous solution is 1kg:5L. Stir and extract at 100r / min for 2h under constant temperature of 25℃. After extraction, separate by vacuum filtration. The vacuum degree of vacuum filtration is controlled between -0.06MPa and -0.08MPa. After filtration, a primary extract and a primary filter residue are obtained. S300. Add 500L of 90% ethanol aqueous solution to the primary filter residue. The amount of 90% ethanol aqueous solution added is 1kg:5L based on the initial mass of the krill raw material powder described in S100. Stir and extract for 2 hours at a constant temperature of 35℃ and a stirring speed of 100r / min. After extraction, separate by vacuum filtration. The vacuum degree of vacuum filtration is controlled between -0.06MPa and -0.08MPa. A secondary extract is obtained after filtration. S400. Transfer all of the primary extract and the secondary extract to a sealed, light-proof container and mix them thoroughly to obtain a combined extract. Place the combined extract in a constant temperature environment at 8°C and let it stand in the dark for 8 hours. S500. After standing, the combined extracts are transferred to a centrifuge and centrifuged at 4000 r / min for 10 min. After centrifugation, the upper clear supernatant is collected and the lower precipitate is discarded. The supernatant is then filtered under pressure using a food-grade polypropylene filter membrane with a pore size of 0.8 μm. The filtration pressure is controlled at 0.2 MPa to obtain a clear filtrate. S600. The clarified filtrate is transferred to a rotary evaporator. The rotation speed of the rotary evaporator is controlled at 70 r / min. The evaporator is concentrated under reduced pressure at 42°C and a vacuum of -0.090 MPa in the dark. The evaporation flask is wrapped with aluminum foil to protect it from light throughout the concentration process until no distillate is produced. Ethanol and water are removed to obtain crude krill oil. S700. The crude krill oil is transferred to a sealed desolventizing tank. Nitrogen gas with a purity of ≥99.9% is introduced at 40°C for bubbling desolventizing treatment for 40 minutes. The nitrogen flow rate is controlled at 0.8L / min. The pressure inside the desolventizing tank is maintained at a slightly positive pressure. After desolventizing, the product is aseptically filled under continuous nitrogen purging and light-proof conditions to obtain food-grade Antarctic krill oil.

[0031] Performance test results The performance indicators of the food-grade Antarctic krill oil product obtained in this embodiment are as follows: yield 8.6%, total phospholipid content 42.1%, EPA content 7.8%, DHA content 3.9%, peroxide value 2.6 meq / kg, and proportion of separable insoluble matter before filtration 0.31%.

[0032] This embodiment employs a dual-concentration gradient extraction method, combining a primary extraction with 75% ethanol and a secondary extraction with 90% ethanol. This allows the two concentrations of ethanol to work synergistically and complement each other. The 75% ethanol efficiently extracts phospholipids, while the 90% ethanol fully recovers the weakly polar lipids remaining in the filter residue. This achieves a stable yield of 8.6% and a high total phospholipid content of 42.1%. By allowing the mixture to stand at 8°C for 8 hours, the proportion of separable insoluble matter before filtration is reduced to 0.31%, utilizing the principle that temperature reduction causes colloidal particles to aggregate and settle. Through continuous operation at temperatures below 42°C, light protection, and nitrogen atmosphere coverage, oil oxidation is effectively controlled.

[0033] Example 2 In this embodiment, 20 kg of Antarctic krill powder with a moisture content of 6.8% was equilibrated at -5°C for 30 min, then pulverized and sieved. The same process conditions as in Example 1 were used for extraction with dual-concentration ethanol, static settling at 8°C for 8 h to remove impurities, centrifugation at 4000 r / min for 10 min, pressure filtration through a 0.8 μm polypropylene membrane, concentration under reduced pressure at 42°C and 0.090 MPa in the dark, and nitrogen desolvation at 40°C for 40 min. This process verified the stability and reproducibility of the invention on a small scale.

[0034] This embodiment provides a method for preparing Antarctic krill oil by low-temperature static purification with food-grade dual-concentration ethanol. The specific steps of this method are as follows: S100. Take 20 kg of Antarctic krill powder with a moisture content of 6.8%, place it in a -5℃ environment for 30 min to equilibrate, and then perform low-temperature pulverization. During the pulverization process, control the temperature of the krill raw material powder to be kept within the range of -5℃ to 0℃. After pulverization, pass it through a 40-mesh standard sieve to obtain krill raw material powder. S200. Add 100L of 75% ethanol aqueous solution to the krill raw material powder. The mass-volume ratio of the krill raw material powder to the ethanol aqueous solution is 1kg:5L. Stir and extract at 100r / min for 2h under constant temperature of 25℃. After extraction, separate by vacuum filtration. The vacuum degree of vacuum filtration is controlled between -0.06MPa and -0.08MPa. After filtration, a primary extract and a primary filter residue are obtained. S300. Add 100L of 90% ethanol aqueous solution to the primary filter residue. The amount of 90% ethanol aqueous solution added is 1kg:5L based on the initial mass of the krill raw material powder mentioned in S100. Stir and extract for 2 hours at a constant temperature of 35℃ and a stirring speed of 100r / min. After extraction, separate by vacuum filtration. The vacuum degree of vacuum filtration is controlled between -0.06MPa and -0.08MPa. The secondary extract is obtained after filtration. S400. Transfer all of the primary extract and the secondary extract to a sealed, light-proof container and mix them thoroughly to obtain a combined extract. Place the combined extract in a constant temperature environment at 8°C and let it stand in the dark for 8 hours. S500. After standing, the combined extracts are transferred to a centrifuge and centrifuged at 4000 r / min for 10 min. After centrifugation, the upper clear supernatant is collected and the lower precipitate is discarded. The supernatant is then filtered under pressure using a food-grade polypropylene filter membrane with a pore size of 0.8 μm. The filtration pressure is controlled at 0.2 MPa to obtain a clear filtrate. S600. The clarified filtrate is transferred to a rotary evaporator. The rotation speed of the rotary evaporator is controlled at 70 r / min. The evaporator is concentrated under reduced pressure at 42°C and a vacuum of -0.090 MPa in the dark. The evaporation flask is wrapped with aluminum foil to protect it from light throughout the concentration process until no distillate is produced. Ethanol and water are removed to obtain crude krill oil. S700. The crude krill oil is transferred to a sealed desolventizing tank. Nitrogen gas with a purity of ≥99.9% is introduced at 40°C for bubbling desolventizing treatment for 40 minutes. The nitrogen flow rate is controlled at 0.8L / min. The pressure inside the desolventizing tank is maintained at a slightly positive pressure. After desolventizing, the product is aseptically filled under continuous nitrogen purging and light-proof conditions to obtain food-grade Antarctic krill oil.

[0035] Performance test results The performance indicators of the food-grade Antarctic krill oil product obtained in this embodiment are as follows: yield 8.5%, total phospholipid content 41.8%, EPA content 7.7%, DHA content 3.8%, peroxide value 2.7 meq / kg, and proportion of insoluble matter that can be separated before filtration 0.33%.

[0036] This embodiment operates under essentially the same process conditions as Embodiment 1, except for changes in the raw material batch and processing scale (reduced from 100kg to 20kg). The performance indicators of the resulting product are highly consistent with those of Embodiment 1, indicating that the method of the present invention has good batch stability and scale adaptability, and can meet the requirements of different raw material sources and production scales in industrial production.

[0037] Example 3 In this embodiment, 50 kg of Antarctic krill powder with a moisture content of 7.2% was equilibrated at -5°C for 30 min, then pulverized and sieved. The same dual-concentration ethanol extraction conditions as in Example 1 were used, but in step S500, a polytetrafluoroethylene filter membrane with a pore size of 0.45 μm was used, and the filtration pressure was controlled at 0.15 MPa. In step S600, the rotary evaporator speed was controlled at 60 r / min. In step S700, the nitrogen desolvation temperature was controlled at 35°C, the processing time was 60 min, and the nitrogen flow rate was controlled at 0.5 L / min. By fine-tuning some process parameters, the robustness of the method of this invention was verified.

[0038] This embodiment provides a method for preparing Antarctic krill oil by low-temperature static purification with food-grade dual-concentration ethanol. The specific steps of this method are as follows: S100. Take 50 kg of Antarctic krill powder with a moisture content of 7.2%, place it in a -5℃ environment for 30 min to equilibrate, and then perform low-temperature pulverization. During the pulverization process, control the temperature of the krill raw material powder to be kept within the range of -5℃ to 0℃. After pulverization, pass it through a 40-mesh standard sieve to obtain krill raw material powder. S200. Add 250L of 75% ethanol aqueous solution to the krill raw material powder. The mass-volume ratio of the krill raw material powder to the ethanol aqueous solution is 1kg:5L. Stir and extract at 100r / min for 2h under constant temperature of 25℃. After extraction, separate by vacuum filtration. The vacuum degree of vacuum filtration is controlled between -0.06MPa and -0.08MPa. After filtration, a primary extract and a primary filter residue are obtained. S300. Add 250L of 90% ethanol aqueous solution to the primary filter residue. The amount of 90% ethanol aqueous solution added is 1kg:5L based on the initial mass of the krill raw material powder described in S100. Stir and extract for 2 hours at a constant temperature of 35℃ and a stirring speed of 100r / min. After extraction, separate by vacuum filtration. The vacuum degree of vacuum filtration is controlled between -0.06MPa and -0.08MPa. A secondary extract is obtained after filtration. S400. Transfer all of the primary extract and the secondary extract to a sealed, light-proof container and mix them thoroughly to obtain a combined extract. Place the combined extract in a constant temperature environment at 8°C and let it stand in the dark for 8 hours. S500. After standing, the combined extracts are transferred to a centrifuge and centrifuged at 4000 r / min for 10 min. After centrifugation, the upper clear supernatant is collected and the lower precipitate is discarded. The supernatant is then pressure filtered using a food-grade polytetrafluoroethylene filter membrane with a pore size of 0.45 μm. The filtration pressure is controlled at 0.15 MPa to obtain a clear filtrate. S600. The clarified filtrate is transferred to a rotary evaporator. The rotation speed of the rotary evaporator is controlled at 60 r / min. The evaporator is concentrated under reduced pressure at 42°C and a vacuum of -0.090 MPa in the dark. The evaporation flask is wrapped with aluminum foil to protect it from light throughout the concentration process until no distillate is produced. Ethanol and water are removed to obtain crude krill oil. S700. The crude krill oil is transferred to a sealed desolventizing tank. Nitrogen gas with a purity of ≥99.9% is introduced at 35°C for 60 minutes for bubble desolventizing treatment. The nitrogen flow rate is controlled at 0.5L / min. The pressure inside the desolventizing tank is maintained at a slightly positive pressure. After desolventizing, the product is aseptically filled under continuous nitrogen purging and light-proof conditions to obtain food-grade Antarctic krill oil.

[0039] Performance test results The performance indicators of the food-grade Antarctic krill oil product obtained in this embodiment are as follows: yield 8.4%, total phospholipid content 42.3%, EPA content 7.9%, DHA content 4.0%, peroxide value 2.5 meq / kg, and proportion of separable insoluble matter before filtration 0.30%.

[0040] In this embodiment, while maintaining the dual-concentration gradient extraction, 8°C low-temperature standing for 8 hours, and 42°C vacuum concentration, the operating parameters such as membrane filter pore size and material, rotary evaporator speed, nitrogen purging desolvation temperature and time were moderately adjusted. The results show that the product yield and key quality indicators remained stable when the parameters were changed, which proves the operating window and good adaptability of the present invention, and makes it easy to make flexible adjustments according to equipment conditions and production needs in actual production.

[0041] Comparative Example 1 This comparative example uses a single volume fraction of 75% ethanol aqueous solution for extraction (the total solvent volume is the same as in Example 1, which is 1000L), without a secondary extraction with 90% ethanol. The remaining steps (standing at 8°C for 8 hours, centrifugation at 4000r / min for 10 minutes, pressure filtration through a 0.8μm polypropylene membrane, concentration under reduced pressure at 42°C and 0.090MPa in the dark, and nitrogen desolvation at 40°C for 40 minutes) are exactly the same as in Example 1, aiming to verify the superiority of the dual concentration gradient extraction of this invention compared with single low-concentration ethanol extraction.

[0042] This comparative example uses a single extraction with a 75% ethanol aqueous solution (volume fraction), without a secondary extraction with 90% ethanol (volume fraction). The specific steps are as follows: S100. Take 100 kg of Antarctic krill powder from the same batch as in Example 1, with a moisture content of 6.5%, place it in a -5℃ environment for 30 min to equilibrate, and then perform low-temperature pulverization. After pulverization, pass it through a 40-mesh standard sieve to obtain krill raw material powder. S200. Add 1000L of 75% ethanol aqueous solution to the krill raw material powder. The mass-volume ratio of the krill raw material powder to the ethanol aqueous solution is 1kg:10L. Stir and extract at 100r / min for 4h under constant temperature of 25℃. After extraction, separate by vacuum filtration. The vacuum degree of vacuum filtration is controlled between -0.06MPa and -0.08MPa. After filtration, a primary extract and a primary filter residue are obtained. S300. Transfer the primary extract to a sealed, light-proof container and place it in a constant temperature environment at 8°C for 8 hours in the dark. S400. Transfer the settled extract to a centrifuge and centrifuge at 4000 r / min for 10 min. After centrifugation, collect the clear supernatant and discard the lower precipitate. Use a food-grade polypropylene filter membrane with a pore size of 0.8 μm to pressurize and filter the supernatant at a pressure of 0.2 MPa to obtain a clear filtrate. S500. The clarified filtrate is transferred to a rotary evaporator. The rotation speed of the rotary evaporator is controlled at 70 r / min. The evaporator is concentrated under reduced pressure at 42°C and a vacuum of -0.090 MPa in the dark. The evaporation flask is wrapped with aluminum foil to protect it from light throughout the concentration process until no distillate is produced. Ethanol and water are removed to obtain crude krill oil. S600. The crude krill oil is transferred to a sealed desolventizing tank. Nitrogen gas with a purity of ≥99.9% is introduced at 40°C for bubbling desolventizing treatment for 40 minutes. The nitrogen flow rate is controlled at 0.8L / min. The pressure inside the desolventizing tank is maintained at a slightly positive pressure. After desolventizing, the product is aseptically filled under continuous nitrogen filling and light protection conditions to obtain the Antarctic krill oil product.

[0043] Performance test results The performance indicators of the Antarctic krill oil product obtained in this comparative example are as follows: yield 7.8%, total phospholipid content 38.7%, EPA content 6.9%, DHA content 3.4%, peroxide value 3.4 meq / kg, and proportion of separable insoluble matter before filtration 0.68%.

[0044] This comparative example uses a single extraction with 75% ethanol aqueous solution, which can effectively extract polar phospholipids. However, due to the low ethanol concentration and high water ratio in the extraction system, water-soluble colloidal impurities such as proteins and polysaccharides are dissolved simultaneously, leading to increased viscosity of the extract and difficulty in solid-liquid separation. The proportion of insoluble matter that can be separated before filtration is as low as 0.68%. At the same time, since the total amount of lipids that can be extracted in a single extraction is limited, and some phospholipids are difficult to be fully recovered due to adsorption and encapsulation by colloidal impurities, the product yield is only 7.8%, and the total phospholipid content is only 38.7%. The large number of impurities in the extract promotes oxidation reactions during subsequent concentration and desolvation, with the peroxide value rising to 3.4 meq / kg. This fully demonstrates that a single extraction with low concentration ethanol cannot simultaneously achieve yield, purity, and oxidative stability.

[0045] Comparative Example 2 This comparative example uses a single extraction with a 90% ethanol aqueous solution (total solvent volume is the same as in Example 1, 1000L), without a single extraction with 75% ethanol. The remaining steps (standing at 8°C for 8 hours, centrifugation at 4000r / min for 10 minutes, pressure filtration through a 0.8μm polypropylene membrane, concentration under reduced pressure at 42°C-0.090MPa in the dark, and nitrogen desolvation at 40°C for 40 minutes) are exactly the same as in Example 1, aiming to verify the superiority of the dual-concentration gradient extraction of this invention compared to single high-concentration ethanol extraction.

[0046] This comparative example uses a single extraction with a 90% ethanol aqueous solution (volume fraction), without a single extraction with 75% ethanol (volume fraction). The specific steps are as follows: S100. Take 100 kg of Antarctic krill powder from the same batch as in Example 1, with a moisture content of 6.5%, place it in a -5℃ environment for 30 min to equilibrate, and then perform low-temperature pulverization. After pulverization, pass it through a 40-mesh standard sieve to obtain krill raw material powder. S200. Add 1000L of 90% ethanol aqueous solution to the krill raw material powder. The mass-volume ratio of the krill raw material powder to the ethanol aqueous solution is 1kg:10L. Stir and extract at 100r / min for 4h under constant temperature of 35℃. After extraction, separate by vacuum filtration. The vacuum degree of vacuum filtration is controlled between -0.06MPa and -0.08MPa. After filtration, a primary extract and a primary filter residue are obtained. S300. Transfer the primary extract to a sealed, light-proof container and place it in a constant temperature environment at 8°C for 8 hours in the dark. S400. Transfer the settled extract to a centrifuge and centrifuge at 4000 r / min for 10 min. After centrifugation, collect the clear supernatant and discard the lower precipitate. Use a food-grade polypropylene filter membrane with a pore size of 0.8 μm to pressurize and filter the supernatant at a pressure of 0.2 MPa to obtain a clear filtrate. S500. The clarified filtrate is transferred to a rotary evaporator. The rotation speed of the rotary evaporator is controlled at 70 r / min. The evaporator is concentrated under reduced pressure at 42°C and a vacuum of -0.090 MPa in the dark. The evaporation flask is wrapped with aluminum foil to protect it from light throughout the concentration process until no distillate is produced. Ethanol and water are removed to obtain crude krill oil. S600. The crude krill oil is transferred to a sealed desolventizing tank. Nitrogen gas with a purity of ≥99.9% is introduced at 40°C for bubbling desolventizing treatment for 40 minutes. The nitrogen flow rate is controlled at 0.8L / min. The pressure inside the desolventizing tank is maintained at a slightly positive pressure. After desolventizing, the product is aseptically filled under continuous nitrogen filling and light protection conditions to obtain the Antarctic krill oil product.

[0047] Performance test results The performance indicators of the Antarctic krill oil product obtained in this comparative example are as follows: yield 8.0%, total phospholipid content 39.2%, EPA content 7.0%, DHA content 3.5%, peroxide value 3.2 meq / kg, and proportion of separable insoluble matter before filtration 0.60%.

[0048] This comparative example used a single extraction with 90% ethanol aqueous solution. Due to the high ethanol concentration and relatively low polarity in the extraction system, the dissolution of colloidal impurities such as water-soluble proteins and polysaccharides was significantly lower than in Comparative Example 1. The proportion of insoluble matter that could be separated before filtration was 0.60%. However, phospholipid molecules are amphiphilic, and the solubility of their polar heads is limited in a high-concentration ethanol environment. As a result, a large amount of phospholipids remained trapped in the filter residue and were not extracted. The total phospholipid content of the product was only 39.2%, and the yield was only 8.0%. At the same time, the high ethanol concentration and extraction temperature aggravated lipid oxidation to some extent, with a peroxide value of 3.2 meq / kg. This fully demonstrates that a single extraction with high-concentration ethanol cannot fully recover phospholipid lipids, and the total phospholipid content of the product is difficult to meet the high-quality requirements.

[0049] Comparative Example 3 This comparative example uses the same dual-concentration ethanol extraction conditions as Example 1 (75% ethanol extraction once + 90% ethanol extraction twice). However, after combining the extracts, the impurity removal step of standing at 8°C for 8 hours is not performed. Instead, the combined extracts are directly centrifuged at 4000 r / min for 10 min, filtered under pressure through a 0.8 μm polypropylene membrane, concentrated under reduced pressure at 42°C -0.090 MPa in the dark, and desolvated under nitrogen at 40°C for 40 min. The remaining steps are exactly the same as in Example 1. This aims to verify the effect of the 8°C low-temperature standing impurity removal step in this invention in reducing the proportion of separable insoluble matter before filtration.

[0050] This comparative example does not include the 8℃ low-temperature standing impurity removal step. The specific steps are as follows: S100. Take 100 kg of Antarctic krill powder from the same batch as in Example 1, with a moisture content of 6.5%, place it in a -5℃ environment for 30 min to equilibrate, and then perform low-temperature pulverization. After pulverization, pass it through a 40-mesh standard sieve to obtain krill raw material powder. S200. Add 500L of 75% ethanol aqueous solution to the krill raw material powder. The mass-volume ratio of the krill raw material powder to the ethanol aqueous solution is 1kg:5L. Stir and extract at 100r / min for 2h under constant temperature of 25℃. After extraction, separate by vacuum filtration. The vacuum degree of vacuum filtration is controlled between -0.06MPa and -0.08MPa. After filtration, a primary extract and a primary filter residue are obtained. S300. Add 500L of 90% ethanol aqueous solution to the primary filter residue. The amount of 90% ethanol aqueous solution added is 1kg:5L based on the initial mass of the krill raw material powder described in S100. Stir and extract for 2 hours at a constant temperature of 35℃ and a stirring speed of 100r / min. After extraction, separate by vacuum filtration. The vacuum degree of vacuum filtration is controlled between -0.06MPa and -0.08MPa. A secondary extract is obtained after filtration. S400. Transfer all of the primary extract and the secondary extract to a sealed, light-proof container and mix them thoroughly to obtain a combined extract. Without allowing it to stand at 8°C in the dark, directly transfer the combined extract to a centrifuge. S500. Centrifuge the combined extracts at 4000 r / min for 10 min. After centrifugation, collect the clear supernatant and discard the lower precipitate. Use a food-grade polypropylene filter membrane with a pore size of 0.8 μm to filter the supernatant under pressure at 0.2 MPa to obtain a clear filtrate. S600. The clarified filtrate is transferred to a rotary evaporator. The rotation speed of the rotary evaporator is controlled at 70 r / min. The evaporator is concentrated under reduced pressure at 42°C and a vacuum of -0.090 MPa in the dark. The evaporation flask is wrapped with aluminum foil to protect it from light throughout the concentration process until no distillate is produced. Ethanol and water are removed to obtain crude krill oil. S700. The crude krill oil is transferred to a sealed desolventizing tank. Nitrogen gas with a purity of ≥99.9% is introduced at 40°C for 40 minutes for bubble desolventizing treatment. The nitrogen flow rate is controlled at 0.8L / min. The pressure inside the desolventizing tank is maintained at a slightly positive pressure. After desolventizing, the product is aseptically filled under continuous nitrogen filling and light protection conditions to obtain the Antarctic krill oil product.

[0051] Performance test results The performance indicators of the Antarctic krill oil product obtained in this comparative example are as follows: yield 8.5%, total phospholipid content 41.6%, EPA content 7.7%, DHA content 3.8%, peroxide value 2.8 meq / kg, and proportion of insoluble matter that can be separated before filtration 0.52%.

[0052] The extraction process of this comparative example is completely consistent with that of Example 1, except that the impurity removal step of standing at 8°C for 8 hours after combining the extracts is omitted. The test results show that after omitting the low-temperature standing step, the proportion of separable insoluble matter before filtration increases from 0.31% in Example 1 to 0.52%. In the extract without low-temperature standing, protein-polysaccharide complexes, fine chitin particles and other high molecular colloidal impurities exist in the form of fine suspended particles, which are difficult to settle effectively when centrifuged directly, increasing the risk of scaling during the concentration process and which is not conducive to the long-term stable operation of industrial production. This fully verifies the role of the 8°C low-temperature standing impurity removal step in this invention in reducing the proportion of insoluble matter before filtration and reducing the load on subsequent filtration.

[0053] To visually compare the performance differences between the various embodiments of the present invention and the comparative examples, and to clearly demonstrate the effects of each core technical feature, the performance test results of all samples are summarized in Table 1 below: Table 1: Comparison of performance test results between each embodiment and the comparative example

[0054] As shown in Table 1, the Antarctic krill oil yield prepared in the three embodiments of this invention remained stable between 8.4% and 8.6%, the total phospholipid content remained stable between 41.8% and 42.3%, the EPA content remained stable between 7.7% and 7.9%, the DHA content remained stable between 3.8% and 4.0%, the peroxide value was no higher than 2.7 meq / kg, and the proportion of separable insoluble matter before filtration was no higher than 0.33%. The performance indicators were highly consistent among the batches. Compared with single extraction with 75% ethanol and single extraction with 90% ethanol, the gradient extraction with dual concentration ethanol can synergistically leverage the extraction advantages of both concentrations of ethanol. This improved the yield from 7.8%-8.0% to 8.5%-8.6%, and the total phospholipid content from 38.7%-39.2% to 41.8%-42.3%. Simultaneously, the proportion of separable insoluble matter before filtration decreased from 0.60%-0.68% to 0.31%-0.33%, achieving a comprehensive improvement in yield, purity, and filtration stability. Compared to omitting the 8℃ low-temperature settling impurity removal step, under identical extraction processes, the proportion of separable insoluble matter before filtration significantly decreased from 0.52% to 0.31%, fully demonstrating that low-temperature settling promotes efficient impurity removal by facilitating the flocculation and sedimentation of colloidal particles.

[0055] In summary, this invention achieves efficient and stable preparation of food-grade Antarctic krill oil from extraction to finished product through a combination of processes including gradient extraction with dual-concentration ethanol, low-temperature static settling at 8°C for impurity removal, low-temperature light-proof and vacuum concentration, and nitrogen-filled desolvation.

[0056] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any way. Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Any person skilled in the art can make some modifications or alterations to the above-disclosed technical content to create equivalent embodiments without departing from the scope of the present invention. Any simple modifications, equivalent changes and alterations made to the above embodiments based on the technical essence of the present invention without departing from the scope of the present invention shall still fall within the scope of the present invention.

Claims

1. A method for preparing Antarctic krill oil by low-temperature static purification with food-grade dual-concentration ethanol, characterized in that, The specific steps of this method are as follows: S100. Take Antarctic krill powder, place it in a -5℃ environment for 30 minutes to equilibrate, and then perform low-temperature pulverization. After pulverization, pass it through a 40-mesh standard sieve to obtain krill raw material powder. S200. Add a 75% ethanol aqueous solution to the krill raw material powder, wherein the mass-volume ratio of the krill raw material powder to the ethanol aqueous solution is 1 kg: 5 L. Stir and extract for 2 hours at a stirring speed of 80 r / min-120 r / min under constant temperature of 25℃. After extraction, separate by vacuum filtration to obtain a primary extract and a primary filter residue. S300. Add a 90% ethanol aqueous solution to the primary filter residue. The amount of the 90% ethanol aqueous solution added is 1 kg: 5 L based on the initial mass of the krill raw material powder mentioned in S100. Stir and extract for 2 hours at a constant temperature of 35°C and a stirring speed of 80 r / min-120 r / min. After extraction, separate by vacuum filtration to obtain the secondary extract. S400. Transfer all of the primary extract and the secondary extract to a sealed, light-proof container and mix them thoroughly to obtain a combined extract. Place the combined extract in a constant temperature environment at 8°C and let it stand in the dark for 8 hours. S500. After standing, transfer the combined extract to a centrifuge and centrifuge at 4000 r / min for 10 min. After centrifugation, collect the upper clear supernatant, discard the lower precipitate, and filter the supernatant under pressure using a food-grade filter membrane with a pore size of 0.45 μm-1.0 μm to obtain a clear filtrate. S600. The clarified filtrate is transferred to a rotary evaporator and concentrated under reduced pressure at 42°C and a vacuum of -0.090MPa in the dark until no more distillate is produced. Ethanol and water are removed to obtain crude krill oil. S700. The crude krill oil is transferred to a sealed desolventizing tank, and nitrogen gas with a purity of ≥99.9% is introduced at a temperature of 35℃-45℃ for bubbling desolventizing treatment for 20min-60min. After desolventizing, the product is aseptically filled under continuous nitrogen purging and light-proof conditions to obtain food-grade Antarctic krill oil.

2. The method for preparing Antarctic krill oil by low-temperature static purification with food-grade dual-concentration ethanol according to claim 1, characterized in that, In the S100, the moisture content of the Antarctic krill powder is <8.0%, and the material temperature is always controlled at <0℃ during the low-temperature grinding process.

3. The method for preparing Antarctic krill oil by low-temperature static purification with food-grade dual-concentration ethanol according to claim 1, characterized in that, In S200 and S300, vacuum filtration is performed using a Buchner funnel in conjunction with qualitative filter paper, and the vacuum degree is controlled between -0.06MPa and -0.08MPa.

4. The method for preparing Antarctic krill oil by low-temperature static purification with food-grade dual-concentration ethanol according to claim 1, characterized in that, In the S500, the food-grade filter membrane is one of polypropylene membrane, polytetrafluoroethylene membrane or polyethersulfone membrane, and the filtration pressure is controlled between 0.1MPa and 0.3MPa.

5. The method for preparing Antarctic krill oil by low-temperature static purification with food-grade dual-concentration ethanol according to claim 1, characterized in that, In the S600, the rotation speed of the rotary evaporator is controlled at 60 r / min-80 r / min, and the evaporation flask is wrapped with aluminum foil to avoid light throughout the concentration process.

6. The method for preparing Antarctic krill oil by low-temperature static purification with food-grade dual-concentration ethanol according to claim 1, characterized in that, In the S700, the nitrogen flow rate for nitrogen bubbling is controlled at 0.5L / min-1.0L / min, and the pressure inside the desolvation tank is maintained at a slightly positive pressure.

7. The method for preparing Antarctic krill oil by low-temperature static purification with food-grade dual-concentration ethanol according to claim 1, characterized in that, This method does not use hexane, petroleum ether, No. 6 solvent oil, cyclohexane, or ethyl acetate as extraction or extraction solvents throughout the entire process.

8. The method for preparing Antarctic krill oil by low-temperature static purification with food-grade dual-concentration ethanol according to claim 1, characterized in that, This method does not employ shear emulsification, countercurrent extraction, or salting-out layering purification steps throughout the entire process.

9. The method for preparing Antarctic krill oil by low-temperature static purification with food-grade dual-concentration ethanol according to claim 1, characterized in that, In the S700, the food-grade Antarctic krill oil product contains 40.5%-43.5% total phospholipids, 7.2%-8.2% EPA, and 3.6%-4.2% DHA.

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