Lipids enriched in sphingomyelin, methods of preparation and phospholipid ingredient for infant formula

Abstract

The application provides a sphingomyelin-rich lipid, a preparation method and a phospholipid ingredient for infant formula milk powder, relates to the technical field of dairy processing, and utilizes supercritical carbon dioxide extraction technology for preliminary separation, and combines secondary extraction of subsequent ethanol as an entraining agent, so that non-polar lipids can be effectively removed, and high-polarity phospholipids, especially sphingomyelin, can be reserved. The ethanol concentration is controlled to be 20% (v / v), which is helpful to improve the solubility of the sphingomyelin-rich lipid and the selective extraction efficiency, so that higher sphingomyelin enrichment can be realized in the final product. Meanwhile, the application can also directly adopt ethanol extraction combined with a freezing crystallization method, in which no other organic solvents are introduced, the method is green and safe, and no complex equipment support is needed; the process flow is short, the operation is simple, the cost is low, and the method is suitable for rapid preparation.

Description

Technical Field

[0001] This invention relates to the field of dairy processing technology, and in particular to a lipid rich in sphingomyelin, its preparation method, and a phospholipid ingredient for use in infant formula. Background Technology

[0002] Breast milk is the gold standard for guiding the IF (Infant Formula) formulation of infant formula. The phospholipids most abundant in breast milk are mainly phosphatidylcholine (PC), phosphatidylethanolamine (PE), sphingomyelin (SM), phosphatidylinositol (PI), and phosphatidylserine (PS). The main fatty acids in breast milk phospholipids are C16:0, C18:0, C18:1, and C18:2. According to Jiang Chenyu et al. ("Detection Technology and Application of Polar Lipids in Breast Milk Based on Ultra-High Performance Supercritical Fluid Chromatography-Mass Spectrometry", Jiang Chenyu, 2022), the content of SM (supercritical fluid lipids) in domestic breast milk is the highest, averaging 29.89%, followed by PE (polysaccharide) and PC (polycarboxylic acid), with average relative contents of 27.59% and 27.38%, respectively. PS (polysaccharide) and PI (polycarboxylic acid) have the lowest contents, at 8.44% and 7.66%, respectively. The main fatty acids in phospholipids are C16:0, C18:0, C18:1, and C18:2, with average relative contents of approximately 40.95%, 17.54%, 12.09%, and 9.75%, respectively. Other fatty acids crucial for infant growth and development include C20:4n-6 (ARA), C22:6n-3 (DHA), C18:3n-3 (ALA), and C20:5n-3 (EPA), with average relative contents of approximately 1.83%, 0.96%, 0.70%, and 0.11%, respectively.

[0003] As research progresses, fibrocystic milk (IF) is becoming increasingly similar to breast milk in terms of nutritional value. Currently, infant formula produced in my country not only macroscopically mimics the fat content of breast milk, but also closely resembles it in fatty acid composition and content, as well as triglyceride configuration. However, because most infant formulas use skim milk powder as a base and vegetable oil to adjust the fat in IF, the phospholipid content in current infant formulas is very low. Some infant formulas add phospholipids, such as soybean phospholipids or milk phospholipid concentrate, but their phospholipid composition and fatty acid composition differ significantly from breast milk, especially the sphingomyelin content, which is far lower than that of breast milk.

[0004] Sphingomyelin is a compound whose concentration can vary between infant formula and breast milk, with breast milk typically containing significantly higher levels than infant formula. This can result in lower sphingomyelin intake in formula-fed infants, which may have negative effects on their growth and development, including neurodevelopment and cognitive development.

[0005] Therefore, it is particularly important to develop infant formula milk powder rich in sphingomyelin.

[0006] In view of this, the present invention is hereby proposed. Summary of the Invention

[0007] The purpose of this invention is to provide a method for preparing lipids rich in sphingomyelin and the prepared lipids rich in sphingomyelin, which are then used as raw materials for phospholipid ingredients in infant formula based on a similarity score evaluation method, thereby at least solving one of the technical problems existing in the prior art.

[0008] In order to achieve the above-mentioned objectives of the present invention, the following technical solution is adopted:

[0009] In a first aspect, the present invention provides a method for preparing lipids rich in sphingomyelin, including supercritical-ethanol extraction and / or ethanol extraction-freeze-crystallization:

[0010] The supercritical-ethanol extraction method includes: using whey protein powder phospholipid concentrate as raw material, performing a first extraction using supercritical carbon dioxide, then adding an entrainer and performing a second extraction using supercritical carbon dioxide, precipitating the aqueous suspension of the extract with alcohol, taking the anhydrous ethanol layer, removing the solvent, and obtaining the lipid SESM rich in sphingomyelin.

[0011] The entrainer includes 20% ± 1% v / v ethanol;

[0012] The alcohol extraction-freeze crystallization method includes: using whey protein powder phospholipid concentrate as raw material, subjecting the aqueous suspension of the whey protein powder phospholipid concentrate to alcohol precipitation, taking the ethanol solution layer, cooling and separating the obtained crystals to obtain the lipid ECSM rich in sphingomyelin;

[0013] The ethanol solution is 80% v / v ethanol.

[0014] Secondly, the present invention provides a sphingomyelin-rich lipid SESM prepared by the above preparation method.

[0015] Thirdly, the present invention provides a sphingomyelin-rich lipid ECSM prepared by the above preparation method.

[0016] Fourthly, the present invention provides the application of the above-mentioned sphingomyelin-rich lipids in the preparation of infant formula.

[0017] Fifthly, the present invention provides a phospholipid ingredient for infant formula milk powder, including soybean phospholipid, sunflower seed phospholipid, milk phospholipid, and the above-mentioned sphingomyelin-rich lipid SESM or sphingomyelin-rich lipid ECSM.

[0018] In a sixth aspect, the present invention provides a method for formulating the above-mentioned phospholipid ingredients, using the composition of phospholipids and phospholipid fatty acids as evaluation indicators, and employing a similarity scoring method to screen out the phospholipids and phospholipid fatty acid compositions with the highest similarity scores to obtain the phospholipid ingredients.

[0019] The evaluation formula is as follows:

[0020] ;

[0021] ;

[0022] ;

[0023] In the formula, Gi (PL / PLFA) The similarity between the phospholipid ingredients and breast milk in terms of phospholipid composition and phospholipid fatty acid composition is given, with each similarity score out of 100.

[0024] Ei (PL / PLFA) For phospholipids and phospholipid fatty acids that differ from each other, the corresponding similarity score should be deducted.

[0025] Di (PL / PLFA) / The ratio of the corresponding phospholipids and phospholipid fatty acids in breast milk to the total phospholipids and total phospholipid fatty acids used in the evaluation.

[0026] Ci (PL / PLFA) This is a fluctuation coefficient, mainly determined by the content of phospholipids and phospholipid fatty acids in the phospholipid ingredients;

[0027] Ai (PL / PLFA) This refers to the upper or lower limit of the corresponding phospholipids or phospholipid fatty acids in breast milk. When the content of phospholipids or phospholipid fatty acids in the phospholipid ingredient exceeds the upper limit of the corresponding phospholipid or phospholipid fatty acid content in breast milk, Ai... (PL / PLFA) Take the upper limit value; when the content of phospholipids or phospholipid fatty acids in the phospholipid ingredients is lower than the corresponding lower limit in breast milk, Ai (PL / PLFA) Take the lower limit value; when the content of phospholipids or phospholipid fatty acids in the phospholipid ingredients is within the corresponding range of breast milk, Ci (PL / PLFA) It is zero.

[0028] Compared with the prior art, the present invention has the following beneficial effects:

[0029] This invention utilizes supercritical carbon dioxide extraction for initial separation, followed by a secondary extraction with ethanol as an entrainer. This effectively removes nonpolar lipids while retaining highly polar phospholipids, especially sphingomyelin. Controlling the ethanol concentration at 20% ± 1% (v / v) helps improve the solubility and selective extraction efficiency of sphingomyelin-rich lipids, resulting in a higher sphingomyelin enrichment in the final product. Furthermore, this invention can also directly employ ethanol extraction combined with freeze-crystallization. This method does not introduce other organic solvents, is environmentally friendly and safe, requires no complex equipment, and features a short, simple, and low-cost process suitable for rapid preparation. It is understood that ethanol is easily prepared artificially in a laboratory setting and may have an error margin of ±1%, which has no substantial impact on the final result.

[0030] The lipids SESM and ECSM rich in sphingomyelin obtained by the preparation method provided by this invention have high purity and can better meet the nutritional needs of infants and young children.

[0031] Furthermore, this invention, referencing the phospholipid and fatty acid composition of Chinese breast milk, and based on phospholipid-rich raw materials and a similarity score evaluation method, uses soybean phospholipid, sunflower seed phospholipid, and whey protein powder phospholipid concentrate as raw materials to prepare infant formula phospholipid ingredients with a phospholipid and fatty acid composition similar to that of Chinese breast milk, enabling them to be better applied to lipid ingredients in infant formula. Attached Figure Description

[0032] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0033] Figure 1 The graph shows the SM yield results of whey protein powder alcohol extraction provided in the experimental example of the present invention and the sphingomyelin-rich lipid SESM in Example 1;

[0034] Figure 2 A graph showing the phospholipid composition of SESM, a lipid rich in sphingomyelin, provided as an experimental example of the present invention;

[0035] Figure 3 The results of sphingomyelin SM yield, phospholipid composition and breast milk similarity in the lipid SESM rich in sphingomyelin provided in Example 1 of the present invention are shown in the figure.

[0036] Figure 4 The similarity score results of Examples 6 and 7 and Comparative Examples 3 and 4 provided for the experimental examples of the present invention with breast milk are shown in the figure. Detailed Implementation

[0037] Unless otherwise defined herein, the scientific and technical terms used in conjunction with this invention shall have the meanings commonly understood by one of ordinary skill in the art. The meaning and scope of terms shall be clear; however, in any case of potential ambiguity, the definitions provided herein shall prevail over any dictionary or foreign definitions. In this application, unless otherwise stated, the use of "or" means "and / or". Furthermore, the use of the term "comprising" and other forms is non-limiting.

[0038] The technical solution of the present invention will be clearly and completely described below with reference to the embodiments. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0039] According to a first aspect of the present invention, a method for preparing sphingomyelin-rich lipids is provided, characterized in that it comprises a supercritical-ethanol extraction method and / or an ethanol extraction-freeze-crystallization method:

[0040] Supercritical-ethanol extraction method The process includes: using whey protein powder phospholipid concentrate as raw material, performing a first extraction using supercritical carbon dioxide, then adding an entrainer and performing a second extraction using supercritical carbon dioxide, precipitating the aqueous suspension of the extract with alcohol, taking the anhydrous ethanol layer, removing the solvent, and obtaining the lipid rich in sphingomyelin, named SESM; the entrainer includes 20% v / v ethanol.

[0041] This method utilizes supercritical carbon dioxide extraction (SC-CO2) for initial separation, combined with a subsequent secondary extraction using ethanol as an entrainer. This effectively removes nonpolar lipids (such as triglycerides and cholesterol) while retaining highly polar phospholipids, especially sphingomyelin. Maintaining an ethanol concentration of 20% (v / v) helps improve the solubility and selective extraction efficiency of sphingomyelin, resulting in a higher SM enrichment in the final product. Furthermore, supercritical extraction operates at low temperatures and without solvent residue, avoiding the damage to heat-sensitive phospholipid components caused by high temperatures, and leaving no toxic solvent residue, ensuring product safety and making it suitable for infant food ingredients.

[0042] Alcohol extraction-freeze crystallization method The method includes: using whey protein powder phospholipid concentrate as raw material, subjecting the aqueous suspension of the whey protein powder phospholipid concentrate to alcohol precipitation, taking the ethanol solution layer, cooling and separating the obtained crystals to obtain the lipid rich in sphingomyelin, named ECSM; the ethanol solution is 80% v / v ethanol.

[0043] This method directly employs ethanol extraction combined with freeze crystallization, requiring no complex equipment. It features a short process flow, simple operation, and low cost, making it suitable for rapid preparation on a small to medium scale or in a laboratory setting. The freeze crystallization process preferentially crystallizes sphingomyelin from mixed phospholipids, further increasing its relative content (reaching 55.29% in the examples), which more closely resembles the high proportion of sphingomyelin in Chinese breast milk (approximately 29.89%), thus better mimicking the composition of breast milk. Furthermore, low-temperature operating conditions (such as cooling to 0°C) effectively prevent structural damage to heat-sensitive phospholipids, maintaining their bioactivity, making it particularly suitable for the preparation of infant formula.

[0044] In some preferred embodiments, in the supercritical-ethanol extraction process:

[0045] The temperature of the first extraction is 40~50℃, for example, but not limited to 40℃, 42℃, 45℃, 48℃ or 50℃, preferably 45℃; the pressure is 25~35 MPa, for example, but not limited to 25 MPa, 28 MPa, 30 MPa, 32 MPa or 35 MPa, preferably 30 MPa; the time is 1.5~2.5 hours, for example, but not limited to 1.5 hours, 1.8 hours, 2 hours, 2.2 hours or 2.5 hours, preferably 2 hours.

[0046] The temperature of the secondary extraction is 40~60℃, for example, but not limited to 40℃, 45℃, 50℃, 55℃ or 60℃, preferably 40℃; the pressure is 25~35 MPa, for example, but not limited to 25 MPa, 28 MPa, 30 MPa, 32 MPa or 35 MPa, preferably 30 MPa; the time is 1.5~2.5 hours, for example, but not limited to 1.5 hours, 1.8 hours, 2 hours, 2.2 hours or 2.5 hours, preferably 2 hours.

[0047] Within the temperature and pressure range of the first extraction, carbon dioxide is in a supercritical state, possessing a liquid-like density and gas-like diffusion capacity, enabling it to effectively penetrate the raw material and improve the extraction efficiency for non-polar or moderately polar components. 40–50°C is a mild heating range, which helps protect the structural integrity of heat-sensitive components such as phospholipids, preventing oxidation or hydrolysis. A time range of 1.5–2.5 hours ensures sufficient extraction while avoiding excessively long operation times that could lead to inefficiency or high energy consumption. Furthermore, the identical pressure range for both extractions facilitates equipment design and operational control, reducing process complexity.

[0048] In some preferred embodiments, the ratio of whey protein powder phospholipid concentrate to entrainer is 1:5 to 7, for example, but not limited to 1:5, 1:6 or 1:7, preferably 1:6.

[0049] A reasonable feed-to-liquid ratio helps the entrainer to fully contact the raw material, improving the extraction and recovery rate of sphingomyelin. A feed-to-liquid ratio of 1:5 to 7 ensures extraction efficiency while avoiding the recovery burden and cost increase caused by excessive solvent.

[0050] In some preferred embodiments, the material-to-liquid ratio of the extract suspension is 1:5 to 7, for example, but not limited to 1:5, 1:6 or 1:7, preferably 1:6.

[0051] A reasonable material-to-liquid ratio in aqueous suspensions can ensure that phospholipid components are fully dispersed in water, providing a good foundation for subsequent alcohol precipitation.

[0052] In some preferred embodiments, the alcohol precipitation includes mixing the aqueous suspension of the extract with anhydrous ethanol at a temperature of 60-80°C for 0.8-1.2 hours, wherein the volume ratio of the aqueous suspension of the extract to anhydrous ethanol is 1:4-6; wherein the temperature can be, for example, but not limited to, 60°C, 65°C, 70°C, 75°C or 80°C, preferably 70°C; the time can be, for example, but not limited to, 0.8 hours, 1 hour or 1.2 hours, preferably 1 hour; and the volume ratio can be, for example, but not limited to, 1:4, 1:5 or 1.6, preferably 1:5.

[0053] Ethanol is a good precipitant for phospholipids. Heating at 60-80℃ can accelerate the precipitation of phospholipids from the aqueous phase and shorten the precipitation time. A stirring time of 0.8-1.2 hours is recommended to ensure sufficient reaction while avoiding excessive stirring that could lead to co-precipitation of impurities. A 1:4-6 ratio effectively precipitates the target component without wasting solvent or losing phospholipids due to excessive ethanol.

[0054] In some preferred embodiments, the anhydrous ethanol layer is obtained by centrifugation at a speed of 4000-5000 r / min, for example, but not limited to 4000 r / min, 4200 r / min, 4500 r / min, 4800 r / min or 5000 r / min, preferably 4500 r / min; and for a time of 25-35 minutes, for example, but not limited to 25 minutes, 28 minutes, 30 minutes, 32 minutes or 35 minutes, preferably 30 minutes.

[0055] Reasonable centrifugation conditions can improve phospholipid recovery rate, and the parameter range is in line with the performance of conventional industrial centrifuges, which is conducive to large-scale application.

[0056] In some preferred embodiments, the solvent is removed by rotary evaporation at a temperature of 50-60°C, for example, but not limited to 50°C, 52°C, 55°C, 58°C or 60°C.

[0057] At this temperature, ethanol is easily volatilized and recycled, which is beneficial to environmental protection and cost control, while not causing phospholipid oxidation or hydrolysis.

[0058] In some preferred embodiments, in the alcohol extraction-freeze crystallization method:

[0059] The liquid-to-solid ratio of the aqueous suspension of the whey protein powder phospholipid concentrate is 1:5 to 7, for example, but not limited to 1:5, 1:6 or 1:7, preferably 1:6.

[0060] The water-liquid ratio of whey protein powder phospholipid concentrate is set to 1:5~7 to ensure that the raw materials and solvents are in full contact, which ensures the full dissolution of phospholipids and avoids excessive solvent, which would lead to excessively long concentration steps.

[0061] In some preferred embodiments, the alcohol precipitation includes mixing an aqueous suspension of whey protein powder phospholipid concentrate with an ethanol solution at a temperature of 60-80°C for 0.8-1.2 hours, wherein the volume ratio of the aqueous suspension of whey protein powder phospholipid concentrate to the ethanol solution is 1:4-6; wherein the temperature can be, for example, but not limited to, 60°C, 65°C, 70°C, 75°C or 80°C, preferably 70°C; the time can be, for example, but not limited to, 0.8 hours, 1 hour or 1.2 hours, preferably 1 hour; and the volume ratio can be, for example, but not limited to, 1:4, 1:5 or 1.6, preferably 1:5.

[0062] Ethanol is a good precipitant for phospholipids. Heating at 60-80℃ can accelerate the precipitation of phospholipids from the aqueous phase and shorten the precipitation time. A stirring time of 0.8-1.2 hours is recommended to ensure sufficient reaction while avoiding excessive stirring that could lead to co-precipitation of impurities. A 1:4-6 ratio effectively precipitates the target component without wasting solvent or losing phospholipids due to excessive ethanol.

[0063] In some preferred embodiments, the ethanol solution layer is obtained by centrifugation at a speed of 4000-5000 r / min, for example, but not limited to 4000 r / min, 4200 r / min, 4500 r / min, 4800 r / min or 5000 r / min, preferably 4500 r / min; and for a time of 25-35 minutes, for example, but not limited to 25 minutes, 28 minutes, 30 minutes, 32 minutes or 35 minutes, preferably 30 minutes.

[0064] This parameter range can effectively separate soluble phospholipids from insoluble impurities in the ethanol phase, while avoiding excessive centrifugal force that could damage the phospholipid structure or increase operating costs.

[0065] Preferably, the process further includes a step of freeze-drying the crystals to a constant weight; preferably, freeze-drying is performed using a low-temperature refrigerated centrifuge; the operating temperature of the low-temperature refrigerated centrifuge is 0°C, and the rotation speed is 5500~6500 rpm / s, for example, but not limited to 5500 r / min, 5800 r / min, 6000 r / min, 6200 r / min or 6500 r / min, preferably 6000 r / min; the centrifugation time is 25~35 minutes, for example, but not limited to 25 minutes, 28 minutes, 30 minutes, 32 minutes or 35 minutes, preferably 30 minutes.

[0066] Under the above conditions, residual moisture and ethanol can be effectively removed to obtain a dry, stable SM-rich phospholipid product (ECSM).

[0067] According to a second aspect of the present invention, a sphingomyelin-rich lipid SESM prepared by the above-described preparation method is provided.

[0068] According to a third aspect of the present invention, a sphingomyelin-rich lipid ECSM prepared by the above-described preparation method is provided.

[0069] The lipids SESM and ECSM rich in sphingomyelin obtained by the preparation method provided by this invention have high purity and can better meet the nutritional needs of infants and young children.

[0070] Based on this, a fourth aspect of the present invention provides the use of the above-mentioned sphingomyelin-rich lipids in the preparation of infant formula.

[0071] According to a fifth aspect of the present invention, a phospholipid ingredient for infant formula is provided, comprising soybean phospholipids, sunflower seed phospholipids, milk phospholipids, and the aforementioned sphingomyelin-rich lipid SESM or sphingomyelin-rich lipid ECSM.

[0072] This invention, referencing the phospholipid and fatty acid composition of Chinese breast milk, and based on phospholipid-rich raw materials and a similarity scoring evaluation method, uses soybean phospholipid, sunflower seed phospholipid, and whey protein powder phospholipid concentrate as raw materials to prepare phospholipid ingredients for infant formula with a phospholipid and fatty acid composition similar to that of Chinese breast milk, making them better suited for use in lipid ingredients of infant formula.

[0073] In some preferred embodiments, the mass ratio of soybean lecithin, sunflower seed lecithin, milk lecithin, and sphingomyelin-rich lipid SESM is 1:11:17:21.

[0074] In some other preferred embodiments, the mass ratio of soybean lecithin, sunflower seed lecithin, milk lecithin, and sphingomyelin-rich lipid ECSM is 5:18:30:47.

[0075] At the above ratio, the fatty acid composition is more balanced, which meets the physiological needs of infants and young children for fatty acids. At the same time, it increases the intake of sphingomyelin, promotes the neural development of infants and young children, and provides a lipid ingredient basis for infant formula that is closer to breast milk.

[0076] Furthermore, according to a sixth aspect of the present invention, a method for formulating the above-mentioned phospholipid ingredients is provided, wherein the composition of phospholipids and phospholipid fatty acids is used as an evaluation index, and a similarity scoring method is employed to screen out the phospholipids and phospholipid fatty acid compositions with the highest similarity scores to obtain the phospholipid ingredients.

[0077] The evaluation formula is as follows:

[0078] ;

[0079] ;

[0080] ;

[0081] In the formula, Gi (PL / PLFA) The similarity between the phospholipid ingredients and breast milk in terms of phospholipid composition and phospholipid fatty acid composition is given, with each similarity score out of 100.

[0082] Ei (PL / PLFA) For phospholipids and phospholipid fatty acids that differ from each other, the corresponding similarity score should be deducted.

[0083] Di (PL / PLFA) / The ratio of the corresponding phospholipids and phospholipid fatty acids in breast milk to the total phospholipids and total phospholipid fatty acids used in the evaluation.

[0084] Ci (PL / PLFA) This is a fluctuation coefficient, mainly determined by the content of phospholipids and phospholipid fatty acids in the phospholipid ingredients;

[0085] Ai (PL / PLFA) This refers to the upper or lower limit of the corresponding phospholipids or phospholipid fatty acids in breast milk. When the content of phospholipids or phospholipid fatty acids in the phospholipid ingredient exceeds the upper limit of the corresponding phospholipid or phospholipid fatty acid content in breast milk, Ai... (PL / PLFA) Take the upper limit value; when the content of phospholipids or phospholipid fatty acids in the phospholipid ingredients is lower than the corresponding lower limit in breast milk, Ai (PL / PLFA) Take the lower limit value; when the content of phospholipids or phospholipid fatty acids in the phospholipid ingredients is within the corresponding range of breast milk, Ci (PL / PLFA) It is zero.

[0086] Phospholipids include SM, PC, PE, PS, and PI; phospholipid fatty acids include C16:0, C18:0, C18:1 n-9, C18:2 n-6, C18:3 n-3 ALA, C20:4 n-6 ARA, C20:5 n-3 EPA, and C22:6 n-3 DHA.

[0087] Using mathematical models to quantify and score phospholipid composition and fatty acid composition enhances scientific rigor and reproducibility. Furthermore, the introduction of fluctuation coefficients and upper / lower limit mechanisms prevents extreme biases of single indicators from affecting the overall score, enabling precise assessment of the contribution of each phospholipid type in the formulation to the overall similarity.

[0088] Based on the method for preparing lipids rich in sphingomyelin and the method for formulating phospholipid ingredients provided by the present invention, the present invention also provides a method for preparing phospholipid ingredients for infant formula, comprising the following steps:

[0089] Step 1: Enrichment of sphingomyelin-rich lipids by supercritical-ethanol extraction: Using whey protein powder phospholipid concentrate as raw material, a first extraction was performed using supercritical carbon dioxide. Then, 20% v / v ethanol was added as an entrainer for a second extraction using supercritical carbon dioxide. The aqueous suspension of the extract was precipitated with alcohol, and the anhydrous ethanol layer was collected. After removing the solvent, the sphingomyelin-rich lipid SESM was obtained.

[0090] Step 2: Enrichment of SM-rich phospholipids by alcohol extraction-freeze-crystallization: Using whey protein powder phospholipid concentrate as raw material, the aqueous suspension of the whey protein powder phospholipid concentrate is subjected to alcohol precipitation. The ethanol solution layer is collected, cooled, and the resulting crystals are separated to obtain the sphingomyelin-rich lipid ECSM. The ethanol solution is 80% v / v ethanol.

[0091] Step 3, Phospholipid Composition Determination and Sample Preparation: The enriched phospholipids were analyzed by ultra-high performance liquid chromatography to determine their composition; and an appropriate amount of phospholipids was weighed to prepare a phospholipid sample; specifically:

[0092] Weigh 25 mg of PC, PE, SM, PI and PS standards respectively and dilute to 10 mL volumetric flasks with chromatographic grade chloroform to prepare a 2.5 mg / mL mixed standard stock solution;

[0093] Accurately measure 0.4 mL, 0.8 mL, 1.6 mL, 2.4 mL, and 3.2 mL of the stock solution into 10 mL volumetric flasks, dilute to volume with chromatographic grade chloroform, and prepare mixed phospholipid standards of 0.1, 0.2, 0.4, 0.6, and 0.8 mg / mL. After passing through a 0.22 μm organic filter membrane, perform ultra-high performance liquid chromatography (UPLC) analysis.

[0094] Ultra-high performance liquid chromatography conditions:

[0095] Column: Waters BEH HILIC, 1.7 μm, 2.1 mm × 100 mm;

[0096] Aqueous phase: 10 mM ammonium formate aqueous solution (adjusted to pH=3 with formic acid);

[0097] Organic phase: acetonitrile;

[0098] Flow rate: 0.3 mL / min;

[0099] Column temperature: 30℃;

[0100] Injection volume: 5 μL;

[0101] Evaporation parameters: drift tube temperature 85℃; gas flow rate 1.5L / min;

[0102] Gradient elution reference conditions: 0 min 95% B, 0.1 min 95% B, 20 min 80% B, 21 min 70% B, 23 min 70% B, 24 min 95% B, 26 min 95% B;

[0103] Add 1 mL of chromatographically pure chloroform to the phospholipid sample, filter through a 0.22 μm organic filter membrane, and transfer to a brown vial for analysis.

[0104] Step 4: Determination of total fatty acid content in phospholipids: First, the phospholipids are purified using soybean phospholipids, sunflower seed phospholipids, and two lipids rich in sphingomyelin as raw materials. Taking advantage of the insolubility of phospholipids in acetone, cold acetone is added to the raw materials. After magnetic stirring, the supernatant is removed. This process is repeated until thin-layer chromatography detects almost no neutral lipids in the precipitate. Then, the fatty acid composition of the phospholipid sample is determined using the sodium methoxide-methanol method. Fatty acid analysis is performed according to standard spectra, and each fatty acid is quantified based on its relative content as a percentage of total fatty acids. Specifically:

[0105] Take 2 mg of phospholipids, dissolve them in 1.5 mL of n-hexane solution, add 40 μL of methyl acetate solution and 100 μL of sodium methoxide solution, vortex mix, react in a 37℃ water bath for 20 min, then place in a -20℃ refrigerator for 10 min, take it out immediately and add 60 μL of oxalate-methyl acetate solution, centrifuge to discard the precipitate, add anhydrous sodium sulfate to dry, blow dry with nitrogen, and analyze the total fatty acid composition of breast milk by gas chromatography.

[0106] Gas chromatography conditions: CP-Si188 fused silica capillary column (100m × 0.25mm, 0.2μm) was used, with H2 and N2 as fuel gas and carrier gas, respectively; the injection port temperature and FID temperature were both 250℃.

[0107] Temperature program: Hold at 45℃ for 4 minutes, increase to 175℃ at 13℃ / min, hold for 27 minutes, then increase to 215℃ at 4℃ / min, hold at 215℃ for 35 minutes, for a total duration of 86 minutes.

[0108] Step 5: Formulation and Evaluation of Phospholipid Ingredients in Infant Formula: Using the phospholipid sample as a base, calculate the formulation ratio, prepare the phospholipid ingredients for infant formula, and evaluate their similarity. Using a similarity scoring method, select the infant formula phospholipid ingredient formula with the highest similarity score, i.e.:

[0109] IFPI1: The mass ratio of soybean lecithin, sunflower seed lecithin, milk lecithin, and sphingomyelin-rich lipids (SESM) is 1:11:17:21;

[0110] IFPI2: The mass ratio of soybean phospholipids, sunflower seed phospholipids, milk phospholipids, and sphingomyelin-rich lipid ECSM is 5:18:30:47.

[0111] It should be noted that breast milk phospholipids contain a relatively high content of sphingomyelin (SM), an animal-specific phospholipid not found in plants. SM was not detected in soybean phospholipids (SPL) and sunflower seed phospholipids (SSPL), and only milk phospholipids (MPL) contained a relatively low content of SM (see Table 1 below), which cannot meet the requirement of breast milk phospholipids for a relatively high SM content. Therefore, in order to obtain IFPI (infant formula phospholipid ingredient) as similar as possible to breast milk phospholipids, this invention prepared SESM and ECSM with relatively high SM content based on MPL.

[0112] Table 1. Phospholipid composition (%) of SPL, SSPL, MPL, SESM and ECSM

[0113]

[0114] Secondly, by adjusting the proportions of the phospholipid samples, this invention yielded a variety of phospholipid ingredients for infant formula. Furthermore, using a similarity score evaluation model, two phospholipid ingredients, IFPI1 and IFPI2, were prepared for infant formula. These ingredients have similarity scores of 91.25 and 91.49, respectively, to Chinese breast milk in terms of phospholipid and phospholipid fatty acid composition and content.

[0115] Table 2. Important phospholipid fatty acid composition (%) of SPL, SSPL, MPL, SESM, and ECSM

[0116]

[0117] The present invention will be further illustrated by the following examples. Unless otherwise specified, the materials in the examples are prepared according to existing methods or purchased directly from the market.

[0118] Example 1

[0119] This embodiment provides a sphingomyelin-rich lipid SESM, which is prepared by the following method:

[0120] Take 60g of whey protein powder phospholipid concentrate, place it in a material bag, and add 60g of glass beads to enhance mass transfer performance. Perform a single extraction with pure carbon dioxide at a temperature of 45℃, an extraction pressure of 30MPa, and an extraction time of 2h. After the first extraction, continuously add 360mL of 20% ethanol through an entrainer pump for a second extraction at a temperature of 40℃, an extraction pressure of 30MPa, and an extraction time of 2h. After two supercritical extractions, 50.43g of raffinate powder is obtained.

[0121] Add 2L of 100% anhydrous ethanol to a beaker and heat to 70℃. Dissolve the extract powder in 300mL of deionized water and mix well to obtain an aqueous suspension of the extract. Then pour the aqueous suspension of the extract into 100% anhydrous ethanol and stir magnetically at a constant temperature for 1h. Centrifuge at 4500r / min for 30min. Take the upper anhydrous ethanol layer and evaporate it using a rotary evaporator at 55℃. When the upper liquid is evaporated to 1mL, collect the concentrated upper liquid into a glass dish and dry it to constant weight using a vacuum freeze dryer to obtain 14.42g of sphingomyelin-rich lipid SESM.

[0122] Example 2

[0123] This embodiment provides a sphingomyelin-rich lipid SESM, which is prepared by the following method:

[0124] Take 60g of whey protein powder phospholipid concentrate, place it in a material bag, and add 60g of glass beads to enhance mass transfer performance. Perform a single extraction with pure carbon dioxide at a temperature of 40℃, an extraction pressure of 35MPa, and an extraction time of 1.5h. After the first extraction, continuously add 420mL of 20% ethanol through an entrainer pump for a second extraction at a temperature of 60℃, an extraction pressure of 35MPa, and an extraction time of 1.5h. After two supercritical extractions, 50.57g of raffinate powder is obtained.

[0125] Add 2L of 100% anhydrous ethanol to a beaker and heat to 80℃. Dissolve the extract powder in 300mL of deionized water and mix well to obtain an aqueous suspension of the extract. Then pour the aqueous suspension of the extract into 100% anhydrous ethanol and stir magnetically at a constant temperature for 0.8h. Centrifuge at 5000r / min for 25min. Take the upper anhydrous ethanol layer and evaporate it using a rotary evaporator at 60℃. When the upper liquid is evaporated to 1mL, collect the concentrated upper liquid into a glass dish and dry it to constant weight using a vacuum freeze dryer to obtain 14.37g of sphingomyelin-rich lipid SESM.

[0126] Comparative Example 1

[0127] This comparative example provides a sphingomyelin-rich lipid SESM, which differs from Example 1 in that the entrainer is replaced with 95% (v / v) ethanol.

[0128] Comparative Example 2

[0129] This comparative example provides a sphingomyelin-rich lipid SESM, which was prepared by the following method:

[0130] Whey protein powder phospholipid concentrate was dissolved in ultrapure water (w:v=1:6) to obtain a whey protein powder solution. The whey protein powder solution was poured into a beaker containing anhydrous ethanol (the volume ratio of whey protein powder solution to ethanol solution was 1:5). The mixture was stirred at 70℃ for 1 hour, centrifuged at 4500 r / min for 30 minutes, and the anhydrous ethanol layer was collected. The ethanol layer was then evaporated using a rotary evaporator at 55℃ until the supernatant was evaporated to 1 mL. The concentrated supernatant was collected into a glass dish and dried to constant weight using a vacuum freeze dryer, which yielded the pure ethanol extract.

[0131] Experimental Example 1: Comparison of Sphingomyelin (SM) Yields

[0132] Sphingomyelin SM yield refers to the ability to recover sphingomyelin SM from whey protein powder raw materials, which is determined by... Figure 1 It can be seen that the sphingomyelin SM yield in Example 1 was 78.40%, significantly higher than that of the ethanol extraction in Comparative Example 2 (61.80%) and Comparative Example 1 (35.88%). This demonstrates that under optimal conditions, adding supercritical extraction with ethanol beforehand can significantly improve the sphingomyelin SM yield. However, this requires specific supercritical extraction conditions, necessitating the use of 20% low-concentration ethanol, while 95% high-concentration ethanol has no effect on improving the sphingomyelin SM yield.

[0133] Understandably, the sphingomyelin SM yield is obtained by multiplying the concentration of sphingomyelin in the extract (unit: SM g / g extract) by the mass of extract that can be extracted from the raw material (unit: extract g / g raw material) to get the content of sphingomyelin extracted from the raw material (unit: SM g / g raw material). This data is then divided by the absolute content of sphingomyelin measured in the raw material (unit: SM g / g raw material) and multiplied by 100 to get the sphingomyelin yield (unit: %).

[0134] Experimental Example 2: Extraction of Phospholipid Components from the Product

[0135] like Figure 2 As shown, in the extract obtained in Example 1, sphingomyelin SM accounted for 16.3% of the total extract mass, while in the extract obtained in Comparative Example 1, sphingomyelin SM accounted for only 9% of the total extract. There was a significant difference in the sphingomyelin SM content in the extracts under the two supercritical extraction conditions.

[0136] The inventors also investigated the content of total phospholipids (PL) in the extract. Under the conditions of Example 1, the proportion of total phospholipids (PL) in the extract was 31.5%, significantly higher than the 25% proportion under the conditions of Comparative Example 1. There was a significant difference in the content of total phospholipids (PL) in the extract under the two supercritical extraction conditions.

[0137] Furthermore, the ratio of sphingomyelin to phospholipids (SM / PL) in the extract was compared. The SM / PL value in the extract of Example 1 was 51.7%, significantly higher than that of Comparative Example 1 (36.2%). The results indicate that Example 1, compared to Comparative Example 1, has superior performance in terms of sphingomyelin (SM), total phospholipids (PL), and the SM / PL ratio of sphingomyelin to phospholipids in the extract.

[0138] Example 3: Comparison of the extract with breast milk phospholipids and phospholipid fatty acids

[0139] Table 3 Comparison of extracts and breast milk phospholipids

[0140]

[0141] Table 4 Comparison of extracts and breast milk phospholipid fatty acids

[0142]

[0143] like Figure 3 As shown, compared with breast milk, the similarity score of the extract of Example 1 to breast milk phospholipids and phospholipid fatty acids was 86.11, which was higher than the score of the extract of Comparative Example 1 (73.27). This indicates that the extract of Example 1 is more similar to breast milk, and 20% ethanol is more effective in supercritical extraction.

[0144] The results above indicate that 20% ethanol supercritical extraction + alcohol extraction is superior to 95% ethanol supercritical extraction + alcohol extraction in terms of sphingomyelin (SM) yield, phospholipid composition in the extracted product, and similarity to breast milk.

[0145] Example 3

[0146] This embodiment provides a lipid ECSM rich in sphingomyelin, which is prepared by the following method:

[0147] Take 60g of whey protein powder phospholipid concentrate, dissolve the powder in 360mL of deionized water and mix well to obtain an aqueous suspension of whey protein powder phospholipid concentrate. Add 2L of 80% v / v ethanol solution to a beaker and heat to 70℃. Then pour the aqueous suspension of whey protein powder phospholipid concentrate into the ethanol solution and stir magnetically at a constant temperature for 1h. Centrifuge at 4500r / min for 30min. Take the supernatant and cool it for crystallization using a low-temperature refrigerated centrifuge at 0℃ for 30min at 6000r / min. Take the precipitated crystals and dry them to constant weight using a vacuum freeze dryer to obtain 8.99g of sphingomyelin-rich lipid ECSM.

[0148] Example 4

[0149] This embodiment provides a lipid ECSM rich in sphingomyelin, which is prepared by the following method:

[0150] Take 60g of whey protein powder phospholipid concentrate, dissolve the powder in 360mL of deionized water and mix well to obtain an aqueous suspension of whey protein powder phospholipid concentrate. Add 2L of anhydrous ethanol to a beaker and heat to 80℃. Then pour the aqueous suspension of whey protein powder phospholipid concentrate into the ethanol solution and stir magnetically at a constant temperature for 1.2h. Centrifuge at 4000r / min for 35min. Take the supernatant and cool it for crystallization using a low-temperature refrigerated centrifuge at 0℃ for 35min. Take the precipitated crystals and dry them to constant weight using a vacuum freeze dryer to obtain 6.33g of sphingomyelin-rich lipid ECSM.

[0151] Example 5

[0152] This embodiment provides a lipid ECSM rich in sphingomyelin, which is prepared by the following method:

[0153] Take 60g of whey protein powder phospholipid concentrate, dissolve the powder in 360mL of deionized water and mix well to obtain an aqueous suspension of whey protein powder phospholipid concentrate. Add 2L of 70% v / v ethanol solution to a beaker and heat to 60℃. Then pour the aqueous suspension of whey protein powder phospholipid concentrate into the ethanol solution and stir magnetically at a constant temperature for 0.8h. Centrifuge at 5000r / min for 25min. Take the supernatant and cool it for crystallization using a low-temperature refrigerated centrifuge at 0℃ for 25min. Take the precipitated crystals and dry them to constant weight using a vacuum freeze dryer to obtain 4.18g of sphingomyelin-rich lipid ECSM.

[0154] Example 6

[0155] This embodiment provides a phospholipid ingredient for infant formula, which is prepared by the following method:

[0156] Take 30g each of soybean lecithin, sunflower seed lecithin and raw milk powder. Dissolve each raw material powder in 180mL of deionized water and mix well. Add 1L of 100% anhydrous ethanol to a beaker and heat to 70℃. Then pour the liquid into the ethanol solution and stir magnetically for 1 hour at a constant temperature. Centrifuge at 4500r / min for 30min. Take the upper liquid and evaporate it using a rotary evaporator at 55℃. When the upper liquid is evaporated to 1mL, collect the concentrated upper liquid into a glass dish and dry it to constant weight using a vacuum freeze dryer to obtain SPL (soybean lecithin), SSPL (sunflower seed lecithin) and MPL (milk lecithin).

[0157] Take 0.6g of SPL, 6.6g of SSPL, 10.2g of MPL, and 12.6g of SESM provided in Example 1, mix them, and you will get an infant formula phospholipid ingredient with a phospholipid and fatty acid composition similar to that of Chinese breast milk phospholipids.

[0158] Example 7

[0159] This embodiment provides a phospholipid ingredient for infant formula, which is prepared by the following method:

[0160] Take 0.5g of SPL, 1.8g of SSPL, 3g of MPL provided in Example 6, and 4.7g of ECSM provided in Example 3, mix them to obtain an infant formula phospholipid ingredient whose phospholipid and fatty acid composition is similar to that of Chinese breast milk phospholipids.

[0161] Comparative Example 3

[0162] This comparative example provides a phospholipid ingredient for infant formula, prepared by the following method:

[0163] Weigh 50g each of soybean lecithin and sunflower seed lecithin, place them in two 500ml beakers, add 150ml of cold acetone, stir magnetically for 30min, remove the supernatant, repeat the above steps until thin-layer chromatography detects almost no neutral lipids in the precipitate, and dry to obtain relatively pure SPL and SSPL.

[0164] Weigh 50g of milk phospholipid concentrate and place it in a 500ml beaker. Add 250ml of anhydrous ethanol and stir magnetically at 30℃ for 2 hours. After standing for 12 hours, collect the anhydrous ethanol layer and rotary evaporate at 45℃ to obtain crude milk phospholipid. Add 150ml of cold acetone to the crude milk phospholipid and stir magnetically for 30 minutes. Remove the supernatant solution and repeat the above steps until thin-layer chromatography shows almost no neutral lipids in the precipitate. After drying, obtain relatively pure MPL. Take 50g of milk phospholipid and place it in a 500ml beaker. Add 250ml of anhydrous diethyl ether and extract statically at 30℃ in a water bath for 30 minutes. Centrifuge at 4500r / min for 15 minutes and remove the anhydrous diethyl ether layer to obtain SMPL (repeated extraction 3 times).

[0165] Take 15.82g of purified SPL, 1.45g of SSPL, 39.17g of MPL, and 43.55g of SMPL, and mix them to obtain the phospholipid ingredients.

[0166] Comparative Example 4

[0167] This comparative example provides a phospholipid ingredient for infant formula, prepared by the following method:

[0168] Take 3.9g of exogenous phospholipids SPL, 1.1g of SSPL, 3g of EPL, and 3.2g of SMPL, and mix them to obtain the phospholipid ingredients.

[0169] SMPL is prepared by the following method:

[0170] Using sphingomyelin from yak ghee as the main raw material for enriching sphingomyelin, yak ghee and chloroform: alcohol were placed in a 45℃ magnetically stirred water bath and shaken at a constant temperature for 60 minutes. The n-hexane layer was then taken and evaporated using a rotary evaporator at 50℃ for 20-60 minutes to obtain SMPL.

[0171] Experiment Example 4

[0172] Using the phospholipid composition determination method provided by the present invention, Tables 5 and 6 below show the formulation composition, phospholipid and its important fatty acid composition of the phospholipid ingredients for infant formula provided in Examples 6 (IFPI1) ​​and 7 (IFPI2) of the present invention. It can be seen that the phospholipid ingredients of these two infant formulas are quite close to their respective breast milk data references in terms of phospholipid and its fatty acid composition, and can meet the nutritional needs of infants.

[0173] Table 5. Phospholipid composition of Chinese breast milk and formulation of phospholipid ingredients in infant formula, and phospholipid composition (%)

[0174]

[0175] Table 6. Key Phospholipid Fatty Acid Composition (%) of Phospholipid Ingredients in Chinese Breast Milk and Infant Formula

[0176]

[0177] Meanwhile, the inventors conducted a corresponding data comparison of Comparative Example 3 and Comparative Example 4, and the results are shown in Tables 7 and 8 below.

[0178] Table 7 Comparison of phospholipids in the Examples and Comparative Examples

[0179]

[0180] Table 8 Comparison of phospholipid fatty acids between the examples and the comparative examples

[0181]

[0182] Furthermore, the similarity scoring method provided by this invention was used to detect the similarity scores between the embodiments and comparative examples and breast milk, and the results are as follows: Figure 4 As shown in the figure, the phospholipid ingredients for infant formula provided in Examples 6 and 7 of this invention have similarity scores of 91.25 and 91.49 with breast milk, respectively, which are higher than those of Comparative Example 2 (90.12) and Comparative Example 3 (88.31). This indicates that the phospholipid ingredients for infant formula prepared in this embodiment have a higher similarity to breast milk.

[0183] Furthermore, Comparative Examples 2 and 3 only focused on the four most abundant fatty acids—C16:0, C18:0, C18:1, and C18:2—when simulating the fatty acid composition of breast milk phospholipids. In contrast, this example incorporates several long-chain polyunsaturated fatty acids crucial for infant growth and development, namely C20:4n-6 (ARA), C22:6n-3 (DHA), C18:3n-3 (ALA), and C20:5n-3 (EPA), into the comprehensive evaluation index. This example incorporates these functional fatty acids, achieving accurate simulation of the physiological functions of breast milk phospholipids, improving the nutritional integrity of the simulation system, and better meeting the actual nutritional needs of infants' growth and development.

[0184] Experimental Example 5

[0185] Experimental grouping and feeding

[0186] Forty-eight male, newly weaned C57bl6 juvenile mice (3 weeks old) were purchased from Nanjing Junke Experimental Animal Co., Ltd., with experimental animal production license SCXK(Su)2016-0010. The rearing environment was clean-grade, with a temperature of (24±2℃), humidity of 50-60%, and a 12h / 12h light / dark cycle. The procedures for using the experimental animals all followed the "Basic Management Regulations for Experimental Animals of Nanchang University" and were approved by the "Ethics Committee for Experimental Animals of Nanchang University" (Approval No.: 86 SYXK (GAN)2015-0001). The mice were randomly divided into four groups according to body weight: control group, SPL group, IFPI1 group, and IFPI2 group, with 12 mice in each group. The diet was formulated based on the AIN93G standard formula, with phospholipids accounting for 1.2% of the energy in each group (see Table 9 for specific formulas). Feed and water were provided freely, with the feed changed weekly and feed intake measured for four weeks. Behavioral experiments were conducted after 4 weeks of free access to food and water. After the behavioral experiments, brain samples from 3 mice in each group were collected for Golgi-Cox staining to investigate the effects of different phospholipid diets on the dendritic spine density of neurons in the CA1 region of the mouse hippocampus.

[0187] Table 9. Feed Formulation Composition (g / kg)

[0188]

[0189] Experimental Results: Compared with the control group, mice in the IFPI1 and IFPI2 groups of infant formula phospholipid formulations showed increased swimming time with added weight in water. In the progressive water maze test, the time to reach the safe platform decreased with increasing training time, but the time taken by the IFPI1 and IFPI2 groups was significantly shorter than that of the control and SPL groups. Control group mice were more active and frequently entered the central area. Compared to the control group, phospholipid group mice tended to move along the box walls, and their entry into the central area was significantly less frequent. This result was particularly pronounced in the IFPI2 group, indicating enhanced environmental awareness and increased avoidance behavior, which is more conducive to their survival in the wild. Furthermore, the total dendritic spine density in the phospholipid groups of both infant formula phospholipid formulations was significantly higher than that of the control group, with the IFPI2 group showing the highest density and the SPL group slightly higher. Both groups showed significant differences from the control group. Moreover, for mature dendritic spines, the IFPI1 and IFPI2 groups had higher dendritic spine density than the control and SPL groups.

[0190] The above results indicate that the phospholipid ingredients in infant formula increased the spatial learning and cognitive abilities of mice, and also increased the density of mature dendritic spines and total dendritic spines in the CA1 region of the hippocampus at the physiological level. This, to some extent, explains why the IFPI1 and IFPI2 groups of mice exhibited stronger learning and memory abilities in behavioral experiments.

[0191] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for the preparation of a lipid rich in sphingomyelin, characterized in that, Including supercritical-ethanol extraction: The supercritical-ethanol extraction method includes: using whey protein powder phospholipid concentrate as raw material, performing a first extraction using supercritical carbon dioxide, then adding an entrainer and performing a second extraction using supercritical carbon dioxide, precipitating the aqueous suspension of the extract with alcohol, taking the anhydrous ethanol layer, removing the solvent, and obtaining the lipid SESM rich in sphingomyelin. The entrainer includes 20% ± 1% v / v ethanol; The first extraction was performed at a temperature of 45°C, a pressure of 30 MPa, and a time of 2 hours; the second extraction was performed at a temperature of 40°C, a pressure of 30 MPa, and a time of 2 hours. The alcohol precipitation involves mixing the aqueous suspension of the extract with anhydrous ethanol at a temperature of 60-80°C for 0.8-1.2 hours, wherein the volume ratio of the aqueous suspension of the extract to anhydrous ethanol is 1:4-6.

2. The production method according to claim 1, characterized by, In the supercritical-ethanol extraction method: The ratio of whey protein powder phospholipid concentrate to entrainer is 1:5~7; The ratio of the material to the aqueous suspension of the extract is 1:5~7; The anhydrous ethanol layer is obtained by centrifugation at a speed of 4000-5000 r / min for 25-35 minutes. Solvents are removed by rotary evaporation at a temperature of 50-60°C.

Images