Construction method and application of lactoferrin-phospholipid-based emulsion with three layers of breast milk fat globule membrane imitating structures
By constructing a three-layer membrane structure of lactoferrin-phospholipid-based emulsion, the problem of fat globule membrane structure destruction during infant formula emulsification was solved, achieving a fat digestion effect similar to breast milk and improving the nutrient absorption efficiency of infant formula.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-12-10
- Publication Date
- 2026-04-10
AI Technical Summary
Existing infant formulas disrupt the three-layer membrane structure of breast milk fat globules during emulsification, leading to competitive adsorption of fat by lipases and proteins, affecting fat digestion, and increasing the risk of childhood obesity and metabolic syndrome.
The method of constructing lactoferrin-phospholipid-based emulsion was adopted. A single-layer membrane structure was formed by ultrasonic treatment. The microporous membrane was constructed by stacking phospholipids and cholesterol, and a three-layer breast milk fat globule membrane structure was formed by electrostatic self-assembly technology, which simulates the natural structure of breast milk fat globules.
It achieves fat globule stability and digestibility characteristics similar to breast milk, improving the digestibility of infant formula and reducing the risk of obesity and metabolic syndrome.
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Figure CN121817271A_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the field of dairy product technology, and particularly relates to a method for constructing a lactoferrin-phospholipid-based emulsion with a three-layered breast milk fat globule membrane structure and its application. Background Technology
[0002] The unique composition and structure of breast milk lipids are fundamental to healthy lipid metabolism in infants, while the physical structure and chemical composition of fats in infant formula differ significantly from those in breast milk. This difference stems from the unique three-layered membrane structure of breast milk fat globules (an outer phospholipid bilayer and an inner monolayer). This membrane, composed of polar lipids, cholesterol, and various proteins, is collectively known as the milk fat globule membrane (MFGM), accounting for over 90% of the dry weight of milk fat globules. The MFGM stabilizes fat globules, preventing their aggregation. Furthermore, the MFGM not only regulates the rate of lipid hydrolysis but also mediates the temporal adsorption of bile salts through sphingomyelin-cholesterol microdomains. The specific distribution of specific components at its interface makes fats more easily digestible and utilized by infants. Infant formula lacking the natural three-layered MFGM structure naturally reduces the effective digestion of fat globules by infants, thus significantly increasing the risk of childhood obesity and metabolic syndrome.
[0003] The emulsification process before spray drying is crucial in the preparation of infant formula. The high-pressure shearing process leads to significant differences in the fat globule interface structure between infant formula and breast milk. Traditional emulsification and homogenization methods cause the disintegration of the natural MFGM structure in cow's milk. The reconstituted interface, due to the disordered embedding of milk proteins, results in an excessively large specific surface area of fat globules, leading to excessive protein loading at the interface. This triggers competitive adsorption of lipases by proteins during digestion, weakening the original activity of lipases for fat. These factors significantly affect fat digestion. This is a key factor affecting the quality of infant formula and a problem that urgently needs to be solved in this field. Summary of the Invention
[0004] To address the aforementioned problems, this invention provides a method for constructing a lactoferrin-phospholipid-based emulsion with a three-layered breast milk fat globule membrane structure and its application. The emulsion membrane structure closely resembles the natural three-layered breast milk fat globule membrane, making it easy for infants to digest and absorb lipids. It is suitable for addition to infant formula and other complementary foods, and has outstanding practical value.
[0005] To achieve the above objectives, the technical solution adopted by the present invention is as follows: The application discloses a method for constructing a lactoferrin-phospholipid-based emulsion with a three-layered fat globule membrane structure similar to breast milk, and the method comprises the following steps: lactoferrin is subjected to ultrasonic treatment to obtain an emulsion with a single-layered membrane structure; phospholipids and cholesterol are stacked on the surface of a microporous membrane to obtain a microporous membrane with a bilayer membrane; and the emulsion with the single-layered membrane structure and the microporous membrane with the bilayer membrane are subjected to secondary covering through electrostatic self-assembly to obtain the lactoferrin-phospholipid-based emulsion with the three-layered fat globule membrane structure similar to breast milk.
[0006] Further, the method comprises the following steps: The emulsion with the single-layered membrane structure is prepared by dissolving lactoferrin in water, adding compound vegetable oil into the obtained lactoferrin solution, and subjecting the obtained suspension to ultrasonic treatment to form a single-layered membrane structure with positive charges. The surface of the microporous membrane is stacked by using a mixture of phospholipids and cholesterol as lipids, and a rotary evaporation method is used to prepare a lipid film with negative charges on the surface of the microporous membrane to obtain the microporous membrane with the bilayer membrane. The secondary covering through electrostatic self-assembly is achieved by using a premix membrane emulsification method, and the bilayer phospholipid layer is formed on the surface of the single-layered membrane through the electrostatic self-assembly to obtain the lactoferrin-phospholipid-based emulsion with the three-layered fat globule membrane structure similar to breast milk.
[0007] Further, the method comprises the following specific steps: The emulsion with the single-layered membrane structure is prepared by dissolving lactoferrin in water, adding compound vegetable oil into the obtained lactoferrin solution, and subjecting the obtained suspension to ultrasonic treatment to form a single-layered membrane structure with positive charges. The surface of the microporous membrane is stacked by using a mixture of phospholipids and cholesterol as lipids, and anhydrous ethanol is added into the lipids to form a lipid solution, and then the microporous membrane is put into the lipid solution and subjected to rotary evaporation, so that the lipid film with negative charges is formed on the surface of the microporous membrane after the ethanol volatilizes to obtain the microporous membrane with the bilayer membrane. The secondary covering through electrostatic self-assembly is achieved by using a premix membrane emulsification method, and the bilayer phospholipid layer is formed on the surface of the single-layered membrane through the electrostatic self-assembly to obtain the lactoferrin-phospholipid-based emulsion with the three-layered fat globule membrane structure similar to breast milk.
[0008] Further, in the process of preparing the emulsion with the single-layered membrane structure, the ultrasonic treatment is performed at a power of 200-1000 W for 50-200 s. In the process of the secondary covering through electrostatic self-assembly, the premix membrane emulsification method is performed at an air inlet pressure of 10-50 kPa.
[0009] Further, the raw materials for preparing the compound vegetable oil include 1,3-dioleic acid-2-palmitic acid triglyceride 30-42 parts by weight, butter fat 15-20 parts by weight, coconut oil 14-19 parts by weight, palm oil 8-12 parts by weight, corn oil 8-12 parts by weight and sunflower oil 8-12 parts by weight.
[0010] Further, the addition amount of lactoferrin is 0.02-1wt% of the single-layer membrane structure emulsion; The total addition amount of phospholipid and cholesterol is 1.45-5wt% of the single-layer membrane structure emulsion; The use amount of the compound vegetable oil is 1-30wt% of the single-layer membrane structure emulsion.
[0011] Further, the weight ratio of phospholipid and cholesterol in the milk fat globule membrane is 4-14:1.
[0012] Further, the preparation process of the single-layer membrane structure emulsion further includes dissolving the core material in the compound vegetable oil, and then adding the compound vegetable oil with the dissolved core material into the lactoferrin solution, so that the core material is included in the single-layer membrane structure.
[0013] Further, the preparation process of the single-layer membrane structure emulsion is to dissolve lactoferrin in water to obtain a lactoferrin solution, dissolve the core material in the compound vegetable oil, and then add the compound vegetable oil with the dissolved core material into the lactoferrin solution, and the obtained suspension is subjected to ultrasonic treatment to form a single-layer membrane structure emulsion coated with positively charged lactoferrin; The core material is a fat-soluble nutrient or a protective active ingredient; the fat-soluble nutrient or the protective active ingredient includes but is not limited to a carrier of fat-soluble vitamins, carotenoids or probiotics.
[0014] The application of a lactoferrin-phospholipid-based emulsion with a three-layered milk fat globule membrane structure prepared by the above construction method in the preparation of infant formula emulsion.
[0015] The construction method of a lactoferrin-phospholipid-based emulsion with a three-layered milk fat globule membrane structure and the beneficial effects of the application are as follows: The application is based on the interface engineering strategy of "electrostatic combination-layer-by-layer covering", and reconstructs the three-layered MFGM membrane structure through membrane emulsification technology, effectively controls the specific surface area of the fat globule, realizes the ordered arrangement of the interface components, and makes it infinitely close to natural breast milk in terms of structure composition and nutritional function, thereby providing a new scheme and technical method for the breakthrough of the key technology of high-end infant formula milk powder production; The application establishes a new type of emulsion construction method for simulating the structure of milk fat globules-microporous membrane stacking-electrostatic self-assembly secondary covering combined with ultrasonic-premixing membrane emulsification technology, which is simple in equipment, easy to operate, green and pollution-free, energy-saving and consumption-reducing, without input of heating and shear force, and suitable for heat-sensitive and mechanically sensitive materials; The present application adopts lactoferrin to form a positively charged inner monolayer membrane, adopts phospholipid and cholesterol to form a negatively charged outer bilayer membrane, and forms a stable three-layer membrane through electrostatic interaction self-assembly; the obtained emulsion membrane structure similar to the breast milk fat globule structure is similar to the three-layer structure of the breast milk fat globule membrane, and the emulsion particle size is close to that of the breast milk fat globule and is uniformly distributed, can embed fat-soluble nutrients, and can be applied to infant formula food; The three-layer emulsion similar to the breast milk fat globule structure provided by the present application realizes the equivalence of infant formula fat digestion characteristics and breast milk through structure bionics, breaks through the technical bottleneck of infant formula powder "close composition but insufficient function", and has great application value in high-end infant formula food. BRIEF DESCRIPTION OF DRAWINGS
[0016] Figure 1 is a process flow diagram of a construction method of a lactoferrin-phospholipid-based emulsion with a three-layer breast milk fat globule membrane structure in the present application; Figure 2 is the particle size determination result of each emulsion to be tested in embodiment 9 of the present application; wherein the simple membrane structure emulsion represents the determination result of the monolayer membrane structure emulsion prepared in step S1 of embodiments 1-8, and the three-layer membrane structure emulsion represents the determination result of the lactoferrin-phospholipid-based emulsion with a three-layer breast milk fat globule membrane structure finally obtained in embodiments 1-8; Figure 3 is the potential determination result of each emulsion to be tested in embodiment 9 of the present application; wherein the simple membrane structure emulsion represents the determination result of the monolayer membrane structure emulsion prepared in step S1 of embodiments 1-8, and the three-layer membrane structure emulsion represents the determination result of the lactoferrin-phospholipid-based emulsion with a three-layer breast milk fat globule membrane structure finally obtained in embodiments 1-8; Figure 4 is the particle size determination result and the potential determination result of breast milk and a commercially available infant formula milk powder in embodiment 9 of the present application; wherein the breast milk represents the determination result of breast milk, and the commercially available milk powder represents the determination result of the commercially available infant formula milk powder; Figure 5 is the fat decomposition degree determination result in embodiment 9 of the present application; wherein embodiments 1-8 represent the determination result of the lactoferrin-phospholipid-based emulsion with a three-layer breast milk fat globule membrane structure finally obtained in embodiments 1-8, breast milk represents the determination result of breast milk, and commercially available milk powder represents the determination result of the commercially available infant formula milk powder. DETAILED DESCRIPTION
[0017] The technical solutions in the embodiments of the present application are clearly and completely described below. In the following description, a large number of specific details are set forth in order to facilitate a thorough understanding of the present application, but the present application can also be implemented in other manners different from those described herein, and those skilled in the art can make similar generalizations without departing from the spirit and scope of the present application, therefore the present application is not limited to the specific embodiments disclosed below.
[0018] The raw material of the lactoferrin-phospholipid-based emulsion with a three-layered fat globule membrane structure similar to breast milk includes lactoferrin, phospholipids, cholesterol, complex vegetable oil and water. Among them, the phospholipids are at least one of common phospholipids such as phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, phosphatidylglycerol and glycerophosphoric acid.
[0019] No core material is added in the above formula, but in the actual application process, fat-soluble nutrients or protective active ingredients can also be added as core materials according to actual needs. The fat-soluble nutrients or protective active ingredients include but are not limited to carriers of fat-soluble vitamins, carotenoids or probiotics.
[0020] A method for constructing a lactoferrin-phospholipid-based emulsion with a three-layered fat globule membrane structure similar to breast milk, as shown in Figure 1 includes the following specific steps: S1, single-layer membrane structure emulsion: dissolve lactoferrin in water, add complex vegetable oil to the obtained lactoferrin solution (if a core material is added, first dissolve the core material in the complex vegetable oil, and then add the complex vegetable oil containing the core material to the lactoferrin solution), and then ultrasonically treat the obtained suspension. By controlling the time (50-200 s) and power (200-1000 W) during ultrasonic treatment, a single-layer membrane structure emulsion coated with positively charged lactoferrin is formed.
[0021] Among them, the complex vegetable oil is a mixture of 1,3-dioleic acid-2-palmitic acid triglyceride 30-42 parts by weight, butter fat 15-20 parts by weight, coconut oil 14-19 parts by weight, palm oil 8-12 parts by weight, corn oil 8-12 parts by weight and sunflower oil 8-12 parts by weight. The amount of complex vegetable oil is 1-30 wt% of the single-layer membrane structure emulsion. The amount of lactoferrin added is 0.02-1 wt% of the single-layer membrane structure emulsion. The fatty acid composition after compounding meets the requirements of the fatty acid ratio required by infants.
[0022] S2, surface stacking of the microporous membrane: take the phospholipid and cholesterol mixed as the lipid in a weight ratio of 4-14:1, add anhydrous ethanol to the lipid to form a lipid solution, then put the microporous membrane into the lipid solution, rotary evaporation, and after the ethanol volatilizes, the lipid film (i.e. phospholipid and cholesterol) is stacked on the surface of the microporous membrane, forming a negatively charged lipid film on the surface of the microporous membrane, i.e. a microporous membrane with a bilayer membrane; wherein the total amount of phospholipid and cholesterol added is 1.45-5 wt% of the single-layer membrane structure emulsion in step S1.
[0023] S3, electrostatic self-assembly secondary coverage: using a conventional membrane emulsification device, the microporous membrane with a bilayer membrane prepared in step S2 is loaded onto the membrane emulsification device as the microporous membrane of the membrane emulsification device, a premix membrane emulsification method (premix membrane emulsification is used to simulate the formation of the outer bilayer phospholipid membrane of the natural breast milk fat globule in the process of exocytosis, thereby realizing the biomimetic construction of the membrane structure evolution from the cell to the secretion process) is adopted, the single-layer membrane structure emulsion obtained in step S1 is pressed through the microporous membrane with a bilayer membrane obtained in step S2 by controlling the inlet pressure (10-50 kPa), the lipid film stacked on the surface of the microporous membrane with a bilayer membrane is swollen, and a bilayer phospholipid layer is formed on the lactoferrin surface of the single-layer membrane structure emulsion by electrostatic self-assembly (i.e. the negatively charged phospholipid and cholesterol on the surface of the microporous membrane are self-assembled on the positively charged lactoferrin surface through electrostatic interaction), i.e. a lactoferrin-phospholipid-based emulsion with a three-layer breast milk fat globule membrane structure (hereinafter referred to as a three-layer membrane structure emulsion, as shown in Figure 1 ).
[0024] The microporous membrane used in the present application is a 1 μm microporous level membrane used in conventional membrane emulsification process, which is purchased from Japan SPG Company.
[0025] The lactoferrin-phospholipid-based emulsion with a three-layer breast milk fat globule membrane structure of the present application can be used as a raw material for preparing high-end infant formula emulsion. The lactoferrin-phospholipid-based emulsion with a three-layer breast milk fat globule membrane structure of the present application realizes the simulation of the three-layer structure of the breast milk fat globule membrane in the infant formula emulsion by coupling membrane emulsification and electrostatic self-assembly technology, which is used to improve the lipid digestion and absorption and the breast milk similarity of the formula milk.
[0026] Example 1, a method for constructing a lactoferrin-phospholipid-based emulsion with a three-layer breast milk fat globule membrane structure and its application The formula of the lactoferrin-phospholipid-based emulsion with a three-layer breast milk fat globule membrane structure in this embodiment includes the following raw materials by weight: lactoferrin 0.02 g, deionized water 90 g, phosphatidylcholine 1.35 g, cholesterol 0.1 g, and compound vegetable oil 10 g; The composite vegetable oil includes the following raw materials by weight: 1,3-dioleic acid-2-palmitic acid glycerol triester 4.2 g, butter fat 2 g, coconut oil 1.4 g, palm oil 0.8 g, corn oil 0.8 g, and sunflower seed oil 0.8 g.
[0027] The method for constructing the lactoferrin-phospholipid-based emulsion with the three-layered mimetic human milk fat globule membrane structure in the embodiment includes the following specific steps: S1, single-layer membrane structure emulsion: 0.2 g of lactoferrin is added to 90 g of deionized water, stirred uniformly until completely dissolved, 10 g of composite vegetable oil is further added to the obtained lactoferrin solution, and then ultrasonic treatment is performed at 700 W for 72 s to form a positively charged single-layer membrane structure emulsion.
[0028] S2, microporous membrane surface stacking: 1.35 g of phosphatidylcholine and 0.1 g of cholesterol are added to 20 mL of anhydrous ethanol to obtain a lipid solution. A microporous membrane is placed in the above lipid solution, and rotary evaporation is performed. After the ethanol is volatilized, a lipid film is stacked on the surface of the microporous membrane to form a microporous membrane with a bilayer membrane. S3, electrostatic self-assembly secondary coverage: using a pre-mixed membrane emulsification method, the single-layer membrane structure emulsion is pressed through the microporous membrane with a bilayer membrane at 40 kPa using nitrogen gas. The lipid film stacked on the surface of the microporous membrane with a bilayer membrane swells, and a bilayer phospholipid layer is formed on the surface of the lactoferrin layer through electrostatic self-assembly secondary coverage, thereby obtaining a lactoferrin-phospholipid-based emulsion with a three-layered mimetic human milk fat globule membrane structure, which is referred to as a three-layered membrane structure emulsion.
[0029] The lactoferrin-phospholipid-based emulsion with the three-layered mimetic human milk fat globule membrane structure prepared in the embodiment can be used as a raw material to prepare a high-end infant formula emulsion. Through the coupling of membrane emulsification and electrostatic self-assembly technology, the three-layered structure of the mimetic human milk fat globule membrane in the infant formula emulsion is realized, which is used to improve lipid digestion and absorption and the human milk similarity of the formula milk.
[0030] In actual application, fat-soluble nutrients or protective active ingredients can also be added as core materials according to actual needs. The fat-soluble nutrients or protective active ingredients include but are not limited to fat-soluble vitamins, carotenoids, or carriers of probiotics. When the core material is added, the core material is first dissolved in the composite vegetable oil, and then the lactoferrin solution is added.
[0031] Embodiment 2: A method for constructing a lactoferrin-phospholipid-based emulsion with a three-layered mimetic human milk fat globule membrane structure The formula of the lactoferrin-phospholipid-based emulsion with the three-layered mimetic human milk fat globule membrane structure in the embodiment includes the following raw materials by weight: lactoferrin 0.5 g, deionized water 70 g, phosphatidylcholine 1.35 g, cholesterol 0.1 g, and composite vegetable oil 30 g. The composite vegetable oil is obtained by mixing 1,3-dioleic acid-2-palmitic acid triglyceride 12.6 g, milk fat 6 g, coconut oil 4.2 g, palm oil 2.4 g, corn oil 2.4 g and sunflower oil 2.4 g.
[0032] The method for constructing the lactoferrin-phospholipid-based emulsion with the three-layered emulsion membrane structure similar to the fat globule in breast milk comprises the following specific steps: S1, single-layered emulsion: 0.5 g of lactoferrin is added to 70 g of deionized water, and stirred until completely dissolved, then 30 g of composite vegetable oil is added to the lactoferrin solution, and ultrasonic treatment is performed at 700 W for 180 s to form a single-layered emulsion with positive charges.
[0033] S2, micro-porous membrane surface stacking: 1.35 g of phosphatidylcholine and 0.1 g of cholesterol are added to 25 mL of anhydrous ethanol to obtain a lipid solution. A micro-porous membrane is placed in the above lipid solution, and rotary evaporation is performed until the ethanol is volatilized. The lipid film is stacked on the surface of the micro-porous membrane to form a micro-porous membrane with a bilayer membrane. S3, electrostatic self-assembly secondary coverage: the single-layered emulsion is pressed through the micro-porous membrane with a bilayer membrane at 40 kPa using nitrogen gas to swell the lipid film stacked on the surface of the micro-porous membrane with a bilayer membrane, and a double-molecular phospholipid layer is formed on the surface of the lactoferrin layer by electrostatic self-assembly secondary coverage to obtain a lactoferrin-phospholipid-based emulsion with a three-layered emulsion membrane structure similar to the fat globule in breast milk, which is referred to as a three-layered emulsion.
[0034] Example 3: A method for constructing a lactoferrin-phospholipid-based emulsion with a three-layered emulsion membrane structure similar to the fat globule in breast milk The formula of the lactoferrin-phospholipid-based emulsion with a three-layered emulsion membrane structure similar to the fat globule in breast milk in this example comprises the following raw materials by weight: lactoferrin 0.02 g, deionized water 90 g, phosphatidylcholine 1.16 g, cholesterol 0.29 g and composite vegetable oil 10 g. The composite vegetable oil is obtained by mixing 1,3-dioleic acid-2-palmitic acid triglyceride 4.2 g, milk fat 2 g, coconut oil 1.4 g, palm oil 0.8 g, corn oil 0.8 g and sunflower oil 0.8 g.
[0035] The method for constructing the lactoferrin-phospholipid-based emulsion with the three-layered emulsion membrane structure similar to the fat globule in breast milk comprises the following specific steps: S1, single-layered emulsion: 0.2 g of lactoferrin is added to 90 g of deionized water, and stirred until completely dissolved, then 10 g of composite vegetable oil is added to the lactoferrin solution, and ultrasonic treatment is performed at 700 W for 72 s to form a single-layered emulsion with positive charges.
[0036] S2, surface stacking of the microporous membrane: 1.16 g of phosphatidylcholine and 0.29 g of cholesterol were added to 20 mL of anhydrous ethanol to obtain a lipid solution. A microporous membrane was placed in the above lipid solution, and rotary evaporation was performed until the ethanol was volatilized. The lipid film was stacked on the surface of the microporous membrane to form a microporous membrane with a bilayer membrane. S3, secondary coverage by electrostatic self-assembly: using the premix membrane emulsification method, the single-layer membrane structure emulsion was pressed through the microporous membrane with a bilayer membrane at 40 kPa using nitrogen. The lipid film stacked on the surface of the microporous membrane with a bilayer membrane swelled, and a double-molecular phospholipid layer was formed on the surface of the lactoferrin layer by secondary coverage through electrostatic self-assembly, thereby obtaining a lactoferrin-phospholipid-based emulsion with a three-layered breast milk fat globule membrane structure, referred to as a three-layer membrane structure emulsion.
[0037] Example 4: A method for constructing a lactoferrin-phospholipid-based emulsion with a three-layered breast milk fat globule membrane structure The formula of the lactoferrin-phospholipid-based emulsion with a three-layered breast milk fat globule membrane structure in this example includes the following raw materials by weight: lactoferrin 0.5 g, deionized water 70 g, phosphatidylcholine 1.16 g, cholesterol 0.29 g, and compound vegetable oil 30 g. The compound vegetable oil is obtained by mixing 1,3-dioleic acid-2-palmitic acid triglyceride 12.6 g, cow milk fat 6 g, coconut oil 4.2 g, palm oil 2.4 g, corn oil 2.4 g, and sunflower seed oil 2.4 g.
[0038] The method for constructing a lactoferrin-phospholipid-based emulsion with a three-layered breast milk fat globule membrane structure in this example includes the following specific steps: S1, single-layer membrane structure emulsion: 0.5 g of lactoferrin was added to 70 g of deionized water, stirred until completely dissolved, and then 30 g of compound vegetable oil was added to the obtained lactoferrin solution. Ultrasonic treatment was performed at 700 W for 180 s to form a positively charged single-layer membrane structure emulsion.
[0039] S2, surface stacking of the microporous membrane: 1.16 g of phosphatidylcholine and 0.29 g of cholesterol were added to 25 mL of anhydrous ethanol to obtain a lipid solution. A microporous membrane was placed in the above lipid solution, and rotary evaporation was performed until the ethanol was volatilized. The lipid film was stacked on the surface of the microporous membrane to form a microporous membrane with a bilayer membrane. S3, secondary coverage by electrostatic self-assembly: using the premix membrane emulsification method, the single-layer membrane structure emulsion was pressed through the microporous membrane with a bilayer membrane at 40 kPa using nitrogen. The lipid film stacked on the surface of the microporous membrane with a bilayer membrane swelled, and a double-molecular phospholipid layer was formed on the surface of the lactoferrin layer by secondary coverage through electrostatic self-assembly, thereby obtaining a lactoferrin-phospholipid-based emulsion with a three-layered breast milk fat globule membrane structure, referred to as a three-layer membrane structure emulsion.
[0040] Example 5: A method for constructing a lactoferrin-phospholipid-based emulsion with a three-layered breast milk fat globule membrane structure. In this embodiment, a lactoferrin-phospholipid-based emulsion with a three-layered breast milk fat globule membrane structure is formulated with the following raw materials by weight: 0.02g lactoferrin, 90g deionized water, 1.35g phosphatidylcholine, 0.1g cholesterol, and 10g compound vegetable oil. The compound vegetable oil is made by mixing 3g of 1,3-dioleoyl-2-palmitoylglycerol triglyceride, 1.5g of milk fat, 1.9g of coconut oil, 1.2g of palm oil, 1.2g of corn oil and 1.2g of sunflower seed oil.
[0041] This embodiment describes a method for constructing a lactoferrin-phospholipid-based emulsion with a three-layered, breast milk-mimicking fat globule membrane structure, comprising the following specific steps: S1. Single-layer membrane structure emulsion: Take 0.2g of lactoferrin and add it to 90g of deionized water. Stir until completely dissolved. Add 10g of compound vegetable oil to the resulting lactoferrin solution and then sonicate at 500W for 72s to form a positively charged single-layer membrane structure emulsion.
[0042] S2. Microporous membrane surface stacking: 1.35g of phosphatidylcholine and 0.1g of cholesterol were added to 20mL of anhydrous ethanol to obtain a lipid solution. A microporous membrane was placed in the above lipid solution and rotary evaporated. After the ethanol evaporated, the lipid film stacked on the surface of the microporous membrane to form a microporous membrane with a bilayer. S3. Electrostatic self-assembly secondary coating: Using a premixed membrane emulsification method, a single-layer membrane structure emulsion is pressed through a microporous membrane with a bimolecular membrane at 30 kPa using nitrogen gas. The lipid film stacked on the surface of the microporous membrane with the bimolecular membrane swells and forms a bimolecular phospholipid layer on the surface of the lactoferrin layer through electrostatic self-assembly, thus obtaining a lactoferrin-phospholipid-based emulsion with a three-layer breast milk fat globule membrane structure, referred to as a three-layer membrane structure emulsion.
[0043] Example 6: A method for constructing a lactoferrin-phospholipid-based emulsion with a three-layered breast milk fat globule membrane structure. In this embodiment, a lactoferrin-phospholipid-based emulsion with a three-layered breast milk fat globule membrane structure is formulated with the following raw materials by weight: 0.5g lactoferrin, 70g deionized water, 1.35g phosphatidylcholine, 0.1g cholesterol, and 30g compound vegetable oil. The compound vegetable oil is made by mixing 9g of 1,3-dioleoyl-2-palmitoyl glycerol, 4.5g of milk fat, 5.7g of coconut oil, 3.6g of palm oil, 3.6g of corn oil and 3.6g of sunflower seed oil.
[0044] A method for constructing a lactoferrin-phospholipid-based emulsion with a three-layered structure of a fat globule membrane mimicking breast milk in the embodiment includes the following specific steps: S1, single-layered membrane structure emulsion: 0.5 g of lactoferrin is added to 70 g of deionized water, stirred uniformly until completely dissolved, 30 g of compound vegetable oil is then added to the obtained lactoferrin solution, and then ultrasonic treatment is performed at 500 W for 180 s to form a single-layered membrane structure emulsion with positive charges.
[0045] S2, surface stacking of microporous membranes: 1.35 g of phosphatidylcholine and 0.1 g of cholesterol are added to 25 mL of anhydrous ethanol to obtain a lipid solution. A microporous membrane is placed in the above lipid solution, and rotary evaporation is performed until the ethanol volatilizes. After the volatilization of ethanol, a lipid film is stacked on the surface of the microporous membrane to form a microporous membrane with a bilayer membrane. S3, electrostatic self-assembly secondary coverage: a premix membrane emulsification method is used to press the single-layered membrane structure emulsion through the microporous membrane with a bilayer membrane at 30 kPa by using nitrogen gas. The lipid film stacked on the surface of the microporous membrane with a bilayer membrane is swollen, and a double-molecular phospholipid layer is formed on the surface of the lactoferrin layer by electrostatic self-assembly secondary coverage to obtain a lactoferrin-phospholipid-based emulsion with a three-layered structure of a fat globule membrane mimicking breast milk, which is referred to as a three-layered membrane structure emulsion.
[0046] A method for constructing a lactoferrin-phospholipid-based emulsion with a three-layered structure of a fat globule membrane mimicking breast milk in the embodiment includes the following specific steps: The formula of the lactoferrin-phospholipid-based emulsion with a three-layered structure of a fat globule membrane mimicking breast milk in the embodiment includes the following raw materials by weight: 0.02 g of lactoferrin, 90 g of deionized water, 1.16 g of phosphatidylcholine, 0.29 g of cholesterol, and 10 g of compound vegetable oil. The compound vegetable oil is obtained by mixing 3 g of 1,3-dioleic acid-2-palmitic acid glycerol triester, 1.5 g of bovine milk fat, 1.9 g of coconut oil, 1.2 g of palm oil, 1.2 g of corn oil, and 1.2 g of sunflower seed oil.
[0047] A method for constructing a lactoferrin-phospholipid-based emulsion with a three-layered structure of a fat globule membrane mimicking breast milk in the embodiment includes the following specific steps: S1, single-layered membrane structure emulsion: 0.2 g of lactoferrin is added to 90 g of deionized water, stirred uniformly until completely dissolved, 30 g of compound vegetable oil is then added to the obtained lactoferrin solution, and then ultrasonic treatment is performed at 500 W for 72 s to form a single-layered membrane structure emulsion with positive charges.
[0048] S2, surface stacking of microporous membranes: 1.16 g of phosphatidylcholine and 0.29 g of cholesterol are added to 20 mL of anhydrous ethanol to obtain a lipid solution. A microporous membrane is placed in the above lipid solution, and rotary evaporation is performed until the ethanol volatilizes. After the volatilization of ethanol, a lipid film is stacked on the surface of the microporous membrane to form a microporous membrane with a bilayer membrane. S3, secondary covering by electrostatic self-assembly: using the premix membrane emulsification method, the single-layer membrane structure emulsion is pressed through the microporous membrane with double-molecule membrane at 30 kPa using nitrogen, the lipid film stacked on the surface of the microporous membrane with double-molecule membrane swells, and a double-molecule phospholipid layer is formed on the surface of the lactoferrin layer by electrostatic self-assembly, i.e. a lactoferrin-phospholipid-based emulsion with a three-layer breast milk fat globule membrane structure, referred to as a three-layer membrane structure emulsion.
[0049] Example 8 A method for constructing a lactoferrin-phospholipid-based emulsion with a three-layer breast milk fat globule membrane structure The formula of the lactoferrin-phospholipid-based emulsion with a three-layer breast milk fat globule membrane structure in this example includes the following raw materials by weight: lactoferrin 0.5 g, deionized water 70 g, phosphatidylcholine 1.16 g, cholesterol 0.29 g, and compound vegetable oil 30 g. The compound vegetable oil is obtained by mixing 1,3-dioleic acid-2-palmitic acid triglyceride 9 g, cow milk fat 4.5 g, coconut oil 5.7 g, palm oil 3.6 g, corn oil 3.6 g, and sunflower seed oil 3.6 g.
[0050] The method for constructing a lactoferrin-phospholipid-based emulsion with a three-layer breast milk fat globule membrane structure in this example includes the following specific steps: S1, single-layer membrane structure emulsion: 0.5 g of lactoferrin is added to 70 g of deionized water, stirred until completely dissolved, 30 g of compound vegetable oil is then added to the lactoferrin solution, and then ultrasonic treatment is performed at 500 W for 180 s to form a single-layer membrane structure emulsion with positive charges.
[0051] S2, microporous membrane surface stacking: 1.16 g of phosphatidylcholine and 0.29 g of cholesterol are added to 25 mL of anhydrous ethanol to obtain a lipid solution. A microporous membrane is placed in the above lipid solution, and rotary evaporation is performed until the ethanol is volatilized. A lipid film is stacked on the surface of the microporous membrane, and a microporous membrane with a double-molecule membrane is formed. S3, secondary covering by electrostatic self-assembly: using the premix membrane emulsification method, the single-layer membrane structure emulsion is pressed through the microporous membrane with double-molecule membrane at 30 kPa using nitrogen, the lipid film stacked on the surface of the microporous membrane with double-molecule membrane swells, and a double-molecule phospholipid layer is formed on the surface of the lactoferrin layer by electrostatic self-assembly, i.e. a lactoferrin-phospholipid-based emulsion with a three-layer breast milk fat globule membrane structure, referred to as a three-layer membrane structure emulsion.
[0052] Example 9 Detection of a lactoferrin-phospholipid-based emulsion with a three-layer breast milk fat globule membrane structure The single-layer membrane structure emulsion prepared in step S1 of Examples 1-8 and the final three-layer membrane structure emulsion are taken as the emulsion to be detected, respectively, and the following detection is performed.
[0053] 1) Particle size determination Referring to Regulating interfacial structure of fat globules based on milkfat globule membrane with milk phospholipids to improve physicochemical properties and fat digestion of infant formula emulsions (Food Hydrocolloids, 2024, 157, 110433.), 0.1 mL of each emulsion to be tested was diluted to 50 mL with ultrapure water, and the volume-weighted particle size distribution and average diameter of the emulsion were determined using a BT-9300S laser particle size analyzer (Dandong Bettersize Instruments Co., Ltd., China). The results are shown in Figure 2 .
[0054] At the same time, the above method was used to determine the particle size of breast milk and a commercially available infant formula milk powder (reconstituted), and the results are shown in Figure 4 .
[0055] 2) Zeta potential determination Referring to Regulating interfacial structure of fat globules based on milkfat globule membrane with milk phospholipids to improve physicochemical properties and fat digestion of infant formula emulsions (Food Hydrocolloids, 2024, 157, 110433.), each emulsion to be tested was diluted 400 times with buffer (5 mM CaCl2, 50 mM NaCl and 20 mM imidazole), and the zeta potential was detected at 25°C using a Nano-Zeta potential analyzer (Malvern Instruments Ltd., Malvern, UK). The results are shown in Figure 3 .
[0056] At the same time, the above method was used to determine the zeta potential of breast milk and a commercially available infant formula milk powder (reconstituted), and the results are shown in Figure 4 .
[0057] Through the average particle size and zeta potential detection, as shown in Figure 2 , 3 , the particle size d3,2 3.85~5.13μm, and zeta potential was 1.61~1.72mV. The emulsion particle size d 3,2 1.77~2.50μm, and zeta potential was -15.9~-16.77mV. This was because lactoferrin was positively charged under neutral conditions, and the single-layer membrane emulsion formed by coating the oil droplets with lactoferrin alone was positively charged. The lipid bilayer was negatively charged, and after being added, it neutralized the positive charge of lactoferrin through electrostatic binding. The excess negative charge caused the system potential to turn negative, confirming that the secondary coating of phospholipids formed a "lactoferrin-phospholipid and cholesterol" three-layer membrane structure. Figure 4 As shown in the figure, the particle size d 3,2 3.5±0.21μm and 0.37±0.01μm, respectively, and the zeta potential was -7.8±0.2mV and -20±0.35mV, respectively. The particle size of the three-layer membrane emulsion of Examples 1~8 was close to that of breast milk and higher than that of the reconstituted milk of the commercially available milk powder, and the zeta potential was between that of breast milk and the reconstituted milk of the commercially available milk powder.
[0058] 3) In vitro simulation of infant gastrointestinal digestion Referring to Regulating interfacial structure of fat globules based on milkfat globule membrane with milk phospholipids to improve physicochemical properties and fat digestion of infant formula emulsions (Food Hydrocolloids, 2024, 157, 110433.), 126 mL of the three-layer membrane structured emulsions obtained in Examples 1-8 were mixed with 74 mL of simulated gastric fluid (30 U / mg gastric lipase, 3000 U / mg pepsin, 94 mM NaCl and 13 mM KCl) during gastric digestion, the pH value was maintained at 5.3 by adding 0.1 M NaOH, and incubated at 37°C for 60 min with shaking. 136 mL of each resulting gastric digestion completed solution was mixed with 64 mL of simulated intestinal fluid (250 U / mg trypsin, 30 U / mg pancreatic lipase, 0.67 mmol / g bile salt, 94 mM NaCl, 13 mM KCl, 85 mM NaHCO3 and 3 mM CaCl2), the pH value of each resulting mixture was adjusted to 6.6 using 0.1 M NaOH, and incubated at 37°C for 120 min with shaking. Samples were collected at 0 min, 30 min, 60 min, 90 min and 120 min of intestinal digestion, and the free fatty acid content, emulsion triglyceride average molar mass, and average fat concentration were determined, and the fat decomposition degree LD was calculated using these values, and the results are shown in Figure 5 The fat decomposition degree LD (%) refers to the percentage of free fatty acids released by hydrolysis in the total initial acyl groups in triglycerides, and the calculation formula is as follows: , wherein FFA is the free fatty acid content (mol), which can be obtained from the consumed NaOH molar amount; MMeq is the emulsion triglyceride average molar mass (g / mol); FC is the average fat concentration (g / mol); and V is the emulsion volume (mL).
[0059] At the same time, the free fatty acid content, emulsion triglyceride average molar mass, and average fat concentration of breast milk and a commercially available infant formula powder (reconstituted) were determined using the above method, and the fat decomposition degree was calculated using these values, and the results are shown in Figure 5
[0060] The three-layer membrane structured emulsions prepared in Examples 1-8 all have a fat decomposition capacity similar to that of breast milk (as shown in Figure 5 The fat decomposition rate of the three-layer membrane structured emulsions prepared in Examples 1, 3, 5, and 7 was much higher than that of the commercial infant formula milk powder and slightly higher than that of the three-layer membrane structured emulsions prepared in Examples 2, 4, 6, and 8 after the simulated gastric digestion and the intestinal digestion. Among them, the fat decomposition degree of the three-layer membrane structured emulsions prepared in Examples 1, 3, 5, and 7 reached 60.5% to 61.9% after the simulated intestinal digestion for 120 min, which was slightly lower than that of the breast milk (67.54±1.92%) and higher than that of the three-layer membrane structured emulsions prepared in Examples 2, 4, 6, and 8 (54.4% to 56.9%) and the commercial infant formula milk powder (42±0.7%). It can be seen that the three-layer membrane structured emulsions are helpful for the digestion and absorption of the internal embedded fat by the digestive juice, and make the digestion behavior of the three-layer membrane structured emulsions closer to that of the breast milk.
[0061] Methods for constructing lactoferrin-phospholipid-based emulsions with three-layer membrane structure of breast milk fat globules Examples 10 to 13 are methods for constructing lactoferrin-phospholipid-based emulsions with three-layer membrane structure of breast milk fat globules, and the steps thereof are basically the same as those of Example 1, except that the process parameters are different. For details, see Table 1: Table 1: List of process parameters in Examples 10 to 13
[0062] The process parameters and steps of other parts of Examples 10 to 13 are the same as those of Example 1.
[0063] Obviously, the described examples are only a part of the examples of the present application, but not all the examples. Based on the examples in the present application, all other examples obtained by those skilled in the art without creative labor fall within the scope of protection of the present application.
Claims
1. A method for constructing a lactoferrin-phospholipid-based emulsion with a three-layered, breast milk-mimicking fat globule membrane structure, characterized in that, The construction method involves treating lactoferrin with ultrasound to obtain a single-layer membrane structure emulsion; stacking phospholipids and cholesterol through a microporous membrane to obtain a microporous membrane with a bilayer membrane; and then electrostatically self-assembling the single-layer membrane structure emulsion and the microporous membrane with the bilayer membrane to obtain the lactoferrin-phospholipid-based emulsion with a three-layer breast milk fat globule membrane structure.
2. The method for constructing a lactoferrin-phospholipid-based emulsion with a three-layered, breast milk-mimicking fat globule membrane structure according to claim 1, characterized in that, The construction method includes the following steps: Single-layer membrane structure emulsion: Lactoferrin is dissolved in water, and compound vegetable oil is added to the resulting lactoferrin solution. The resulting suspension is then treated with ultrasound to form a positively charged single-layer membrane structure emulsion. Microporous membrane surface stacking: Using a mixture of phospholipids and cholesterol as lipids, a negatively charged lipid film is prepared on the surface of a microporous membrane by rotary evaporation, resulting in a microporous membrane with a bilayer. Electrostatic self-assembly secondary coating: A phospholipid bilayer is formed by premixing a monolayer emulsion with a microporous membrane containing a bilayer membrane and then forming a secondary coating on the outside of the monolayer membrane through electrostatic self-assembly, thus obtaining the lactoferrin-phospholipid-based emulsion with a three-layer breast milk fat globule membrane structure.
3. The method for constructing a lactoferrin-phospholipid-based emulsion with a three-layered, breast milk-mimicking fat globule membrane structure according to claim 2, characterized in that, The construction method includes the following specific steps: Single-layer membrane structure emulsion: Lactoferrin is dissolved in water, and compound vegetable oil is added to the resulting lactoferrin solution. The resulting suspension is then ultrasonically treated to form a positively charged single-layer membrane structure emulsion. Microporous membrane surface stacking: Phospholipids and cholesterol are mixed as lipids, anhydrous ethanol is added to the lipids to form a lipid solution, and then the microporous membrane is placed in the lipid solution and rotary evaporated. After the ethanol evaporates, a negatively charged lipid film is formed on the surface of the microporous membrane, resulting in a microporous membrane with a bilayer. Electrostatic self-assembly secondary coating: Using a premixed membrane emulsification method, a monolayer membrane structure emulsion is pressed through a microporous membrane with a bilayer membrane. The lipid film stacked on the surface of the microporous membrane with the bilayer membrane swells and forms a bilayer phospholipid layer on the surface of the monolayer membrane structure emulsion through electrostatic self-assembly, thus obtaining the lactoferrin-phospholipid-based emulsion with a three-layer breast milk fat globule membrane structure.
4. The method for constructing a lactoferrin-phospholipid-based emulsion with a three-layered, breast milk-mimicking fat globule membrane structure according to claim 3, characterized in that, In the process of preparing a single-layer film emulsion, the ultrasonic treatment power is 200~1000W and the time is 50~200s; During the electrostatic self-assembly secondary covering process, the inlet pressure of the premixed membrane emulsification method is 10~50kPa.
5. The method for constructing a lactoferrin-phospholipid-based emulsion with a three-layered, breast milk-mimicking fat globule membrane structure according to any one of claims 2-4, characterized in that, The raw materials for making compound vegetable oil include: 30-42 parts by weight of 1,3-dioleoyl-2-palmitoyl glycerol triglyceride, 15-20 parts by weight of cow's milk fat, 14-19 parts by weight of coconut oil, 8-12 parts by weight of palm oil, 8-12 parts by weight of corn oil and 8-12 parts by weight of sunflower seed oil.
6. The method for constructing a lactoferrin-phospholipid-based emulsion with a three-layered, breast milk-mimicking fat globule membrane structure according to any one of claims 2-4, characterized in that, The amount of lactoferrin added is 0.02~1 wt% of the monolayer membrane emulsion; The total amount of phospholipids and cholesterol added is 1.45~5 wt% of the single-layer membrane structure emulsion; The amount of compound vegetable oil used is 1 to 30 wt% of the single-layer film emulsion.
7. The method for constructing a lactoferrin-phospholipid-based emulsion with a three-layered, breast milk-mimicking fat globule membrane structure according to any one of claims 1-4, characterized in that, The weight ratio of phospholipids to cholesterol in milk fat globule membranes is 4-14:
1.
8. The method for constructing a lactoferrin-phospholipid-based emulsion with a three-layered, breast milk-mimicking fat globule membrane structure according to any one of claims 1-4, characterized in that, The preparation process of the single-layer membrane structure emulsion also includes first dissolving the core material in a composite vegetable oil, and then adding the composite vegetable oil containing the core material to the lactoferrin solution, thereby encapsulating the core material within the single-layer membrane structure.
9. The method for constructing a lactoferrin-phospholipid-based emulsion with a three-layered, breast milk-mimicking fat globule membrane structure according to claim 8, characterized in that, The preparation process of the single-layer membrane structure emulsion is to dissolve lactoferrin in water to obtain a lactoferrin solution, dissolve the core material in a composite vegetable oil, and then add the composite vegetable oil containing the core material to the lactoferrin solution. The resulting suspension is then treated with ultrasound to form a positively charged lactoferrin-coated single-layer membrane structure emulsion. The core material is a carrier of fat-soluble nutrients or protective active ingredients; fat-soluble nutrients or protective active ingredients include, but are not limited to, fat-soluble vitamins, carotenoids, or probiotics.
10. The use of a lactoferrin-phospholipid-based emulsion with a three-layered breast milk fat globule membrane structure prepared by the construction method according to any one of claims 1-9 in the preparation of infant formula milk.