Construction method of emulsion with three-layer breast milk fat globule membrane imitating structure and application of emulsion in preparation of high-end infant formula emulsion

Abstract

The invention provides a construction method of an emulsion with a three-layer breast milk-imitating fat globule membrane structure and application of the emulsion in preparation of a high-end infant formula emulsion, and belongs to the technical field of dairy products, the construction method comprises the following steps: carrying out membrane emulsification on lactoferrin and sphingosine to obtain an emulsion with a single-layer membrane structure; stacking the milk fat globule membrane phospholipid and cholesterol through a microporous membrane to obtain a microporous membrane with a bimolecular membrane; and carrying out electrostatic self-assembly secondary covering on the emulsion with the single-layer membrane structure and a microporous membrane with a bimolecular membrane to obtain the emulsion with the three-layer breast milk imitating fat globule membrane structure. The emulsion with the three-layer breast milk-imitating fat globule membrane structure is used for preparing high-end infant formula emulsion. The invention establishes a novel breast milk fat globule structure imitating emulsion construction method, which combines microporous membrane piling-electrostatic self-assembly secondary covering with direct membrane emulsification-premixed membrane emulsification technology, has the advantages of simple equipment, simplicity and convenience in operation, greenness, no pollution, energy conservation, consumption reduction, no temperature rise and high shear force input, and is suitable for heat-sensitive and mechanical force-sensitive materials.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of dairy products, and particularly relates to a construction method of an emulsion with a three-layer milk fat globule membrane structure and application thereof in preparation of high-end infant formula emulsion. BACKGROUND

[0002] Milk fat globules are secretory products accumulated through different biological membranes multiple times during the lactation process of a mother, and are wrapped by a natural milk fat globule membrane (MFGM) with a three-layer ordered membrane structure: (1) glycerol triesters are synthesized by the action of rough endoplasmic reticulum of mammary gland epithelial secretory cells and released into the cytoplasm, and a monolayer film is formed on the surface of the glycerol triesters, which is composed of a variety of complex components such as proteins (milk fat protein, xanthine dehydrogenase / oxidase, mucin, fat droplet binding protein and lactadherin, etc.), glycerophospholipids (phosphatidylethanolamine PE and phosphatidylinositol PI, etc.), sphingomyelin (SM) and cholesterol, etc.; (2) the fat globules migrate to the apical plasma membrane (i.e. a double-layer phospholipid including phosphatidylcholine PC, phosphatidylserine PS and SM, etc.) of the cell, and are coated by the double-layer phospholipid of the apical cytoplasmic membrane through the process of exocytosis and secretion, so as to form milk fat globules covered by the three-layer MFGM membrane structure. In order to simulate the interface structure and nutritional value of milk fat globules, improve the fat digestibility of infants and promote the growth and development of infants and young children, it has become a new trend to improve the function of infant formula powder by adding bovine milk-derived MFGM components. However, the interface membrane of the milk fat globule simulated by adding MFGM does not have the reasonable arrangement effect of the milk fat globule membrane. This is mainly because the traditional homogenization method can only form a relatively thick milk protein (casein and whey protein) complex, and cannot reproduce the ordered topological structure of the three-layer membrane of the milk fat globule, which naturally weakens the effective digestion of the fat globules by infants, and further significantly increases the risk of childhood obesity and metabolic syndrome. In view of this, the present application provides a new technology to solve the existing problem. SUMMARY

[0003] In view of the above problems, the present application provides a construction method of an emulsion with a three-layer milk fat globule membrane structure and application thereof in preparation of high-end infant formula emulsion. The emulsion membrane structure is close to the natural three-layer milk fat globule membrane, and the particle size is close to that of the milk fat globule, which is easy for infants to digest and absorb lipids, and is suitable for being added to infant formula milk powder and other complementary foods, and has good practical use value.

[0004] To achieve the above object, the technical scheme adopted by the present application is as follows: A method for constructing an emulsion with a three-layered breast milk fat globule membrane structure, wherein the method comprises emulsifying lactoferrin and sphingosine via a membrane to obtain a single-layer membrane structure emulsion; stacking milk fat globule membrane phospholipids and cholesterol via a microporous membrane to obtain a microporous membrane with a bilayer membrane; and electrostatically self-assembling the single-layer membrane structure emulsion and the microporous membrane with the bilayer membrane to obtain the emulsion with the three-layered breast milk fat globule membrane structure.

[0005] Furthermore, the construction method includes the following steps: Single-layer membrane structure emulsion: Lactoferrin and sphingosine are directly used for membrane emulsification to form a positively charged single-layer membrane structure emulsion; Microporous membrane surface stacking: Using a mixture of milk fat globule membrane phospholipids and cholesterol as lipids, a negatively charged lipid film is prepared on the surface of the microporous membrane by rotary evaporation, resulting in a microporous membrane with a bilayer. Electrostatic self-assembly secondary coating: A monolayer emulsion and a microporous membrane with a bilayer are emulsified by premixing the membrane, and a phospholipid bilayer is formed on the outside of the monolayer membrane by electrostatic self-assembly, thus obtaining the emulsion with a three-layer breast milk fat globule membrane structure.

[0006] Furthermore, the construction method includes the following specific steps: Single-layer membrane structure emulsion: Lactoferrin is dissolved in water as the aqueous phase, and sphingosine is dissolved in a compound vegetable oil as the oil phase; the oil phase is pressed through a microporous membrane and dispersed in the continuously stirred aqueous phase using a direct membrane emulsification method to form a positively charged single-layer membrane structure emulsion. Microporous membrane surface stacking: Take milk fat globule membrane phospholipids and cholesterol as lipids, add anhydrous ethanol to the lipids to form a lipid solution, then put the microporous membrane into the lipid solution and rotary evaporate. 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 bimolecular membrane. 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 monolayer membrane structure emulsion through electrostatic self-assembly, thus obtaining the emulsion with a three-layer breast milk fat globule membrane structure.

[0007] Furthermore, in the process of preparing monolayer membrane structure emulsions, the inlet pressure of the direct membrane emulsification method is 5~20 kPa; the continuous stirring speed of the aqueous phase is 300~1200 rpm. During the electrostatic self-assembly secondary covering process, the inlet pressure of the premixed membrane emulsification method is 20~50kPa.

[0008] Furthermore, the raw materials for making the 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.

[0009] Furthermore, the total amount of lactoferrin and sphingosine added was 0.02~1 wt% of the single-layer membrane structure emulsion; The total amount of milk fat globule membrane phospholipids and cholesterol added is 1.5~5 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.

[0010] Furthermore, the weight ratio of lactoferrin to sphingosine is 4~11:1; The weight ratio of phospholipids to cholesterol in milk fat globule membranes is 4~15:1.

[0011] Furthermore, the preparation process of the single-layer membrane structure emulsion also includes adding a core material together with sphingosine, thereby encapsulating the core material within the single-layer membrane structure.

[0012] Furthermore, the preparation process of the single-layer membrane structure emulsion involves dissolving lactoferrin in water as the aqueous phase, dissolving the core material and sphingosine together in a composite vegetable oil as the oil phase, and using a direct membrane emulsification method to press the oil phase through a microporous membrane and disperse it in the continuously stirred aqueous phase to form a positively charged 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.

[0013] Application of an emulsion with a three-layered breast milk fat globule membrane structure prepared by the above construction method in the preparation of high-end infant formula emulsions.

[0014] The beneficial effects of the present invention on the construction method of an emulsion with a three-layered breast milk fat globule membrane structure and its application in the preparation of high-end infant formula emulsions are as follows: The present invention aims to introduce a biomimetic interface engineering strategy to simulate the preparation of MFGM three-layer membrane structure-coated breast milk fat globules using the principle of "electrostatic self-assembly-secondary covering". The specific method is as follows: (1) First, the phase to be dispersed is pushed into the membrane pore and contacted with the cross-flow or stirred continuous phase to form an emulsion through direct membrane emulsification. The emulsifier (lipid or protein) is positively charged by pH-induced emulsification to prepare a single-layer premixed water-in-oil (o / w) emulsion fat globules; (2) Then, the premixed emulsion droplets are squeezed and broken through a porous structure to obtain a fine emulsion premixed membrane emulsification. The negatively charged phospholipid bilayer is coated on the surface of the primary positively charged oil droplets in an electrostatic bonding manner, so that its formation method is similar to the classic method of desorbing dry phospholipids from the membrane surface into water to form liposomes, thereby obtaining a three-layer protein-phospholipid membrane-coated breast milk fat globules; Compared with traditional homogenization methods, the advantages of membrane emulsification methods are mainly: (1) a crude emulsion can be formed by direct membrane emulsification, and then a finely controlled emulsion can be made by premixed membrane emulsification. The biomimetic effect of a biological membrane can be achieved by using a series of multi-scale membrane physical assembly methods; (2) the low shear force process is suitable for heat or shear force sensitive bioactive substances, which is conducive to preserving the integrity of the natural structure of MFGM and avoiding the loss of natural membrane components; (3) the mass transfer rate of membrane emulsification method is relatively slow, which is conducive to the orderly assembly of natural MFGM macromolecules on the surface of fat globules to reduce entropy change and free energy change, thereby achieving interface stabilization; (4) (5) By examining the interfacial adsorption capacity of MFGM components, the particle size of the actual dispersed droplets can be predicted based on the theoretical model, thus providing a theoretical basis for regulating the structure of fat globules; (6) By screening condition parameters such as membrane pore size, feed pressure, and stirring speed, it is easier to control the particle size and surface area of ​​fat globules to be close to those of breast milk fat globules; (7) The lower energy density requirement improves the high precision of fat globules and also enhances the quality and functionality of fine emulsion components; By achieving the equivalent digestibility of infant formula fat with breast milk through structural biomimicry, the technical bottleneck of "composition similar but function insufficient" for infant formula is broken, which has a positive effect on promoting the development of this industry; This invention establishes a novel method for constructing emulsions with a breast milk-like fat globule structure—a combination of microporous membrane stacking, electrostatic self-assembly, secondary covering, direct membrane emulsification, and premixed membrane emulsification technology. The equipment is simple, easy to operate, green and pollution-free, energy-saving and consumption-reducing, and requires no heating or high shear force input, making it suitable for heat-sensitive and mechanically sensitive materials. This invention employs lactoferrin and sphingosine to form a positively charged inner monolayer membrane, and milk fat globule membrane phospholipids and cholesterol to form a negatively charged outer bilayer membrane. Through electrostatic interaction, they self-assemble to form a stable three-layer membrane. The resulting breast milk fat globule-like emulsion membrane has a structure similar to the three-layer structure of breast milk fat globule membrane. At the same time, the composition of the breast milk fat globule-like emulsion is similar to that of breast milk fat globules, with a particle size close to that of breast milk fat globules and a uniform distribution. It can encapsulate fat-soluble nutrients and can be applied to infant formula. The three-layer breast milk fat globule structure emulsion provided by this invention achieves the same fat digestibility characteristics as breast milk through structural biomimicry, breaking through the technical bottleneck of "similar composition but insufficient function" in infant formula, and has great application value in high-end infant formula foods. Attached Figure Description

[0015] Figure 1 This is a simplified process flow diagram of a method for constructing an emulsion with a three-layered breast milk fat globule membrane structure according to the present invention. Figure 2 These are the particle size measurement results of each emulsion to be tested in Example 9 of the present invention; wherein, the simple membrane structure emulsion represents the measurement results of the single-layer membrane structure emulsion prepared in step S1 of Examples 1 to 8, and the three-layer membrane structure emulsion represents the measurement results of the emulsion with three layers of breast milk fat globule membrane structure finally obtained in Examples 1 to 8. Figure 3 These are the potential measurement results of each emulsion to be tested in Example 9 of the present invention; wherein, the simple membrane structure emulsion represents the measurement results of the single-layer membrane structure emulsion prepared in step S1 of Examples 1 to 8, and the three-layer membrane structure emulsion represents the measurement results of the emulsion with three layers of breast milk fat globule membrane structure finally obtained in Examples 1 to 8. Figure 4 The results of particle size measurement and potential measurement of breast milk and a commercially available infant formula are from Example 9 of this invention; wherein, the results of measurement of breast milk as a substitute for breast milk and the results of measurement of commercially available infant formula are from commercially available infant formula. Figure 5 These are the results of the fat decomposition degree measurement in Example 9 of the present invention; wherein, Examples 1 to 8 represent the measurement results of the emulsion with a three-layer imitation breast milk fat globule membrane structure finally obtained in Examples 1 to 8, the measurement results of breast milk substitute for breast milk, and the measurement results of commercially available milk powder represent the measurement results of commercially available infant formula. Detailed Implementation

[0016] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Many specific details are set forth in the following description to provide a thorough understanding of the present invention. However, the present invention may also be implemented in other ways different from those described herein. Those skilled in the art can make similar extensions without departing from the spirit of the present invention. Therefore, the present invention is not limited to the specific embodiments disclosed below.

[0017] The raw materials for an emulsion with a three-layered breast milk fat globule membrane structure include lactoferrin, sphingosine, milk fat globule membrane phospholipids, cholesterol, compound vegetable oils, and water.

[0018] The above formula does not include a core material. However, in practical applications, fat-soluble nutrients or protective active ingredients can be added as a core material as needed. Fat-soluble nutrients or protective active ingredients include, but are not limited to, fat-soluble vitamins, carotenoids, or carriers of probiotics.

[0019] A method for constructing an emulsion with a three-layered, breast milk-mimicking fat globule membrane structure, such as... Figure 1 As shown, the specific steps include the following: S1. Single-layer membrane structure emulsion: Lactoferrin is dissolved in water as the aqueous phase, and sphingosine is dissolved in the composite vegetable oil as the oil phase (if a core material is added, the core material and sphingosine are dissolved together in the composite vegetable oil as the oil phase).

[0020] Using a conventional membrane emulsification device, a direct membrane emulsification method (which simulates the formation of a single-layer membrane of natural breast milk fat globules in mammary epithelial secretory cells) is employed. The oil phase is forced through a microporous membrane on the membrane emulsification device and dispersed in a continuously stirred aqueous phase. By controlling the inlet pressure (5~20 kPa) and stirring speed (300~1200 rpm), a positively charged single-layer membrane structure emulsion is formed. This single-layer membrane structure emulsion utilizes lactoferrin and sphingosine to form a positively charged inner single-layer membrane. When a core material is added, the core material is encapsulated within the single-layer membrane structure, thereby promoting the absorption of fat-soluble nutrients or protecting the encapsulation of active ingredients.

[0021] The compound vegetable oil is composed of 30-42 parts by weight of 1,3-dioleoyl-2-palmitoylglycerol triglyceride, 15-20 parts by weight of 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. The amount of compound vegetable oil used is 1-30 wt% of the weight of the single-layer film emulsion. The fatty acid composition of the compounded oil meets the requirements of infants for fatty acid ratios.

[0022] The total amount of lactoferrin and sphingosine added is 0.02~1wt% of the single-layer membrane structure emulsion, and the weight ratio of lactoferrin to sphingosine is 4~11:1.

[0023] S2. Microporous membrane surface stacking: A mixture of milk fat globule membrane phospholipids and cholesterol in a weight ratio of 4~15:1 is taken as lipids. Anhydrous ethanol is added to the lipids to form a lipid solution. The microporous membrane is then placed in the lipid solution and rotary evaporated. After the ethanol evaporates, the lipid film (i.e., milk fat globule membrane phospholipids and cholesterol) stacks on the surface of the microporous membrane, forming a negatively charged lipid film on the surface of the microporous membrane, which is a microporous membrane with a bilayer membrane. The total amount of milk fat globule membrane phospholipids and cholesterol added is 1.5~5wt% of the single-layer membrane structure emulsion in step S1.

[0024] 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 device. A premixed membrane emulsification method is employed (premixed membrane emulsification is used to simulate the formation of the bilayer phospholipid membrane on the outer layer of natural breast milk fat globules during exocytosis, thereby achieving biomimetic construction of the membrane structure evolution from intracellular to secretory processes). By controlling the inlet pressure (20~50 kPa), the monolayer membrane structure obtained in step S1 is... The emulsion is passed through the microporous membrane with a bilayer obtained in step S2. The lipid film stacked on the surface of the microporous membrane with the bilayer swells and forms a bilayer phospholipid layer on the surface of the monolayer membrane structure emulsion through electrostatic self-assembly. (That is, the negatively charged milk fat globule membrane phospholipids and cholesterol on the surface of the microporous membrane self-assemble through electrostatic interaction onto the surface of the positively charged inner monolayer membrane composed of lactoferrin and sphingosine.) This yields an emulsion with a three-layered breast milk fat globule membrane structure (referred to as a three-layered membrane structure emulsion, such as...). Figure 1 (As shown). In the three-layer membrane structure of the emulsion with a three-layer structure that mimics the fat globule membrane of breast milk, the inner monolayer membrane is composed of lactoferrin and sphingosine, and the outer bilayer membrane is composed of milk fat globule membrane phospholipids and cholesterol. The overall structure simulates the three-layer structure of the natural breast milk fat globule membrane.

[0025] The microporous membrane used in this invention is a 1μm microporous membrane used in conventional membrane emulsification processes, purchased from SPG Corporation of Japan.

[0026] This invention discloses an emulsion with a three-layered, breast milk-mimicking fat globule membrane structure, which can be used as a raw material to prepare high-end infant formula. This emulsion, through the coupling of membrane emulsification and electrostatic self-assembly technology, achieves the simulation of the three-layered structure of the breast milk fat globule membrane in infant formula, thereby improving lipid digestibility and absorption and enhancing the breast milk-likeness of the formula.

[0027] Example 1: A method for constructing an emulsion with a three-layered breast milk fat globule membrane structure and its application. In this embodiment, the formulation of an emulsion with a three-layered breast milk fat globule membrane structure includes the following raw materials by weight: 0.02g lactoferrin, 99g deionized water, 0.002g sphingosine, 1g compound vegetable oil, 1.4g milk fat globule membrane phospholipids, and 0.1g cholesterol. The compound vegetable oil is made by mixing 0.42g of 1,3-dioleoyl-2-palmitoylglycerol triglyceride, 0.2g of milk fat, 0.14g of coconut oil, 0.08g of palm oil, 0.08g of corn oil and 0.08g of sunflower seed oil.

[0028] This embodiment describes a method for constructing an emulsion with a three-layered, breast milk-mimicking fat globule membrane structure, comprising the following specific steps: S1. Single-layer membrane emulsion: 0.02g lactoferrin was dissolved in 99g deionized water as the aqueous phase, and 0.002g sphingosine was dissolved in 1g of compound vegetable oil as the oil phase. Using direct membrane emulsification, the oil phase was pressed through a microporous membrane at 10kPa and dispersed in the aqueous phase stirred at 500rpm, forming a positively charged single-layer membrane emulsion.

[0029] S2. Microporous membrane surface stacking: 1.4g of milk fat globule membrane phospholipids and 0.1g of cholesterol were added to 30mL 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 35 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 single-layer membrane through electrostatic self-assembly, thus obtaining an emulsion with a three-layer breast milk fat globule membrane structure, referred to as a three-layer membrane structure emulsion.

[0030] The emulsion with a three-layered, breast milk-mimicking fat globule membrane structure prepared in this embodiment can be used as a raw material to prepare high-end infant formula emulsions. By coupling membrane emulsification with electrostatic self-assembly technology, the three-layered structure of the breast milk fat globule membrane is simulated in the infant formula emulsion, which can improve lipid digestion and absorption and the breast milk similarity of the formula emulsion.

[0031] In practical applications, fat-soluble nutrients or protective active ingredients can be added as core materials as needed. These include, but are not limited to, fat-soluble vitamins, carotenoids, or carriers of probiotics. When adding core materials, the core materials and sphingosine are dissolved together in a compound vegetable oil as the oil phase.

[0032] Example 2: A method for constructing an emulsion with a three-layered breast milk fat globule membrane structure. In this embodiment, the formulation of an emulsion with a three-layered breast milk fat globule membrane structure includes the following raw materials by weight: 0.2g lactoferrin, 90g deionized water, 0.02g sphingosine, 10g compound vegetable oil, 1.4g milk fat globule membrane phospholipids, and 0.1g cholesterol. The compound vegetable oil is made by mixing 4.2g of 1,3-dioleoyl-2-palmitoylglycerol triglyceride, 2g of milk fat, 1.4g of coconut oil, 0.8g of palm oil, 0.8g of corn oil and 0.8g of sunflower seed oil. This embodiment describes a method for constructing an emulsion with a three-layered, breast milk-mimicking fat globule membrane structure, comprising the following specific steps: S1. Single-layer membrane structure emulsion: 0.2g lactoferrin was dissolved in 90g deionized water as the aqueous phase, and 0.02g sphingosine was dissolved in 10g compound vegetable oil as the oil phase. The direct membrane emulsification method was used to press the oil phase through a microporous membrane at 10kPa and disperse it in the aqueous phase stirred at 500rpm to form a positively charged single-layer membrane structure emulsion.

[0033] S2. Microporous membrane surface stacking: 1.4g of milk fat globule membrane phospholipids and 0.1g of cholesterol were added to 30mL 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 35 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 single-layer membrane through electrostatic self-assembly, thus obtaining an emulsion with a three-layer breast milk fat globule membrane structure, referred to as a three-layer membrane structure emulsion.

[0034] Example 3: A method for constructing an emulsion with a three-layered breast milk fat globule membrane structure. In this embodiment, the formulation of an emulsion with a three-layered breast milk fat globule membrane structure includes the following raw materials by weight: 0.02g lactoferrin, 99g deionized water, 0.002g sphingosine, 1g compound vegetable oil, 1.16g milk fat globule membrane phospholipids, and 0.29g cholesterol. The compound vegetable oil is made by mixing 0.42g of 1,3-dioleoyl-2-palmitoylglycerol triglyceride, 0.2g of milk fat, 0.14g of coconut oil, 0.08g of palm oil, 0.08g of corn oil and 0.08g of sunflower seed oil. This embodiment describes a method for constructing an emulsion with a three-layered, breast milk-mimicking fat globule membrane structure, comprising the following specific steps: S1. Single-layer membrane structure emulsion: 0.02g lactoferrin was dissolved in 99g deionized water as the aqueous phase, and 0.002g sphingosine was dissolved in 1g compound vegetable oil as the oil phase. The direct membrane emulsification method was used to press the oil phase through a microporous membrane at 10kPa and disperse it in the aqueous phase stirred at 500rpm to form a positively charged single-layer membrane structure emulsion.

[0035] S2. Microporous membrane surface stacking: 1.16g of milk fat globule membrane phospholipids and 0.29g of cholesterol were added to 30mL 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, a thin lipid film was 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 35 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 single-layer membrane through electrostatic self-assembly, thus obtaining an emulsion with a three-layer breast milk fat globule membrane structure, referred to as a three-layer membrane structure emulsion.

[0036] Example 4: A method for constructing an emulsion with a three-layered breast milk fat globule membrane structure. In this embodiment, the formulation of an emulsion with a three-layered breast milk fat globule membrane structure includes the following raw materials by weight: 0.2g lactoferrin, 90g deionized water, 0.02g sphingosine, 10g compound vegetable oil, 1.16g milk fat globule membrane phospholipids, and 0.29g cholesterol. The compound vegetable oil is made by mixing 4.2g of 1,3-dioleoyl-2-palmitoylglycerol triglyceride, 2g of milk fat, 1.4g of coconut oil, 0.8g of palm oil, 0.8g of corn oil and 0.8g of sunflower seed oil. This embodiment describes a method for constructing an emulsion with a three-layered, breast milk-mimicking fat globule membrane structure, comprising the following specific steps: S1. Single-layer membrane structure emulsion: 0.2g lactoferrin was dissolved in 90g deionized water as the aqueous phase, and 0.02g sphingosine was dissolved in the composite vegetable oil as the oil phase. The direct membrane emulsification method was used to press the oil phase through a microporous membrane at 10kPa and disperse it in the aqueous phase stirred at 500rpm to form a positively charged single-layer membrane structure emulsion.

[0037] S2. Microporous membrane surface stacking: 1.16g of milk fat globule membrane phospholipids and 0.29g of cholesterol were added to 30mL 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 35 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 single-layer membrane through electrostatic self-assembly, thus obtaining an emulsion with a three-layer breast milk fat globule membrane structure, referred to as a three-layer membrane structure emulsion.

[0038] Example 5: A method for constructing an emulsion with a three-layered breast milk fat globule membrane structure. In this embodiment, the formulation of an emulsion with a three-layered breast milk fat globule membrane structure includes the following raw materials by weight: 0.02g lactoferrin, 99g deionized water, 0.002g sphingosine, 1g compound vegetable oil, 1.4g milk fat globule membrane phospholipids, and 0.1g cholesterol. The compound vegetable oil is made by mixing 0.3g of 1,3-dioleoyl-2-palmitoylglycerol triglyceride, 0.15g of cow's milk fat, 0.19g of coconut oil, 0.12g of palm oil, 0.12g of corn oil and 0.12g of sunflower seed oil. This embodiment describes a method for constructing an emulsion with a three-layered, breast milk-mimicking fat globule membrane structure, comprising the following specific steps: S1. Single-layer membrane structure emulsion: 0.02g lactoferrin was dissolved in 99g deionized water as the aqueous phase, and 0.002g sphingosine was dissolved in 1g of compound vegetable oil as the oil phase. The direct membrane emulsification method was used to press the oil phase through a microporous membrane at 7.5kPa and disperse it in the aqueous phase stirred at 300rpm to form a positively charged single-layer membrane structure emulsion.

[0039] S2. Microporous membrane surface stacking: 1.4g of milk fat globule membrane phospholipids and 0.1g of cholesterol were added to 30mL 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 25 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 single-layer membrane through electrostatic self-assembly, thus obtaining an emulsion with a three-layer breast milk fat globule membrane structure, referred to as a three-layer membrane structure emulsion.

[0040] Example 6: A method for constructing an emulsion with a three-layered breast milk fat globule membrane structure. In this embodiment, the formulation of an emulsion with a three-layered breast milk fat globule membrane structure includes the following raw materials by weight: 0.2g lactoferrin, 90g deionized water, 0.02g sphingosine, 10g compound vegetable oil, 1.4g milk fat globule membrane phospholipids, and 0.1g cholesterol. The compound vegetable oil is composed of 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. This embodiment describes a method for constructing an emulsion with a three-layered, breast milk-mimicking fat globule membrane structure, comprising the following specific steps: S1. Single-layer membrane structure emulsion: 0.02g lactoferrin was dissolved in 99g deionized water as the aqueous phase, and 0.002g sphingosine was dissolved in the composite vegetable oil as the oil phase. The direct membrane emulsification method was used to press the oil phase through a microporous membrane at 7.5kPa and disperse it in the aqueous phase stirred at 300rpm to form a positively charged single-layer membrane structure emulsion.

[0041] S2. Microporous membrane surface stacking: 1.4g of milk fat globule membrane phospholipids and 0.1g of cholesterol were added to 30mL 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 25 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 single-layer membrane through electrostatic self-assembly, thus obtaining an emulsion with a three-layer breast milk fat globule membrane structure, referred to as a three-layer membrane structure emulsion.

[0042] Example 7: A method for constructing an emulsion with a three-layered breast milk fat globule membrane structure. In this embodiment, the formulation of an emulsion with a three-layered breast milk fat globule membrane structure includes the following raw materials by weight: 0.02g lactoferrin, 99g deionized water, 0.002g sphingosine, 1g compound vegetable oil, 1.16g milk fat globule membrane phospholipids, and 0.29g cholesterol. The compound vegetable oil is composed of 0.3g of 1,3-dioleoyl-2-palmitoyl glycerol, 0.15g of milk fat, 0.19g of coconut oil, 0.12g of palm oil, 0.12g of corn oil and 0.12g of sunflower seed oil. This embodiment describes a method for constructing an emulsion with a three-layered, breast milk-mimicking fat globule membrane structure, comprising the following specific steps: S1. Single-layer membrane structure emulsion: 0.02g lactoferrin was dissolved in 99g deionized water as the aqueous phase, and 0.002g sphingosine was dissolved in the composite vegetable oil as the oil phase. The direct membrane emulsification method was used to press the oil phase through a microporous membrane at 7.5kPa and disperse it in the aqueous phase stirred at 300rpm to form a positively charged single-layer membrane structure emulsion.

[0043] S2. Microporous membrane surface stacking: 1.16g of milk fat globule membrane phospholipids and 0.29g of cholesterol were added to 30mL 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 25 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 single-layer membrane through electrostatic self-assembly, thus obtaining an emulsion with a three-layer breast milk fat globule membrane structure, referred to as a three-layer membrane structure emulsion.

[0044] Example 8: A method for constructing an emulsion with a three-layered breast milk fat globule membrane structure. In this embodiment, the formulation of an emulsion with a three-layered breast milk fat globule membrane structure includes the following raw materials by weight: 0.2g lactoferrin, 90g deionized water, 0.02g sphingosine, 10g compound vegetable oil, 1.16g milk fat globule membrane phospholipids, and 0.29g cholesterol. The compound vegetable oil is composed of 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. This embodiment describes a method for constructing an emulsion with a three-layered, breast milk-mimicking fat globule membrane structure, comprising the following specific steps: S1. Single-layer membrane structure emulsion: 0.02g lactoferrin was dissolved in 99g deionized water as the aqueous phase, and 0.002g sphingosine was dissolved in the composite vegetable oil as the oil phase. The direct membrane emulsification method was used to press the oil phase through a microporous membrane at 7.5kPa and disperse it in the aqueous phase stirred at 300rpm to form a positively charged single-layer membrane structure emulsion.

[0045] S2. Microporous membrane surface stacking: 1.16g of milk fat globule membrane phospholipids and 0.29g of cholesterol were added to 30mL 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 25 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 single-layer membrane through electrostatic self-assembly, thus obtaining an emulsion with a three-layer breast milk fat globule membrane structure, referred to as a three-layer membrane structure emulsion.

[0046] Example 9: Detection of an emulsion with a three-layered breast milk fat globule membrane structure. The single-layer membrane structure emulsion prepared in step S1 of Examples 1-8 and the finally obtained emulsion with a three-layer breast milk fat globule membrane structure were taken as test emulsions and subjected to the following tests.

[0047] 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 test emulsion was diluted to 50 mL with ultrapure water. The volume-weighted particle size distribution and average diameter of the emulsions were determined using a BT-9300S laser particle size analyzer (Dandong Better Instruments Co., Ltd., China). The results are as follows: Figure 2 As shown.

[0048] Meanwhile, particle size was determined using the above method for breast milk and a commercially available infant formula (reconstituted), and the results are as follows: Figure 4 As shown.

[0049] 2) Potential measurement 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 test emulsion was diluted 400-fold with buffer solutions (5 mM CaCl2, 50 mM NaCl, and 20 mM imidazole). The zeta potential was measured at 25°C using a Nano-Z zeta potential analyzer (Malvern Instruments Ltd., Malvern, UK). The results are as follows: Figure 3 As shown.

[0050] Simultaneously, the above method was used to measure the potential of breast milk and a commercially available infant formula (reconstituted), and the results are as follows: Figure 4 As shown.

[0051] Detection was performed using average particle size and zeta potential, such as Figure 2 , 3 As shown, the particle size d of the monolayer film emulsions obtained in Examples 1-8 3,2With a particle size of 6.49~6.99μm and a zeta potential of 21.57~21mV, the emulsion has a three-layered fat globule membrane structure that mimics breast milk. 3,2 The potential ranges from 3.39 to 3.90 μm, and the zeta potential is -28.53 to -27.07 mV. This is because lactoferrin and sphingosine carry a positive charge under neutral conditions, resulting in a positive potential for the monolayer emulsion formed by their individual coating of oil droplets. The lipid bilayer carries a negative charge, which, upon addition, neutralizes the positive charge of lactoferrin or sphingosine through electrostatic binding. The excess negative charge causes the system potential to become negative, confirming that the secondary coating of phospholipids forms a three-layer membrane structure of "lactoferrin and sphingosine - milk fat globule membrane phospholipids and cholesterol." Figure 4 The particle size d of breast milk and commercially available infant formula reconstituted milk powder 3,2 The particle sizes were 3.5±0.21μm and 0.37±0.01μm, respectively, and the zeta potentials were -7.8±0.2mV and -20±0.35mV, respectively. The emulsions with a three-layered, breast milk-mimicking fat globule membrane structure in Examples 1-8 had particle sizes close to breast milk and larger than those in commercially available reconstituted milk powder. The zeta potentials of these emulsions were also higher than those of both breast milk and commercially available reconstituted milk powder.

[0052] 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.), during gastric digestion, 126 mL of the emulsion with the three-layered breast milk fat globule membrane structure obtained in Examples 1-8 was mixed with 74 mL of simulated gastric juice (30 U / mg gastric lipase, 3000 U / mg pepsin, 94 mM NaCl and 13 mM KCl). The pH was maintained at 5.3 by adding 0.1 M NaOH, and the mixture was incubated at 37°C with shaking for 60 min. 136 mL of the digested solutions from each stomach were mixed with 64 mL of simulated intestinal fluid (250 U / mg trypsin, 30 U / mg pancreatic lipase, 0.67 mmol / g bile salts, 94 mM NaCl, 13 mM KCl, 85 mM NaHCO3, and 3 mM CaCl2). The pH of each mixture was adjusted to 6.6 using 0.1 M NaOH, and the mixtures were incubated at 37°C with shaking for 120 min. Samples were collected at 0, 30, 60, 90, and 120 min of intestinal digestion. The free fatty acid content, average molar mass of triglycerides in the emulsion, and average fat concentration were measured. The degree of fat breakdown (LD) was calculated using these values. The results are as follows: Figure 5 As shown. The degree of fat breakdown (LD) refers to the percentage of free fatty acids released during hydrolysis relative to the initial total acyl groups in the triglycerides. The calculation formula is as follows: , Wherein, FFA is the free fatty acid content (mol), which can be obtained from the molar amount of NaOH consumed; MMeq emulsion average molar mass of triglycerides (g / mol); FC is the average fat concentration (g / mol); and V is the emulsion volume (mL).

[0053] Simultaneously, the free fatty acid content, average molar mass of triglycerides in the emulsion, and average fat concentration were determined in breast milk and a commercially available infant formula (reconstituted) using the above method. These values ​​were then used to calculate the degree of fat breakdown. The results are as follows: Figure 5 As shown.

[0054] The emulsions with a three-layered, breast milk-mimicking fat globule membrane structure prepared in Examples 1-8 all exhibited fat-decomposing capabilities similar to those of breast milk (e.g., Figure 5(As shown). After simulating gastric digestion and entering the intestinal digestion stage, the fat decomposition rate of breast milk and the emulsions with a three-layered breast milk-mimicking fat globule membrane structure prepared in Examples 1-8 was higher than that of commercially available infant formula. Specifically, after 120 minutes of simulated intestinal digestion, the fat decomposition rate of the emulsions with a three-layered breast milk-mimicking fat globule membrane structure prepared in Examples 1, 3, 5, and 7 reached 64.9%~65.2%, slightly lower than breast milk (67.54±1.92%), and slightly higher than the emulsions in Examples 2, 4, 6, and 8 (61.3%~61.8%) and commercially available infant formula (42±0.7%). Therefore, the emulsion with a three-layered breast milk-mimicking fat globule membrane structure of the present invention promotes the digestion and absorption of internally embedded fat, making its digestive behavior closer to that of breast milk.

[0055] Examples 10-13: Methods for constructing emulsions with a three-layered breast milk fat globule membrane structure Examples 10-13 are methods for constructing an emulsion with a three-layered, breast milk-mimicking fat globule membrane structure. Their steps are basically the same as in Example 1, differing only in the process parameters, as detailed in Table 1. Table 1. Summary of process parameters in Examples 10-13

[0056] The process parameters and steps for the other parts of Examples 10-13 are the same as those for Example 1.

[0057] Obviously, the described embodiments are only some, not all, of the embodiments of the present invention. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without inventive effort are within the scope of protection of the present invention.

Claims

1. A method for constructing an emulsion with a three-layered, breast milk-mimicking fat globule membrane structure, characterized in that, The construction method involves emulsifying lactoferrin and sphingosine through a membrane to obtain a single-layer membrane structure emulsion; stacking milk fat globule membrane 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 emulsion with a three-layer breast milk fat globule membrane structure.

2. The method for constructing an 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 and sphingosine are directly used for membrane emulsification to form a positively charged single-layer membrane structure emulsion; Microporous membrane surface stacking: Using a mixture of milk fat globule membrane phospholipids and cholesterol as lipids, a negatively charged lipid film is prepared on the surface of the microporous membrane by rotary evaporation, resulting in a microporous membrane with a bilayer. Electrostatic self-assembly secondary coating: A monolayer emulsion and a microporous membrane with a bilayer are emulsified by premixing the membrane, and a phospholipid bilayer is formed on the outside of the monolayer membrane by electrostatic self-assembly, thus obtaining the emulsion with a three-layer breast milk fat globule membrane structure.

3. The method for constructing an emulsion with a three-layered, breast milk-mimicking fat globule membrane structure according to claim 1 or 2, characterized in that, The construction method includes the following specific steps: Single-layer membrane structure emulsion: Lactoferrin is dissolved in water as the aqueous phase, and sphingosine is dissolved in a compound vegetable oil as the oil phase; the oil phase is pressed through a microporous membrane and dispersed in the continuously stirred aqueous phase using a direct membrane emulsification method to form a positively charged single-layer membrane structure emulsion. Microporous membrane surface stacking: Take milk fat globule membrane phospholipids and cholesterol as lipids, add anhydrous ethanol to the lipids to form a lipid solution, then put the microporous membrane into the lipid solution and rotary evaporate. 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 bimolecular membrane. 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 monolayer membrane structure emulsion through electrostatic self-assembly, thus obtaining the emulsion with a three-layer breast milk fat globule membrane structure.

4. The method for constructing an emulsion with a three-layered, breast milk-mimicking fat globule membrane structure according to claim 3, characterized in that, In the preparation of monolayer membrane structure emulsions, the inlet pressure of the direct membrane emulsification method is 5~20 kPa; the continuous stirring speed of the aqueous phase is 300~1200 rpm. During the electrostatic self-assembly secondary covering process, the inlet pressure of the premixed membrane emulsification method is 20~50kPa.

5. The method for constructing an emulsion with a three-layered, breast milk-mimicking fat globule membrane structure according to claim 3, 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 an emulsion with a three-layered, breast milk-mimicking fat globule membrane structure according to claim 3, characterized in that, The total amount of lactoferrin and sphingosine added was 0.02~1 wt% of the monolayer membrane structure emulsion; The total amount of phospholipids and cholesterol added to the milk fat globule membrane was 1.5–5 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 an emulsion with a three-layered, breast milk-mimicking fat globule membrane structure according to claim 1, 2, 4, 5, or 6, characterized in that, The weight ratio of lactoferrin to sphingosine is 4~11:1; The weight ratio of phospholipids to cholesterol in milk fat globule membranes is 4~15:

1.

8. The method for constructing an emulsion with a three-layered, breast milk-mimicking fat globule membrane structure according to claim 1, 2, 4, 5, or 6, characterized in that, The preparation process of the single-layer membrane structure emulsion also includes adding a core material together with sphingosine, thereby encapsulating the core material within the single-layer membrane structure.

9. The method for constructing an 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 involves dissolving lactoferrin in water as the aqueous phase, dissolving the core material and sphingosine together in a composite vegetable oil as the oil phase, and using a direct membrane emulsification method to press the oil phase through a microporous membrane and disperse it in the continuously stirred aqueous phase to form a positively charged single-layer membrane structure emulsion. The core material consists of fat-soluble nutrients or protective active ingredients.

10. The application of an 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 emulsion.

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