Bilayer membrane lipid droplet for simulating breast milk as well as construction method and application of bilayer membrane lipid droplet

Abstract

The invention provides a double-layer membrane lipid droplet simulating breast milk and a construction method and application thereof, milk phospholipid and a membrane protein concentrate are separated from MFGM-10, butter or cream is used as an oil phase, and phospholipid double-layer membrane lipid droplets and / or phospholipoprotein double-layer membrane lipid droplets are prepared from the milk phospholipid and / or the membrane protein concentrate. Through detection, the steric hindrance is increased due to the existence of cholesterol in the interface of the double-layer membrane lipid droplet, the specific surface area of the double-layer membrane lipid droplet is larger, the interface contains more emulsifiers, and the long-term stability and emulsibility of the double-layer membrane lipid droplet are superior to those of a single-layer membrane lipid droplet with the same components. The double-layer membrane lipid droplet is closer to the interface structure of breast milk fat globules than the single-layer membrane lipid droplet, the long-term stability of dairy products can be improved by adding the double-layer membrane lipid droplet into the dairy products including infant formula milk powder, and more unsaturated fatty acids can be released in the digestion process.

Description

Technical Field

[0001] The invention belongs to the technical field of dairy products, and particularly relates to a double-layer membrane lipid droplet simulating breast milk and a construction method and application thereof. Background Art

[0002] Breast milk (HM) is known as the safest and most ideal natural food for infant growth and development. It contains the nutrients needed for infant growth and provides a solid foundation for the healthy growth of infants. It is worth mentioning that breast milk is also rich in a variety of known or unknown bioactive substances, which play a pivotal role in protecting infants from adapting to the natural environment and promoting healthy growth. The study of bioactive substances in breast milk has become a key breakthrough in exploring early human development, especially brain, intestinal and immune development. These substances not only contribute to the normal development of infants' bodies, but also play an indispensable role in promoting infants' intelligence, immunity and intestinal health.

[0003] Lipids are important nutrients in breast milk and exist in the form of milk fat globules (MFG). The fat globules are wrapped in a biological membrane called milk fat globule membrane (MFGM). The milk fat globule membrane is a three-layer membrane structure composed of phospholipids and proteins, which wrap around the outside of the fat globules and maintain their stability, which can promote digestion and absorption, intestinal health and immunity of infants and children.

[0004] At present, the reconstruction of monolayer lipid droplets has become the main trend of simulating breast milk lipid droplets. Researchers have long been aware that there are huge differences between the lipid droplet interface components in traditional infant formula and breast milk, and are gradually adding components of milk fat globule membrane to the interface of infant formula and its lipid droplets. Although the interface composition has gradually approached the composition of breast milk fat globule membrane, it still remains at the monolayer membrane stage and still has a large difference in interface structure with the three-layer membrane of breast milk fat globules. Summary of the invention

[0005] In view of the problems existing in the prior art, the purpose of the present invention is to provide a double-layer membrane lipid droplet simulating breast milk, and a construction method and application thereof, separate milk phospholipids and membrane protein concentrates from MFGM-10, use butter or cream as the oil phase, and use milk phospholipids and / or membrane protein concentrates to prepare phospholipid bilayer membrane lipid droplets and / or phospholipid protein bilayer membrane lipid droplets, and the long-term stability and emulsification of the double-layer membrane lipid droplets are better than those of single-layer membrane lipid droplets with the same components.

[0006] The objective of the present invention is achieved through the following technical solutions: The first aspect of the present invention provides a method for constructing a bilayer membrane lipid droplet simulating breast milk, wherein the bilayer membrane lipid droplet is a phospholipid bilayer membrane lipid droplet and / or a phospholipid protein bilayer membrane lipid droplet; The method for constructing the phospholipid bilayer membrane lipid droplets comprises the following steps: Add milk lecithin and cholesterol to anhydrous ethanol and fully dissolve them using a magnetic stirrer; then transfer the solution to a round-bottom flask, perform vacuum rotary evaporation to completely evaporate the ethanol, and leave the lecithin and cholesterol on the flask wall in the form of a thin film; add butter to the simulated milk ultrafiltrate to completely melt it; transfer the simulated milk ultrafiltrate containing butter or cream to a round-bottom flask to rehydrate the liposomes; and then perform ultrasonic crushing to obtain phospholipid bilayer membrane lipid droplets; The phospholipid bilayer membrane lipid droplets are further constructed to obtain phospholipid protein bilayer membrane lipid droplets, comprising the following steps: Firstly, the phospholipid membrane protein concentrate is dissolved in simulated milk ultrafiltrate to obtain a membrane protein concentrate solution; Then, Triton X-10 and membrane protein concentrate solution are added to the lipid droplets of the phospholipid bilayer membrane, and the mixture is mixed evenly on a shaker; Bio-beads (polystyrene microbeads) are added in small amounts for several times and mixed evenly on a shaker; after removing the Bio-beads, the phospholipid protein bilayer membrane is collected and obtained.

[0007] Further, the processing method of Bio-beads is: Add Bio-beads to methanol, shake on a shaker at room temperature, and then remove the methanol; repeat the addition of methanol and then remove the methanol, and finally wash with ultrapure water; store the washed Bio-beads in PBS solution to prevent drying.

[0008] The second aspect of the present invention provides a double-layer membrane lipid droplet simulating breast milk, wherein the double-layer membrane lipid droplet is constructed by the method of claim 1 or 2.

[0009] The third aspect of the present invention provides an application of the double-layer membrane lipid droplets simulating breast milk, wherein the double-layer membrane lipid droplets simulating breast milk are added to dairy products to improve the long-term stability of the emulsion and release more unsaturated fatty acids.

[0010] Furthermore, the dairy product includes infant formula milk powder.

[0011] Compared with the prior art, the present invention has the following beneficial effects: Based on the existing monolayer membrane lipid droplets, the present invention uses milk phospholipids and / or membrane protein concentrates to prepare phospholipid bilayer membrane lipid droplets and phospholipid protein bilayer membrane lipid droplets with similar specific surface areas, and characterizes and compares them with monolayer membrane lipid droplets with the same interface components. The main conclusions are as follows: (1) The size of the bilayer lipid droplets of different interface components can be regulated by controlling the ultrasonic power and duration during the preparation of liposomes. The experimental results show that the lipid droplet size is monodisperse, and the size of the bilayer lipid droplets is smaller than that of the monolayer lipid droplets.

[0012] (2) By comparing the lipid droplet interface structure, component distribution and content, the number of membrane layers at the lipid droplet interface was confirmed, and the membrane thickness of the double-layer membrane lipid droplets was significantly higher than that of the single-layer membrane lipid droplets. It was also confirmed that there were differences in the components of the lipid droplet interface, that is, the interface of the phospholipid membrane lipid droplets did not contain protein, while the interface of the phospholipid protein membrane lipid droplets contained both milk phospholipids and proteins. There was no difference in the interface protein composition of the phospholipid protein membrane lipid droplets, but the proportion of membrane proteins and caseins in the interface of the phospholipid protein double-layer membrane lipid droplets was higher than that of the phospholipid protein single-layer membrane lipid droplets, while the whey protein content decreased.

[0013] (3) Through the analysis of stability and emulsification, the long-term stability of phospholipid bilayer membrane lipid droplets is better than that of phospholipid protein bilayer membrane lipid droplets, while the short-term stability and emulsification of phospholipid protein bilayer membrane lipid droplets are better than those of phospholipid bilayer membrane lipid droplets. Since the presence of cholesterol in the interface of bilayer membrane lipid droplets increases steric hindrance, and the bilayer membrane lipid droplets have a larger specific surface area and contain more emulsifiers at the interface, the long-term stability and emulsification of bilayer membrane lipid droplets are better than those of monolayer membrane lipid droplets with the same components.

[0014] (4) The interaction between milk lecithin and membrane proteins can cause fat droplets to release less saturated fatty acids and more unsaturated fatty acids during gastric digestion. The phospholipid monolayer and bilayer membrane lipid droplets increase the release of polyunsaturated fatty acids during the digestion stage. Due to the increase in the content of interfacial emulsifiers, the bilayer membrane structure increases the release of saturated fatty acids from lipid droplets during intestinal digestion. The interaction between milk lecithin and proteins can protect lipid droplets during digestion, reduce the occurrence of aggregation, and maintain them in a relatively stable state. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] The present invention will be further described below in conjunction with the accompanying drawings and embodiments: Figure 1 The particle size distribution of lipid droplets with different interface structures in Test Example 1 is shown; Figure 2 The structural morphology of lipid droplets with different interface structures in Test Example 2 is shown; A is a phospholipid monolayer lipid droplet, B is a phospholipid protein monolayer lipid droplet, C is a phospholipid bilayer lipid droplet, and D is a phospholipid protein bilayer lipid droplet; the white solid arrows point to the bilayer lipid droplets, and the white dotted arrows point to the monolayer lipid droplets in the bilayer lipid droplet emulsion; Figure 3 The interface distribution of lipid droplets with different interface structures in Test Example 3 is shown; Figure 4The coverage of the interface components of lipid droplets with different structures in Test Example 4 is shown; Mean ± SD, ac / AD, indicates significant difference, n=3, P < 0.05; Figure 5 The SDS gel electrophoresis diagram of the lipid droplet interface protein of the bilayer membrane of different interface components in Test Example 5 is shown; wherein, lane M is a marker; lane 1 is a phospholipid monolayer membrane; lane 2 is a phospholipid protein monolayer membrane; lane 3 is a phospholipid bilayer membrane; lane 4 is a phospholipid protein bilayer membrane; Figure 6 The Zeta potential and stability constant of lipid droplets with different interface structures in Test Example 6 are shown ( K E ); Mean ± SD, ac / AD, indicates significant difference, n = 3, P < 0.05; Figure 7 The lipid droplet emulsification activity index and emulsion stability index of different interface structures in Test Example 7 are shown; wherein, Mean±SD, ac / AD, indicates significant difference, n=3, P < 0.05; Figure 8A-8C The release patterns of fatty acids in the phospholipid monolayer membrane lipid droplets, protein monolayer membrane lipid droplets, and phospholipid protein monolayer membrane lipid droplets of Test Example 8 during in vitro simulated digestion are shown respectively; Figure 9A-9B The release patterns of fatty acids from the phospholipid bilayer membrane lipid droplets and phospholipid protein bilayer membrane lipid droplets of Test Example 8 during in vitro simulated digestion are shown respectively. DETAILED DESCRIPTION Example 1

[0016] This embodiment provides a phospholipid bilayer membrane lipid droplet, the preparation method is: 1. Separate milk lecithin (1) Total lipid extraction Total lipids were extracted from MFGM-10 powder: MFGM-10 and chloroform / methanol (2:1, v / v) were thoroughly mixed at a ratio of 1:20 (w / v). The extraction was repeated three times through filter paper, and the extracts were mixed and concentrated using a rotary evaporator under vacuum at 35°C. The residual solvent was removed by nitrogen blowing at room temperature (25°C) to obtain a total lipid sample.

[0017] (2) Solid phase extraction The total lipid sample (400 mg) was dissolved in 1 mL of chloroform-methanol (2:1, v / v). 1 mL of the lipid solution was added to different LC-Si solid phase extraction tubes for solid phase extraction. After the total lipid sample was completely absorbed by the adsorbent, non-polar lipids were eluted with 6 mL of n-hexane / ethyl ether (8:2, v / v) and 6 mL of n-hexane / ethyl ether (1:1, v / v). 4 mL of methanol and 4 mL of chloroform / methanol / water (3:5:2, v / v / v) were used to recover milk phospholipids. The recovered fractions were dried under nitrogen and stored at 4 °C.

[0018] 2. Prepare butter Fresh raw milk was pasteurized at 63°C for 30 min and then rapidly cooled to 10°C in an ice water bath. Pasteurized milk was centrifuged at 4000 g, 10°C for 20 min and the upper layer of cream was collected. Cream with a fat content of 30-40% was stirred at high speed at 10°C using a multifunctional chef machine until butter and buttermilk were separated and the butter solid particles were collected. Butter was melted at 65°C and centrifuged at 4000 g for 5 min to remove butter whey and obtain butter, which was stored at -20°C.

[0019] 3. Preparation of phospholipid bilayer membrane droplets Add 1% (w / v) milk lecithin and 1 / 6 of the mass of cholesterol to anhydrous ethanol and use a magnetic stirrer to fully dissolve them. Then transfer the solution to a round-bottom flask and perform vacuum rotary evaporation at 50°C to completely evaporate the ethanol and leave the phospholipid cholesterol on the flask wall in the form of a thin film. Add 1.14% butter to the simulated milk ultrafiltrate and melt it completely. Transfer the simulated milk ultrafiltrate containing butter to a round-bottom flask to rehydrate the liposomes. Ultrasonication at 150 w for 5 min will yield phospholipid bilayer membrane lipid droplets. Example 2

[0020] This embodiment provides a phospholipid protein bilayer membrane lipid droplet, the preparation method is: 1. Isolation of membrane protein concentrate MFGM-10 powder has removed most of the casein and contains a higher proportion of membrane proteins, but the whey proteins are difficult to remove. Dissolve MFGM-10 powder in ultrapure water at a ratio of 1:10. Centrifuge the sample at 1500 g for 10 min to obtain the surface cream. Wash the cream with PBS (0.1 M, pH 6.8), centrifuge at 1500 g for 10 min, and repeat 3 times. Mix the washed cream with 0.4% SDS (1:1, v / v), sonicate for 1 min, and centrifuge at 1500 g for 10 min to obtain the membrane protein concentrate.

[0021] 2. Prepare the cream The raw milk was centrifuged at 350×g for 5 min at room temperature. The top layer of milk rich in large fat globules was collected. The milk rich in large fat globules was preheated to 40°C and centrifuged at 4000 rpm for 5 min to obtain cream.

[0022] 3. Preparation of phospholipid protein bilayer membrane lipid droplets Add 5 g of Bio-beads (Bio-Rad, USA) to 37.5 mL of methanol and shake on a shaker at 30 rpm / min at room temperature for 20 min. After removing the methanol, add 100 mL of methanol, then remove the methanol and wash with 625 mL of ultrapure water. Store the washed Bio-beads in PBS solution to prevent drying.

[0023] 1% (w / v) membrane protein concentrate was dissolved in simulated milk ultrafiltrate. 1% (w / v) milk lecithin and 1 / 6 of the milk lecithin mass of cholesterol were added to anhydrous ethanol and fully dissolved using a magnetic stirrer. The solution was then transferred to a round-bottom flask and vacuum-rotated at 50°C to completely evaporate the ethanol and leave the phospholipid cholesterol on the flask wall in the form of a thin film. 2% cream was added to the simulated milk ultrafiltrate and completely melted. The simulated milk ultrafiltrate containing cream was transferred to a round-bottom flask to rehydrate the liposomes and ultrasonically disrupted at 100 w for 3 min. 10% TritonX-10 was added at a ratio of 2:1 to lipid, and the same volume of membrane protein concentrate solution as the liposomes was added, and mixed on a shaker at 300 rpm at 24°C for 30 min. Add Bio-beads twice the weight of Triton X-100, mix on a shaker at 300 rpm for 30 min at 24°C, repeat 3 times, and add Bio-beads twice the weight for the last time. After removing the Bio-beads, collect and obtain the phospholipid protein bilayer membrane lipid droplets.

[0024] Comparative example: Monolayer lipid droplets with different interface components In this comparative example, milk lecithin and membrane protein concentrate were used as raw materials to prepare monolayer membrane lipid droplets, and the specific method was as follows: Milk lecithin and membrane protein concentrate were dissolved in ultrapure water at a concentration of 1% (w / v) and stirred overnight at 500 rpm at 25°C with a magnetic stirrer to fully hydrate. Phospholipid monolayer droplets were obtained by adding 1.14% (w / v) butter to the milk lecithin solution and shearing at 30,000 rpm for 15 min at 25°C using a handheld shearing instrument, followed by secondary shearing at 16,800 rpm for 3 min.

[0025] Using the same method, by adding cream with the same amount of butter fat content (i.e. 2% (w / v)), shearing was performed at 30,000 rpm for 12 min using a handheld shearing instrument and secondary shearing at 16,800 rpm for 3 min to obtain protein monolayer lipid droplets.

[0026] Then, 2% (w / v) cream was added to the membrane protein concentrate solution, pre-sheared at 30,000 rpm for 1 min, and then an equal volume of milk lecithin solution was added, sheared at 30,000 rpm for 12 min, and secondary sheared at 16,800 rpm for 3 min to obtain phospholipid protein monolayer membrane lipid droplets.

[0027] The preparation methods of the raw materials such as milk lecithin, membrane protein concentrate, cream, butter, etc. used in the comparative example are the same as those in Examples 1 and 2.

[0028] Test Example 1: Comparison of lipid droplet size and specific surface area with different interface structures This test example measures the particle size and specific surface area of ​​different emulsions in Examples 1, 2 and Comparative Examples. The particle size distribution and specific surface area of ​​lipid droplets with different interface structures and different components are as follows: Figure 1 As shown in Table 1. The particle size of fat globules in breast milk ranges from 0.1 µm to 15 µm, while the particle size of fat droplets in infant formula is about 1 / 10 of that of milk fat globules, with an average particle size of about 0.4 µm. 4,3 The D values ​​of lipid droplets in phospholipid monolayer membrane and phospholipid protein monolayer membrane were 0.61 ± 0.01 µm and 0.57 ± 0.02 µm, respectively. 4,3 4.90 ± 0.01 µm and 4.87 ± 0.06 µm, respectively, which are significantly larger than the particle size of the double membrane lipid droplet ( P < 0.05). Figure 1 It can be concluded that the particle size distribution of bilayer lipid droplets is monodisperse, while the particle size distribution of monolayer lipid droplets is polydisperse, with the two main peaks at approximately 0.5 µm and 5 µm. This is mainly because the particle size distribution in bilayer lipid droplets is relatively concentrated, while the monolayer lipid droplets contain large and small lipid droplets, resulting in polydispersity.

[0029] D 4,3 is the volume average diameter, which is sensitive to changes in the number of large particles in the emulsion. 3,2 is the average diameter of the surface area, which is sensitive to the change in the number of small particles in the emulsion. The protein bound to phospholipids and their interaction force will reduce the particle size of the emulsion, so the particle size of the lipid droplets of the phospholipid protein bilayer membrane is slightly smaller than that of the lipid droplets of the phospholipid bilayer membrane. 3,2In the process of preparing double-layer lipid droplets, the power and time of ultrasonic treatment of liposomes can be adjusted to regulate the D 3,2 The particle size makes the specific surface area of ​​the bilayer membrane lipid droplets close to that of the phospholipid bilayer membrane lipid droplets and the phospholipid protein bilayer membrane lipid droplets, which are 17.08 ± 0.91 m 2 / g and 17.35 ± 0.25 m 2 / g. Due to the D 3,2 Significantly larger than the bilayer membrane, that is, the specific surface area is significantly smaller than the bilayer membrane lipid droplets ( P < 0.05), the specific surface areas of lipid droplets in phospholipid monolayer membrane and phospholipid protein monolayer membrane were 11.43 ± 0.51 m 2 / g and 13.55 ± 0.18 m 2 / g.

[0030] Test Example 2: Comparison of lipid droplet interface structures with different interface structures In this test example, Cryo-TEM was used to observe the interface structure of lipid droplets.

[0031] Figure 2 The structural morphology of lipid droplets with different interface structures in Examples 1 and 2 and the comparative example is shown. Figure 2 It can be seen that both single-layer lipid droplets and double-layer lipid droplets are spherical. In A and B, it can be seen that the lipid droplet interface is a single-layer membrane, and in C and D, it can be seen that the lipid droplet interface is a double-layer membrane, and there is an obvious difference in thickness between the single-layer and double-layer membranes.

[0032] Figure 2 The phospholipid bilayer membrane in C is formed by phospholipid bilayers, its morphology and membrane thickness are relatively uniform, and the bilayers are tightly connected; Figure 2 The solid white arrow in D points to the lipid droplet of the phospholipid bilayer membrane. It may be because the protein is inserted into the phospholipid bilayer membrane and the distribution of the protein is not uniform, resulting in inconsistent thickness of the interface membrane of the same ball. From the white dotted arrow in the figure, it can be seen that there are still lipid droplets with a single membrane structure in the bilayer membrane lipid droplet emulsion, so not all lipid droplets can form a bilayer membrane structure.

[0033] Table 2 shows the ash analysis of the membrane thickness / density of lipid droplets with different interface structures. The ash value represents the number of particles per unit area in the image, so it can be seen whether there are differences in membrane thickness / density between different samples. As mentioned above, the membrane thickness / density of double-layer membrane lipid droplets is significantly higher than that of single-layer membrane lipid droplets ( P< 0.05), because the increase in the number of membrane layers and the larger specific surface area of ​​the bilayer membrane than the monolayer membrane will result in a higher adsorption of emulsifier. Due to the special structure of the protein, the membrane thickness / density of the lipid droplets of the phospholipid protein monolayer membrane is significantly higher than that of the phospholipid monolayer membrane, but there is no significant difference in the membrane thickness / density between the lipid droplets of the phospholipid bilayer membrane and the lipid droplets of the phospholipid protein bilayer membrane ( P > 0.05), which may be due to the uneven distribution of proteins in the bilayer membrane, resulting in uneven membrane thickness / density of lipid droplets in the phospholipid bilayer membrane, so that the average membrane thickness / density is similar to that of the phospholipid bilayer membrane.

[0034] Test Example 3: Comparison of distribution of lipid droplet interface components with different interface structures In order to verify whether milk phospholipids or membrane proteins exist on the interface of the prepared emulsion lipid droplets, this test example uses a laser scanning confocal microscope for verification.

[0035] Figure 3 The interface distribution of lipid droplets with different interface structures in Examples 1 and 2 and the comparative example, wherein WGA-488 dye stains glycoprotein to show green fluorescence, Rh-DOPE dye stains milk phospholipids to show red fluorescence, and orange fluorescence is the superposition of green fluorescence and red fluorescence.

[0036] Depend on Figure 3 As shown in Figures B and J, there is no green fluorescence from WGA-488 in the phospholipid monolayer and phospholipid bilayer, but only red fluorescence from Rh-DOPE, which means that the phospholipid monolayer and phospholipid bilayer do not contain glycoproteins but only milk phospholipids. Figure 3 In EH and MP, the green fluorescence emitted by WGA-488, the red fluorescence emitted by Rh-DOPE, and the orange fluorescence of the combination of the two can be clearly seen, indicating that glycoproteins and milk phospholipids exist simultaneously on the phospholipid monolayer and the phospholipid bilayer.

[0037] Test Example 4: Comparison of the coverage of lipid droplet interface components with different interface structures In this test example, the interfacial protein coverage (or interfacial milk phospholipid coverage) is calculated by dividing the amount of interfacial protein (or the amount of phospholipid) per gram of separated lipid by the specific surface area of ​​the fat globule.

[0038] Figure 4The coverage of interfacial phospholipids and proteins in lipid droplets with different interfacial structures in Examples 1 and 2 and Comparative Examples is shown. Consistent with the results of laser scanning confocal microscopy in Test Example 3, no protein content was detected at the interface of lipid droplets of phospholipid monolayer membrane and lipid droplets of phospholipid bilayer membrane, while there were both milk phospholipids and proteins at the interface of lipid droplets of phospholipid protein monolayer membrane and phospholipid protein bilayer membrane. Since part of the phospholipids in the phospholipid protein membrane is replaced by proteins, the coverage of interfacial phospholipids in the phospholipid protein membrane is significantly reduced compared with the phospholipid membrane ( P < 0.05).

[0039] Compared with single and bilayer lipid droplets of the same composition, the interfacial phospholipid coverage and interfacial protein coverage in bilayer lipid droplets are significantly increased ( P < 0.05), which is because the bilayer membrane is composed of two molecular layers, in which the molecules are tightly arranged through hydrophobic interactions and hydrogen bonds, thereby increasing the coverage of phospholipids or proteins on the interface.

[0040] Test Example 5: Comparison of composition and content of lipid droplet interface proteins with different interface structures In this test example, SDS-PAGE polyacrylamide gel electrophoresis was used to separate and identify the interface proteins, and the gel electrophoresis image of the protein was obtained by gel imaging, and then the main protein types of the interface proteins were analyzed.

[0041] Figure 5 The protein composition of the lipid droplet interface of the bilayer membrane with different interface components in Examples 1 and 2 and the comparative example is shown. As with the monolayer membrane, no protein bands were detected at the lipid droplet interface of the phospholipid bilayer membrane, which is consistent with the results of the interface component coverage. In the phospholipid protein bilayer membrane, in addition to casein and whey protein, multiple membrane proteins such as MUC1, XO, PAS III, CD36, BTN, ADPH and PAS 6 / 7 were also detected.

[0042] As can be seen from Table 3, in the lipid droplet interface of the phospholipid bilayer membrane, the proportion of membrane proteins is the largest, reaching 47.68 ± 2.06%, while the proportion of membrane proteins in the lipid droplet interface of the phospholipid monolayer membrane is 45.86 ± 0.40%. The content of membrane proteins in the bilayer membrane is increased compared with the monolayer membrane. Among them, the proportion of MUC1, CD 36, and ADPH in the total membrane protein on the lipid droplet interface of the phospholipid bilayer membrane is increased compared with the phospholipid monolayer membrane. Compared with the phospholipid monolayer membrane, in addition to the increase in membrane protein content, the content of casein in the phospholipid bilayer membrane also increased from 13.77 ± 0.78% to 22.29 ± 1.37%, but the content of whey protein decreased from 40.37 ± 3.28% to 30.03 ± 0.69%.

[0043] Test Example 6: Comparison of Zeta Potential and Stability Constants of Lipid Droplets with Different Interface Structures Zeta potential describes the potential difference between the inner and outer interfaces of the diffusion layer in the double electric layer. Its absolute value can reflect whether the system tends to be stable or flocculated, and is a key indicator for evaluating the stability of the system. In this test example, the Zeta potential of the emulsion was measured using the Malvern ZetasizerNano ZS nanoparticle size analyzer.

[0044] The physical stability of simulated MFG emulsion is an important indicator to examine the stability of the constituent fat globules. The stability constant ( K E ) is calculated using the following formula:

[0045] The sample (2 mL) was centrifuged at 3000 × g for 15 min. After discarding the upper layer, 0.5 mL of the remaining liquid was diluted to 10 mL with distilled water. The absorbance (A) was recorded at 450 nm using a UV-visible near-infrared spectrophotometer. Then, another 0.5 mL of the uncentrifuged sample was diluted to 10 mL with distilled water, and the absorbance value A0 was recorded at 450 nm. K E The smaller the value, the higher the stability. Since centrifugal treatment accelerates the reaction process, that is, K E The value reflects the long-term stability of the emulsion.

[0046] The Zeta potential and stability constant of lipid droplets with different interface structures in Examples 1 and 2 and Comparative Examples are as follows: Figure 6 The absolute value of the Zeta potential of the lipid droplets in the phospholipid bilayer membrane is significantly higher than that of the lipid droplets in the phospholipid protein bilayer membrane ( P < 0.05), and the K E The value is also significantly lower than that of phospholipid bilayer membrane lipid droplets ( P < 0.05), indicating that the long-term stability of phospholipid bilayer membrane lipid droplets is better than that of phospholipid protein bilayer membrane lipid droplets. Compared with lipid droplets of the same composition but different structures, the absolute value of the potential of bilayer membrane lipid droplets is significantly lower than that of monolayer membrane lipid droplets ( P < 0.05).

[0047] The stability constants of bilayer lipid droplets are significantly lower than those of monolayer lipid droplets of the same composition ( P< 0.05), that is, the long-term stability of the bilayer membrane is better than that of the monolayer membrane. This may be because cholesterol is added during the preparation of the bilayer membrane, and cholesterol has the function of stabilizing the bilayer membrane structure and produces a steric hindrance effect, which increases the stability of the bilayer membrane. In addition, since the bilayer membrane lipid droplets have a larger specific surface area, there are more emulsifiers on the bilayer membrane interface. The increase in emulsifiers also increases the electrostatic repulsion between lipid droplets, making the long-term stability of the bilayer membrane lipid droplets better than that of the monolayer membrane lipid droplets.

[0048] Test Example 7: Comparison of emulsification properties of lipid droplets with different interface structures This test example measures the emulsification activity index (EAI) and emulsification stability index (ESI) of lipid droplets with different interface structures in Examples 1 and 2 and the comparative example. The specific method is: After the sample was formed, 50 μL of the sample was immediately added to a centrifuge tube containing 5 mL of sodium dodecyl sulfate (SDS) solution (0.1 g / L), and the absorbance of the mixture (A0) was recorded at 500 nm using a UV-visible near-infrared spectrophotometer at 25°C. The sample was allowed to stand for 30 min, and 50 μL of the sample was taken to measure the absorbance (A 30 ).

[0049] Emulsifying activity index (EAI, m 2 The calculation formulas of emulsion stability index (ESI, %) are as follows: Where: N——dilution factor, which is 250; ρ——emulsion concentration, g / mL; φ——Volume fraction of oil phase in emulsion.

[0050] Figure 7 It is the emulsification activity index of lipid droplets and the emulsion stability index of different interface structures. Figure 7 It can be seen that the EAI value of the lipid droplets of the phospholipid bilayer membrane is significantly higher than that of the phospholipid bilayer membrane ( P < 0.05); while the EAI value of phospholipid monolayer lipid droplets was also significantly higher than that of phospholipid monolayer lipid droplets ( P < 0.05). That is, the interaction between the appropriate amount of phospholipids and proteins can effectively reduce the interfacial tension between the oil phase and the water phase, thereby improving the stability of the lipid droplets of the phospholipid protein monolayer / bilayer membrane; and the lipid droplets of the phospholipid protein bilayer membrane have the strongest ability to stabilize the water-oil interface area per unit mass. Whether it is a monolayer or a bilayer membrane, the ESI value of the phospholipid protein membrane lipid droplets is significantly higher than that of the phospholipid membrane lipid droplets ( P < 0.05), that is, compared with phospholipid membrane lipid droplets, phospholipid protein membrane lipid droplets have better short-term stability. The ESI value of bilayer membrane lipid droplets is significantly lower than that of monolayer membrane lipid droplets ( P< 0.05), that is, the short-term stability of monolayer lipid droplets is better than that of bilayer lipid droplets. This may be because the bilayer lipid droplets have a larger specific surface area and a larger interfacial tension than the monolayer lipid droplets, resulting in poor stability in a short period of time.

[0051] Test Example 8: Effects of different interface components and structures on fatty acid release patterns This test example used pH-stat to simulate the gastrointestinal digestive environment of infants and young children in vitro to study the effects of different interface components and structures on the digestive process in five groups of lipid droplets: comparison example phospholipid monolayer membrane lipid droplets / protein monolayer membrane lipid droplets / phospholipid protein monolayer membrane lipid droplets, example 1 phospholipid bilayer membrane lipid droplets, and example 2 phospholipid protein bilayer membrane lipid droplets.

[0052] The fatty acid patterns of lipid droplets with different interface components and structures during in vitro simulated digestion of the infant gastrointestinal tract are shown in Figures 8 and 9. From the results, it can be seen that, in terms of monolayer membranes, the main fatty acids released by lipid droplets during digestion are caprylic acid (C8:0), capric acid (C10:0), lauric acid (C12:0), tridecanoic acid (C13:0), myristic acid (C14:1 n5c), myristic acid (C14:0), pentadecanoic acid (C15:0), palmitoleic acid (C16:1), palmitic acid (C16:0), heptadecanoic acid (C17:0), linoleic acid (C18:2 n6c), oleic acid (C18:1 n9c), stearic acid (C18:0), arachidic acid (C20:0), etc., most of which are long-chain fatty acids containing more than 12 carbons. Due to different interface components, the release patterns of fatty acids are different.

[0053] Phospholipid monolayer droplets released a total of 23 fatty acids during digestion, including 15 saturated fatty acids and 8 unsaturated fatty acids. After C16:0 and C18:0 were released in the stomach stage, their release rate gradually slowed down. Three fatty acids, 13-tridecanoic acid (13Methyl C14:0), 14-methylpentadecanoic acid (14Methyl C15:0) and eicosantrienoic acid (C20:3, n6c), were concentrated in the intestine stage. The release priority of C12:0 and C18:2, n6c was always low.

[0054] A total of 28 fatty acids were released from protein monolayer lipid droplets during digestion, including 17 saturated fatty acids and 11 unsaturated fatty acids. At 60 min of gastric digestion, 15 fatty acids, including C14:1 n5c, C14:0, 13-methyltetradecanoic acid (13Methyl C14:0), 9-methyltetradecanoic acid (9 Methyl C14:0), C15:0, C16:1 n9c, C16:1 n7c, 15-methylhexadecanoic acid (15Methyl C16:0), 14-methylhexadecanoic acid (14 Methyl C16:0), heptadecaenoic acid (C17:1 n7c), C18:2n6c, C18:1 n9c, arachidonic acid (C20:4 n6c), C20:0, and erucic acid (C22:1 n9c), were released preferentially. Except for C18:1 n9c, the release rates of the remaining fatty acids were relatively stable during the gastric digestion stage. When switching to the intestinal digestive environment, the release rates of C16:0 and C18:0 were accelerated, while the release rate of C12:0 was accelerated in the later stage of intestinal digestion.

[0055] Phospholipid monolayer lipid droplets released a total of 26 fatty acids during digestion, including 17 saturated fatty acids and 9 unsaturated fatty acids. At 60 min of gastric digestion, 8 fatty acids, including C12:0, 12-methyltridecanoic acid (12 MethylC13:0), 13MethylC14:0, 9 Methyl C14:0, 14 Methyl C15:0, 15 Methyl C16:0, C18:2 n6c, and C18:1n9c, were released preferentially. Except for C12:0 and C18:1 n9c, the other 6 fatty acids were released at a stable rate during gastric digestion. When switching to the intestinal digestive environment, the release rates of C16:0 and C18:0 accelerated, while the release rates of 13Methyl C14:0 and C18:2 n6c accelerated in the late intestinal digestion.

[0056] As for the double membrane lipid droplets, as shown in Figure 9, the main fatty acids released from the lipid droplets during digestion are caproic acid (C6:0), caprylic acid (C8:0), capric acid (C10:0), lauric acid (C12:0), tridecanoic acid (C13:0), myristic acid (C14:1n5c), myristic acid (C14:0), pentadecanoic acid (C15:0), palmitoleic acid (C16:1), palmitic acid (C16:0), heptadecanoic acid (C17:0), γ-linolenic acid (C18:3 n6c), linoleic acid (C18:2 n6c), oleic acid (C18:1 n9c), stearic acid (C18:0), arachidic acid (C20:0), etc. Due to the different interface components, the release patterns of fatty acids are different.

[0057] Phospholipid bilayer membrane lipid droplets release a total of 30 fatty acids during digestion, including 17 saturated fatty acids and 13 unsaturated fatty acids. 19 fatty acids, including C6:0, C13:0, 12Methyl C13:0, C14:1 n5c, 13Methyl C14:0, 12Methyl C14:0, 14Methyl C15:0, C16:1 n7c, C16:0, C18:3 n6c, C18:2 n6c, C18:1 n9c, elaidic acid (C18:1n9t), C18:0, nonadecenoic acid (C19:1 n9c), C20:4 n6c, eicosanoid trienoic acid (C20:3 n6c), C20:0, and C22:1 n9c, are preferentially released during gastric digestion. Among them, the release rates of 10 fatty acids, including 14Methyl C15:0, C16:1 n7c, C16:0, C18:2 n6c, C18:1 n9c, C18:1 n9t, C18:0, C20:4 n6c, C20:0, and C22:1 n9c, slowed down in the stomach. In the intestinal digestive environment, the release rates of 13Methyl C14:0, C16:0, C17:0, and C18:0 accelerated.

[0058] Phospholipid bilayer membrane lipid droplets release a total of 24 fatty acids during digestion, including 16 saturated fatty acids and 8 unsaturated fatty acids. Four fatty acids, including C15:0, C16:0, C17:0, and C18:0, are preferentially released in the early stage of gastric digestion, and their release rate slows down during gastric digestion. Seven fatty acids, including 12Methyl C13:0, 12Methyl C14:0, C17:1 n7c, C18:2 n6c, C18:1n9c, C20:3 n6c, and C20:0, are released in the late stage of gastric digestion. In the intestinal digestion stage, the release rate of six fatty acids, including 13Methyl C14:0, 14Methyl C15:0, C16:0, C17:0, C18:0, and C22:1 n9c, accelerates.

[0059] During gastrointestinal digestion, the most released fatty acid from monolayer lipid droplets was C16:0, followed by C18:1 and C14:0, while the most released fatty acids from bilayer lipid droplets were C16:0, C18:1 n9c and C14:0. Table 4 shows the release of saturated or unsaturated fatty acids at the end of digestion of different groups of emulsions. At the end of gastric digestion, phospholipid monolayer lipid droplets released the least saturated fatty acids (SFA) and the most monounsaturated fatty acids (MUFA), while phospholipid bilayer lipid droplets released the most polyunsaturated fatty acids (PUFA). This may be because SFA is usually located in the core of TAG, while unsaturated fatty acids (USFA) are generally present in the outer layer. The interaction between milk phospholipids and MP attracts gastric lipase to act on the outer layer of TAG. Long-chain unsaturated fatty acids are usually found in glycerophospholipids, and there is a certain degree of positive correlation between the release of total fatty acids and the types of phosphatidylcholine (PC) and sphingomyelin (SM) in milk, leading to the release of more USFA. Protein monolayer lipid droplets release the most SFA and the least USFA. At the end of intestinal digestion, phospholipid monolayer lipid droplets release the most MUFA, phospholipid protein monolayer lipid droplets release the most PUFA, and the amount of SFA released from bilayer lipid droplets is significantly more than that from monolayer lipid droplets ( P < 0.05), which may be caused by the higher emulsifier content at the interface of the double-layer structure.

[0060] Unsaturated fatty acids contain one or more double bonds, are highly soluble and easily absorbed by the body. Long-chain unsaturated fatty acids, such as C18:1, C18:2, C18:3, C20:3 and C20:5, play a vital role in the growth and development of infants. C18:1 is an important energy source for infants, while C18:2 is an essential fatty acid precursor for other long-chain fatty acids. In addition, higher levels of USFA are beneficial to the survival and development of infant retinal and brain cells. During digestion, the USFA content (such as C18:1 and C18:2) in lipid droplet emulsions containing milk lecithin was higher than that in lipid droplet emulsions without milk lecithin (protein monolayer lipid droplet emulsions), indicating that the addition of milk lecithin can promote the release of USFA. Phospholipid-protein bilayer lipid droplets can release less SFA and more PUFA during the gastric digestion stage. These results suggest that regulating the interfacial composition and structure of lipid droplets can further affect lipolysis by affecting the release pattern of fatty acids.

[0061] In summary, the interaction between phospholipids and membrane proteins can improve the emulsification properties of lipid droplet emulsions and release more unsaturated fatty acids; double-layer membrane lipid droplets are closer to the interface structure of breast milk fat globules than single-layer membrane lipid droplets, and can also improve the long-term stability of lipid droplet emulsions and the release rate of fatty acids, and release more polyunsaturated fatty acids during the digestion stage.

[0062] Finally, it should be noted that the above is only used to illustrate the technical solution of the present invention rather than to limit it. Although the present invention has been described in detail with reference to the preferred arrangement scheme, a person skilled in the art should understand that the technical solution of the present invention may be modified or replaced by equivalents without departing from the spirit and scope of the technical solution of the present invention.

Claims

1. A method for constructing double-layer membrane lipid droplets simulating breast milk, characterized in that: The bilayer membrane lipid droplets are phospholipid bilayer membrane lipid droplets and / or phospholipid protein bilayer membrane lipid droplets; The method for constructing the phospholipid bilayer membrane lipid droplets comprises the following steps: Add milk lecithin and cholesterol to anhydrous ethanol and fully dissolve them using a magnetic stirrer; then transfer the solution to a round-bottom flask, perform vacuum rotary evaporation to completely evaporate the ethanol, and leave the lecithin and cholesterol on the flask wall in the form of a thin film; add butter to the simulated milk ultrafiltrate to completely melt it; transfer the simulated milk ultrafiltrate containing butter or cream to a round-bottom flask to rehydrate the liposomes; and then perform ultrasonic crushing to obtain phospholipid bilayer membrane lipid droplets; The phospholipid bilayer membrane lipid droplets are further constructed to obtain phospholipid protein bilayer membrane lipid droplets, comprising the following steps: Firstly, the phospholipid membrane protein concentrate is dissolved in simulated milk ultrafiltrate to obtain a membrane protein concentrate solution; Then, Triton X-10 and membrane protein concentrate solution are added to the lipid droplets of the phospholipid bilayer membrane, and the mixture is mixed evenly on a shaker; Bio-beads are added in small amounts for several times, and the mixture is mixed evenly on a shaker; after removing the Bio-beads, the phospholipid protein bilayer membrane is collected.

2. The construction method according to claim 1, characterized in that: The processing method of Bio-beads is: Add Bio-beads to methanol, shake on a shaker at room temperature, and then remove the methanol; repeat the addition of methanol and then remove the methanol, and finally wash with ultrapure water; store the washed Bio-beads in PBS solution to prevent drying.

3. A double-layer membrane lipid droplet simulating breast milk, characterized in that: The double-layer membrane lipid droplets are constructed by the method described in claim 1 or 2.

4. An application of the double-layer membrane lipid droplets simulating breast milk as claimed in claim 3, characterized in that: The double-layer membrane lipid droplets simulating breast milk are added to dairy products to improve the long-term stability of the emulsion and release more unsaturated fatty acids.

5. The use of the double-layer membrane lipid droplets simulating breast milk according to claim 4, characterized in that: The dairy product includes infant formula.

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