A method for preparing a human milk-like three-layer membrane fat globule emulsion

Abstract

The application discloses a preparation method of breast milk three-layer membrane fat ball emulsion and belongs to the technical field of infant formula food preparation. The preparation method of the breast milk three-layer membrane fat ball emulsion disclosed by the application takes breast milk as a reference object, adds fat ball membranes and triglycerides into skimmed milk, and then prepares single-layer membrane fat ball emulsion through magnetic stirring, hydration, ultrasonic, shearing and homogenization; the single-layer membrane fat ball emulsion is dropped on the top of an oil / water column (the upper layer is a soybean oil lipid molecular layer, and the lower layer is sucrose), and then is centrifuged after being static; the upper oil phase is removed, and the lower emulsion is subjected to ultrasonic treatment to obtain three-layer membrane fat ball emulsion. The application can provide technical reference for the production process of the breast milk three-layer membrane fat ball added in infant formula milk powder. Meanwhile, the emulsion simulating the structure of the breast milk three-layer membrane fat ball can improve the insufficient lipid utilization of infants fed with infant formula milk powder and is easier for lipid digestion and absorption of infants.

Description

Technical Field

[0001] This invention relates to the field of infant formula preparation technology, and more specifically to a method for preparing a humanized three-layer membrane fat globule emulsion. Background Technology

[0002] Breast milk has long been recognized as the most suitable source of nutrition for newborns. Its unique composition, including proteins, lipids, carbohydrates, and bioactive substances, provides all the nutrients needed for infant growth and development. Among these components, the fat globule membrane is a crucial element of breast milk. Enclosing the triglyceride core, it is a three-layered membrane structure primarily composed of phospholipids and proteins. It exhibits both hydrophilic and lipophilic properties, effectively preventing the aggregation of fat globules. Natural milk fat globules range in diameter from 0.2 to 15 μm, with an average diameter of approximately 4 μm. As the outer layer of the milk fat globules, the fat globule membrane ensures their stability and structural support, playing a vital role in infant intellectual development, immune function, and gut health.

[0003] While infant formula can meet the nutritional needs of infants to some extent, its composition differs significantly from breast milk, especially in the structure and composition of the fat globule membrane. Traditional formula often has a simple lipid structure, lacking the three-layer membrane structure and various bioactive components of the fat globule membrane, resulting in infants' lower digestibility and absorption compared to breast milk. Currently, the field of infant formula technology is dedicated to finding methods that can mimic the functional components and structure of breast milk and improve infants' absorption and utilization of nutrients. The three-layer membrane structure of the fat globules in breast milk is crucial for infant nutrient absorption and growth, but current infant formula often fails to fully mimic this structure, leading to insufficient utilization of lipids and other nutrients, thus affecting healthy growth. By adding fat globule membranes or mimicking their composition and structure to formula, the structure and nutritional composition of the formula can be improved, increasing its similarity to breast milk and thus better meeting the nutritional needs of infants.

[0004] Therefore, providing a method for preparing a humanized three-layer membrane fat globule emulsion is a problem that urgently needs to be solved by those skilled in the art. Summary of the Invention

[0005] In view of this, the present invention provides a method for preparing a breast milk-like three-layered membrane fat globule emulsion, which is used to simulate the three-layered membrane fat globule structure of breast milk. The present invention employs a layer-by-layer deposition method, continuously depositing membrane layers of different compositions to simulate the particle size, potential, and structural composition of the three-layered membrane fat globules.

[0006] This invention improves upon the traditional single-layer fat globule membrane structure by proposing a novel method for preparing a three-layer fat globule emulsion that mimics breast milk. By simulating the structure of the fat globule membrane in breast milk through layer-by-layer deposition, it effectively improves the nutritional value and similarity of formula milk powder, showing promising market prospects and broad application potential. It is of great significance in improving the nutritional value of infant formula and enhancing infant health.

[0007] To achieve the above objectives, the present invention adopts the following technical solution:

[0008] A method for preparing a human milk-like three-layer membrane fat globule emulsion includes the following steps:

[0009] 1) Construction of a single-layer membrane fat globule emulsion:

[0010] Mix 0.15-0.5% fat globule membrane and 3-5% triglycerides (preferably 0.32% fat globule membrane and 4% triglycerides) of the skim milk with the skim milk. Stir the mixture in a water bath at 30-50°C at 100-500 rpm for 2-5 hours (preferably 3 hours in a water bath at 45°C at 200 rpm). Hydrate the mixture at 4°C for 6-12 hours (preferably 10 hours) to fully absorb water. Ultrasonicate the mixture for 5-15 minutes (preferably 10 minutes). The ultrasonic conditions are as follows: frequency 10-30 Hz (preferably 20 Hz), duration 5 seconds, interval 3 seconds. Shear the mixture using a shear emulsifier at 10000-15000 rpm for 3-7 minutes (preferably 5 minutes at 13000 rpm). The emulsion is preheated to 35-55°C (preferably 45°C) and then homogenized using a two-stage homogenizer at 150-300 bar for 5-15 cycles (preferably 10 cycles at 200 bar).

[0011] 2) Construction of a three-layer membrane fat globule emulsion:

[0012] To form a three-layered fat globule emulsion, an oil / water column is first prepared in a 50 mL centrifuge tube. The lower aqueous phase is sucrose with a concentration of 0.2-0.8 M, added at a rate of 2-8 mL (preferably sucrose concentration of 0.5 M, added at a rate of 5 mL). The upper oil phase is sunflower oil or soybean oil, added at a rate of 1-2 mL (preferably soybean oil, added at a rate of 1.5 mL). The column is stabilized for 15-30 min (preferably 20 min) to allow the formation of a lipid molecular layer at the water / oil interface. Then, 1.5 mL of a monolayered fat globule emulsion (oil-in-water) is dropwise spread on the top of the column and allowed to stand for 15-30 min (preferably 20 min). The column is then centrifuged at a speed of 7000-12000 g (preferably 9000 g) for 10-30 min (preferably 30 min) at a temperature of 20-30°C (preferably 25°C); a three-layered fat globule emulsion is formed in the lower layer. Remove the upper oil phase and sonicate the lower clear liquid at a frequency of 20-30 Hz (preferably 20 Hz) for 5 seconds with a 3-second interval; sonicate for 5-15 minutes (preferably 10 minutes) to obtain a three-layer membrane fat globule emulsion.

[0013] As can be seen from the above technical solution, compared with the prior art, this invention discloses a method for preparing a human milk-like three-layered fat globule emulsion. By simulating the three-layered fat globule structure of breast milk, the emulsion prepared by this invention can improve the utilization of lipids by infant formula, promote the development of the infant's brain and nervous system, enhance cognitive ability and anti-infection ability, and at the same time, make it easier for infants to digest and absorb lipids. This can provide a technical reference for the production process of human milk-like infant formula. Attached Figure Description

[0014] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on the provided drawings without creative effort.

[0015] Figure 1 The attached figure is a schematic diagram and characterization method of forming a three-layer membrane fat globule emulsion from a single-layer membrane fat globule emulsion by density-driven phase transfer according to the present invention; the inner membrane (red) is a single-layer membrane fat globule emulsion stained with a 16:0 Liss Rhod PE fluorescent probe, and the outer membrane (green) is a three-layer membrane fat globule emulsion stained with an 18:1 PE CF fluorescent probe.

[0016] Figure 2 The attached figure shows the effects of different homogenization conditions on the emulsion particle size (a) and potential (b) of the present invention;

[0017] Figure 3 The attached figure shows the effect of different homogenization conditions on the microstructure of the emulsion according to the present invention; red indicates Nile red staining, and green indicates PE-CF staining; the scale bar is 10 μm, and the magnification is 40×.

[0018] Figure 4 The attached figure shows the effect of different fat globule membrane addition ratios on emulsion particle size (a) and potential (b) of the present invention; 1×MFGM, one fat globule membrane; 2×MFGM, two fat globule membranes; 3×MFGM, three fat globule membranes;

[0019] Figure 5 The attached figure illustrates the effect of different fat globule membrane addition ratios on the microstructure of the emulsion according to the present invention. Red indicates Nile red staining, and green indicates PE-CF staining; the scale bar is 10 μm, and the magnification is 40×; 1×MFGM represents one layer of fat globule membrane; 2×MFGM represents two layers of fat globule membrane; 3×MFGM represents three layers of fat globule membrane.

[0020] Figure 6 The attached figure shows the microstructure of a three-layered fat globule emulsion prepared by layer-by-layer deposition of different oil phases (sunflower oil and soybean oil) according to the present invention; the inner membrane (red) is a single-layered fat globule emulsion stained with a 16:0 Liss Rhod PE fluorescent probe, and the outer membrane (green) is a three-layered fat globule emulsion stained with an 18:1 PE CF fluorescent probe; the scale bar is 10 μm; the magnification is 40×. Detailed Implementation

[0021] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0022] In this invention, all instruments and other equipment whose manufacturers are not specified are conventional products that can be purchased through legitimate channels. Unless otherwise specified, all methods used are conventional methods, and all raw materials used are commercially available. Where specific techniques or conditions are not specified in the embodiments, they are performed in accordance with the techniques or conditions described in the literature in this field, or according to the product instructions.

[0023] Example 1

[0024] A method for preparing a human milk-like three-layer membrane fat globule emulsion includes the following steps:

[0025] 1) Construction of a single-layer membrane fat globule emulsion:

[0026] Purchase 10L of fresh raw milk from the local market, preheat to 45°C, and then separate twice using a cream separator to collect cream and skim milk for later use. Wash the obtained cream with 3 volumes of phosphate wash buffer (PBS, 0.1 mol / L, pH 6.8) and centrifuge at 3000g for 30 min to obtain the upper fat globules. Disrupt the fat globules using an ultrasonic cell disruptor under the following conditions: sonication time 30 min, frequency 20 Hz, sonication duration 5 s, interval 3 s. Add an equal volume of PBS to the disrupted fat globules and slowly stir in a 45°C water bath until a homogeneous melt is obtained. Collect the lower buttermilk layer by centrifuging at 1000g for 10 min at 25°C. The upper layer is triglycerides, which is reserved. Adjust the pH of the buttermilk to 4.8 with 0.1 mol / L hydrochloric acid and let stand for 30 min. Then centrifuge at 3000g for 15 min. Collect the lower layer and adjust the pH to 6.8 using 0.1 mol / L sodium hydroxide to obtain a fat globule membrane solution. The fat globule membrane solution was freeze-dried to obtain fat globule membrane lyophilized powder for later use.

[0027] 40 mL of triglycerides and 3.2 g of fat globule membrane were mixed with 1 L of skim milk. The mixture was stirred with a magnetic stirrer in a 45°C water bath at 200 rpm for 3 h, and then dissolved at 4°C for 10 h to allow for complete water absorption. The sample mixture was sonicated for approximately 10 min under the following conditions: frequency 20 Hz, duration 5 s, interval 3 s. Shear emulsifier was used to shear the mixture at 13000 rpm for 5 min. The emulsion was preheated to 45°C and then homogenized 10 times at 200 bar using a two-stage homogenizer. The resulting monolayer fat globule emulsion was stored at 4°C.

[0028] 2) Construction of a three-layer membrane fat globule emulsion:

[0029] To form a three-layered fat globule emulsion, an oil / water column was first prepared in a 50 mL centrifuge tube, consisting of sucrose (0.5 M, 5 mL) and soybean oil (1.5 mL) from bottom to top. The column was stabilized for 20 min to allow the formation of a lipid molecular layer at the water / oil interface. Then, 1.5 mL of a monolayered fat globule emulsion (oil-in-water) was dropwise added to the top of the column and allowed to stand for 20 min. The column was then centrifuged at 9000 g for 30 min at 25 °C, resulting in a three-layered fat globule emulsion in the lower layer. The upper oil phase was removed, and the lower supernatant was sonicated at 20 Hz for 5 s, with 3 s intervals, for a total sonication time of 10 min, yielding the three-layered fat globule emulsion (e.g., [image of emulsion name]). Figure 1 (As shown).

[0030] Test method:

[0031] 1. Particle size determination:

[0032] The volume-weighted particle size distribution and average diameter of fat globules in 1 mL of emulsion were measured using a laser particle size analyzer at room temperature. The refractive indices were set to 1.46 (466 nm) and 1.458 (633 nm), and the refractive index of the dispersed phase (water) was set to 1.33.

[0033] 2. Zeta potential measurement:

[0034] Dilute 15 μL of sample 400-fold with buffer (20 mM imidazole, 50 mM NaCl, 5 mM CaCl2) and measure using a potentiometer at 25 °C. Each sample was measured three times.

[0035] 3. Microstructural observation using laser confocal microscopy

[0036] The microstructure of the three-layered fat globule emulsion was imaged using a laser confocal microscope.

[0037] The effects of different homogenization conditions and the proportion of fat globule membrane added on the microstructure were observed: 200 μL of the prepared milk fat globule emulsion sample was taken. 20 μL of 0.1 mg / mL Nile Red (prepared with ethanol) was mixed with the sample at a ratio of 1:10 (v / v), and 1 mg / mL PE CF fluorescent probe was mixed with the sample at a ratio of 1:40 (v / v). The mixture was placed at room temperature in the dark for 30 min, vortexed to mix, and observed using laser confocal microscopy.

[0038] Observation of the microstructure of the three-layer membrane: 200 μL of the prepared three-layer membrane fat globule emulsion sample was taken. 5 μL of 1 mg / mL 16:0 Liss Rhod PE fluorescent probe was mixed with the monolayer membrane fat globule emulsion sample at a ratio of 1:40 (v / v). After layer-by-layer deposition, 5 μL of 1 mg / mL 18:1 PE CF fluorescent probe was mixed with the oil phase in the oil / water column at a ratio of 1:40 (v / v). The prepared three-layer membrane fat globule emulsion sample was observed by laser confocal microscopy.

[0039] Example 2 Homogenization Pressure Optimization

[0040] The effects of different homogenization conditions on reconstructed fat globules were investigated using homogenization at 200 bar for 5 cycles, at 200 bar for 10 cycles, and at 300 bar for 15 cycles. Other conditions were the same as in Example 1. Figure 2 It can be seen that the particle size D[4,3] is 9.910 μm after homogenization at 200 bars for 5 times; D[4,3] is 11.047 μm after homogenization at 200 bars for 10 times; and D[4,3] is 11.153 μm after homogenization at 300 bars for 15 times. Furthermore, there is no significant difference in Zeta potential under different homogenization conditions. Figure 3 It can be seen that different homogenization conditions affect the microstructure. The results show that when homogenized 10 times at 200 bars, the outer layer of the fat globule is better wrapped with the fat globule membrane.

[0041] Example 3: Optimization of the proportion of fat globule membrane added

[0042] The effect of fat globule membrane addition ratio on reconstructed fat globules was investigated when the addition amounts were one times (0.16% fat globule membrane by weight of skim milk), two times (0.32% fat globule membrane by weight of skim milk), and three times (0.48% fat globule membrane by weight of skim milk). Homogenization was performed 10 times at 200 bar pressure, with other conditions the same as in Example 1. Figure 4 It can be seen that the particle size D[4,3] is 5.120 μm when the particle size is one times that of a fat globule membrane, 5.594 μm when the particle size is two times that of a fat globule membrane, and 10.332 μm when the particle size is three times that of a fat globule membrane. Furthermore, different amounts of fat globule membrane added did not significantly affect the Zeta potential. Figure 5 It can be seen that different amounts of fat globule membrane have an effect on the microstructure. The results show that the microstructure of the double and triple fat globule membranes can completely encapsulate the fat globules.

[0043] Example 4: Optimization of oil phase selection in oil / water columns

[0044] The effect of oil phase selection in an oil / water column on the formation of a three-layer emulsion fat globule emulsion was investigated, with other conditions identical to those in Example 1. An oil / water column was prepared in a 50 mL centrifuge tube. The lower aqueous phase consisted of sucrose at a concentration of 0.5 M, added in 5 mL volumes. The upper oil phase consisted of sunflower oil or soybean oil, added in 1.5 mL volumes. The column was stabilized for 30 min to allow the formation of a lipid molecular layer at the water / oil interface. Figure 6 It is known that the three-layer film emulsion fat globule emulsion prepared with soybean oil has better effect, and since soybean oil is a food raw material, soybean oil is preferred as the oil phase of the oil / water column.

[0045] The above description of the disclosed embodiments enables those skilled in the art to make or use the invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the invention. Therefore, the invention is not to be limited to the embodiments shown herein, but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A method for preparing a human milk-like three-layer membrane fat globule emulsion, characterized in that, Includes the following steps: 1) Construction of a single-layer membrane fat globule emulsion: A single-layer fat globule emulsion was prepared by adding fat globule membrane and triglycerides to skim milk, stirring until dissolved under constant temperature, hydrating at 4°C, and then sonicating, shearing, and homogenizing. The amount of fat globule membrane added is 0.15-0.5% of the mass of skim milk; the amount of triglycerides added is 3-5% of the mass of skim milk. 2) Construction of a three-layer membrane fat globule emulsion: An oil / water column was prepared, with an oil phase on top and an aqueous phase on the bottom, and the oil / water column was stabilized at room temperature. A monolayer fat globule emulsion was dropwise spread on the top of the oil phase, allowed to stand, and then centrifuged. The upper oil phase was removed, and the lower emulsion was ultrasonically treated to obtain a three-layer fat globule emulsion. The stirring conditions described in step 1) are as follows: a magnetic stirrer is used, the stirring speed is 100-500 r / min, the stirring time is 2-5 h, and the stirring temperature is 30-50℃; The hydration time is 6-12 hours; The ultrasound conditions are: frequency 20 kHz, duration 5 s, interval 3 s, and ultrasound time 5-15 min. The shearing conditions are 10000-15000 r / min, and the shearing time is 3-7 min; The homogenization conditions are as follows: the emulsion is preheated to 35-55°C before homogenization, the homogenization pressure is 150-300 bars, and the number of homogenization cycles is 5-15. Step 2) The preparation of the oil / water column is as follows: an oil / water column is prepared in a 50 mL centrifuge tube, the lower aqueous phase is sucrose with a concentration of 0.2-0.8 M and an addition amount of 2-8 mL; the upper oil phase is sunflower oil or soybean oil and an addition amount of 1-2 mL. The oil / water column stabilization time is 15-30 min; The settling time is 15-30 minutes; 1.5 mL of monolayer membrane fat globule emulsion was dropwise spread on the top of the oil phase, allowed to stand, and then centrifuged. The centrifugation speed was 7000-12000 g, the centrifugation time was 10-30 min, and the centrifugation temperature was 20-30℃. The ultrasonic treatment frequency is 20 kHz, lasts for 5 seconds, with a 3-second interval, and the ultrasonic time is 5-15 minutes.

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