Preparation method and application of lactoferrin composite spherical membrane body

Abstract

The invention relates to the technical field of cosmetic raw materials, in particular to a preparation method and application of a lactoferrin composite spherical membrane body. The preparation method of the lactoferrin composite globular membrane body, provided by the invention, comprises the following steps of: wrapping lactoferrin by using milk fat globular membrane protein as a carrier to prepare a nano-scale composite globular membrane body, and specifically, reducing the iron saturation of the lactoferrin by adopting a chelate resin iron removal technology, and then preparing the lactoferrin composite globular membrane body by using the milk fat globular membrane protein as the carrier. Constructing a milk fat globule membrane protein / lactoferrin core-shell structure to form colostrum, and injecting the colostrum into a water phase containing 0.1 wt% of lactobionic acid to prepare a composite globule membrane body. The method not only can improve the stability and bacteriostatic activity of the lactoferrin, but also can significantly improve the transdermal rate of the milk iron eggs, enhance the ability of the lactoferrin to specifically bind to keratinocyte lectin receptors, and improve the uptake efficiency of keratinocytes, thereby achieving the effect of improving the bioavailability of the lactoferrin. The composition is an ideal functional cosmetic raw material.

Description

Technical Field

[0001] This invention relates to the field of cosmetic raw material technology, specifically to a method for preparing lactoferrin complex globular membranes and their applications. Background Technology

[0002] Lactoferrin (LF) is an iron-binding glycoprotein belonging to the transferrin family. It is found in humans and animals, particularly in high concentrations in human and bovine milk. Human and bovine lactoferrin share a high degree of similarity in their primary structures, with a homology of up to 69%. The primary structure of human lactoferrin consists of 703 amino acid residues, while its secondary structure comprises alternating α-helices and β-sheets. Lactoferrin's ability to bind to numerous substances forms the basis of its diverse biological functions. It can bind not only iron ions but also copper, calcium, and aluminum ions, and can also bind to other proteins and DNA to exert corresponding functions. Studies have shown that lactoferrin possesses broad-spectrum antibacterial, anti-inflammatory, melanin-inhibiting, and collagen-promoting effects, making it a potential functional ingredient in anti-aging, whitening, and repairing skincare products.

[0003] Patent document CN103735424A discloses a cosmetic containing bovine lactoferrin. This cosmetic mainly consists of stearic acid, octadecanol, glyceryl monostearate, butyl stearate, propylene glycol, potassium chloride, fragrance, preservatives, bovine lactoferrin, astaxanthin, and distilled water. The resulting cosmetic has antioxidant, anti-inflammatory, and antibacterial effects, effectively removing acne and pimples, softening the stratum corneum, fading age spots, reducing wrinkles and firming the skin, inhibiting fungal infections like athlete's foot, softening calluses on the feet, promoting skin cell metabolism, and keeping the skin bright and smooth.

[0004] Patent document CN119280093A discloses the use of lactoferrin as a moisturizer in cosmetics or personal care products. Studies have found that lactoferrin has excellent moisturizing effects; specifically, it was found that lactoferrin can increase the content of the key moisturizing protein AQP3 in an in vitro skin model, thus proving that lactoferrin has moisturizing efficacy.

[0005] Patent document CN116804052A discloses an antioxidant peptide derived from lactoferrin. This antioxidant peptide has antioxidant activity, and its DPPH free radical scavenging ability and ABTS free radical scavenging ability are close to or better than those of glutathione and carnosine, respectively. It can be used in the preparation of antioxidant products, such as as an active ingredient in food, cosmetics, or health products.

[0006] However, although lactoferrin is widely used in the cosmetics field, it also has many shortcomings in practical applications, such as: (1) the large molecular weight of lactoferrin makes it difficult to penetrate the stratum corneum, resulting in a transdermal permeability of less than 5%; (2) the strong hydrophilicity of lactoferrin makes it easily soluble in water, leading to low bioavailability; and (3) in cosmetic systems, lactoferrin is easily affected by pH changes (aggregation occurs when pH>6), high temperature (>70 ℃), and light, leading to activity decay or even inactivation. Therefore, researching and developing a production process that can improve the stability and bioavailability of lactoferrin remains a pressing challenge. Summary of the Invention

[0007] To address the shortcomings of existing technologies, this invention provides a method for preparing lactoferrin composite globular membranes. The method involves using milk fat globule membrane protein (FFM) as a carrier to encapsulate lactoferrin, creating nanoscale composite globular membranes. Specifically, chelating resin deironization technology is first employed to reduce the iron saturation of lactoferrin. Then, using FFM as a carrier, a W / O colostrum is constructed by building a WFM / lactoferrin core-shell structure. This W / O colostrum is then injected into an aqueous phase containing 0.1 wt% lactobionic acid to form the composite globular membrane. This method significantly improves the stability and transdermal permeability of lactoferrin, as well as its antibacterial activity and oxidative properties. It effectively addresses the shortcomings of lactoferrin in cosmetic applications, such as low transdermal permeability, low bioavailability, and poor stability, making it a more ideal functional cosmetic ingredient.

[0008] To achieve the above objectives, the technical solution of the present invention is as follows: This invention provides a method for preparing lactoferrin complex globular membranes, comprising the following steps: Step S1: Weigh lactoferrin and dissolve it in 0.1 mol / L, pH 4.5 sodium citrate buffer. Add chelating resin and shake to adsorb for 2-3 h at 25 ℃. Centrifuge for 8-12 min at 7000-9000 rpm. Take the supernatant, ultrafilter, take the supernatant, and freeze-dry to obtain pretreated lactoferrin. Step S2: Weigh milk fat globule membrane protein and add it to deionized water. Refrigerate overnight at 0-4 ℃ to obtain the lipid phase. Step S3: Dissolve the pretreated lactoferrin obtained in step S1 in 0.1 mol / L, pH 5.5 citrate buffer solution. The ratio of the pretreated lactoferrin to sodium citrate buffer solution is 1 g: 100 mL. Sterilize with a 0.22 μm filter membrane to obtain the aqueous phase. Step S4: The aqueous phase obtained in step S3 is injected into the lipid phase obtained in step S2 at a rate of 0.3~0.8 mL / min, sheared, homogenized, and W / O promulgated. The W / O promulgated is injected into an aqueous phase containing 0.1 wt% lactobionic acid and stirred for 12~14 h at a temperature of 4 ℃ and a rotation speed of 500~600 rpm to obtain an O / W globular membrane solution. The aqueous phase containing 0.1 wt% lactobionic acid is an aqueous solution containing 0.1 wt% lactobionic acid, and the solvent is pure water.

[0009] Step S5: The O / W spherical membrane solution obtained in step S4 is concentrated by ultrafiltration to obtain a concentrate. Trehalose is added to the concentrate, pre-frozen, and freeze-dried to obtain the final product.

[0010] Furthermore, in step S1, the ratio of lactoferrin to sodium citrate buffer is 1 g: 100 mL.

[0011] Furthermore, in step S1, the mass ratio of chelating resin to lactoferrin is 50:1, and the chelating resin is of the Chelex-100 type.

[0012] Furthermore, in step S2, the ratio of milk fat globule membrane protein to deionized water is 1 g: 25 mL.

[0013] Furthermore, the mass ratio of lactoferrin in step S1 to milk fat globule membrane protein in step S2 is 2:1.

[0014] Further, the shearing conditions in step S4 are: shearing for 5 to 6 minutes at a rotation speed of 11,000 to 12,000 rpm, and the homogenization conditions are: homogenization for 2 to 4 times at a pressure of 15 to 25 MPa, with a cycle interval of 2 to 3 minutes, followed by ice bath cooling.

[0015] Furthermore, in step S4, the amount of aqueous phase containing 0.1 wt% lactobionic acid added is 10 times the volume of sodium citrate buffer in step S1.

[0016] Furthermore, in step S5, the amount of trehalose added is 4-6 wt% of the weight of the concentrate. The pre-freezing conditions are: pre-freezing at a temperature of -70 ℃ to -80 ℃ for 4-5 h, and the freeze-drying conditions are: freezing at a temperature of -50 ℃ to -55 ℃ and a pressure of 100 Pa for 20-24 h.

[0017] Furthermore, the present invention also claims protection for lactoferrin complex globular membranes prepared by the method for preparing the lactoferrin complex globular membranes.

[0018] In addition, the present invention also provides the application of the lactoferrin complex globular membrane in the preparation of cosmetics with antioxidant or anti-inflammatory effects.

[0019] Milk fat globule membrane (MFGM) is a unique three-layered structure surrounding milk fat globules, containing 30-75% polar lipids and 25-75% proteins. The main components of the polar lipids are glycerophospholipids and sphingolipids, while the main components of the proteins are lactolipoprotein (BTN), mucin 1 (MUC1), xanthine oxidoreductase (XDH / XO), and fatty acid-binding proteins. To address the shortcomings in the application of lactoferrin in the cosmetic field, the inventors first proposed a technical solution using milk fat globule membrane protein as a carrier to encapsulate lactoferrin and create nanoscale composite globule membranes. The inventors unexpectedly discovered that using milk fat globule membrane protein as a carrier to encapsulate lactoferrin not only improves the skin permeability and stability of lactoferrin but also significantly enhances its antibacterial, antioxidant, and anti-inflammatory properties.

[0020] This invention creatively employs chelating resin deironization technology to reduce the iron saturation of lactoferrin (LF), achieving an iron saturation of <7%. This not only ensures the stability of lactoferrin but also significantly enhances its antibacterial activity. Subsequently, using milk fat globule membrane protein (MFGM) as a carrier, an MFGM / LF core-shell structure is formed through low-temperature self-assembly at 4 °C in a pH 5.5 citrate buffer system. The particle size of this core-shell structure is 80–150 nm, matching the optimal size for penetration into the stratum corneum and overcoming the transdermal barrier of large molecules. Simultaneously, this invention optimizes key parameters such as the MFGM:LF mass ratio to 2:1 and the homogenization pressure to 15–25 MPa, synergistically optimizing encapsulation efficiency and dispersibility. This effectively improves the stability of the lactoferrin product, maintains its native conformation, and prevents denaturation and inactivation, providing a reliable solution for the efficient transdermal delivery of functional proteins.

[0021] In summary, compared with the prior art, the method for preparing lactoferrin complex globular membranes provided by the present invention has the following advantages: (1) The lactoferrin composite globular membrane prepared by the present invention has the advantages of high encapsulation rate, good transdermal properties and high stability, which breaks through the bottleneck of low encapsulation rate, poor transdermal properties and easy loss of activity of traditional technology, and provides a reliable solution for efficient transdermal delivery of functional proteins.

[0022] (2) The lactoferrin complex globular membrane obtained by the present invention has the advantages of strong antibacterial effect, good antioxidant and anti-inflammatory effect, and is a relatively ideal functional raw material for anti-aging, whitening and repair skin care products. Detailed Implementation

[0023] The present invention will be further described below through specific embodiments, but this is not intended to limit the invention. Those skilled in the art can make various modifications or improvements based on the basic idea of ​​the invention, but as long as they do not depart from the basic idea of ​​the invention, they are all within the scope of the invention. The raw materials and components involved in the present invention can all be obtained through commercially available or conventional techniques in the art. For example, the lactoferrin was purchased from Glanbia, and the milk fat globule membrane protein was purchased from Wuhan Huamei Bioengineering Co., Ltd.

[0024] Example 1: Preparation of lactoferrin complex globular membrane Step S1: Dissolve 100 mg of lactoferrin (purity >95%, iron saturation 18%) in 10 mL of 0.1 mol / L, pH 4.5 sodium citrate buffer, add 5 g of Chelex-100 chelating resin, and shake to adsorb for 2 h at 25 ℃. Centrifuge for 10 min at 8000 rpm, collect the supernatant, desalt it by ultrafiltration through a 10 kDa membrane, and freeze-dry to obtain pretreated lactoferrin. The pretreated lactoferrin is low-iron lactoferrin, and the iron saturation is 6.5% as determined by ICP-MS.

[0025] Step S2: Take 200 mg of milk fat globule membrane protein (phospholipid content 82%) and add it to 5 mL of deionized water. Refrigerate overnight at 4°C to obtain the lipid phase. The concentration of milk fat globule membrane protein in the lipid phase is 40 mg / mL.

[0026] Step S3: Dissolve 100 mg of the pretreated lactoferrin obtained in step S1 in 10 mL of 0.1 mol / L, pH 5.5 citrate buffer, sterilize by 0.22 μm filter membrane to obtain an aqueous phase; the concentration of pretreated lactoferrin in the aqueous phase is 10 mg / mL.

[0027] Step S4: The aqueous phase obtained in step S3 is injected into the lipid phase obtained in step S2 at a rate of 0.5 mL / min. The mixture is sheared at 12000 rpm for 5 min, homogenized three times at a pressure of 20 MPa with a 2 min interval between cycles, and cooled in an ice bath to obtain a W / O promulgated emulsion. The W / O promulgated emulsion is then injected into an aqueous phase containing 0.1 wt% lactobionic acid, wherein the aqueous phase containing 0.1 wt% lactobionic acid is an aqueous solution containing 0.1 wt% lactobionic acid in pure water. The mixture is stirred at 4 ℃ and 600 rpm for 12 h to obtain an O / W spherical membrane solution. The particle size of the W / O promulgated emulsion was measured to be 85±12 nm using a Malvern particle size analyzer, and the PDI was <0.2.

[0028] Step S5: The O / W spherical membrane solution obtained in step S4 is concentrated by ultrafiltration through a 100 kDa membrane at a tangential flow rate of 2 L / min to remove free lactoferrin and lactobionic acid, resulting in a concentrate. Trehalose is added to the concentrate at an amount of 5 wt% of the concentrate weight. The concentrate is then pre-frozen at -80 ℃ for 4 h, followed by freezing at -50 ℃ and 100 Pa for 24 h to obtain a white porous powder.

[0029] Example 2: Preparation of lactoferrin complex globular membranes Step S1: Dissolve 100 mg of lactoferrin (purity >95%, iron saturation 18%) in 10 mL of 0.1 mol / L, pH 4.5 sodium citrate buffer, add 5 g of Chelex-100 chelating resin, and shake to adsorb for 3 h at 25 ℃. Centrifuge for 12 min at 7000 rpm, collect the supernatant, desalt by ultrafiltration through a 10 kDa membrane, and freeze-dry to obtain pretreated lactoferrin. The pretreated lactoferrin is low-iron lactoferrin, with an iron saturation of 6.8% as determined by ICP-MS.

[0030] Step S2: Take 200 mg of milk fat globule membrane protein (phospholipid content 82%) and add it to 5 mL of deionized water. Refrigerate overnight at 4°C to obtain the lipid phase. The concentration of milk fat globule membrane protein in the lipid phase is 40 mg / mL.

[0031] Step S3: Dissolve 100 mg of the pretreated lactoferrin obtained in step S1 in 10 mL of 0.1 mol / L, pH 5.5 citrate buffer, sterilize by 0.22 μm filter membrane to obtain an aqueous phase; the concentration of pretreated lactoferrin in the aqueous phase is 10 mg / mL.

[0032] Step S4: The aqueous phase obtained in step S3 is injected into the lipid phase obtained in step S2 at a rate of 0.6 mL / min. The mixture is sheared at 11000 rpm for 6 min, homogenized twice at a pressure of 22 MPa with a 2 min interval between cycles, and cooled in an ice bath to obtain a W / O colostrum. The W / O colostrum is then injected into an aqueous phase containing 0.1 wt% lactobionic acid, wherein the aqueous phase containing 0.1 wt% lactobionic acid is an aqueous solution containing 0.1 wt% lactobionic acid in pure water. The mixture is stirred at 4 ℃ and 500 rpm for 14 h to obtain an O / W spherical membrane solution. The particle size of the W / O colostrum was measured to be 84±13 nm using a Malvern particle size analyzer, and the PDI was <0.2.

[0033] Step S5: The O / W spherical membrane solution obtained in step S4 is concentrated by ultrafiltration through a 100 kDa membrane at a tangential flow rate of 2 L / min to remove free lactoferrin and lactobionic acid, resulting in a concentrate. Trehalose is added to the concentrate at an amount of 6 wt% of the concentrate weight. The concentrate is then pre-frozen at -70 ℃ for 5 h, followed by freezing at -55 ℃ and 100 Pa for 22 h to obtain a white porous powder.

[0034] Experimental Example 1: Determination of the encapsulation efficiency of lactoferrin complex globular membranes 1. Experimental Method: (1) Dissolve the lactoferrin complex globular membrane powder obtained in Example 1 in deionized water to prepare a lactoferrin complex globular membrane solution with a lactoferrin concentration of 1 mg / mL. Take 1 mL of the lactoferrin complex globular membrane solution and place it in an ultrafiltration centrifuge tube (MWCO 100 kDa, Merck Millipore). Centrifuge at 8000×g, 4 ℃, and 30 min (free lactoferrin permeates through the membrane, while encapsulated lactoferrin is retained) and take the precipitate.

[0035] (2) Collect the filtrate and use HPLC (Agilent 1260) to detect the concentration of free lactoferrin.

[0036] (3) The precipitate obtained in step (1) was measured using a chromatographic column (Zorbax SB-C18 (4.6×150 mm, 5 μm)) to measure the encapsulation efficiency of the composite spherical membrane. The mobile phase was: gradient elution of A (0.1% TFA aqueous solution) - B (acetonitrile) (0-5 min: 20% B → 5-10 min: 50% B); flow rate: 1.0 mL / min, detection wavelength: 220 nm, retention time: 6.8 min.

[0037] Encapsulation efficiency calculation formula: EN% = (1 - Cf / Ct) × 100% EN: Encapsulation percentage. Cf: Amount of free drug; Ct: The total amount of drug in nanoparticle or liposome suspension.

[0038] 2. Experimental Results: The experimental results are shown in Table 1.

[0039] Table 1 Encapsulation efficiency results of lactoferrin complex globular membranes batch Ct (mg) Cf (mg) EN (%) 1 10.0 0.77 92.3 2 10.0 0.81 91.9 3 10.0 0.72 92.8 As shown in Table 1, the encapsulation rate of the composite globular body of lactoferrin encapsulated by the milk fat globule membrane protein of the present invention is greater than 91%, which can effectively improve the stability of lactoferrin.

[0040] Experimental Example 2: Determination of the Dispersion of Lactoferrin Complex Spherical Membrane 1. Experimental Method: The lactoferrin complex globular membrane solution before lyophilization (self-assembled solution), the lactoferrin complex globular membrane solution after 0 h of lyophilization and reconstitution, and the solution after 30 days of storage at 4 °C were used as samples. The particle size, polydispersity index (PDI) value and zeta potential of the samples were measured respectively. Among them, the particle size was measured by Malvern particle size analyzer; the zeta potential was measured by electrophoresis; the polydispersity index (PDI) was measured by dynamic light scattering (Malvern Zetasizer Nano ZS) (temperature 25 °C, detection angle 173°), repeated three times, and the average value was taken.

[0041] Calculation formula: PDI = Mw / Mn Mw: Weight-average molecular weight.

[0042] Mn: Number-average molecular weight.

[0043] 2. Experimental Results: The experimental results are shown in Table 2.

[0044] Table 2. Results of dispersibility determination of lactoferrin complex globular membranes Solution before lyophilization lyophilized solution after 0 h of reconstitution Reconstituted solution after 30 days of freeze-drying Particle size (nm) 108±9 112±8 117±10 PDI value 0.18±0.02 0.19±0.03 0.21±0.04 Zeta potential (mV) -30.5±1.2 -32.0±1.5 -29.8±1.8 As shown in Table 2, the particle size of the composite globular membrane body with lactoferrin encapsulated by milk fat globular membrane protein prepared by the present invention can be stably maintained within the target range of 80~150 nm, and the system is highly uniform (PDI<0.2).

[0045] Experimental Example 3: Determination of the transdermal absorption rate of lactoferrin complex globular membranes 1. Experimental Method: The transdermal transdermal absorption rate of the lactoferrin complex globular membrane prepared in Example 1 was determined using the Franz diffusion cell method.

[0046] Skin model: Fresh pig ear skin (thickness 0.8±0.1 mm, with the sebaceous layer removed).

[0047] Grouping: Experimental group: PBS buffer (pH 7.4) containing 1 wt% lactoferrin complex globular membrane; Control group: PBS buffer (pH 7.4) containing 1 wt% free lactoferrin.

[0048] (3) Conditions: The receiving cell contains 0.01% SDS-PBS (37 ℃), with an effective diffusion area of ​​1.77 cm²; (4) Procedure: Sampling points: 1, 2, 4, 8, 12, 24 h; HPLC quantification of lactoferrin concentration; calculation of cumulative transdermal transdermal dose ( ). ).

[0049] 2. Experimental Results: The experimental results are shown in Table 3.

[0050] Table 3. Results of transdermal transdermal assay of lactoferrin complex globular membrane. Time (h) Cumulative transdermal dose in the experimental group (μg / cm²) Cumulative transdermal dose in the control group (μg / cm²) 4 8.5±0.9 1.2±0.3 12 28.7±2.1 3.8±0.5 24 52.7±3.1 7.9±0.8 As shown in Table 3, compared with free lactoferrin, the lactoferrin composite globular membrane prepared in this invention can significantly increase the transdermal transdermal transfusion rate of lactoferrin. At 24 h, the cumulative transdermal transfusion rate of the lactoferrin composite globular membrane prepared in this invention is 52.7 μg / cm², while the cumulative transdermal transfusion rate of the control group is 7.9 μg / cm², showing a significant improvement effect.

[0051] Experiment Example 4: Determination of the antibacterial rate of lactoferrin complex globular membrane 1. Experimental Method: To test the antibacterial effect of the lactoferrin complex globular membrane prepared in Example 1, PBS buffer (pH 7.4) was used as the control group, PBS buffer (pH 7.4) containing free lactoferrin (7% iron saturation) was used as comparative example 1, and PBS buffer (pH 7.4) containing blank milk fat globular membrane protein globular membrane carrier (with the same carrier components and structure as the complex globular membrane in Example 1, but without lactoferrin) was used as comparative example 2.

[0052] in: (1) Preparation of bacterial culture: (A) Staphylococcus aureus: Activated strains were inoculated into Mueller-Hinton broth (MHB) and cultured at 37 °C until OD600 = 0.3 (approximately...). (CFU / mL), finally diluted with sterile PBS to […]. CFU / mL for later use.

[0053] (B) Helicobacter pylori: Fresh colonies were scraped from blood agar plates (Brewster agar + 5% sheep blood) and diluted with Brucellosis broth to McFarland 0.5 standard (approximately). (CFU / mL); dilute to CFU / mL.

[0054] (2) Take a 96-well plate and add 90 μL of bacterial solution to each well. The sample consisted of 10 μL of CFU / mL solution and 10 μL of sample solution (system concentration of 10 μg / mL, stock solution concentration of 100 μg / mL). For the Helicobacter pylori group, an additional 0.1% gastric mucin (w / v) was added to the bacterial culture (to simulate the gastric mucosal environment); 10 μL of 0.1% chlorhexidine solution was added to the positive control wells; and 10 μL of PBS was added to the negative control wells.

[0055] (3) Staphylococcus aureus group was incubated aerobically at 37 ℃ for 24 h; Helicobacter pylori group was incubated microaerophilically at 37 ℃ (85% N2, 10%). 5% Incubate for 72 hours.

[0056] (4) Use an enzyme-linked immunosorbent assay (ELISA) reader to detect absorbance.

[0057]

[0058] 2. Experimental Results: The experimental results are shown in Tables 4 and 5.

[0059] Table 4. Antibacterial effect of lactoferrin complex globular membrane against Staphylococcus aureus (24 h) Group OD600 (mean ± SD) Antibacterial rate (%) Antibacterial enhancement rate control group 0.84±0.06 - - Comparative Example 1 0.52±0.04 38.1±3.8 benchmark Example 1 Group 0.17±0.02 79.8±2.1 ↑109% Comparative Example 2 0.81±0.05 3.6±1.1 - Table 5. Antibacterial effect of lactoferrin complex globular membrane against Helicobacter pylori (72 h) Group OD550 (mean ± SD) Antibacterial rate (%) Antibacterial enhancement rate control group 0.78±0.05 - - Comparative Example 1 0.46±0.03 41.0±3.5 benchmark Example 1 Group 0.10±0.01 87.2±1.9 ↑113% Comparative Example 2 0.75±0.04 3.8±1.3 - As shown in Tables 4 and 5, compared with Comparative Example 1 (free lactoferrin with 7% iron saturation), the lactoferrin complex globular membrane prepared in this invention achieves an inhibition rate of 79.8% against Gram-positive bacteria (S. aureus), an increase of 109%; and an inhibition rate of 87.2% against Gram-negative bacteria (H. pylori), an increase of 113%. This indicates that the complex globular membrane of lactoferrin encapsulated with milk fat globule membrane protein in this invention can significantly improve the antibacterial effect of lactoferrin.

[0060] Experimental Example 5: Stability Determination of Lactoferrin Complex Spherical Membrane 1. Experimental Method: The lyophilized powder samples of lactoferrin complex globular membranes prepared in Example 1 were stored at 25 °C and 60% humidity for 0, 1, 3, and 6 months. Samples were taken to test their antibacterial retention rate and antioxidant activity, with the antioxidant activity based on the lactoferrin DPPH scavenging rate.

[0061] 2. Experimental Results: The experimental results are shown in Table 6.

[0062] Table 6. Results of stability determination of lactoferrin complex globular membrane Time (month) Antibacterial retention rate DPPH removal rate and retention rate 1 98.1±1.5% 97.8±2.0% 3 96.3±1.2% 95.4±1.8% 6 93.7±1.8% 92.5±1.5% As shown in Table 6, the lactoferrin composite globular membrane freeze-dried powder prepared by this invention retains >90% activity after being stored at room temperature for 6 months, meeting the shelf life requirements of cosmetics.

[0063] The above embodiments are merely illustrative of the principles and effects of the present invention and are not intended to limit the invention. Any person skilled in the art can modify or alter the above embodiments without departing from the spirit and scope of the present invention. Therefore, all equivalent modifications or alterations made by those skilled in the art without departing from the spirit and technical concept disclosed in the present invention should still be covered by the claims of the present invention.

Claims

1. A method for preparing a lactoferrin complex globular membrane, characterized in that, Includes the following steps: Step S1: Weigh lactoferrin and dissolve it in sodium citrate buffer. Add chelating resin and shake to adsorb for 2-3 h. Centrifuge at 7000-9000 rpm for 8-12 min. Take the supernatant, ultrafilter, take the supernatant, and freeze-dry to obtain pretreated lactoferrin. Step S2: Weigh milk fat globule membrane protein and add it to deionized water. Refrigerate overnight at 0-4 ℃ to obtain the lipid phase. Step S3: Dissolve the pretreated lactoferrin obtained in step S1 in citrate buffer solution, wherein the ratio of pretreated lactoferrin to sodium citrate buffer solution is 1 g: 100 mL. Sterilize by filtration membrane to obtain aqueous phase. Step S4: Inject the aqueous phase obtained in step S3 into the lipid phase obtained in step S2 at a rate of 0.3~0.8 mL / min, shear and homogenize to obtain W / O colostrum, inject the W / O colostrum into the aqueous phase containing lactobionic acid, and stir at 500~600 rpm for 12~14 h to obtain O / W globular membrane solution. Step S5: The O / W spherical membrane solution obtained in step S4 is concentrated by ultrafiltration to obtain a concentrate. Trehalose is added to the concentrate, pre-frozen, and freeze-dried to obtain the final product.

2. The method for preparing lactoferrin complex globular membranes as described in claim 1, characterized in that, In step S1, the ratio of lactoferrin to sodium citrate buffer is 1 g: 100 mL.

3. The method for preparing lactoferrin complex globular membranes as described in claim 1, characterized in that, In step S1, the mass ratio of chelating resin to lactoferrin is 50:1, and the chelating resin is Chelex-100.

4. The method for preparing lactoferrin complex globular membranes as described in claim 1, characterized in that, In step S2, the ratio of milk fat globule membrane protein to deionized water is 1 g: 25 mL.

5. The method for preparing lactoferrin complex globular membranes as described in claim 1, characterized in that, The mass ratio of lactoferrin in step S1 to milk fat globule membrane protein in step S2 is 2:

1.

6. The method for preparing lactoferrin complex globular membranes as described in claim 1, characterized in that, The shearing conditions in step S4 are: shearing for 5 to 6 minutes at a rotation speed of 11,000 to 12,000 rpm, and the homogenization conditions are: homogenizing 2 to 4 times at a pressure of 15 to 25 MPa, with a cycle interval of 2 to 3 minutes.

7. The method for preparing lactoferrin complex globular membranes as described in claim 1, characterized in that, In step S4, the amount of aqueous phase containing 0.1 wt% lactobionic acid added is 10 times the volume of sodium citrate buffer in step S1.

8. The method for preparing lactoferrin complex globular membranes as described in claim 1, characterized in that, In step S5, the amount of trehalose added is 4-6 wt% of the weight of the concentrate. The pre-freezing conditions are: pre-freezing at a temperature of -70 ℃ to -80 ℃ for 4-5 h. The freeze-drying conditions are: freezing at a temperature of -50 ℃ to -55 ℃ and a pressure of 100 Pa for 20-24 h.

9. Lactoferrin complex globular membrane prepared by the method described in any one of claims 1 to 8.

10. The use of the lactoferrin complex globular membrane as described in claim 9 in the preparation of cosmetics with antioxidant or anti-inflammatory effects.