Phosphogroup-modified lactoferrin and preparation method and application thereof

Abstract

The application belongs to the field of daily chemicals, and discloses a preparation method of phosphoric acid group modified lactoferrin. In the presence of a thickening agent, a modified agent with a phosphoric acid polar group is mixed with lactoferrin to obtain phosphoric acid group modified lactoferrin. The modified agent is one or a combination of lecithin, hydrogenated lecithin and glycerophosphocholine. The method uses a modified agent with a phosphoric acid polar group and emulsion protein to react, and the permeability and stability of the modified emulsion protein are obviously improved. Another purpose of the application is to provide the application of the phosphoric acid group modified lactoferrin.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of cosmetics, in particular to a phospho group modified lactoferrin and its preparation method and application. BACKGROUND

[0002] Lactoferrin is a glycosylated protein of 80 kDa consisting of about 700 amino acids, with high homology among species. Lactoferrin has two symmetrical homologous globular domains: N-loop and C-loop; the N-loop corresponds to the 1-333 amino acid residues in the peptide chain, and the C-loop corresponds to the 345-692 amino acid residues, and the two ends of the two domains are connected by a short chain of alpha-helix. Each loop has two subdomains (subdomains): N1, N2 and C1, C2, which respectively contain an iron binding site and a glycosylation site; lactoferrin can bind to various metal ions: Fe 2+ , Fe 3+ , Cu 2+ , Zn 2+ and Mn 2+ , etc., is a non-heme iron binding protein, belonging to the transferrin family. Lactoferrin is abundant in the body fluids of mammalian organisms and is produced by mucosal epithelial cells, and is also an important component of the body's humoral immune system, connecting innate and adaptive immune functions.

[0003] Lactoferrin has multiple effects on the skin, especially in improving skin barrier function and anti-inflammatory. Lactoferrin can improve skin barrier function and reduce irritation by inhibiting the secretion of inflammatory factors. Lactoferrin can bind to bacterial walls, oxidize bacteria by forming peroxide, increase the permeability of bacterial cell membranes, and cause bacterial lipopolysaccharide to leak out of the outer membrane, thereby playing a bactericidal role. In addition, lactoferrin can also inhibit pathogenic fungi that cause skin diseases, such as Trichophyton mentagrophytes and Candida albicans, which are associated with various skin diseases.

[0004] However, the molecular weight of lactoferrin is large, and in the application process of cosmetic products, due to the high molecular weight, there is a problem of poor permeability, and the stability is not outstanding.

[0005] Therefore, the technical problem to be solved by the present application is how to improve the permeability and storage stability of lactoferrin in the application of cosmetics. SUMMARY

[0006] One of the purposes of the present application is to provide a preparation method of phospho group modified lactoferrin, which uses a modifier with a phospho polar group and a milk protein to react, and the permeability and stability of the modified milk protein obtained are significantly improved.

[0007] Another object of the present application is to provide the application of the phosphorus group modified lactoferrin.

[0008] To achieve the above-mentioned object, the present application provides a preparation method of the phosphorus group modified lactoferrin, which comprises the following steps: mixing and reacting the lactoferrin and a modifier with a phosphorus polar group in the presence of a thickening agent to obtain the phosphorus group modified lactoferrin.

[0009] The modifier is one or a combination of lecithin, hydrogenated lecithin and glycerophosphocholine.

[0010] The present application can improve the permeability and stability of the modified emulsion protein by using the modifier with the phosphorus polar group. In some researches, such as the FT-IR research on the interaction between phosphatidylcholine and bovine serum albumin in the document of Physical Chemistry, March 4, 2004, it is mentioned that the glycerophosphocholine is used for the research on the secondary structure of the bovine serum albumin, the content of alpha helix and the order of the carbon chain of the phosphatidylcholine.

[0011] In the research process of the present application, the glycerophosphocholine is further expanded to the application research of lecithin and hydrogenated lecithin, and it is surprisingly found that the permeability and stability of the lactoferrin are improved after the effective modification of the lactoferrin and the modifier with the phosphorus polar group.

[0012] It is found through further research that, among the above-mentioned three modifiers, the improvement effect of hydrogenated lecithin and glycerophosphocholine on the stability is better than that of lecithin, and the improvement effect of glycerophosphocholine on the permeability is better than that of lecithin and hydrogenated lecithin. One of the possible reasons is that the stability and emulsifying capacity of the hydrogenated lecithin are improved after the hydrogenation treatment, which has a positive effect on the stability maintenance. The structure of the glycerophosphocholine is simpler than that of lecithin and hydrogenated lecithin, and the molecular weight is small, which is helpful to the improvement of the stability and permeability.

[0013] In the above-mentioned preparation method of the phosphorus group modified lactoferrin, the weight ratio of the lactoferrin to the modifier is 1:0.2-1.

[0014] In the above-mentioned preparation method of the phosphorus group modified lactoferrin, the thickening agent is xanthan gum or guar gum.

[0015] In the actual application, the optional thickening agent can also be modified starch, carboxymethyl cellulose, carboxymethyl chitosan and the like.

[0016] In the above-mentioned preparation method of the phosphorus group modified lactoferrin, the ratio of the thickening agent to the lactoferrin is 0.1-0.3:1.

[0017] In the above-mentioned preparation method of the phosphorus group modified lactoferrin, the preparation method comprises the following steps:

[0018] Step 1: Add the modifier and thickener to the water to dissolve them;

[0019] Step 2: Add lactoferrin to the solution from Step 1 at 30–50°C, stir and keep warm for 3–5 hours; then homogenize and filter, and the resulting filtrate contains the phosphate-modified lactoferrin.

[0020] In the above-described method for preparing phosphate-modified lactoferrin, in step 1, the weight ratio of the modifier to water is 0.2–1:50–85.

[0021] Meanwhile, this invention also discloses a phosphate-modified lactoferrin, which is prepared using any of the methods described above.

[0022] Meanwhile, the present invention also discloses the use of the above-mentioned phosphate-modified lactoferrin in the preparation of cosmetics.

[0023] Finally, the present invention also discloses a cosmetic containing lactoferrin modified with phosphate groups as described above.

[0024] The aforementioned cosmetics contain 0.1–3 wt% of phosphate-modified lactoferrin.

[0025] In the application of this invention, the cosmetic product can be selected as a lotion or cream.

[0026] Beneficial effects

[0027] This invention uses a modifier with a phosphate polar group to react with emulsion protein, resulting in a modified emulsion protein with significantly improved permeability and stability. Attached Figure Description

[0028] Figure 1 These are the results of stability tests on each sample at 45℃ for 4 weeks. Detailed Implementation

[0029] The present invention will be further described below with reference to embodiments, but this does not constitute any limitation on the present invention. Any limited modifications made within the scope of the claims of the present invention are still within the scope of the claims of the present invention.

[0030] To illustrate the technical content of the present invention in detail, the following description is provided in conjunction with the embodiments.

[0031] Examples and Comparative Examples

[0032] Step 1: Preparation of the modifier solution: Slowly dissolve xanthan gum in deionized water, mix and stir at room temperature, add a certain amount of modifier, heat to 80℃ to fully dissolve the modifier and xanthan gum, homogenize at 5000r / min for 2min to disperse evenly, and cool to 40℃.

[0033] Step 2: Add the lyophilized lactoferrin powder to the solution in Step 1 above, stir at 40°C for 1 hour to fully dissolve the lactoferrin and modifier, and keep warm for 4 hours to allow the components to interact.

[0034] A primary emulsion was prepared by pre-emulsification at 8000 rpm for 2 minutes using an emulsifier, followed by secondary homogenization using a high-pressure homogenizer at 40 MPa. After homogenization, the solution was filtered, and the filtrate was a solution containing phosphate-modified lactoferrin.

[0035] The formula can be found in Table 1.

[0036] Table 1 Formula Table (Unit: g)

[0037]

[0038] Performance testing

[0039] High and low temperature stability testing: Referencing the "Technical Guidelines for Stability Testing and Evaluation of Cosmetics" (China National Institutes for Food and Drug Control, July 2024), the stability of Examples 1-9 and Comparative Example 1 was examined at 5°C, room temperature, and 45°C for 4 weeks. The results are shown in Table 2 below. Figure 1 ;

[0040] Table 2 Stability Test Results

[0041] 5°C (2 weeks) 5°C (4 weeks) Room temperature (2 weeks) Room temperature (4 weeks) 45°C (2 weeks) 45°C (4 weeks) Example 1 √ √ √ √ √ Slightly delaminated Example 2 √ √ √ √ √ √ Example 3 √ √ √ √ √ √ Example 4 √ √ √ √ Slightly delaminated Delaminated Example 5 √ √ √ √ √ Slightly delaminated Example 6 √ √ √ √ √ √ Example 7 √ √ √ √ √ √ Example 8 √ √ √ √ √ √ Example 9 √ √ √ √ √ √ Comparative Example 1 √ √ √ √ Slightly delaminated Slightly delaminated

[0042] Note: √ indicates no abnormality.

[0043] As can be seen from the table, in Comparative Example 1 without modification, slight stratification occurred at 45°C for both 2 and 4 weeks. This is related to the inherent stability of the lactoferrin solution. The stratification phenomenon improved after adding the modifier and changing the amount of thickener. A comparison of Examples 1-3 and Examples 4-6 shows that hydrogenated lecithin is more stable than lecithin, which is related to the improved stability and emulsifying properties of lecithin after hydrogenation. Examples 7-9 remained stable, indicating that glycerophosphate choline helps maintain the stability of the lactoferrin solution.

[0044] Permeability test

[0045] The permeability test was conducted in accordance with GB / T 27818-2011 "In vitro test method for skin absorption of chemicals". Based on the pig skin-Franz diffusion cell system, the test sample was applied to the surface of isolated skin, and then the cumulative permeation, recovery rate and cumulative permeability were calculated to evaluate the transdermal permeability of the test sample.

[0046] The test results are shown in Table 3 below;

[0047] Table 3. Permeability Test Results

[0048]

[0049] The results show that the permeability of Examples 2, 5, and 8 was improved, and this improvement was related to the amount of modifier. The results varied among different modifiers. Overall, with the same amount of modifier added, glycerophosphocholine > hydrogenated lecithin > lecithin. The permeability of hydrogenated lecithin and lecithin is related to their ability to form liposomes, which helps lactoferrin permeate. Glycerophosphocholine lacks a fatty acid structure and does not form liposomes, but it also has a significant permeation-enhancing effect. This may be related to its small molecular weight; for the same mass, it has more phosphocholine polar groups, which have a greater impact on the secondary structure of lactoferrin.

[0050] The embodiments presented herein are merely selected implementations based on combinations of all possible embodiments. The appended claims should not be limited to the embodiments described herein. Some numerical ranges used in the claims include sub-ranges within them, and variations within these ranges should also be covered by the appended claims.

Claims

1. A method for preparing phosphate-modified lactoferrin, characterized in that, In the presence of a thickener, lactoferrin and a modifier with a phosphate polar group are mixed and reacted to obtain phosphate-modified lactoferrin. The modifier is glycerophosphate choline; the weight ratio of lactoferrin to the modifier is 1:0.2-1.

2. The method for preparing phosphate-modified lactoferrin according to claim 1, characterized in that, The thickener is xanthan gum or guar gum.

3. The method for preparing phosphate-modified lactoferrin according to claim 2, characterized in that, The ratio of the thickener to lactoferrin is 0.1 to 0.3:

1.

4. The method for preparing phosphate-modified lactoferrin according to claim 1, characterized in that, Includes the following steps: Step 1: Add the modifier and thickener to the water to dissolve them; Step 2: Add lactoferrin to the solution from Step 1 at 30–50°C, stir and keep warm for 3–5 hours; then homogenize and filter, and the resulting filtrate contains the phosphate-modified lactoferrin.

5. The method for preparing phosphate-modified lactoferrin according to claim 4, characterized in that, In step 1, the weight ratio of the modifier to water is 0.2-1:50-85.

6. A phosphate-modified lactoferrin, characterized in that, It is prepared by the method described in any one of claims 1 to 5.

7. Use of the modified emulsion protein as described in claim 6 in the preparation of cosmetics.

8. A cosmetic product, characterized in that, Lactoferrin containing the phosphate group modified as described in claim 6.

9. The cosmetic product according to claim 8, characterized in that, Lactoferrin containing 0.1–3 wt% phosphate-modified lactoferrin.

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