Preparation method and application of composite antibody IgY for preventing and treating acne

Abstract

The invention discloses a preparation method and application of a composite antibody IgY for preventing and treating acne, and belongs to the technical field of cosmetics. The preparation method comprises the following steps: (1) preparing immunogens: respectively mixing inactivated propionibacterium acnes liquid, staphylococcus epidermidis liquid, staphylococcus aureus liquid and escherichia coli liquid with an ISA 78 adjuvant to obtain the immunogens of the four bacteria; (2) immunizing: respectively injecting immunogens of the four bacteria to laying hens, and collecting to obtain immunized eggs of the four bacteria; and (3) collection and preparation of a composite antibody IgY: mixing immunized eggs of the four bacteria, separating yolk, and carrying out acid treatment and impurity removal to obtain the composite antibody IgY. The prepared composite antibody IgY is high in titer, has the effect of preventing and treating acne and acne infection, is high in safety and remarkable in curative effect, and provides a new treatment strategy for preventing and treating acne.

Description

Technical Field

[0001] The present invention relates to the technical field of cosmetics, and particularly to a preparation method and application of a composite antibody IgY for preventing and treating acne. Background Art

[0002] Acne is a common skin disease, mainly manifested as comedones, papules, pustules, etc. on the face, back and other parts. It is a symbol of entering adolescence. However, with the increasing proportion of women entering the society, the subsequent mental stress is intensifying day by day. Therefore, the number of women who develop acne for the first time in their 20s or 30s is gradually increasing. The reasons for inducing acne are as follows: First, it is because the sebum secretion is overly vigorous. Under the regulation of endocrine glands, excessive sebum secreted from the skin sebaceous glands leads to continuous generation of sebaceous glands, and the sebum is secreted from the sebaceous gland ducts through the infundibulum and secreted on the skin surface, resulting in excessive sebum secretion. In addition, testosterone also promotes sebum secretion and activates the biosynthesis and secretion of lipids. Therefore, in the second growth period - adolescence, the sebaceous glands are very developed. Generally speaking, testosterone secreted from the testicles in adult men keeps sebum secretion stable, while in a certain period after ovulation in women, the increase in hormones will stimulate the sebaceous glands, resulting in increased sebum secretion, and acne will deteriorate after menstruation. Second, it is because the pores are blocked. In the first reason above, excessive sebum secretion will cause the infundibulum to be blocked, resulting in the occlusion or narrowing of the hair follicle pores and sebaceous gland openings, so that sebum cannot be excreted normally, and the sebum remains in the hair funnel, turning into blackhead comedones and increasing the reproduction of acne bacteria.

[0003] Acne occurs throughout the year and can occur repeatedly. It often naturally alleviates or heals after puberty. However, patients often leave permanent damage such as facial connective tissue hyperplasia bumps and depressions, seriously affecting the appearance and causing psychological trauma. Clinically, acne is classified into 3 degrees and 4 grades according to the nature and severity of acne lesions. Grade 1: Only comedones; Grade 2: In addition to comedones, there are also some inflammatory papules; Grade 3: In addition to comedones, there are more inflammatory papules or pustules; Grade 4: In addition to comedones, inflammatory papules and pustules, there are also nodules, cysts or scars. The non-inflammatory skin lesions of acne are manifested as open and closed comedones. The typical skin lesion of closed comedones is a skin-colored papule about 1 mm in size, without an obvious hair follicle opening. Open comedones are manifested as dome-shaped papules with a significantly dilated hair follicle opening. Comedones will further develop into various inflammatory skin lesions, manifested as inflammatory papules, pustules, nodules and cysts. Inflammatory papules are red, with a diameter ranging from 1 to 5 mm; pustules are of the same size and filled with white pus; nodules have a diameter greater than 5 mm, with a hard mass and pain when touched; cysts are deeper in position and filled with a mixture of pus and blood. These skin lesions can also fuse to form large inflammatory plaques and fistulas, etc. After the inflammatory skin lesions subside, pigmentation, persistent erythema, pitted or hypertrophic scars are often left.

[0004] As sebum components turn into black clots containing melanin, the attachment of anaerobic bacteria - Propionibacterium acnes, Staphylococcus epidermidis, Staphylococcus aureus, and Escherichia coli will gradually increase. Propionibacterium acnes is a typical Gram-positive anaerobic short rod-shaped bacterium with relatively slow growth, intracellular parasitism, and belongs to the normal flora of the skin. It generally inhabits the hair follicles and sebaceous glands of the skin. The genes of Propionibacterium acnes control the production of enzymes that break down skin and immunogenic proteins. When pores are blocked, they grow wildly, break down saturated fatty acids, and produce a large amount of free fatty acids. These fatty acids seep into the skin through the pores, causing skin stress reactions, such as acne, swelling, etc. Then, bacteria such as staphylococci stick to the skin through the flowing pus, resulting in damage to skin tissues. The mucus-producing ability of Staphylococcus epidermidis plays an important role in the pathogenicity of the bacteria. The mucous substance will cover the bacterial cells and form a mucus layer through external polysaccharides. This mucus layer can resist antibodies, protect the bacterial cells, and prevent the host's immune response - phagocytosis. According to relevant data, it can also inhibit hemagglutinin and damage the host's immune system. Staphylococcus aureus is a common skin bacterium. When it multiplies in hair follicles, it can cause hair follicle inflammation and infection, thus triggering acne. This bacterium has strong pathogenicity and adaptability and is easy to survive in humid and warm environments. Research also shows that in acne lesion areas, the abundance of Staphylococcus aureus is significantly higher than that in non-lesion areas, and its quantity increases with the increase in severity. Therefore, Staphylococcus aureus is considered an important factor in the pathogenesis of acne. Escherichia coli in secondary infections in unclean skin will exacerbate the inflammation of acne.

[0005] Currently, the treatment methods for acne mainly include: (1) Antibacterial therapy: such as antibiotics (e.g., quinolones, azelaic acid) are used to inhibit Propionibacterium acnes and Staphylococcus aureus, but long-term use is likely to lead to drug resistance (e.g., methicillin-resistant Staphylococcus aureus, MRSA) and skin microecological imbalance. (2) Regulating sebum secretion: such as 5α-reductase inhibitors (e.g., candidate drugs developed by Merck & Co., Inc.) can inhibit the conversion of testosterone to dihydrotestosterone (DHT), reducing excessive sebum secretion by sebaceous glands. (3) Anti-inflammatory and immunomodulatory: such as retinoids and androgen antagonists can improve follicular keratinization and inhibit inflammatory responses. However, the existing therapies still have the following problems: (1) Risk of drug resistance: Long-term use of antibiotics (e.g., vancomycin) may lead to dysbacteriosis and the proliferation of drug-resistant strains (e.g., MRSA). (2) Secondary infection and permanent skin lesions: Severe acne (e.g., inflammatory nodules, cysts) is prone to secondary Staphylococcus infections, resulting in irreversible damage such as scars. (3) Large individual differences: The pathogenesis of acne is closely related to immune function, and it is difficult for existing drugs to balance both efficacy and microecology. Therefore, there is an urgent need to develop a new treatment strategy that can effectively inhibit Propionibacterium acnes, Staphylococcus epidermidis, Staphylococcus aureus, and Escherichia coli, avoid disrupting the skin microecology, and at the same time regulate sebum secretion and immune responses, thereby reducing the risks of drug resistance and permanent skin lesions. Summary of the Invention

[0006] The objective of the present invention is to provide a method for preparing a composite antibody IgY for preventing and treating acne and its application to solve the problems existing in the above-mentioned prior art. The composite antibody IgY prepared by the present invention has a high titer, can specifically bind to Propionibacterium acnes, Staphylococcus epidermidis, Staphylococcus aureus, and Escherichia coli, playing a role in preventing and treating acne and acne infections, and has high safety and remarkable efficacy, providing a new treatment strategy for preventing and treating acne.

[0007] To achieve the above objective, the present invention provides the following solutions:

[0008] The present invention provides a method for preparing a composite antibody IgY for preventing and treating acne, including the following steps:

[0009] (1) Preparation of immunogens: Respectively mix inactivated Propionibacterium acnes bacterial solution, Staphylococcus epidermidis bacterial solution, Staphylococcus aureus bacterial solution, and Escherichia coli bacterial solution with ISA78 adjuvant to obtain immunogens of the four bacteria.

[0010] (2) Immunization: Inoculate the immunogens of the four bacteria into laying hens respectively, and collect immunized eggs of the four bacteria.

[0011] (3) Collection and preparation of composite antibody IgY: Mix the immunized eggs of the four bacteria, separate the egg yolks, and obtain the composite antibody IgY through acid treatment and impurity removal treatment.

[0012] Further, the volume ratio of the inactivated Propionibacterium acnes bacterium solution, Staphylococcus epidermidis bacterium solution, Staphylococcus aureus bacterium solution, and Escherichia coli bacterium solution to the ISA78 adjuvant is 3:7 when they are mixed;

[0013] The bacterial concentrations of the inactivated Propionibacterium acnes bacterium solution, Staphylococcus epidermidis bacterium solution, Staphylococcus aureus bacterium solution, and Escherichia coli bacterium solution are all 8×10 8 -8×10 10 CFU / mL.

[0014] Further, the mixing method is high-speed emulsification; during high-speed emulsification, the rotation speed is 9000 - 12000 rpm, and the time is 3 - 6 minutes.

[0015] Further, the inoculation method is 3-point injection, with 3 primary immunizations at intervals of 28 days each, and 1 mL is injected each time; eggs are collected 14 days after the third immunization.

[0016] Further, when the immune eggs of the four bacteria are mixed, the immune eggs of Propionibacterium acnes, Staphylococcus epidermidis, Staphylococcus aureus, and Escherichia coli are mixed according to a mass ratio of 4:2:2:1.

[0017] Further, the methods of acid treatment and impurity removal are as follows: the egg yolk and water are mixed according to a volume ratio of 1:9 to obtain an egg yolk solution; the pH of the egg yolk solution is adjusted to 5.2, and after standing overnight at 4°C, the supernatant A is collected; after removing impurities from the supernatant A using 1% n-octanoic acid, the supernatant B is collected; the supernatant B is successively subjected to vacuum filtration, ultrafiltration with a molecular weight cut-off of 50,000, and ultrafiltration concentration to obtain the composite antibody IgY.

[0018] The present invention also provides the composite antibody IgY prepared by the above preparation method.

[0019] The present invention also provides the application of the above composite antibody IgY in the preparation of products for preventing and treating acne.

[0020] Optionally, the products include sprays, rubbing agents, and facial mask liquids.

[0021] The present invention also provides a product for preventing and treating acne, which contains the above composite antibody IgY.

[0022] The present invention discloses the following technical effects:

[0023] The present invention provides a method for preparing a composite antibody IgY for preventing and treating acne. This composite antibody IgY can specifically bind to acne pathogenic bacteria, namely Propionibacterium acnes, Staphylococcus epidermidis, Staphylococcus aureus, and Escherichia coli, and has a high titer. The acne pathogenic bacteria, Propionibacterium acnes, Staphylococcus epidermidis, Staphylococcus aureus, and Escherichia coli, are highly sensitive to it and can effectively prevent bacteria from invading the skin. Using this composite antibody IgY, products such as composite antibody IgY sprays, liniments, special sprays for women, and facial mask liquids can be prepared to prevent and treat acne, acne infections, acute and chronic inflammations, suppurations, boils, and subcutaneous carbuncles caused by skin breakage. Through human clinical trials, it is found that the products related to the composite antibody IgY can effectively prevent and treat acne, acne infections, acute and chronic inflammations, suppurations, boils, and subcutaneous carbuncles caused by skin breakage, with an effective rate of over 90%, which is much higher than 1% of metronidazole in the control group, and has the significant advantages of high efficacy and high safety. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required to be used in the embodiments. Obviously, the following described drawings are only some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can be obtained based on these drawings.

[0025] Figure 1 It is the SDS-PAGE electrophoresis pattern of the IgY antibody isolated and purified in Example 3;

[0026] Figure 2 It is a clinical trial case of the composite antibody IgY spray for preventing and treating acne in Example 9; the left figure is before use, and the right figure is after use;

[0027] Figure 3 It is a clinical trial case of the composite antibody IgY spray for preventing and treating acne in Example 9; the left figure is before use, and the right figure is after use;

[0028] Figure 4 It is a clinical trial case of the special composite antibody IgY spray for women to prevent and treat acne in Example 9; the left figure is before use, and the right figure is after use;

[0029] Figure 5 It is a clinical trial case of the composite antibody IgY liniment for preventing and treating acne in Example 9; the left figure is before use, and the right figure is after use;

[0030] Figure 6 It is a clinical trial case of the composite antibody IgY liniment for preventing and treating acne in Example 9; the left figure is before use, and the right figure is after use;

[0031] Figure 7This is a clinical trial case of the egg yolk antibody mask solution encapsulated with nano-gelatin particles for preventing and treating acne in Example 9. The left picture is before use, and the right picture is after use.

[0032] Figure 8 This is a clinical trial case of the egg yolk antibody mask solution encapsulated with nano-gelatin particles for preventing and treating acne in Example 9. The left picture is before use, and the right picture is after use. Detailed implementation manners

[0033] Now, various exemplary implementation manners of the present invention will be described in detail. This detailed description should not be regarded as a limitation of the present invention, but should be understood as a more detailed description of certain aspects, characteristics and implementation schemes of the present invention.

[0034] It should be understood that the terms described in the present invention are only for describing specific implementation manners and are not used to limit the present invention. Additionally, for the numerical ranges in the present invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any intermediate value within any stated value or stated range, as well as each smaller range between any other stated value or intermediate value within the stated range, is also included in the present invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0035] Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the present invention pertains. Although the present invention only describes preferred methods and materials, any methods and materials similar or equivalent to those described herein can also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials related to the documents. In case of conflict with any incorporated document, the content of this specification shall prevail.

[0036] Without departing from the scope or spirit of the present invention, various improvements and changes can be made to the specific implementation manners of the present invention specification, which are obvious to those skilled in the art. Other implementation manners obtained from the specification of the present invention are obvious to those skilled in the art. The specification and examples of the present invention are only exemplary.

[0037] Regarding the terms "comprising", "including", "having", "containing", etc. used herein, they are all open-ended terms, meaning including but not limited to.

[0038] The technical solution of the present invention is as follows:

[0039] Immunize laying hens with Propionibacterium acnes, Staphylococcus epidermidis, Staphylococcus aureus and Escherichia coli to induce the production of specific IgY antibodies against Propionibacterium acnes, Staphylococcus epidermidis, Staphylococcus aureus and Escherichia coli, thereby preparing a composite IgY antibody, which can not only produce specific immune reactions with the immunized Propionibacterium acnes, Staphylococcus epidermidis, Staphylococcus aureus and Escherichia coli, but also can produce specific immune reactions with Propionibacterium acnes, Staphylococcus epidermidis, Staphylococcus aureus and Escherichia coli of different species and types with common antigenic determinants.

[0040] When the IgY antibody undergoes an immune reaction with the bacterial surface antigen, it can prevent Propionibacterium acnes from entering cells and Staphylococcus epidermidis, Staphylococcus aureus and Escherichia coli from adhering to the inflamed site, promote phagocytes to eliminate Propionibacterium acnes, Staphylococcus epidermidis, Staphylococcus aureus and Escherichia coli, thereby reducing infections and achieving the effect of preventing and treating diseases. Moreover, when the IgY antibody undergoes an immune reaction with the bacterial surface antigen, it changes the bacterial surface structure, inhibits some functional activities of the bacteria, inhibits bacterial growth, reduces and inhibits the adhesion of bacteria to cells, and promotes phagocytosis and clearance of bacteria by phagocytes such as monocytes and macrophages.

[0041] The composite IgY antibody against Propionibacterium acnes, Staphylococcus epidermidis, Staphylococcus aureus and Escherichia coli has a different mechanism of action from current antibiotics. It specifically binds to the corresponding antigen on the bacterial surface, inhibits the activity of the bacteria, prevents the bacteria from adhering to tissue cells, excludes the presence of bacteria at the inflamed site, changes the bacterial surface structure and electric field, and promotes the phagocytosis and clearance of bacteria by cells. At the same time, it does not induce the generation of drug-resistant strains at all, does not disrupt the local and overall microecological balance, and also avoids the abuse of antibiotics and the occurrence of a wide range of drug-resistant strains, showing the importance and feasibility of the composite IgY antibody preparation against Propionibacterium acnes, Staphylococcus epidermidis, Staphylococcus aureus and Escherichia coli. According to the mechanism of action of the above-mentioned specific 1gY antibody, the composite 1gY antibody preparation against Propionibacterium acnes, Staphylococcus epidermidis, Staphylococcus aureus and Escherichia coli can effectively treat all acne and skin infectious inflammations caused by bacteria such as Propionibacterium acnes, Staphylococcus epidermidis, Staphylococcus aureus and Escherichia coli, and bacteria with common antigenic determinants with these bacteria, such as acne and acne infections, acute and chronic inflammations, suppuration, furuncles and subcutaneous carbuncles caused by skin breaks.

[0042] Example 1 Cultivation of Propionibacterium acnes, Staphylococcus epidermidis, Staphylococcus aureus and Escherichia coli

[0043] 1. Cultivation of Propionibacterium acnes

[0044] Select Cutibacterium acnes, ATCC 11827. Inoculate the strain into Brain Heart Infusion Medium (BHI), place it in an anaerobic environment (85% N2, 10% H2, 5% CO2), and culture it at 37°C for 72 h, then measure the OD of the bacterial solution 600 value.

[0045] 2. Cultivation of Staphylococcus epidermidis

[0046] Select Staphylococcus epidermidis, ATCC 35983. Inoculate the strain into Tryptic Soy Broth (TSB) containing 0.5% sucrose and 0.5% ethylene diamine - DI - O - resorcinol acetate, and culture it at 37°C for 36 h, then measure the OD of the bacterial solution 600 value.

[0047] 3. Cultivation of Staphylococcus aureus

[0048] Select Staphylococcus aureus, ATCC 25923. Inoculate the strain into Tryptic Soy Broth (TSB), and culture it at 37°C for 24 h, then measure the OD of the bacterial solution 600 value.

[0049] 4. Cultivation of Escherichia coli

[0050] Select Escherichia coli O157:H7, ATCC 35150. Inoculate the strain into Tryptic Soy Broth (TSB), and culture it at 37°C for 48 h, then measure the OD of the bacterial solution 600 value.

[0051] Example 2 Preparation and Immunization of Antigens from Cutibacterium acnes, Staphylococcus epidermidis, Staphylococcus aureus and Escherichia coli

[0052] 1. Antigen Preparation

[0053] Respectively inactivate the Cutibacterium acnes, Staphylococcus epidermidis, Staphylococcus aureus and Escherichia coli obtained from the culture in Example 1 with 0.2% formaldehyde for 24 h. Then wash them 3 times with 0.9% NaCl at 4000 rpm for 20 minutes, and measure the OD of the bacterial solution 600 value, adjust the OD of the bacterial solution according to the need 600 value, the OD 600 value is 1, and the cell content is about 8×10 8 / ml; adjust the OD of the bacterial solution according to the need respectively 600 value. For Staphylococcus epidermidis, do not wash it and directly measure the OD of the bacterial solution 600Value, adjust the OD of the bacterial solution as needed respectively 600 value

[0054] 2. Immunogen preparation - Adjuvant type optimization

[0055] Scheme 1: ISA78 adjuvant

[0056] Adjuvant phase: 7 volumes of ISA78 adjuvant

[0057] Antigen aqueous phase: 3 volumes of Propionibacterium acnes, Staphylococcus epidermidis, Staphylococcus aureus, and Escherichia coli bacterial solutions (each bacterium is 8×10 8 CFU / mL).

[0058] Take 3 volumes of Propionibacterium acnes, Staphylococcus epidermidis, Staphylococcus aureus, and Escherichia coli bacterial solutions (each bacterium is 8×10 8 CFU / mL) (before mixing, sonicate for 30 minutes) and mix with 7 volumes of adjuvant ISA 78, and emulsify by high-speed stirring (high-speed homogenizer, stirring head 5 cm, 10000 rpm, 4 minutes) to prepare the immunogens of Propionibacterium acnes, Staphylococcus epidermidis, Staphylococcus aureus, and Escherichia coli.

[0059] Scheme 2: ISA70 adjuvant

[0060] Take 3 volumes of Propionibacterium acnes, Staphylococcus epidermidis, Staphylococcus aureus, and Escherichia coli bacterial solutions (each bacterium is 8×10 8 CFU / mL) (before mixing, sonicate for 30 minutes) and mix with 7 volumes of adjuvant ISA 70, and emulsify by high-speed stirring (high-speed homogenizer, stirring head 5 cm, 10000 rpm, 4 minutes) to prepare the immunogens of Propionibacterium acnes, Staphylococcus epidermidis, Staphylococcus aureus, and Escherichia coli.

[0061] Inject and immunize laying hens with the immunogens of Propionibacterium acnes, Staphylococcus epidermidis, Staphylococcus aureus, and Escherichia coli in Scheme 1 and Scheme 2 respectively. Use intramuscular injection (pectoral muscle), inject 1 mL intramuscularly into laying hens, immunize at 3 points, immunize initially 3 times, with an interval of 28 days each time, and collect eggs 14 days after the third immunization to extract IgY antibodies. Strengthen the immunization once every 2 months after the third immunization, and the antigen dose and immunization program are the same as the initial immunization.

[0062] After extracting IgY antibodies, use an ELISA kit (Shanghai Boman Biotechnology Co., Ltd.) to measure the titer of antibody IgY. The detection steps are as follows:

[0063] (1) Dilute each bacterial strain to OD 600It is 0.05. Add 100 μL to each well and coat at 4°C for 16 h.

[0064] (2) Block with 3% BSA / TBS, add 200 μL to each well, and incubate at room temperature for 1 h.

[0065] (3) Wash three times with TBST.

[0066] (4) Except for row H, add 100 μL of 1% BSA / TBST to each well, dilute the IgY sample according to the recommended concentration, and then serially dilute it two-fold. Add 200 μL of sample diluent to each well in row H, then take 100 μL of the diluent from each well in row H to row G, mix well and then serially dilute to row B. After mixing well in row B, aspirate 100 μL. Row A serves as the negative control. Incubate at room temperature for 2 h.

[0067] (5) Wash three times with TBST.

[0068] (6) Dilute the secondary antibody with 1% BSA / TBST and add 100 μL to each well. Incubate at room temperature for 1 h.

[0069] (7) Wash five times with TBST.

[0070] Add 50 μL of substrate solution (10 mL Citrate Buffer, 0.4 mL TMB, 33 μL 3% H2O2) to each well and mix well.

[0071] (8) Wait until it changes color, then add 50 μL of stop solution (1 M HCl or 1 M H2SO4) to each well. The time taken for color change is 2 - 5 minutes.

[0072] (9) Measure and record the OD value at a wavelength of 450 nm. Consider the sample OD / negative control average OD > 2.1 as positive, and the highest dilution factor when the sample is positive as the ELISA titer.

[0073] The results showed that the ELISA titers (IgY: 1 mg / mL) of the egg yolk antibodies IgY against Propionibacterium acnes, Staphylococcus epidermidis, Staphylococcus aureus, and Escherichia coli obtained by immunizing with the immunogens prepared using ISA78 adjuvant were: 2 13 , 2 12 , 2 14 , 2 15 . The ELISA titers (IgY: 1 mg / mL) of the egg yolk antibodies IgY against Propionibacterium acnes, Staphylococcus epidermidis, Staphylococcus aureus, and Escherichia coli obtained by immunizing with the immunogens prepared using ISA 70 adjuvant were: 2 11 , 2 10 , 2 12 , 2 13It can be seen that the immunization potency of the novel adjuvant ISA78 for IgY antibody is 4 times higher than that of the traditional adjuvant ISA70. Therefore, the immunization potency of the novel adjuvant ISA78 for IgY antibody is better than that of the traditional adjuvant ISA70. The novel adjuvant ISA78 was selected for subsequent experiments.

[0074] 3. Preparation of Immunogen - Optimization of Antigen Combination

[0075] Protocol 1: After separately preparing immunogens from Propionibacterium acnes, Staphylococcus epidermidis, Staphylococcus aureus, and Escherichia coli antigens and immunizing them respectively, the bacterial content of each antigen was 8×10 8 CFU / mL. The preparation process of the immunogen and the immunization process of laying hens are shown in "2. Preparation of Immunogen - Optimization of Adjuvant Types". Immunized eggs of Propionibacterium acnes, Staphylococcus epidermidis, Staphylococcus aureus, and Escherichia coli were harvested respectively.

[0076] Protocol 2: The bacterial content of Propionibacterium acnes, Staphylococcus epidermidis, Staphylococcus aureus, and Escherichia coli antigens was adjusted to 3.2×10 9 CFU / mL respectively; then they were mixed at a volume ratio of 1:1:1:1 to prepare an immunogen for immunization. The preparation process of the immunogen and the immunization process of laying hens are shown in "2. Preparation of Immunogen - Optimization of Adjuvant Types". Mixed immunized eggs of Propionibacterium acnes, Staphylococcus epidermidis, Staphylococcus aureus, and Escherichia coli were harvested.

[0077] After extracting IgY antibody, the IgY potency of each bacterium was measured by ELISA. The detection steps are shown in "2. Preparation of Immunogen - Optimization of Adjuvant Types".

[0078] The results showed that the IgY potencies (IgY: 1mg / mL) of Propionibacterium acnes, Staphylococcus epidermidis, Staphylococcus aureus, and Escherichia coli egg yolk antibodies in Protocol 1 were respectively: 2 13 ,2 12 ,2 14 ,2 15 。The IgY potencies (IgY: 1mg / mL) of Propionibacterium acnes, Staphylococcus epidermidis, Staphylococcus aureus, and Escherichia coli egg yolk antibodies in Protocol 2 were respectively: 2 12 ,2 11 ,2 13 ,2 14 。It can be seen that immunizing with Propionibacterium acnes, Staphylococcus epidermidis, Staphylococcus aureus, and Escherichia coli antigens separately resulted in an IgY antibody potency approximately 2 times higher than that obtained by immunizing them after mixing. Therefore, the immunization effect of separately immunizing with Propionibacterium acnes, Staphylococcus epidermidis, Staphylococcus aureus, and Escherichia coli antigens is better.

[0079] Example 3 Extraction, Purification of IgY Antibody and Preparation of Freeze-dried Composite IgY Antibody

[0080] 1. Extraction and Purification of IgY Antibody - Optimization of Antibody Mixing Ratio

[0081] 1.1 Collect the eggs of the four immunized hens in Scheme 1 of "3. Preparation of Immunogen - Optimization of Antigen Combination" in Example 2 respectively. Mix the four kinds of immunized eggs according to the mass ratio of eggs of Propionibacterium acnes, Staphylococcus epidermidis, Staphylococcus aureus and Escherichia coli of 4:2:2:1. Separate the egg yolks of the immunized hens' eggs with an egg yolk sieve, add 9 times distilled water, stir and mix evenly to prepare an egg yolk solution. Adjust the pH to 5.2 with 1 moL HCl and let it stand and precipitate overnight at 4°C. Centrifuge at 8,000 rpm for 20 minutes and discard the precipitate. Collect the supernatant, slowly add n-octanoic acid to 1% in the supernatant for impurity removal, stir and mix evenly. Let it stand at 4°C for 2 h, centrifuge at 4°C and 8,000 rpm for 30 minutes, collect the supernatant for ultrafiltration concentration. After concentrating to 1 / 10 of the original volume, add diatomaceous earth with a final concentration of 3% as a filter aid, and perform vacuum filtration (0.09 MPa) with a 0.45 μm mixed cellulose ester membrane to obtain a filtrate. Select a 50,000 molecular weight cut-off ultrafiltration membrane, feed at room temperature, and perform ultrafiltration concentration on the filtrate in a single-stage batch operation mode, concentrating to 20% of the volume after the first concentration (a total of 50 times of concentration after two ultrafiltration concentrations). After pasteurization and filtration sterilization with a 0.22 μm membrane, the separated and purified IgY antibody is obtained.

[0082] Perform SDS-PAGE electrophoresis analysis on the separated and purified IgY antibody. Sample preparation: 3% stacking gel, 8% separating gel, load 8 μL of sample, voltage 80 V, for 60 minutes. Stop electrophoresis when the indicator reaches 1-2 cm in front of the separating gel. Use Coomassie brilliant blue staining and decolorization. Take pictures. The results are as Figure 1 shown. It can be seen that the heavy chain of the IgY antibody is 67-70 KD, the light chain is 22-30 KD, and the purity can reach 51%.

[0083] 1.2 Collect the eggs of the four kinds of immunized hens in Protocol 1 of "3. Immunogen Preparation - Antigen Combination Optimization" in Example 2 respectively. Mix the four kinds of immunized eggs according to the mass ratio of eggs of Propionibacterium acnes, Staphylococcus epidermidis, Staphylococcus aureus and Escherichia coli of 1:1:1:1. Separate the egg yolks of the immunized hens with an egg yolk sieve, add 9 times distilled water, stir and mix evenly to prepare an egg yolk solution, adjust the pH to 5.2 with 1 moL HCl, and let it stand and precipitate overnight at 4°C. Centrifuge at 8,000 rpm for 20 minutes and discard the precipitate. Collect the supernatant, slowly add n-octanoic acid to 1% in the supernatant for impurity removal, stir and mix evenly. Let it stand at 4°C for 2 h, centrifuge at 4°C and 8,000 rpm for 30 minutes, and collect the supernatant for ultrafiltration and concentration to 1 / 10 of the original volume; add diatomaceous earth with a final concentration of 3% as a filter aid, and perform vacuum filtration (0.09 MPa) with a 0.45 μm mixed cellulose ester membrane to obtain a filtrate. Select a 50,000 molecular weight cut-off ultrafiltration membrane, feed at room temperature, and perform ultrafiltration and concentration on the filtrate in a single-stage intermittent operation mode until it is concentrated to 20% of the volume after the first concentration. After pasteurization and filtration and sterilization with a 0.22 μm membrane, the separated and purified IgY antibody is obtained.

[0084] Use ELISA to measure the titers of the above two ratios of the antibody IgY. The detection steps are shown in "2. Immunogen Preparation - Adjuvant Type Optimization".

[0085] The results showed that the titers (IgY: 1 mg / mL) of the egg yolk antibody IgY extracted by mixing the eggs immunized with Propionibacterium acnes, Staphylococcus epidermidis, Staphylococcus aureus and Escherichia coli antigens at 1:1:1:1 were respectively: 2 13 , 2 12 , 2 14 , 2 15 . And the titers (IgY: 1 mg / mL) of the egg yolk antibody IgY extracted by mixing at a ratio of 4:2:2:1 were respectively: 2 15 , 2 12 , 2 14 , 2 14 . It can be seen that the titer of the egg yolk antibody IgY of Propionibacterium acnes, the main pathogen of acne, extracted by mixing at a ratio of 4:2:2:1 is higher than that of the egg yolk antibody IgY extracted by mixing at 1:1:1:1. Select the egg yolk antibody IgY prepared by mixing at a ratio of 4:2:2:1 to prepare a lyophilized powder for subsequent experiments.

[0086] 2. Preparation of composite IgY antibody lyophilized powder

[0087] Use 5% trehalose, 3% mannitol, 0.1% poloxamer 188, 2% sucrose, 3% glycine, 0.5% Tween 80 and 0.2% gelatin as cryoprotectants to perform lyophilization treatment on the IgY antibody. The lyophilization steps are as follows:

[0088] a) Pre-freezing: Place the vial containing the IgY antibody solution on the shelf of the freeze dryer. Set the temperature to 5°C for pre-freezing. Set the time to 30 minutes to reach the preset temperature, and maintain for 1 hour. Then cool down to -60°C, set the time to 10 minutes to reach the preset temperature, and maintain for 6 hours for freezing.

[0089] b) Sublimation drying: Raise the temperature of the shelf system to -25°C. Set the time to 10 hours to reach the preset temperature, and maintain for 25 hours. At the same time, maintain the vacuum degree at 0.2 m Bar to complete sublimation drying.

[0090] c) Re-drying: Raise the temperature of the shelf system to 30°C. Set the time to 100 minutes to reach the preset temperature, and maintain for 10 hours for drying. At the same time, maintain the vacuum degree at 0.2 m Bar to end the freeze-drying. This is the freeze-dried powder of the composite IgY antibody for preventing and treating acne. The content is 500 mg IgY / g freeze-dried powder, and the purity is 50%.

[0091] Example 4 In vitro antibacterial experiment

[0092] The minimum inhibitory concentration (MIC) is an index for evaluating the sensitivity of microorganisms to drugs. Using the freeze-dried powder of the composite IgY antibody in Example 3 as the test drug, and Propionibacterium acnes, Staphylococcus epidermidis, Staphylococcus aureus, and Escherichia coli as the test strains. The specific test process is as follows:

[0093] 1. Preparation of bacterial suspension

[0094] (1) Inoculate the bacteria into Mueller-Hinton broth (MHB) medium respectively and culture until the logarithmic growth phase.

[0095] (2) Adjust the bacterial solution concentration to 0.5 McFarland standard (1.5×10 8 CFU / mL) with sterile normal saline.

[0096] (3) Further dilute the bacterial solution with the medium to 1×10 6 CFU / mL (working concentration).

[0097] 2. Drug dilution

[0098] Prepare the stock solution of the composite IgY antibody (1024 μg / mL). In a 96-well plate, perform 2-fold serial dilution of the IgY antibody with the medium, from 512 μg / mL to 2 μg / mL. Add 100 μL of the diluted IgY antibody solution to each well.

[0099] 3. Inoculate bacteria

[0100] Add 100 μL of the bacterial suspension at the working concentration to each well. At the same time, set up controls: positive control: add only the bacterial suspension without the drug; negative control: add only the culture medium without bacteria and the drug.

[0101] 4. Incubation

[0102] Place the 96-well plate in an incubator at 37°C. Propionibacterium acnes is cultured anaerobically, and other bacteria are cultured under normal conditions for 24 hours.

[0103] 5. Result observation

[0104] Use an enzyme-linked immunosorbent assay (ELISA) reader to measure the turbidity of each well. The lowest drug concentration that completely inhibits bacterial growth is the MIC value. The results are shown in Table 1.

[0105] Table 1 Results of MIC value determination

[0106] Bacterial species MIC value (μg / mL) Propionibacterium acnes <![CDATA[2 2 > Staphylococcus epidermidis <![CDATA[2 5 > Staphylococcus aureus <![CDATA[2 3 > Escherichia coli <![CDATA[2 3 >

[0107] Example 5 Preparation of a composite antibody IgY spray for preventing and treating acne for women

[0108] The formula of the composite antibody IgY spray for preventing and treating acne for women is shown in Table 2.

[0109] Table 2 Formula of the composite antibody IgY spray for preventing and treating acne for women

[0110]

[0111]

[0112] The functions of the components in Table 2 are as follows:

[0113] 1. Freeze-dried composite IgY antibody for preventing and treating acne: Specifically binds to acne-related pathogens such as Propionibacterium acnes, Staphylococcus epidermidis, Staphylococcus aureus, and Escherichia coli, and inhibits their growth.

[0114] 2. Tretinoin: Promotes keratin metabolism, reduces follicular occlusion, and inhibits acne formation.

[0115] 3. Cyproterone acetate: Regulates hormone levels and reduces sebum secretion.

[0116] 4. Glycerol: A humectant that maintains skin moisture.

[0117] 5. Sodium guaiacolsulfonate: Anti-inflammatory and soothes the skin.

[0118] 6. Disodium EDTA: A chelating agent that stabilizes the formula.

[0119] 7. Propolis: Antibacterial, anti-inflammatory, and promotes skin repair.

[0120] 8, Hydroxypropyl-β-cyclodextrin: Solubilizer, which helps dissolve hydrophobic ingredients (such as retinoic acid, propolis).

[0121] 9, EGF (Epidermal Growth Factor): Promotes skin repair and regeneration.

[0122] 10, Rose essence: Provides fragrance and enhances the usage experience.

[0123] 11, Chlorhexidine gluconate: Antibacterial agent, preventing microbial contamination.

[0124] 12, Salicylic acid: Keratoregulation.

[0125] 13, Hyaluronic acid: Moisturizing.

[0126] The preparation process of the compound antibody IgY spray for preventing and treating acne for women only is as follows:

[0127] 1. Prepare raw materials:

[0128] Ensure that all raw materials meet the quality standards, especially the active ingredients (such as IgY antibody, retinoic acid, EGF, etc.).

[0129] 2. Dissolve the aqueous phase components:

[0130] Dissolve disodium EDTA, sodium guaiacolsulfonate, salicylic acid and chlorhexidine gluconate in appropriate amount of water in sequence, and stir evenly to obtain an aqueous phase solution.

[0131] 3. Add moisturizer:

[0132] Add glycerol and hyaluronic acid to the aqueous phase solution and stir until completely dissolved.

[0133] 4. Solubilize hydrophobic ingredients:

[0134] Mix propolis, retinoic acid with hydroxypropyl-β-cyclodextrin and stir until completely dissolved to form a uniform solubilizing solution. Then add it to the aqueous phase solution and stir evenly.

[0135] 5. Add active ingredients:

[0136] Add the freeze-dried powder of the compound IgY antibody for preventing and treating acne and EGF to the aqueous phase solution, and gently stir to avoid damaging the protein structure.

[0137] 6. Add other ingredients:

[0138] Add cyproterone acetate and rose essence to the aqueous phase solution and stir evenly.

[0139] 7. Adjust the pH value:

[0140] Adjust the pH value of the solution to 5.5 - 6.5 with appropriate amount of citric acid to adapt to the skin pH environment.

[0141] 8. Fixed volume:

[0142] Add water to the final weight and stir evenly. All water is injection water.

[0143] 9. Filtration and filling:

[0144] Filter the solution with a sterile filter membrane of 0.22 μm to remove impurities and microorganisms. Fill the filtered solution into a sterile spray bottle.

[0145] 10. Quality inspection:

[0146] Conduct microbial detection, pH value determination and active ingredient content determination on the finished product to ensure compliance with quality standards.

[0147] Precautions:

[0148] 1. Aseptic operation: The preparation process needs to be carried out in an aseptic environment to avoid microbial contamination.

[0149] 2. Stability of active ingredients: IgY antibody and EGF are sensitive to temperature and need to be stored and operated at low temperature.

[0150] 3. pH value control: Ensure that the pH value of the final product is suitable for skin use (usually 5.5 - 6.5).

[0151] 4. Skin tolerance test: Conduct skin irritation test on the finished product to ensure safety.

[0152] 5. Regulatory compliance: Ensure that all ingredients and dosages comply with the regulatory requirements of cosmetics or drugs.

[0153] Preparation of a compound antibody IgY spray for preventing and treating acne

[0154] The formula of the compound antibody IgY spray for preventing and treating acne is shown in Table 3.

[0155] Table 3 Formula of the compound antibody IgY spray for preventing and treating acne

[0156] The functions of each component in Table 3 are as follows:

[0157] 1. Compound IgY antibody freeze-dried powder for preventing and treating acne: Specifically binds to acne-related pathogens such as Propionibacterium acnes, Staphylococcus epidermidis, Staphylococcus aureus, and Escherichia coli, and inhibits their growth.

[0158] 2. Tretinoin: Promotes keratin metabolism, reduces follicular blockage, and inhibits acne formation.

[0159] 3. Glycerol: A humectant that keeps the skin moisturized.

[0160] 4. Sodium guaiacolsulfonate: Anti-inflammatory, soothes the skin.

[0161] 5. Disodium EDTA: Chelating agent, stabilizes the formulation.

[0162] 6. Propolis: Antibacterial, anti-inflammatory, promotes skin repair.

[0163] 7. Hydroxypropyl-β-cyclodextrin: Solubilizer, helps dissolve hydrophobic components (such as retinoic acid, propolis).

[0164] 8. EGF (Epidermal Growth Factor): Promotes skin repair and regeneration.

[0165] 9. Osmanthus fragrance essence: Provides aroma, enhances the usage experience.

[0166] 10. Chlorhexidine gluconate: Antibacterial agent, prevents microbial contamination.

[0167] 11. Salicylic acid: Keratolytic regulation.

[0168] 12. Hyaluronic acid: Moisturizing.

[0169] The preparation process of the composite antibody IgY spray for preventing and treating acne is as follows:

[0170] 1. Prepare raw materials:

[0171] Ensure that all raw materials meet the quality standards, especially the active ingredients (such as IgY antibody, retinoic acid, EGF, etc.).

[0172] Ingredient Weight ratio % Anti-acne composite IgY antibody freeze-dried powder (Example 3) 0.30 Tretinoin 0.08 Water 90.62 Glycerol 4.00 Salicylic acid 0.1 Hyaluronic acid 1 Sodium guaiacolsulfonate 0.40 Disodium EDTA 0.10 Propolis 1.50 Hydroxypropyl-β-cyclodextrin 1.00 EGF 0.70 Osmanthus fragrans essence 0.10 Chlorhexidine gluconate 0.10

[0173] 2. Dissolve the aqueous phase components:

[0174] Dissolve disodium EDTA, salicylic acid, sodium guaiacolsulfonate, and chlorhexidine gluconate in appropriate amount of water in sequence, stir evenly to obtain an aqueous phase solution.

[0175] 3. Add the moisturizer:

[0176] Add glycerol and hyaluronic acid to the aqueous phase solution, stir until completely dissolved.

[0177] 4. Solubilize the hydrophobic components:

[0178] Mix propolis and retinoic acid with hydroxypropyl-β-cyclodextrin, stir until completely dissolved to form a uniform solubilized solution. Then add it to the aqueous phase solution and stir evenly.

[0179] 5. Add the active ingredients:

[0180] Add the freeze-dried powder of the composite IgY antibody for preventing and treating acne and EGF to the aqueous phase solution, gently stir to avoid damaging the protein structure.

[0181] 6. Add other ingredients:

[0182] Add osmanthus fragrance essence and stir evenly.

[0183] 7. Adjust the pH value:

[0184] Adjust the pH value of the solution to 5.5 - 6.5 with an appropriate amount of citric acid to adapt to the skin pH environment.

[0185] 8. Make up the volume:

[0186] Add water to the final weight and stir evenly. All water is injection water.

[0187] 9. Filtration and filling:

[0188] Filter the solution with a sterile filter membrane of 0.22 μm to remove impurities and microorganisms. Fill the filtered solution into a sterile spray bottle.

[0189] 10. Quality inspection:

[0190] Conduct microbial detection, pH value determination and active ingredient content determination on the finished product to ensure compliance with quality standards.

[0191] Precautions:

[0192] 1. Aseptic operation: The preparation process needs to be carried out in a sterile environment to avoid microbial contamination.

[0193] 2. Stability of active ingredients: IgY antibody and EGF are sensitive to temperature and need to be stored and operated at low temperature.

[0194] 3. pH value control: Ensure that the pH value of the final product is suitable for skin use (usually 5.5 - 6.5).

[0195] 4. Skin tolerance test: Conduct a skin irritation test on the finished product to ensure safety.

[0196] 5. Regulatory compliance: Ensure that all ingredients and dosages comply with the regulatory requirements of cosmetics or drugs.

[0197] Preparation of the Compound Antibody IgY Liniment for Preventing and Treating Acne

[0198] The formula of the compound antibody IgY liniment for preventing and treating acne is shown in Table 4.

[0199] Table 4 Formula of the Compound Antibody IgY Liniment for Preventing and Treating Acne

[0200] Ingredient Weight ratio % Anti-acne composite IgY antibody freeze-dried powder (Example 3) 0.30 Tretinoin 0.08 Water 12.02 Salicylic acid 0.1 Propolis extract 1.50 Glycerol 19.00 Sorbitol 16.00 Hyaluronic acid 1 Sodium carboxymethyl cellulose 1.50 Calcium hydrogen phosphate 45.00 K12 1.50 EGF 0.80 Sophora japonica flower essence 0.10 Hydroxypropyl-β-cyclodextrin 1.00 Chlorhexidine gluconate 0.10

[0201] The functions of each ingredient in Table 4 are as follows:

[0202] 1. Acne prevention and treatment compound IgY antibody freeze-dried powder: Specifically binds to acne-related pathogens such as Propionibacterium acnes, Staphylococcus epidermidis, Staphylococcus aureus, and Escherichia coli, and inhibits their growth.

[0203] 2. Retinoic acid: Promotes keratin metabolism, reduces follicular blockage, and inhibits acne formation.

[0204] 3. Propolis extract: Antibacterial, anti-inflammatory, and promotes skin repair.

[0205] 4. Glycerol and sorbitol: Humectants that keep the skin hydrated.

[0206] 5. Sodium carboxymethyl cellulose: A thickening agent that provides an appropriate paste texture.

[0207] 6. Calcium hydrogen phosphate: A filler that increases the volume of the paste and improves spreadability.

[0208] 7. K12 (sodium lauryl sulfate): A surfactant that helps to evenly disperse the ingredients.

[0209] 8. EGF (epidermal growth factor): Promotes skin repair and regeneration.

[0210] 9. Sophora japonica flower essence: Provides fragrance and enhances the usage experience.

[0211] 10. Hydroxypropyl-β-cyclodextrin: A solubilizing agent that helps to dissolve hydrophobic ingredients (such as retinoic acid and propolis extract).

[0212] 11. Chlorhexidine gluconate: An antibacterial agent that prevents microbial contamination.

[0213] 12. Salicylic acid: Keratin regulation.

[0214] 13. Hyaluronic acid: Moisturizing.

[0215] The preparation process of the antibody IgY liniment for acne prevention and treatment is as follows:

[0216] 1. Prepare the raw materials:

[0217] Ensure that all raw materials meet the quality standards, especially the active ingredients (such as IgY antibody, retinoic acid, EGF, etc.).

[0218] 2. Dissolve the aqueous phase components:

[0219] Dissolve sodium carboxymethyl cellulose, chlorhexidine gluconate, and K12 in an appropriate amount of water in sequence, and stir evenly.

[0220] 3. Add the humectants:

[0221] Add glycerol and sorbitol to the aqueous phase solution and stir until completely dissolved.

[0222] 4. Solubilize hydrophobic components:

[0223] Mix propolis extract and retinoic acid with hydroxypropyl-β-cyclodextrin, and stir until completely dissolved to form a uniform solubilizing solution. Then add it to the aqueous solution and stir evenly.

[0224] 5. Add active ingredients:

[0225] Add the freeze-dried powder of acne-combating composite IgY antibody and EGF to the aqueous solution, and gently stir to avoid damaging the protein structure.

[0226] 6. Add other ingredients:

[0227] Add Sophora japonica flower essence and stir evenly.

[0228] 7. Add solid ingredients:

[0229] Slowly add calcium hydrogen phosphate to the solution and stir evenly to form a uniform paste.

[0230] 8. Adjust the pH value:

[0231] Adjust the pH value of the paste to 5.5 - 6 with an appropriate amount of citric acid to adapt to the skin pH environment.

[0232] 9. Homogenize:

[0233] Use a homogenizer to homogenize the paste to ensure a delicate and uniform texture.

[0234] 10. Fill:

[0235] Fill the homogenized paste into sterilized aluminum foil and plastic hoses. The water used is injection water.

[0236] 11. Quality inspection:

[0237] Conduct microbiological detection, pH value determination, and active ingredient content determination on the finished product to ensure compliance with quality standards.

[0238] Precautions:

[0239] 1. Aseptic operation: The preparation process needs to be carried out in an aseptic environment to avoid microbial contamination.

[0240] 2. Stability of active ingredients: IgY antibody and EGF are sensitive to temperature and need to be stored and operated at low temperature.

[0241] 3. pH value control: Ensure that the pH value of the final product is suitable for skin use (usually 5.5 - 6.5).

[0242] 4. Skin tolerance test: Conduct a skin irritation test on the finished product to ensure safety.

[0243] 5. Regulatory Compliance: Ensure that all ingredients and dosages comply with the regulatory requirements for cosmetics or pharmaceuticals.

[0244] Preparation of a Facial Mask Liquid Containing IgY Antibody Encapsulated in Nanoscale Gelatin Particles for the Prevention and Treatment of Acne

[0245] The formulation of the facial mask liquid containing IgY antibody encapsulated in nanoscale gelatin particles for the prevention and treatment of acne is shown in Table 5.

[0246] Table 5 Facial Mask Liquid Containing IgY Antibody Encapsulated in Nanoscale Gelatin Particles for the Prevention and Treatment of Acne

[0247]

[0248] The preparation process of the facial mask liquid containing IgY antibody encapsulated in nanoscale gelatin particles for the prevention and treatment of acne is as follows:

[0249] 1. Preparation of Part D:

[0250] 1) Prepare raw materials:

[0251] Ensure that the composite IgY antibody lyophilized powder (Example 3), gelatin, and water meet the quality standards. Use high-purity water (water for injection).

[0252] 2) Dissolve gelatin:

[0253] Add 3.0 g of gelatin to 50 mL of water, heat to 40 - 50 °C, and stir until completely dissolved.

[0254] 3) Dissolve egg yolk antibody:

[0255] Add 0.3 g of the composite IgY antibody lyophilized powder to the remaining water (46.7 mL), and gently stir until completely dissolved.

[0256] 4) Mix the solutions:

[0257] Slowly add the dissolved gelatin solution to the egg yolk antibody solution while stirring to avoid local high concentration.

[0258] 5) Homogenize:

[0259] Use a homogenizer to homogenize the mixed solution to ensure that the solution is uniform and free of particles.

[0260] 6) Adjust the volume:

[0261] Add water to make up the final volume to 100 mL and stir evenly.

[0262] 7) Filter:

[0263] Filter the solution with a 0.22 - μm sterile filter membrane to remove impurities and microorganisms.

[0264] 8) Subpackage:

[0265] Dispense the filtered solution into sterile containers.

[0266] 9) Quality inspection:

[0267] Perform microbial detection, pH value determination, and antibody activity determination on the finished product to ensure compliance with quality standards.

[0268] Precautions:

[0269] 1) Aseptic operation: The preparation process needs to be carried out in an aseptic environment to avoid microbial contamination.

[0270] 2) Temperature control: When dissolving gelatin, the temperature needs to be controlled at 40 - 50 °C to avoid overheating and damaging the yolk antibody.

[0271] 3) Stirring speed: The stirring speed should not be too fast to avoid damaging the antibody structure.

[0272] 4) pH value control: Ensure that the pH value of the final solution is suitable for antibody stability (usually 6.5 - 7.5).

[0273] 5) Storage conditions: The prepared solution should be stored at 4 °C to avoid repeated freezing and thawing.

[0274] 2. Preparation of the remaining parts

[0275] 1) Prepare raw materials:

[0276] Ensure that all raw materials meet the quality standards and accurately weigh them according to the given proportions.

[0277] 2) Preparation of the oil phase (Part A):

[0278] Mix the oil phase components such as 1,3 - butanediol, 1,2 - pentanediol, glycerol, and panthenol uniformly by volume percentage. In another container, disperse the powdered components such as hydroxyethyl cellulose, carbomer, xanthan gum, allantoin, trehalose, disodium EDTA, and serine in 50% water for injection to form a uniform suspension. Slowly mix the liquids obtained from the above two steps and continuously stir to ensure uniformity. Heat to an appropriate temperature (70 °C) to promote dissolution and dispersion.

[0279] 3) Preparation of the aqueous phase (Part B, C, D, F and part of the water for injection):

[0280] Dissolve the water - soluble components such as betaine, glutamic acid, and mussel adhesive protein in Part B in an appropriate amount of 40% water for injection by mass percentage. Add PEG (Part C) to the above solution and stir evenly. Then slowly add the nano - yolk antibody solution (Part D) to ensure uniform dispersion of the antibody particles. Finally, add chlorhexidine gluconate and antibacterial peptides (Part F).

[0281] 4) Mix the oil phase and the water phase

[0282] At 60°C, slowly pour the water phase into the oil phase, stirring while pouring to avoid local overheating or coagulation. Use a homogenizer or high-speed stirrer to mix thoroughly until a uniform and delicate emulsion is formed.

[0283] 5) Add flavor and remaining water for injection

[0284] When the emulsion cools to room temperature or close to room temperature, add lavender essence (Part E) and stir well. Add the remaining water for injection to adjust the consistency and volume of the final product, adjust the pH to 6.5-7.0 with citric acid, and stir well.

[0285] 6) Quality inspection and packaging

[0286] The prepared facial mask liquid is subjected to quality inspection, including the inspection of indicators such as appearance, stability, pH value, microbial limit, etc. After passing the quality inspection, the facial mask is filled into sterile plastic or glass bottles and sealed.

[0287] Note:

[0288] 1) During the entire preparation process, the temperature, stirring speed and mixing time should be strictly controlled to ensure the quality and stability of the product.

[0289] 2) All raw materials should be fully pre-treated and sterilized before use to avoid contamination.

[0290] 3) Personal protection should be taken during the preparation process to avoid direct contact of raw materials with skin and eyes.

[0291] 4) After preparation, the equipment and work area should be thoroughly cleaned and disinfected.

[0292] Example 9 Clinical Trial

[0293] This example conducts a clinical validation test on the products prepared in Examples 5-8. The process is as follows:

[0294] 1. Inclusion criteria of trial population

[0295] 1. Age range: 12-40 years old, no gender restrictions.

[0296] 2. Acne diagnosis: Meet the clinical acne diagnostic criteria (acne vulgaris) and be classified as moderate (grade II) to severe (grade IV) according to the Global Acne Grading System (GEA).

[0297] Grade II (moderate): mainly papules and pustules, with a total of 20-50 facial lesions.

[0298] Grade III (severe): numerous papules, pustules, and nodules (≤5).

[0299] Grade IV (extremely severe): Extensive inflammatory skin lesions (nodules, cysts).

[0300] 3. Baseline requirements: No systemic antibiotics, retinoids or hormone therapy within 4 weeks before enrollment.

[0301] 4. Compliance: Sign the informed consent form and promise to cooperate with follow-up (guardian consent is required for adolescents).

[0302] II. Exclusion criteria for the trial population

[0303] 1. Other skin diseases: Complicated with rosacea, hormone-dependent dermatitis, folliculitis and other diseases that may interfere with the assessment.

[0304] 2. Severe systemic diseases: Hepatic and renal insufficiency, immunodeficiency diseases, malignant tumors or pregnant / lactating women.

[0305] 3. History of allergy: A clear history of allergy to the components of the test product.

[0306] 4. Recent treatment history:

[0307] 1) Received photodynamic therapy, chemical peeling or laser treatment within 3 months before enrollment.

[0308] 2) Topically used topical medications containing retinoids, salicylic acid or antibiotics within 1 month before enrollment.

[0309] 5. Others: Unable to cooperate with follow-up or patients with mental diseases.

[0310] III. Efficacy verification criteria

[0311] 1. Primary endpoint (12 weeks after treatment)

[0312] 1) Cured:

[0313] The reduction of inflammatory skin lesions (papules, pustules, nodules) is ≥90%;

[0314] The investigator's global assessment (IGA) score ≤ grade 1 (almost no acne).

[0315] 2) Effective:

[0316] The reduction of inflammatory skin lesions is ≥50% but <90%;

[0317] The IGA score is reduced by ≥2 grades (e.g., from grade IV to grade II).

[0318] 3) Ineffective:

[0319] The reduction of inflammatory skin lesions is <50% or aggravated;

[0320] The IGA score showed no improvement or deterioration.

[0321] 2. Secondary endpoints (at 4 weeks and 8 weeks after treatment)

[0322] Change in lesion count: Reduction rate of non-inflammatory lesions (comedones).

[0323] Quality of life score: Subjective improvement of patients was evaluated through the Acne-Specific Quality of Life Questionnaire (Acne-QoL).

[0324] Safety indicators: Incidence of adverse reactions (such as erythema, desquamation, stinging).

[0325] The table for efficacy evaluation is shown in Table 6.

[0326] Table 6 Table for Efficacy Evaluation

[0327] Therapeutic grade Reduction rate of inflammatory skin lesions Change in IGA score Clinical significance Cured ≥90％ ≤ Grade 1 (none / very mild) Symptoms completely or nearly completely eliminated Effective 50％-89％ Reduction ≥ Grade 2 Significantly improved but not completely cleared Ineffective <50% or aggravated No change or increase Treatment did not meet the expected goal

[0328] IV. Supplementary Instructions

[0329] Blinding design: A double-blind randomized controlled trial (RCT) was adopted, and the IGA score was evaluated by independent assessing physicians to reduce bias.

[0330] Follow-up period: Baseline (0 week), 4 weeks, 8 weeks, 12 weeks. Lesion photos and patient feedback were recorded.

[0331] Statistical method: Intention-to-treat analysis (ITT) was used for the primary endpoint; Chi-square test or Fisher's exact test was used for comparing the effective rates (α = 0.05).

[0332] V. Trial Results

[0333] 1. Usage of the composite antibody IgY spray for preventing and treating acne (Example 6)

[0334] Usage method: Clean the face, and spray the composite antibody IgY spray for preventing and treating acne on the comedones and acne areas once in the morning, once at noon, and once in the evening for 3 months.

[0335] A clinical population experiment of the composite antibody IgY spray for preventing and treating acne was conducted in a tertiary hospital. 600 people were enrolled, aged 12 - 40 years old, with an equal number of males and females. 200 people in the positive control group used 1% metronidazole spray, 200 people in the negative placebo group used normal saline, and 200 people in the experimental group used the composite antibody IgY spray. They were used according to the instructions, spraying once in the morning, once at noon, and once in the evening. After 3 months, the results were statistically analyzed. It was found that the total effective rate of the positive control group was 60%, the total effective rate of the negative placebo group was 6%, and the total effective rate of the experimental group was 90%.

[0336] Specific cases:

[0337] (1) Jiang XX, female, 21 years old. Initial skin condition: Severe acne on the forehead and cheeks. Skin condition after using this product: The skin has restored its tenderness and smoothness, obvious improvement in skin peeling, and significant reduction in acne on the forehead and cheeks. As Figure 2 。

[0338] (2) Qian XX, male, 18 years old. Initial skin condition: Rough, red, and greasy skin, with a large number of acne on the face. Skin condition after using this product: Obvious improvement in the redness phenomenon, the skin becomes dry and non-greasy, and a large number of acne have disappeared. As Figure 3 。

[0339] 2. Usage of the composite antibody IgY spray for preventing and treating acne for women (Example 5)

[0340] Usage method: Clean the face, spray the composite antibody IgY spray for preventing and treating acne for women on the acne and comedone areas, once in the morning, once at noon, and once in the evening, for 3 months.

[0341] A clinical trial of the composite antibody IgY spray for preventing and treating acne for women was conducted in a Class III Grade A hospital. 450 people were enrolled, aged 12 - 40 years old, with an equal number of men and women. 150 people in the positive control group used 1% metronidazole spray, 150 people in the negative placebo group used normal saline, and 150 people in the test group used the composite antibody IgY spray. They were used according to the instructions, sprayed once in the morning, once at noon, and once in the evening. The results were statistically analyzed after 3 months. It was found that the total effective rate of the positive control group was 60%, the total effective rate of the negative placebo group was 4%, and the total effective rate of the test group was 94%.

[0342] Specific cases:

[0343] Liu XX, female, 19 years old. Initial skin condition: Sensitive skin, with a series of skin problems left after skin allergy, and obvious acne. After continuous use of this product: The skin has restored its luster, and the acne has basically disappeared. As Figure 4 。

[0344] 3. Usage of the composite antibody IgY liniment for preventing and treating acne (Example 7)

[0345] Usage method: Clean the face, apply the composite antibody IgY liniment for preventing and treating acne on the acne and comedone areas, once in the morning, once at noon, and once in the evening, for 3 months.

[0346] A clinical population experiment of the composite antibody IgY liniment for preventing and treating acne was conducted in a Class III Grade A hospital. 480 people were enrolled, aged 12 - 40, with an equal number of males and females. 160 people in the positive control group used 1% metronidazole gel, 160 people in the negative placebo group used normal saline, and 160 people in the experimental group used the composite antibody IgY liniment. They were used according to the instructions, applied once in the morning, once at noon, and once in the evening. The results were statistically analyzed after 3 months. It was found that the total effective rate of the positive control group was 65%, the total effective rate of the negative placebo group was 5%, and the total effective rate of the experimental group was 95%.

[0347] Specific cases:

[0348] (1) Feng XX, female, 23 years old. Initial skin condition: Severe acne on the forehead, cheeks, and chin. Skin condition after using this product: The skin has returned to being smooth and hydrated, the skin desquamation has been significantly improved, and the acne on the forehead, cheeks, and chin has significantly reduced. As Figure 5 。

[0349] (2) Ma XX, male, 21 years old. Initial skin condition: Rough, reddened, and oily skin, with a large number of acne on the face. After using this product: The reddening phenomenon has been significantly improved, the skin has become dry and non-greasy, and the number of acne has significantly reduced. As Figure 6 。

[0350] 4. Usage of the nano-gelatin particle encapsulated egg yolk antibody facial mask liquid (Example 8) for preventing and treating acne

[0351] Usage method: Clean the face, take out the supporting compressed facial mask, soak it in the facial mask liquid for more than 10 seconds, apply it on the face for 20 to 30 minutes, and then remove it. Use it once every night for 3 months.

[0352] A clinical population experiment of the nano-gelatin particle encapsulated egg yolk antibody facial mask liquid for preventing and treating acne was conducted in a Class III Grade A hospital. 900 people were enrolled, aged 12 - 40 years old. 300 people in the positive control group used 1% metronidazole solution, 300 people in the negative placebo group used normal saline, and 300 people in the experimental group used the facial mask liquid. They were used according to the instructions, applied the facial mask once every night. The results were statistically analyzed after 3 months. The total effective rate of the positive control group was 67%, the total effective rate of the negative placebo group was 8%, and the total effective rate of the experimental group was 97%.

[0353] Specific cases:

[0354] (1) Dong XX, female, 21 years old. Initial skin condition: Dry, dull, with skin desquamation due to water shortage in winter, and severe acne on the forehead, cheeks, and chin. Skin condition after using this product: The skin has returned to being smooth and hydrated, the skin desquamation has been significantly improved, and the acne on the forehead, cheeks, and chin has significantly reduced. As Figure 7 。

[0355] (2) Yang Moumou, male, 24 years old. Initial skin condition: rough, red, and greasy skin, with a large number of acne on the face. After using this product: The redness phenomenon has been significantly improved, the skin has become dry and non-greasy, and a large number of acne have disappeared. As Figure 8 。

[0356] In summary, the composite IgY antibodies against Propionibacterium acnes, Staphylococcus epidermidis, Staphylococcus aureus, and Escherichia coli prepared by the method of the present invention have a purity of more than 51% as determined by SDS-PAGE; the biological titer of the composite IgY antibodies, as determined by the ELISA method, the titers of specific binding to Propionibacterium acnes, Staphylococcus epidermidis, Staphylococcus aureus, and Escherichia coli are respectively: 2 15 ,2 12 ,2 14 ,2 14 (IgY: 1mg / ml). When the specific composite IgY antibodies against Propionibacterium acnes, Staphylococcus epidermidis, Staphylococcus aureus, and Escherichia coli are topically applied to the facial and whole body skin, there are neither the limitations and side effects of systemic medication, nor the effects of rapid onset, small dosage, good curative effect, non-absorption, non-destruction of the microecological environment of the normal flora of the facial and whole body skin, and non-causing local flora imbalance of local medication, and there are also no toxic and side effects, and no problems such as the easy generation of drug resistance in the local use of antibiotics; the composite IgY antibodies do not activate the complement system, do not non-specifically bind to rheumatoid factors, do not fix protein A of Staphylococcus aureus, do not bind to protein G, and the Fc receptor of human cells, so they are not prone to allergic reactions; the composite IgY antibodies do not produce acute and chronic toxicity reactions, belong to products with actual non-toxicity, no teratogenicity, carcinogenicity, and mutagenicity, and at the same time have no irritation to the conjunctiva, mucosa, and skin, and have higher safety.

[0357] The above-described embodiments are only descriptions of the preferred embodiments of the present invention, and do not limit the scope of the present invention. Without departing from the design spirit of the present invention, various deformations and improvements made by those of ordinary skill in the art to the technical solutions of the present invention should fall within the protection scope determined by the claims of the present invention.

Claims

1. A method for preparing a composite antibody IgY for preventing and treating acne, characterized in that, It includes the following steps: (1) Immunogen preparation: Inactivate Propionibacterium acnes bacteria solution, Staphylococcus epidermidis bacteria solution, Staphylococcus aureus bacteria solution, and Escherichia coli bacteria solution are respectively mixed with ISA78 adjuvant to obtain immunogens of the four kinds of bacteria; (2) Immunization: The immunogens of the four kinds of bacteria are respectively inoculated into laying hens, and immunized eggs of the four kinds of bacteria are collected; (3) Collection and preparation of composite antibody IgY: The immunized eggs of the four kinds of bacteria are mixed, the egg yolks are separated, and after acid treatment and impurity removal treatment, the composite antibody IgY is obtained.

2. The preparation method according to claim 1, wherein The volume ratio of the inactivated Propionibacterium acnes bacterial solution, Staphylococcus epidermidis bacterial solution, Staphylococcus aureus bacterial solution, and Escherichia coli bacterial solution to the ISA 78 adjuvant is all 3:7; the bacterial concentration of the inactivated Propionibacterium acnes bacterial solution, Staphylococcus epidermidis bacterial solution, Staphylococcus aureus bacterial solution, and Escherichia coli bacterial solution is all 8×10 8 -8×10 10 CFU / mL.

3. The preparation method according to claim 1, wherein, The mixing method is high-speed emulsification; when performing high-speed emulsification, the rotation speed is 9000 - 12000 rpm and the time is 3 - 6 minutes.

4. The preparation method according to claim 1, wherein The inoculation method is 3-point injection, with primary immunization 3 times, at intervals of 28 days each time, and 1 mL is injected each time; eggs are collected 14 days after the third immunization.

5. The preparation method according to claim 1, characterized in that, When the immunized eggs of the four kinds of bacteria are mixed, the immunized eggs of Propionibacterium acnes, Staphylococcus epidermidis, Staphylococcus aureus, and Escherichia coli are mixed according to a mass ratio of 4:2:2:

1.

6. The preparation method according to claim 1, characterized in that, The acid treatment and impurity removal treatment method is to mix the egg yolk and water according to a volume ratio of 1:9 to obtain an egg yolk solution; the pH of the egg yolk solution is adjusted to 5.2, and after standing overnight at 4°C, supernatant A is collected; after removing impurities from supernatant A using 1% n-octanoic acid, supernatant B is collected; supernatant B is successively subjected to vacuum filtration, ultrafiltration with a molecular weight cut-off of 50,000, and ultrafiltration concentration to obtain the composite antibody IgY.

7. The composite antibody IgY prepared by the preparation method according to any one of claims 1 - 6.

8. Use of the composite antibody IgY according to claim 7 in the preparation of a product for preventing and treating acne.

9. The application according to claim 8, characterized in that, The product includes spray, liniment, and facial mask liquid.

10. A product for preventing and treating acne, characterized in that, The product contains the composite antibody IgY according to claim 7.

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