Use of milk peptide fermentation product in regulating skin microecological balance

Abstract

The present invention relates to the field of biological fermentation application technology, and in particular, to use of a milk peptide fermentation product in regulating skin microecological balance. The milk peptide fermentation product selectively regulates the growth of Staphylococcus epidermidis and inhibits the growth of Staphylococcus aureus and Escherichia coli. It was discovered for the first time that the milk peptide fermentation product has a selective bacteriostatic effect on skin microorganisms. The experimental results show that the milk peptide fermentation product has an inhibitory effect on Escherichia coli and Staphylococcus aureus, but has no effect on the normal growth of Staphylococcus epidermidis. This characteristic is obviously different from common broad-spectrum chemical bacteriostatic agents, and the use of the milk peptide fermentation product is more conducive to maintaining the balance of skin microbiota.

Description

Application of a milk peptide ferment in regulating the skin's microecological balance Technical Field

[0001] This invention belongs to the field of bio-fermentation application technology, specifically relating to the application of a milk peptide ferment in regulating the skin microecological balance. Background Technology

[0002] Skin microecology refers to an ecosystem composed of microorganisms such as bacteria, fungi, and viruses, as well as tissues, cells, secretions, and microenvironments on the skin surface. These microorganisms interact with the host and the external environment to maintain the balance of the skin microecology, which is crucial for maintaining skin health.

[0003] The skin's surface microbiome is generally divided into resident and transient microbiota. Resident microbiota are a group of microorganisms that reside on healthy skin. Transient microbiota refer to microorganisms acquired through contact with the external environment. Under normal circumstances, the composition and relative abundance of the skin microbiome maintain a dynamic balance within a certain range, playing a role in skin defense, repair, and barrier function. It not only acts as a biological and physical defense against pathogen invasion and produces chemical substances to inhibit the growth of harmful bacteria, but also regulates the body's immune response and participates in the establishment of the skin's innate and adaptive immune barriers, which is crucial for maintaining healthy skin. If the skin microbiome is imbalanced, the skin barrier function is reduced, resistance is weakened, leading to the growth of harmful bacteria and causing skin problems such as inflammation, itching, infection, and recurring acne. Therefore, only by balancing the skin's microbiome can we maintain healthy skin, enhance the skin barrier's ability, and improve various skin problems from the source.

[0004] Currently, bactericides such as isocyanate, alizarin, limonene, and astragalin are commonly added to skincare products to kill harmful bacteria.

[0005] However, long-term use of these broad-spectrum bactericides can easily disrupt the skin's microecological balance, leading to various skin problems. Furthermore, existing technologies only focus on the most basic research on microecological balance, such as promoting the growth of probiotics and inhibiting harmful bacteria, without proving whether the effects of promoting the growth of beneficial bacteria and inhibiting harmful bacteria can be achieved separately when beneficial and harmful bacteria co-grow.

[0006] Therefore, how to effectively regulate and maintain the skin's microecological balance and verify it through experiments is a technical problem that urgently needs to be solved by those skilled in the art. Summary of the Invention

[0007] The purpose of this section is to outline some aspects of embodiments of the present invention and to briefly describe some preferred embodiments. Simplifications or omissions may be made in this section, as well as in the abstract and title of this application, to avoid obscuring the purpose of these documents; however, such simplifications or omissions should not be construed as limiting the scope of the invention.

[0008] In view of the problems existing in the above and / or prior art, the present invention is proposed.

[0009] Therefore, the purpose of this invention is to overcome the shortcomings of the prior art and provide an application of milk peptide fermentation in regulating the skin microecological balance.

[0010] To solve the above-mentioned technical problems, the present invention provides the following technical solution: the application of milk peptide ferment in regulating the skin microecological balance, wherein the milk peptide ferment selectively regulates the growth of Staphylococcus epidermidis, while inhibiting the growth of Staphylococcus aureus and Escherichia coli;

[0011] The preparation methods of milk peptide fermentation products include,

[0012] Milk powder is dissolved in deionized water, protease is added, the pH is adjusted to 7-10, and enzymatic hydrolysis is carried out to obtain milk peptides.

[0013] The obtained milk peptides are mixed with glucose to obtain the fermentation substrate;

[0014] Lactobacillus plantarum was inoculated into the fermentation substrate, fermented, sterilized, and the supernatant was collected by centrifugation and freeze-dried to obtain milk peptide fermentation product.

[0015] The Lactobacillus plantarum has the following preservation number: CCTCC No: M 20232264.

[0016] Another objective of this invention is to overcome the shortcomings of the prior art and provide an application of milk peptide fermentation products in the preparation of cosmetics that regulate the skin's microecological balance.

[0017] In a preferred embodiment of the application described in this invention, the method for preparing the milk fermentation product includes,

[0018] Milk powder is dissolved in deionized water, protease is added, the pH is adjusted to 7-10, and enzymatic hydrolysis is carried out to obtain milk peptides.

[0019] The obtained milk peptides are mixed with glucose to obtain the fermentation substrate;

[0020] Lactobacillus plantarum was inoculated into the fermentation substrate, fermented, sterilized, and the supernatant was collected by centrifugation and freeze-dried to obtain milk peptide fermentation product.

[0021] The Lactobacillus plantarum has the following preservation number: CCTCC No: M 20232264.

[0022] As a preferred embodiment of the application described in this invention, the cosmetics include moisturizing creams, toners, lotions, serums, and masks.

[0023] As a preferred embodiment of the application described in this invention, the moisturizing cream comprises polydimethylsiloxane, squalane, isopropyl myristate, glyceryl stearate, sorbitan monostearate, glycerin, butylene glycol, disodium EDTA, tocopherol, carbomer, milk peptide ferment, phenoxyethanol, ethylhexylglycerin, and water.

[0024] In a preferred embodiment of the application described in this invention, the milk peptide fermentation product accounts for 10-15% of the total mass of the moisturizing cream ingredients.

[0025] As a preferred embodiment of the application described in this invention, the cosmetic product has the effects of purifying, conditioning, and moisturizing.

[0026] In a preferred embodiment of the application described in this invention, the purification process involves inhibiting the colonization of harmful bacteria.

[0027] In a preferred embodiment of the application described in this invention, the conditioning is to maintain the skin's microecological balance.

[0028] As a preferred embodiment of the application described in this invention, the cosmetic is used for daily skin care, sensitive skin care, acne-prone skin care, infant skin care, or scalp care.

[0029] Beneficial effects of this invention:

[0030] This invention is the first to discover that milk peptide fermentation products have a selective antibacterial effect on skin microorganisms. Experimental results show that milk peptide fermentation products have an inhibitory effect on Escherichia coli and Staphylococcus aureus, but have no effect on the normal growth of Staphylococcus epidermidis. This characteristic is significantly different from common broad-spectrum chemical antibacterial agents. Using milk peptide fermentation products is more conducive to maintaining the balance of skin flora. Attached Figure Description

[0031] To more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings used in the description of the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort. Wherein:

[0032] Figure 1 shows the results of the competitive antibacterial experiment of Example 6, Comparative Example 8 and Comparative Example 11 after 12 hours. Detailed Implementation

[0033] To make the above-mentioned objects, features and advantages of the present invention more apparent and understandable, the specific embodiments of the present invention will be described in detail below with reference to the examples in the specification.

[0034] Many specific details are set forth in the following description in order to provide a full understanding of the invention. However, the invention may also be practiced in other ways different from those described herein, and those skilled in the art can make similar extensions without departing from the spirit of the invention. Therefore, the invention is not limited to the specific embodiments disclosed below.

[0035] Secondly, the term "one embodiment" or "embodiment" as used herein refers to a specific feature, structure, or characteristic that may be included in at least one implementation of the present invention. The phrase "in one embodiment" appearing in different places in this specification does not necessarily refer to the same embodiment, nor is it a single or selective embodiment that is mutually exclusive with other embodiments.

[0036] Unless otherwise specified, all chemical reagents and materials used in the following examples are obtained commercially.

[0037] The *Lactobacillus plantarum* used in the preparation of milk peptide fermentation products in the following examples was deposited at the China Center for Type Culture Collection on November 17, 2023, with accession number CCTCC No: M20232264, the same as *Lactobacillus plantarum* in CN118146346A.

[0038] Example 1

[0039] Methods for sample antibacterial testing:

[0040] Staphylococcus epidermidis (BNCC 102555), Escherichia coli (ATCC 8099), and Staphylococcus aureus (ATCC 6538) were selected as test bacteria;

[0041] Staphylococcus epidermidis is a normal flora found on human skin, and it can secrete exogenous antimicrobial peptides to enhance the skin's immune defense. Escherichia coli and Staphylococcus aureus are common pathogenic bacteria on the skin, usually causing papules or pustules.

[0042] (1) Preparation of bacterial suspension: After the bacterial strain was cultured in a shaker at 37°C to the logarithmic growth phase, it was centrifuged at 3500 rpm for 3 minutes, the original culture medium was discarded, and the suspension was resuspended in NB medium to make the colony count 10. 6 CFU / mL.

[0043] (2) Turbidimetric test: The antibacterial test was conducted using the turbidimetric method.

[0044] In the sample group, 100 μL of bacterial culture with adjusted concentration and 100 μL of prepared sample were added to a 96-well plate;

[0045] Add 100 μL of culture medium and 100 μL of the prepared sample to the blank sample group.

[0046] 100 μL of bacterial suspension with adjusted concentration and 100 μL of NB medium were added to the negative control group.

[0047] Add 200 μL of sterile NB medium to the negative control blank group.

[0048] After incubating the 96-well plate in a 37°C incubator for 24 hours, the absorbance at 600 nm was measured using a microplate reader.

[0049] (3) Calculate bacterial survival rate:

[0050] Survival rate (%) = (Absorbance value of sample group - Absorbance value of blank sample group) / (Absorbance value of negative control group - Absorbance value of negative control blank group) × 100%

[0051] Example 2

[0052] Preparation method of milk peptide fermentation products:

[0053] 1) Preparation of milk peptides:

[0054] Add milk powder to deionized water at a ratio of 7:50g / mL, and add protease to deionized water at a ratio of 1:200g / mL. Dissolve the protease in a 50°C water bath until fully dissolved.

[0055] Mix milk and protease solution at a ratio of 6:1 (v / v), adjust pH to 8.0, and incubate in a 50°C water bath for 3.5 hours.

[0056] After enzymatic hydrolysis, the temperature is raised to 90°C and the mixture is placed in a water bath for 10 minutes to inactivate the enzyme. Then, it is cooled to room temperature and the pH is adjusted to 6.5 to obtain the milk peptide.

[0057] 2) Preparation of milk peptide fermentation products:

[0058] Dissolve 5.6g of glucose in 40mL of deionized water. Place the glucose solution and milk peptides separately in an autoclave and sterilize at 115℃ for 15min. Then, take 10mL of the sterilized glucose solution and add it to 60mL of milk peptide solution to obtain the fermentation substrate.

[0059] Take 20 mL of Lactobacillus plantarum (CCTCC No: M 20232264) in the logarithmic growth phase into a 50 mL sterile centrifuge tube, centrifuge at 5000 rpm for 5 min, remove the supernatant, resuspend the bacterial cells in 0.9% sterile physiological saline, repeat the above centrifugation and washing steps twice, and resuspend the centrifuged bacterial cells in 20 mL of 0.9% physiological saline.

[0060] Take 3.5 mL of the resuspended bacterial solution and inoculate it into the fermentation substrate prepared above. Ferment at 37℃ and 200 rpm for 48 h in a shaker. After fermentation, centrifuge the fermentation broth at 10000 rpm for 10 min, collect the supernatant, filter it with a 0.22 μm filter membrane to remove bacterial cells and impurities, and freeze dry to obtain milk peptide fermentation product.

[0061] Example 3:

[0062] In this embodiment, the milk peptide fermentation product obtained in Example 2 was dissolved in sterile NB medium to prepare a sample solution with a concentration of 20% (w / v). After sterilization by filtration through a 0.22 μm aqueous filter membrane, an antibacterial experiment was conducted.

[0063] Example 4:

[0064] In this embodiment, the milk peptide fermentation product obtained in Example 2 was dissolved in sterile NB medium to prepare a sample solution with a concentration of 30% (w / v). After sterilization by filtration through a 0.22 μm aqueous filter membrane, an antibacterial experiment was conducted.

[0065] Comparative Example 1:

[0066] This comparative example uses milk peptides, and the only difference from Example 2 is that Lactobacillus plantarum was not used for fermentation. The obtained samples were then dissolved in sterile NB medium to prepare sample solutions with a concentration of 20% (w / v). After sterilization by filtration through a 0.22 μm aqueous filter membrane, antibacterial experiments were conducted.

[0067] Comparative Example 2:

[0068] This comparative example uses Lactobacillus plantarum culture supernatant, and the only difference from Example 2 is that milk peptides were not used as fermentation substrate;

[0069] The fermentation substrate consists of 10g peptone, 5g beef extract, 5g sodium chloride, 10g glucose, 4g yeast extract, 2g dipotassium hydrogen phosphate, and 1000mL deionized water, with a pH of 6.8-7.0.

[0070] The fermentation supernatant was then dissolved in sterile NB medium to prepare sample solutions with a concentration of 20% (w / v). After sterilization by filtration through a 0.22 μm aqueous filter membrane, antibacterial experiments were conducted.

[0071] Comparative Example 3:

[0072] This comparative example uses direct fermentation of milk powder, differing from Example 2 only in that the milk powder is not enzymatically hydrolyzed. The resulting milk fermentation supernatant was then dissolved in sterile NB medium to prepare sample solutions with a concentration of 20% (w / v). These solutions were then filtered through a 0.22 μm aqueous filter membrane for sterilization before antibacterial experiments.

[0073] Comparative Example 4:

[0074] In this embodiment, the milk peptide fermentation product obtained in Example 2 was dissolved in sterile NB medium to prepare a sample solution with a concentration of 10% (w / v). After sterilization by filtration through a 0.22 μm aqueous filter membrane, an antibacterial experiment was conducted.

[0075] Comparative Example 5:

[0076] In this embodiment, the milk peptide fermentation product obtained in Example 2 was dissolved in sterile NB medium to prepare a sample solution with a concentration of 40% (w / v). After sterilization by filtration through a 0.22 μm aqueous filter membrane, an antibacterial experiment was conducted.

[0077] Comparative Example 6:

[0078] This comparative example used milk peptides, differing from Example 2 only in that it used *Lactobacillus rhamnosus* (CICC6141) for fermentation. The resulting samples were then dissolved in sterile NB medium to prepare 20% (w / v) sample solutions. After sterilization by filtration through a 0.22 μm aqueous filter membrane, antibacterial experiments were conducted.

[0079] Comparative Example 7:

[0080] This comparative example uses milk peptides, and the only difference from Example 2 is that it uses Lactobacillus plantarum (CICC24936) for fermentation.

[0081] The prepared samples were then dissolved in sterile NB medium to prepare sample solutions with a concentration of 20% (w / v). After sterilization by filtration through a 0.22μm aqueous filter membrane, antibacterial experiments were conducted.

[0082] Table 1

[0083] As shown in Table 1, the milk peptide ferments prepared in Examples 3 and 4 significantly inhibited Escherichia coli and Staphylococcus aureus, but had no significant inhibitory effect on the dermatophyte Staphylococcus epidermidis. The ferments of a single strain (Comparative Example 2) showed no significant inhibitory effect on Staphylococcus epidermidis, but exhibited a proliferative effect on Escherichia coli. Meanwhile, the milk peptide (Comparative Example 1) showed a significant growth-promoting effect on all three bacterial strains.

[0084] Furthermore, the unenzymatically hydrolyzed milk fermentation broth (Comparative Example 3) showed significant inhibitory effects on all three strains. The fermentation products from *Lactobacillus rhamnosus* (Comparative Example 6) and *Lactobacillus plantarum* (Comparative Example 7) did not significantly promote the growth of *Staphylococcus epidermidis*, and their inhibition of *Staphylococcus aureus* and *Escherichia coli* was also not significant. These results indicate that the milk peptide fermentation products of Examples 3 and 4 possess selective inhibitory effects against common microorganisms and are potential active ingredients for maintaining skin microecological balance.

[0085] Example 5: Method for competitive inhibition of pathogenic bacteria

[0086] Select Staphylococcus epidermidis (BNCC 102555) and Escherichia coli (ATCC 8099) or Staphylococcus aureus (ATCC 6538) as resident bacteria and combine them with Gram-positive or Gram-negative pathogenic bacteria, as follows:

[0087] (1) Preparation of bacterial suspension: After the bacterial strain was cultured in a shaker at 37°C to the logarithmic growth phase, it was centrifuged at 3500 rpm for 3 minutes, the original culture medium was discarded, and the suspension was resuspended in NB medium to make the colony count of resident bacteria 10. 8 CFU / mL, pathogenic bacteria colony count is 10. 7 CFU / mL.

[0088] (2) Co-culture: The co-culture medium was NB medium. For Staphylococcus epidermidis and Escherichia coli, Staphylococcus epidermidis, Escherichia coli and the sample were added to the shake tube in a 1:1:1 ratio and mixed thoroughly; after incubation at 37℃ for 0, 6 h and 12 h, the colony count of each strain was counted respectively.

[0089] For Staphylococcus epidermidis and Staphylococcus aureus, a co-culture system was established using Transwell insert plates with a 0.4 μm permeable membrane;

[0090] The upper chamber was filled with a 1:2 ratio of sample and Staphylococcus epidermidis, and the lower chamber was filled with a 1:2 ratio of sample and Staphylococcus aureus.

[0091] Incubate all cultures at 37°C with gentle shaking to promote the diffusion of metabolites between compartments. Dilute and plate at 0, 6, and 12 hours, and count colonies.

[0092] The results are shown in Table 3; the control group consisted of NB medium without samples, co-incubated with Staphylococcus epidermidis and Escherichia coli or Staphylococcus aureus.

[0093] Example 6:

[0094] In this embodiment, the milk peptide fermentation product obtained in Example 2 was dissolved in sterile NB medium to prepare a sample solution with a concentration of 30% (w / v). After sterilization by filtration through a 0.22 μm aqueous filter membrane, a competitive antibacterial experiment was conducted.

[0095] Comparative Example 8:

[0096] This comparative example uses milk powder peptides, and the only difference from Example 2 is that Lactobacillus plantarum was not used for fermentation.

[0097] The prepared samples were then dissolved in sterile NB medium to prepare sample solutions with a concentration of 30% (w / v). After sterilization by filtration through a 0.22 μm aqueous filter membrane, competitive inhibition experiments were conducted.

[0098] Comparative Example 9:

[0099] This comparative example uses Lactobacillus plantarum culture supernatant, and the only difference from Example 2 is that milk powder hydrolysate was not used as the fermentation substrate.

[0100] The fermentation substrate consists of 10g peptone, 5g beef extract, 5g sodium chloride, 10g glucose, 4g yeast extract, 2g dipotassium hydrogen phosphate, and 1000mL deionized water, with a pH of 6.8-7.0.

[0101] The fermentation supernatant was then dissolved in sterile NB medium to prepare sample solutions with a concentration of 30% (w / v). After sterilization by filtration through a 0.22 μm aqueous filter membrane, a competitive inhibition experiment was conducted.

[0102] Comparative Example 10:

[0103] This comparative example uses direct fermentation of milk powder, differing from Example 2 only in that the milk powder is not enzymatically hydrolyzed. The resulting milk fermentation supernatant was then dissolved in sterile NB medium to prepare sample solutions with a concentration of 30% (w / v). After sterilization by filtration through a 0.22 μm aqueous filter membrane, a competitive antibacterial experiment was conducted.

[0104] Comparative Example 11:

[0105] This comparative example used sterile NB medium, filtered through a 0.22 μm aqueous filter membrane, and then conducted a competitive antibacterial experiment.

[0106] The results of the competitive antibacterial experiment after 12 hours for Examples 6, 8, and 11 are shown in Figure 1.

[0107] Comparative Example 12:

[0108] This comparative example uses milk peptides, and the only difference from Example 2 is that it uses Lactobacillus rhamnosus (CICC6141) for fermentation. The obtained samples are then dissolved in sterile NB medium to prepare sample solutions with a concentration of 30% (w / v). After sterilization by filtration through a 0.22 μm aqueous filter membrane, a competitive antibacterial experiment is conducted.

[0109] Comparative Example 13:

[0110] This comparative example uses milk peptides, and the only difference from Example 2 is that it uses Lactobacillus plantarum (CICC24936) for fermentation. The obtained samples are then dissolved in sterile NB medium to prepare sample solutions with a concentration of 20% (w / v). After filtration and sterilization using a 0.22 μm aqueous filter membrane, a competitive antibacterial experiment is conducted.

[0111] Table 2

[0112] As shown in Table 2, the milk peptide fermentation product of Example 6 can inhibit Staphylococcus aureus and Escherichia coli in the co-culture system, while promoting the growth of Staphylococcus epidermidis, a commensal bacterium of the skin.

[0113] Milk peptides (Comparative Example 8) showed the opposite results. The *Lactobacillus plantarum* ferment broth alone (Comparative Example 9), and the ferment broth fermented with *Lactobacillus rhamnosus* (Comparative Example 12) and *Lactobacillus plantarum* (Comparative Example 13), failed to significantly promote the growth of *Staphylococcus epidermidis* or regulate the ratio of harmful to beneficial bacteria. Unenzymatically hydrolyzed milk powder ferment broth (Comparative Example 10) showed inhibitory effects on all three strains. In summary, milk peptide ferment broth possesses selective inhibitory effects on common microorganisms on the skin, a characteristic not found in milk peptides and milk ferment broths. Unlike known broad-spectrum antibacterial agents, milk peptide ferment broth helps regulate the skin's microbiome balance and maintain healthy skin homeostasis.

[0114] Example 7: Method for irritation test of chicken embryo chorioallantoic membrane

[0115] The chicken embryo chorioallantoic membrane test can detect whether a product is irritating. The procedure is as follows:

[0116] (1) Preparation of chicken embryo chorioallantoic membrane (CAM): After incubating SPF eggs for nine days, candling is performed to select eggs rich in blood vessels and check the location of the air cell of the chicken embryo. The shell is gently broken with a punch, and the part of the shell corresponding to the air cell is peeled off with curved tweezers to expose the white egg membrane. 0.9% physiological saline is added to wet the inner membrane. The inner membrane is carefully removed with tweezers, ensuring that the vascular membrane is not damaged.

[0117] (2) Test: Gently place the test ring onto the surface of the blood vessel membrane with tweezers, add 200 μL of sample into the ring, and start timing at the same time;

[0118] Observe and record the bleeding time (secH), clotting time (secC), and vascularization time (secL) of the membrane at 0, 0.5 min, 3 min, and 5 min. Five parallel experiments were conducted for each sample.

[0119] Bleeding: Blood flows out from the blood vessels or capillaries of the CAM.

[0120] Coagulation: Protein denaturation inside and outside blood vessels, usually manifested as slowed blood flow or thrombosis inside the blood vessels, the blood vessels appear brownish-black, and the outside of the blood vessels appears cloudy and opaque.

[0121] Vascular dissolution: The walls of small blood vessels on the CAM membrane rupture, and the blood vessels dissolve and disappear.

[0122] (3) Calculation and evaluation of results: The results are expressed in terms of stimulus score (IS). The IS is calculated according to the following formula, and the stimulus intensity is judged according to Table 4.

[0123] Table 3

[0124] Example 8:

[0125] In this embodiment, the milk peptide fermentation product obtained in Example 2 was dissolved in sterile deionized water to prepare a sample solution with a mass fraction of 10% for subsequent chicken embryo chorioallantoic membrane irritation test.

[0126] Comparative Example 14:

[0127] This comparative example used milk powder peptides, differing from Example 2 only in that it did not use *Lactobacillus plantarum* for fermentation. The resulting samples were then dissolved in sterile deionized water to prepare 10% (w / w) sample solutions for subsequent chicken embryo chorioallantoic membrane irritation tests.

[0128] Comparative Example 15:

[0129] This comparative example used *Lactobacillus plantarum* culture supernatant, differing from Example 2 only in that milk peptides were not used as the fermentation substrate. The fermentation substrate consisted of 10g peptone, 5g beef extract, 5g sodium chloride, 10g glucose, 4g yeast extract, 2g dipotassium hydrogen phosphate, and 1000mL deionized water, with a pH of 6.8-7.0. The obtained fermentation supernatant was then dissolved in sterile deionized water to prepare 10% (w / w) sample solutions for subsequent chicken embryo chorioallantoic membrane irritation experiments.

[0130] Comparative Example 16:

[0131] This comparative example uses direct fermentation of milk powder, differing from Example 2 only in that the milk powder is not enzymatically hydrolyzed. The resulting milk fermentation supernatant is then dissolved in sterile deionized water to prepare sample solutions with a mass fraction of 10% for subsequent chicken embryo chorioallantoic membrane irritation experiments.

[0132] Table 4

[0133] Experimental conclusion: This milk peptide fermentation product is not irritating.

[0134] Example 9:

[0135] The preparation method of moisturizing face cream specifically includes the following steps:

[0136] Table 5

[0137] 1. Place phases A and B separately in an 85°C water bath and heat until completely dissolved.

[0138] 2. Homogenize phase B using a homogenizer for 3 minutes (9000-10000 rpm).

[0139] 3. Slowly add phase A to the homogenized phase B and continue homogenizing for 5 minutes (9000-10000 rpm).

[0140] 4. When the temperature of the above mixture drops to 45°C, add phase C, cool to room temperature with low-speed stirring, and let stand for 24 hours.

[0141] Comparative Example 17:

[0142] The preparation method of the face cream differs from that of Example 9 in that the milk peptide fermentation product is replaced with milk peptides, and specifically includes the following steps:

[0143] Table 6:

[0144] 1. Place phases A and B separately in an 85°C water bath and heat until completely dissolved.

[0145] 2. Homogenize phase B using a homogenizer for 3 minutes (9000-10000 rpm).

[0146] 3. Slowly add phase A to the homogenized phase B and continue homogenizing for 5 minutes (9000-10000 rpm).

[0147] 4. When the temperature of the above mixture drops to 45°C, add phase C, cool to room temperature with low-speed stirring, and let stand for 24 hours.

[0148] Comparative Example 18:

[0149] The preparation method of the face cream differs from that of Example 9 in that the milk peptide fermentation product is replaced with the supernatant of Lactobacillus plantarum culture, and specifically includes the following steps:

[0150] Table 7:

[0151] 1. Place phases A and B separately in an 85°C water bath and heat until completely dissolved.

[0152] 2. Homogenize phase B using a homogenizer for 3 minutes (9000-10000 rpm).

[0153] 3. Slowly add phase A to the homogenized phase B and continue homogenizing for 5 minutes (9000-10000 rpm).

[0154] 4. When the temperature of the above mixture drops to 45°C, add phase C, cool to room temperature with low-speed stirring, and let stand for 24 hours.

[0155] Comparative Example 19:

[0156] The preparation method of the face cream differs from that of Example 9 in that the milk peptide fermentation product is replaced with milk fermentation liquid, and specifically includes the following steps:

[0157] Table 8:

[0158] 1. Place phases A and B separately in an 85°C water bath and heat until completely dissolved.

[0159] 2. Homogenize phase B using a homogenizer for 3 minutes (9000-10000 rpm).

[0160] 3. Slowly add phase A to the homogenized phase B and continue homogenizing for 5 minutes (9000-10000 rpm).

[0161] 4. When the temperature of the above mixture drops to 45°C, add phase C, cool to room temperature with low-speed stirring, and let stand for 24 hours.

[0162] Comparative Example 20:

[0163] The preparation method of the face cream differs from that of Example 9 in that the milk peptide fermentation product is replaced with water, and specifically includes the following steps:

[0164] Table 9:

[0165] 1. Place phases A and B separately in an 85°C water bath and heat until completely dissolved.

[0166] 2. Homogenize phase B using a homogenizer for 3 minutes (9000-10000 rpm).

[0167] 3. Slowly add phase A to the homogenized phase B and continue homogenizing for 5 minutes (9000-10000 rpm).

[0168] 4. When the temperature of the above mixture drops to 45°C, add phase C, cool to room temperature with low-speed stirring, and let stand for 24 hours.

[0169] Example 10: Experimental Method for Influence of Samples on Skin Moisturization

[0170] The skin hydration of 20 volunteers (5 in each group) was tested using a skin moisture meter. A 4cm x 4cm test area was marked on the flexor surface of each subject's left and right forearms, and samples were applied to each area. The application amount was 3.0 mg / cm². 2 Gently massage until the sample is absorbed. Measure the skin moisture content 4 hours before and after application, and calculate the increase in skin moisture content using the following formula.

[0171] Skin moisture content increase rate (%) = (value measured after application - baseline value before application) / baseline value before application × 100%.

[0172] The experiment was conducted using face creams prepared with the addition of Example 9 and Comparative Examples 12-15. The experimental results are shown in Table 10.

[0173] Table 10

[0174] As shown in Table 10, compared with the comparative example, Example 9 significantly increased the skin moisture content of the subjects.

[0175] In summary, the milk peptide fermentation product of this invention can regulate the balance of the skin microbiome, has a strong moisturizing effect, and is safe and non-irritating, thus alleviating skin problems.

[0176] It should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit it. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention, and all such modifications or substitutions should be covered within the scope of the present invention.

Claims

1. Use of a milk peptide ferment for regulating the skin microecological balance, characterized in that: The milk peptide ferment selectively regulates the growth of Staphylococcus epidermidis while inhibiting the growth of Staphylococcus aureus and Escherichia coli. The preparation methods of milk peptide fermentation products include, Milk powder is dissolved in deionized water, protease is added, the pH is adjusted to 7-10, and enzymatic hydrolysis is carried out to obtain milk peptides. The obtained milk peptides are mixed with glucose to obtain the fermentation substrate; Lactobacillus plantarum was inoculated into the fermentation substrate, fermented, sterilized, and the supernatant was collected by centrifugation and freeze-dried to obtain milk peptide fermentation product. The Lactobacillus plantarum has the following preservation number: CCTCC No: M 20232264.

2. The application of a milk peptide ferment in the preparation of cosmetics that regulate the skin's microecological balance.

3. Use according to claim 2, wherein: The method for preparing the milk peptide ferment includes: Milk powder is dissolved in deionized water, protease is added, the pH is adjusted to 7-10, and enzymatic hydrolysis is carried out to obtain milk peptides. The obtained milk peptides are mixed with glucose to obtain the fermentation substrate; Lactobacillus plantarum was inoculated into the fermentation substrate, fermented, sterilized, and the supernatant was collected by centrifugation and freeze-dried to obtain milk peptide fermentation product. The Lactobacillus plantarum has the following preservation number: CCTCC No: M 20232264.

4. Use according to claim 2 or 3, characterized in that: The cosmetics include moisturizing creams, toners, lotions, serums, and face masks.

5. The use according to claim 4, wherein: The moisturizing cream comprises dimethicone, squalane, isopropyl myristate, glyceryl stearate, sorbitan monostearate, glycerin, butylene glycol, disodium EDTA, tocopherol, carbomer, milk peptide ferment, phenoxyethanol, ethylhexylglycerin, and water.

6. Use according to claim 5, wherein: The milk peptide fermentation product accounts for 10-15% of the total mass of the moisturizing cream ingredients.

7. The use according to claim 2, wherein: The cosmetics have purifying, conditioning, and moisturizing effects.

8. Use according to claim 7, wherein: The purification process involves inhibiting the colonization of harmful bacteria.

9. The use according to claim 7, wherein: The aforementioned conditioning aims to maintain the skin's microecological balance.

10. The use according to claim 2, characterized in that: The cosmetics are used for daily skin care, sensitive skin care, acne-prone skin care, infant skin care, or scalp care.

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