Application of metagen extract in improvement of skin aging or preparation of preparation for improving skin aging and preparation

Abstract

The invention relates to an effect of a metagen extract for improving skin photoaging. Lactobacillus bulgaricus is fermented, inactivated, centrifuged, filtered and dried to obtain a metagen extract, and the main components of the metagen extract are active substances such as polypeptide, polysaccharide, protein and the like; the metagen extract has the functions of resisting oxidation, promoting human skin cell proliferation, promoting collagen synthesis, reducing inflammatory response and the like, also has the effects of improving UVB-induced skin photoaging and the like, can be used as a medicine and / or a skin care product for resisting aging, promoting skin repair and reducing inflammatory response, and has a wide application prospect. The compound is expected to be applied to novel therapeutic gel, ointment, essence, face cream and functional masks.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology and relates to a postbiotic extract. The extract is prepared by fermentation of Lactobacillus bulgaricus, inactivation of the bacteria, membrane filtration and the like, and has the functions of anti-oxidation, anti-inflammation, cell proliferation promotion and the like, and improves skin aging (such as photoaging). Background Art

[0002] As the body's largest organ, the skin is constantly exposed to a variety of environmental factors, with ultraviolet radiation being one of the main causes of premature skin aging. With an aging population and increasing environmental pollution, skin photoaging has become a significant health concern, not only affecting appearance but also potentially leading to more serious skin diseases. Currently, most anti-aging products on the market suffer from issues such as short-lasting effects and high irritation, necessitating the development of safer and more effective prevention and treatment options.

[0003] Skin photoaging is a complex phenomenon involving multiple pathological processes. Its development can be divided into three main stages: early oxidative stress damage, mid-stage inflammatory response cascade, and late-stage extracellular matrix degradation. UVB radiation produces reactive oxygen species (ROS) that cause oxidative damage to cells, triggering the release of inflammatory factors and ultimately leading to the degradation of structural components such as collagen. Traditional anti-aging products typically target only a single mechanism and are unable to effectively block the multi-step progression of photoaging. Therefore, the development of active substances with multiple functions has become a research priority.

[0004] Postbiotics, as defined by the International Scientific Association of Probiotics and Prebiotics, are "non-living microbial cells, bacterial lysates, and fermentation metabolites that provide a health benefit to the host." Compared to traditional live bacterial preparations, postbiotics offer significant advantages, including a stable composition, enhanced safety, and less stringent processing and storage requirements. Postbiotics contain a variety of active ingredients, including teichoic acids, proteins, peptides, short-chain fatty acids, exopolysaccharides, and organic acids. These substances work synergistically to exhibit antioxidant, anti-inflammatory, and cell proliferation-promoting properties. These properties hold great promise for their application in skin health, particularly as an effective intervention for the prevention and treatment of photoaging. Summary of the Invention

[0005] The present invention provides a postbiotic extract based on Lactobacillus bulgaricus and its use in medicines and / or skin care products for preventing and improving UVB-induced skin photoaging, anti-inflammatory effects, promoting collagen repair, and anti-oxidation. The postbiotic extract is prepared using a Lactobacillus bulgaricus-specific fermentation process and is rich in active substances such as antimicrobial peptides and antioxidant peptides. Its active ingredients are significantly superior to postbiotics derived from other strains in inhibiting the secretion of inflammatory factors and activating collagen synthesis.

[0006] To achieve the above objectives, the present invention provides a postbiotic extract prepared from milk protein via microbial fermentation using Lactobacillus bulgaricus. The fermentation broth is inactivated and purified using multi-stage membrane separation to yield an extract rich in various active peptides. The extract exhibits significant inhibitory activity against both Gram-positive and Gram-negative bacteria and possesses antioxidant, anti-inflammatory, and cell proliferation-promoting properties, improving skin aging.

[0007] The preparation method of the postbiotic extract comprises the following steps:

[0008] A fermentation medium with a protein content of 5% to 20% is prepared, and lactic acid bacteria are inoculated after sterilization; fermentation is carried out at 30 to 60°C for 10 to 60 hours, and sterilization is carried out after the fermentation is completed to inactivate the bacteria; impurities are removed from the fermentation liquid by centrifugation, and the supernatant is separated and purified by ultrafiltration and nanofiltration membranes, and dried into powder to obtain the postbiotic extract.

[0009] Bulgarian lactic acid bacteria are fermented, inactivated, centrifuged, filtered and dried to obtain postbiotic extracts, whose main components are active substances such as peptides, polysaccharides, and proteins. Postbiotic extracts have functions such as antioxidant, promoting human skin cell proliferation, promoting collagen synthesis, and reducing inflammatory responses. They also have the effect of improving UVB-induced skin photoaging. They can be used as anti-aging, skin repair and inflammatory response-reducing drugs and / or skin care products, and are expected to be used in new therapeutic gels, ointments, essences, creams and functional facial masks.

[0010] Its anti-inflammatory effect and collagen repair efficiency are significantly better than those of postbiotics derived from Streptococcus thermophilus, Lactobacillus paracasei and Lactobacillus helveticus prepared by the same process.

[0011] Compared with the prior art, the present invention has the following beneficial effects:

[0012] (1) By comparing the active ingredients of the fermentation products of Lactobacillus bulgaricus, Streptococcus thermophilus, Lacticaseibacillus paracasei and Lactobacillus helveticus, it was confirmed that the types and functional diversity of active peptides in the fermentation products of Lactobacillus bulgaricus were significantly better than those of other strains, and it could simultaneously and efficiently express antioxidant peptides, antimicrobial peptides and anti-inflammatory peptides.

[0013] (2) The prepared postbiotic extract is fermented by Lactobacillus bulgaricus to produce unique strain-dependent active ingredients, which have multi-dimensional anti-aging functions such as strong antibacterial, antioxidant, anti-inflammatory and cell proliferation promotion. Its anti-inflammatory and collagen repair effects are significantly better than other bacterial-derived postbiotics;

[0014] (3) The prepared postbiotic extract has outstanding broad-spectrum antibacterial ability and can simultaneously inhibit the growth of Gram-positive bacteria, Gram-negative bacteria and common skin pathogens (such as Escherichia coli, Staphylococcus aureus, and Pseudomonas aeruginosa). Its antibacterial efficacy (IC50 value) and safety are superior to those of similar fermentation products. It also has high solubility and stable purity, making it suitable for the development of various dosage forms. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 Determination of total antioxidant capacity of postbiotic extracts.

[0016] Figure 2 Effects of postbiotic extracts on HaCaT cell proliferation. *P<0.05, **P<0.01 vs 100%.

[0017] Figure 3 Effects of postbiotic extracts on UVB-induced I-COL secretion in HaCaT cells. *P<0.05, **P<0.01 vs. model group.

[0018] Figure 4 Effect of postbiotic extract on UVB-induced IL-6 secretion in HaCaT cells (*P<0.05, **P<0.01 vs. model group). DETAILED DESCRIPTION

[0019] Example 1 Preparation method of postbiotic extract

[0020] (1) Culture medium preparation and fermentation culture

[0021] An aqueous solution of skimmed milk powder (cow's milk) (New Zealand New Milk Ltd) with a milk protein content of 8% was prepared as a fermentation medium. After being fully mixed, it was sterilized at 110° C. for 15 minutes to obtain a sterile skimmed milk powder medium.

[0022] Bacterial species: Lactobacillus bulgaricus (China Industrial Microbiological Culture Collection Center, strain number: CICC20247) and Tianyi Biological Co., Ltd.), Streptococcus thermophiles (strain number: CICC20364), Lacticaseibacillus paracasei (strain number: CICC0546K), Lactobacillus shelveticus (CICC, strain number: CICC 6024). The strains were stored at −80°C using glycerol.

[0023] Resuscitation: Before the experiment, 200 μL of bacterial suspension of each strain was taken from a -80°C glycerol cryovial and inoculated into 10 mL of the sterile skim milk powder medium prepared in the above step (inoculation volume 2% (v / v)). The culture was incubated in air at 37°C for 8 h to resuscitate the strain. The above recovery operation (inoculation and incubation) was repeated twice to restore the strain to its original state.

[0024] Fermentation culture: 2% inoculum volume (V / V) of the bacteria (restored viability strain) was inoculated into the fermentation medium, and the bacterial content in the fermentation broth was 1×10 7 CFU / mL, ferment in air at 200 rpm and 30-60°C (specifically 38°C) for 10-60 h (specifically 48 h), sterilize at 121°C for 20 min to inactivate the bacteria, and terminate the fermentation.

[0025] (2) Purification of postbiotic extracts

[0026] The fermentation broth is centrifuged at 3000-8000 rpm (specifically 6000 rpm) at 4°C for 20 minutes to remove impurities and insoluble matter from the fermentation broth, and the supernatant is retained. The fermentation supernatant is diluted with pure water to 3-8 times its volume (specifically 5 times its volume) and separated through an ultrafiltration membrane with a molecular weight cutoff of 3-30 kDa (specifically 10 kDa), and the ultrafiltrate (the solution that passes through the ultrafiltration membrane) is collected. The fermentation ultrafiltrate is then separated through a nanofiltration membrane with a molecular weight cutoff of ≤1 kDa (specifically 0.6 kDa) and concentrated to the initial volume of the fermentation broth, and the nanofiltration retentate (the solution that does not pass through the ultrafiltration membrane) is collected.

[0027] (3) The nanofiltration retentate was collected and dried (40°C, 12 h) to obtain a powder to obtain the postbiotic extract, which was then stored frozen at -20°C.

[0028] Example 2 Determination of Minimum Inhibitory Concentration

[0029] (1) Operation steps:

[0030] Sample Preparation: The postbiotic extracts obtained from Example 1 after fermentation with different strains were dissolved in deionized water to prepare a 1 mg / ml solution. Further dilutions were performed using the appropriate culture medium to achieve postbiotic extract concentrations of 102.4, 51.2, 25.6, 12.8, 6.4, 3.2, 1.6, 0.8, 0.4, and 0.2 mg / mL, depending on experimental requirements.

[0031] The Staphylococcus aureus, Pseudomonas aeruginosa and Escherichia coli used in the experiment were all preserved at -80°C using the glycerol preservation method. Before the experiment, a small amount (one ring) of bacteria was taken out from the preservation tube using an inoculation loop and inoculated into 5 mL of liquid LB medium (for Escherichia coli), RCM medium (for Pseudomonas aeruginosa) and liquid TSB medium (for Staphylococcus aureus). The inoculated bacteria were cultured in a constant temperature shaker at 37°C and 200 revolutions per minute (rpm) for 12 hours to revive the strain. In order to ensure that the activity of the strain was fully restored, 1 ring of bacteria was taken from the culture solution after the first recovery and re-inoculated into 5 mL of fresh liquid culture medium of the same kind, and cultured for another 12 hours at 37°C and 200 rpm. Repeat the above recovery (inoculation, culture) operation 3 times.

[0032] Take the logarithmic phase Escherichia coli, Pseudomonas aeruginosa and Staphylococcus aureus liquid and dilute them with LB (Escherichia coli), RCM (Pseudomonas aeruginosa) and TSB (Staphylococcus aureus) blank culture medium respectively to prepare 1×10 6 CFU / mL of bacterial solution. The culture medium containing the postbiotic extract (the postbiotic extract prepared in Example 1 was added to LB (Escherichia coli), RCM (Pseudomonas aeruginosa) and TSB (Staphylococcus aureus) culture medium) was mixed and added to the bacterial solution to 200uL per well of a 96-well culture plate, and the final concentration of the bacterial solution in each well was 5×10 5 CFU / mL, and the postbiotic extract culture medium was diluted with the corresponding culture medium. The final concentration of the postbiotic extract in each well was prepared into the following concentration gradient: 102.4, 51.2, 25.6, 12.8, 6.4, 3.2, 1.6, 0.8, 0.4 and 0.2 mg / mL, respectively. Three replicate wells were set for each concentration. The 96-well culture plate was placed in a 37 ° C incubator and incubated for 18 hours. After being taken out, it was blown evenly and the OD600 value was measured using a microplate reader. A curve was drawn with the postbiotic extract concentration as the horizontal axis and the OD600 value as the vertical axis. The concentration close to the inflection point of the curve was the MIC value of the postbiotic extract.

[0033] (2) Experimental results

[0034] As shown in Table 1, Lactobacillus bulgaricus from CICC and Tianyi Biotech showed similar MIC values ​​(25.6 mg / mL) for Staphylococcus aureus, and IC 50 The MIC values ​​of Streptococcus thermophilus and Lactobacillus paracasei were also similar (25.4 mg / mL (CICC) and 26.3 mg / mL (Tianyi Biotechnology Co., Ltd.), showing good inhibitory effects. In contrast, the MIC values ​​of Streptococcus thermophilus and Lactobacillus paracasei were higher (51.2 mg / mL), while the MIC value of Lactobacillus helveticus was the same as that of Lactobacillus bulgaricus, but the IC 50 The value was slightly higher (29.0 mg / mL).

[0035] As shown in Table 2, in terms of Escherichia coli, Lactobacillus bulgaricus from CICC and Tianyi Biotechnology also showed a low MIC value (6.4 mg / mL), and IC 50 The MIC values ​​of Streptococcus thermophilus, Lacticaseibacillus paracasei and Lactobacillus helveticus were all 12.8 mg / mL, but the IC 50 The values ​​vary, among which the IC 50 The value was the lowest (6.1 mg / mL).

[0036] As shown in Table 3, the MIC values ​​of Lactobacillus bulgaricus from CICC and Tianyi Biotechnology Co., Ltd. were both 51.2 mg / mL and IC 50 The MIC values ​​of Streptococcus thermophiles Lactobacillus helveticus and Lacticaseibacillus paracasei were also similar (28.4 mg / mL and 28.9 mg / mL, respectively).

[0037] In summary, lactic acid bacteria from different sources and species exhibited varying inhibitory effects on different bacteria, with Lactobacillus bulgaricus showing stronger inhibitory effects in some cases, particularly against Escherichia coli. These results suggest that postbiotic extracts have potential antibacterial applications, but the effects vary between different strains.

[0038] Table 1. MIC and IC of postbiotic extracts against Staphylococcus aureus 50 .

[0039]

[0040] Table 2. MIC and IC of postbiotic extracts against Escherichia coli 50 .

[0041]

[0042] Table 3. MICs and ICs of postbiotic extracts against Pseudomonas aeruginosa 50 .

[0043]

[0044]

[0045] Example 3 Antioxidant capacity of postbiotic extracts

[0046] Sample preparation: The postbiotic extracts obtained by fermentation with different strains in Example 1 were dissolved in deionized water to prepare a 600 mg / ml solution. Depending on the experimental requirements, further dilutions were performed with deionized water to achieve postbiotic extract concentrations of 50.0, 100.0, 200.0, 300.0, and 600.0 mg / mL.

[0047] The antioxidant capacity of postbiotic extracts was detected using a total antioxidant capacity detection kit (Biyuntian Biotechnology Co., Ltd.).

[0048] (1) ABTS (2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)) method operation steps:

[0049] Prepare ABTS working solution: Mix 100 μL of 5 mM ABTS solution with 100 μL of 10 mM oxidant solution (potassium persulfate) to prepare the ABTS working stock solution. Store in the dark at room temperature for 12 hours before use. Dilute the ABTS working stock solution to 35 times its volume with phosphate-buffered saline (PBS) to prepare the ABTS working solution.

[0050] Standard curve determination: 10 mM Trolox (6-hydroxy-2,5,7,8-tetramethylchromane-2-carboxylic acid) standard solution was diluted with PBS to a concentration gradient of 0.15, 0.3, 0.6, 0.9, 1.2 and 1.5 mM.

[0051] Total antioxidant capacity assay: 200 μL of ABTS working solution and 10 μL of postbiotic extract at various concentrations (50.0, 100.0, 200.0, 300.0, and 600.0 mg / mL) were added to each well of a 96-well plate. 10 μL of PBS was added to the blank control wells, and 10 μL of Trolox standard solution at various concentrations (0.15, 0.3, 0.6, 0.9, 1.2, and 1.5 mM) was added to the standard curve wells. Incubate at room temperature for 2-6 minutes (5 minutes in this example) before measuring A734.

[0052] ABTS free radical scavenging rate (A%) is expressed as: A% = (A blank - A measurement) / A blank × 100%

[0053] (2) Experimental results

[0054] like Figure 1 In an ABTS experiment, the antioxidant capacity of four lactic acid bacteria strains (Lactobacillus bulgaricus (CICC), Streptococcus thermophilus, Lactobacillus paracasei, and Lactobacillus shelveticus) was investigated at different postbiotic extract concentrations (50.0, 100.0, 200.0, 300.0, and 600.0 mg / mL). The results showed that the ABTS free radical scavenging rate of each strain showed a significant upward trend with increasing concentration, indicating that higher concentrations increase antioxidant capacity. Among all strains, Lactobacillus bulgaricus exhibited the strongest antioxidant capacity, with a scavenging rate reaching 80.73% at 600.0 mg / mL. Lactobacillus paracasei also exhibited a high scavenging rate at high concentrations, approaching 78%. In contrast, Streptococcus thermophilus and Lactobacillus helveticus had relatively low antioxidant capacity, but also showed good clearance rates at high concentrations, reaching 76.53% and 79.59%, respectively. Overall, Lactobacillus bulgaricus performed the best, showing high application potential.

[0055] Example 4 Cell proliferation activity of postbiotic extracts

[0056] Sample Preparation: The postbiotic extracts obtained by fermentation with different strains in Example 1 were dissolved in cell culture medium (see the cell culture section for specific culture medium ratios) to prepare a 1 mg / ml solution. Depending on the experimental requirements, further dilutions were performed using cell culture medium to achieve final concentrations of 0.5, 2, 5, and 10 μg / mL of the postbiotic extracts.

[0057] (1) Cell culture

[0058] Cells were cultured in DMEM basal medium (hereinafter referred to as cell culture medium) containing 10% fetal bovine serum and 1% double-antibody (final concentration of penicillin was 100 μg / mL and streptomycin was 50 μg / mL) (Procell Life Science & Technology Co., Ltd.). After human keratinocytes (HaCaT) (Procell Life Science & Technology Co., Ltd.) were revived, T25 cell culture flasks (25 cm 2 ) Place the cells in a 37°C cell culture incubator containing 5% CO2 by volume for 24 hours, observe the cell adhesion under an inverted microscope, and change the medium (change to the same new medium as before). Observe the presence of contamination and the number of cells attached under an inverted microscope. When the cell confluence reaches more than 80%, consider passaging the cells. Cell passaging steps: discard the culture medium, wash the cells with 1 mL of sterile PBS buffer each time, wash the cells twice, and then add 0.5 mL of 0.25% (w / v, g / ml) trypsin-EDTA digestion solution (Seven Innovation (Beijing) Biological Technology Co., Ltd) to digest the cells. Centrifuge after digestion and subculture in two bottles. The subculture cycle is 2 to 3 days (3 days in this case).

[0059] (2) CCK8 (Cell Counting Kit-8) assay to detect the effects of postbiotic extracts on human keratinocyte proliferation

[0060] HaCaT cells in the logarithmic phase of culture at the 3rd to 4th generation (here the 3rd generation) were digested and resuspended in the same new culture medium to prepare a cell suspension, which was used as the experimental cells. The cell suspension was diluted to 1.5×10 4 / mL cell density, 100uL per well was inoculated into a 96-well culture plate. After 6h, the cell adhesion was observed. If the cells were spindle-shaped with clear cytoplasm under the light microscope, it meant that the state was stable, and 10uL of cell culture medium containing postbiotic extracts was added to the experimental group, so that the final concentrations of postbiotic extracts in each well were 0.5, 2, 5, and 10μg / mL, respectively, and 5 replicate wells were set for each concentration. 10uL of cell culture medium without postbiotic extracts was added to the control group wells (the same cell culture medium as before). The 96-well culture plate was placed in an incubator for culture. After 24h, 10uL of CCK8 solution (5mM) (Sevenbio) was added to each well, and the 96-well culture plate was placed in an incubator for culture for 2.5h. Finally, the absorbance A at 450mm was measured with a microplate reader to calculate the proliferation activity of human fibroblasts.

[0061] Proliferation activity (%) = [A (experimental group) - A (blank well)] / [A (control group) - A (blank well)] × 100

[0062] (3) Experimental results

[0063] The effects of different bacterial species (including Lactobacillus bulgaricus (CICC), Streptococcus thermophilus, Lactaseibacillus paracasei and Lactobacillus shelveticus) on cell proliferation at different concentrations were detected by CCK-8 assay and compared with the control group. Figure 2 ), all bacterial species showed varying degrees of proliferation-promoting effects at various concentrations, and their absorbance values ​​were all higher than those of the control group. Under low concentration conditions (0.5 and 2.0 ug / mL), the proliferation-promoting effects of Lactobacillus bulgaricus and Streptococcus thermophilus were the most significant, with relative proliferation rates exceeding 150%, indicating that these two bacterial species have a strong promoting effect on cell proliferation at low concentrations. As the concentration increases, the proliferation-promoting effects of each bacterial species gradually weakened, but at a concentration of 30.0, they were still significantly higher than those of the control group. The proliferation-promoting abilities of Lactobacillus paracasei and Lactobacillus shelveticus were relatively low, but overall they still showed a certain proliferation-promoting effect. Overall, these bacterial species can significantly promote cell proliferation at different concentrations, especially Lactobacillus bulgaricus, which performed more prominently.

[0064] Example 5 Effect of Postbiotic Extracts on Photoaging Human Type I Collagen (I-COL) and IL-6 (Interleukin-6) The sample preparation process and conditions were the same as those in Example 3, and the cell culture method process and conditions were the same as those in Example 4 (1)

[0065] (1) Establishment of UVB photoaging model

[0066] First, HaCaT cells were used to construct a photoaging cell model. The third generation HaCaT cells were selected as experimental cells to ensure that the cells were in the best condition for the experiment. 1×10 4 The well-grown HaCaT cells were inoculated into 96-well culture plates with the cell number and cell plating density of 0.1 mL of culture medium per well. After cell inoculation, they were placed in a cell culture incubator and cultured overnight to allow the cells to fully adhere to the wall. Subsequently, 100 uL of culture medium containing postbiotic extracts of different concentrations was added to the experimental group, so that the final concentration of postbiotic extracts in each well was 0.5, 2, 5, 10, and 30 μg / mL, respectively. 100 uL of culture medium without postbiotic extracts was added to the model group and the control group and cultured for 24 hours. The culture medium was aspirated, and the cells were rinsed 3 times with PBS buffer, and the PBS was discarded. A UVB irradiation system was used, consisting of two parallel UVB lamps (300 nm-420 nm) (Dongguan Chuangu Lighting Technology Co., Ltd.) placed 10 cm above the cell layer, with an irradiation dose of 50 mJ / cm 2 Irradiation time was calculated according to the formula: irradiation dose = irradiation power x irradiation time. Before each irradiation, the power was measured using an ultraviolet irradiator (Shantou Wenguang Yuehui Co., Ltd.) to ensure accurate dosing. After irradiation, 100 μL of culture medium was added to each well. A non-UVB group (cultured in the same environment as the other groups) served as a control for model validation; this group did not receive UVB irradiation or other treatments.

[0067] The specific grouping and treatment measures are as follows:

[0068] Control group: did not receive UVB irradiation and was only treated with 100uL PBS solution.

[0069] Model group: treated with 100uL of PBS solution before exposure to UVB radiation.

[0070] Postbiotic extract group: treated with different concentrations of postbiotic extracts before exposure to UVB radiation.

[0071] (2) ELISA assay to detect the effects of postbiotic extracts on I-COL and IL-6 in human keratinocytes

[0072] First, the cell culture supernatant was treated, the supernatant was aspirated, and then centrifuged at 4000 rpm and 4°C for 5 minutes to effectively separate the supernatant cell particles from other potential complexes. The protein concentration was determined by the BCA method (Severn Innovation (Beijing) Biotechnology Co., Ltd.). After the protein concentration was normalized, the IL-6 and Ⅰ-COL concentrations in the cell supernatant were accurately measured using Elisa according to the detailed operating instructions provided by the human Ⅰ-COL and IL-6 Elisa kits (Shanghai Jianglai Biological Industry Co., Ltd.). This step was carried out strictly in accordance with the requirements of the kit instructions to ensure the reliability and accuracy of the experimental results. The operation is roughly as follows:

[0073] 1) Prepare the working solution according to the instructions and set up wells for standards, sample diluent, blank, and sample. Add 50 μL of standard solution of varying concentrations to the standard wells, 50 μL of sample diluent to the sample diluent wells, and 50 μL of the sample to be tested to the sample wells. Add 100 μL of biotinylated antibody to each well, except the blank well. Seal the plate and incubate in a 37°C water bath or incubator in the dark for 60 minutes.

[0074] 2) Then, discard the liquid, pat dry, fill each well with washing solution and let it stand for 1 minute. Repeat the washing process 5 times.

[0075] After washing, pat the plate dry on a clean tissue.

[0076] 3) Add 100 μL of HRP-labeled avidin to each well except the blank well. Seal the plate and incubate in a 37°C water bath or incubator in the dark for 20 minutes. Repeat the wash step.

[0077] 4) Add 100 μL of substrate mixture to each well, seal the plate, and incubate in a 37°C water bath or incubator in the dark for 15 minutes. Finally, add 50 μL of stop solution to each well and read the absorbance (OD) of each well on a microplate reader at 450 nm. Create a standard curve equation and calculate the sample concentration using the sample OD values.

[0078] (3) Experimental results

[0079] This study evaluated the anti-inflammatory efficacy of the strain by detecting the IL-6 secretion level in a UVB-induced skin inflammation model. Figure 3), UVB irradiation successfully induced an inflammatory response, and the IL-6 concentration in the model group was significantly increased to 23.34pg / ml compared with the untreated control group (3.34pg / ml in the control group). Lactobacillus bulgaricus (CICC) showed a dose-dependent inhibitory effect: when the strain concentration was increased to 10.0ug / mL, the IL-6 concentration in the postbiotic extract group decreased to 7.38pg / ml, recovering to 91.6% of the level of the normal control group; even at low concentrations, its anti-inflammatory activity was still significantly higher than that of other strains. Further comparative analysis showed that the anti-inflammatory effect of Lactobacillus bulgaricus (CICC) at the same concentration (10.0ug / mL) was significantly better than that of Lactobacillus helveticus (6.39pg / ml), and it exhibited the strongest concentration-responsive characteristics among all tested strains, confirming its ability to stably regulate inflammation.

[0080] The repair effect of the strain on UVB skin damage was verified by testing the concentration of type I collagen. The results showed that ( Figure 4 ). UVB damage reduced collagen concentration in the model group to 23.34 ng / ml (compared to 37.32 ng / ml in the control group). In the Lactobacillus bulgaricus (CICC) group, collagen synthesis significantly rebounded to 38.1 ng / ml, exceeding normal physiological levels by 2%, demonstrating significantly superior repair efficiency to other strains. In comparative experiments, the collagen concentrations of Lactobacillus paracasei, Streptococcus thermophilus, and Lactobacillus helveticus were 37.83 ng / ml, 35.09 ng / ml, and 35.76 ng / ml, respectively, all significantly different from those of Lactobacillus bulgaricus. These experimental data fully confirm the unique advantage of Lactobacillus bulgaricus in specifically activating collagen synthesis.

[0081] Example 6 Analysis of the components of the postbiotic extract prepared in Example 1

[0082] (1) Sample preparation and treatment: Take 2 mg of the postbiotic extract obtained by fermentation with Lactobacillus bulgaricus (CICC) in Example 1 and add 1 mL of water to fully dissolve it. Use an ultrafiltration tube with a molecular weight cutoff of 10 kDa to ultrafilter at 14,000 × g and 20°C for 20 minutes, and collect the ultrafiltrate as a sample for mass spectrometry analysis. Desalt the sample: Use a Waters Oasis HLB SPE column (C18 column, 10 mg) (Waters, USA), first activate it with 0.5 mL of methanol (Merck KGaA), and then add 0.5 mL of 0.1% TFA-H2O solution (Merck KGaA) for equilibrium. The sample is re-dissolved with 1 mL of 0.1% TFA-H2O solution and added to the C18 column. Elute with 0.5 mL of 80% ACN / 0.1% TFA-H2O (volume concentration) solution, collect the eluate, and freeze-dry it in aliquots and store at -80°C.

[0083] (2) Mass spectrometry analysis: The lyophilized sample was reconstituted with a 0.1% FA-H2O solution (Merck KGaA) to a concentration of 0.2 mg / mL. The sample was loaded in a 4 μL volume with an injection volume of 400 ng. Nano LC-MS / MS analysis was performed using a Triple TOF 7600 mass spectrometer (ABSCIEX, USA) in IDA mode. The analytical column was a Kinetex 2.6 μm XB C18 100A (length × inner diameter 150 × 0.3 mm) (Waters, USA), and the pretreatment column was a C18 Micro Trap (length × inner diameter 10 × 0.3 mm, filler particle size 3 μm) (Waters, USA). The mass spectrometry scan range was m / z 350-1250 (full scan) and m / z 100-1500 (MS / MS scan). Mobile phase A consisted of 0.1% formic acid in water (volume concentration), and mobile phase B consisted of 0.1% formic acid (Merck KGaA) + 98% acetonitrile in water (Merck KGaA) (volume concentration). The flow rate was 5 μL / min. The gradient elution (volume ratio) program was: 0-1 min, 4% B; 1-56 min, 4%-18% B; 56-70 min, 18%-40% B; 70-70.5 min, 40%-80% B; 70.5-75.5 min, 80% B; 75.5-76 min, 80%-4% B; 76-80 min, 4% B.

[0084] (3) Data retrieval: After analysis, the *.wiff files collected using Xcalibur software were searched against the Milk Bioactive Peptide Database (MBPDB) and the A Comprehensive Database for Antioxidants (AODB). Enzyme digestion, maximum missed cleavage, and fixed modification were not set during the search. The variable modification was set to methionine oxidation (+15.9949 Da). The parent ion mass tolerance was 50 ppm.

[0085] (4) Experimental results: Examples 1-5 used postbiotic extracts produced by fermentation of five strains, Lactobacillus bulgaricus (CICC and Tianyi Biotechnology Co., Ltd.), Streptococcus thermophilus (CICC), Lactaseibacillus paracasei (CICC), and Lactobacillus helveticus (CICC). The experimental results showed that Lactobacillus bulgaricus was the most promising strain in this study. The postbiotic extracts fermented with Lactobacillus bulgaricus were analyzed for components, and 19 antioxidant peptides and 2 anti-inflammatory peptides were successfully identified. Their sequence characteristics, source proteins, start and end positions, and relative abundance are shown in Tables 1 and 2, respectively. Among them, the two most abundant antioxidant peptides were VVPPFLQPE (1.02%) and VYPFPGPIPN (0.73%), both derived from β-casein. In addition, this study also identified 11 known antimicrobial peptides, accounting for 4.69% of the total peptide abundance. Among them, the two most abundant antimicrobial peptides were YQEPVLGPVRGPFPIIV (3.47%) and DVENLHLPLPL (0.51%).

[0086] Table 4. Antioxidant peptides in postbiotic extracts

[0087]

[0088]

[0089] Table 5. Anti-inflammatory peptides in postbiotic extracts

[0090]

[0091] Table 6. Antimicrobial peptides in postbiotic extracts

[0092]

Claims

1. A postbiotic extract for use in improving skin aging or preparing a preparation for improving skin aging, characterized in that: The postbiotic extract is a postbiotic extract derived from Lactobacillus bulgaricus.

2. The use according to claim 1, characterized in that: The postbiotic extract is prepared by fermenting milk protein (preferably milk protein from mammals such as cows, sheep, chickens, ducks, shrimps, horses and camels) using Lactobacillus bulgaricus as the fermentation strain (fermentation culture in a milk protein fermentation medium), and contains active peptides with a molecular weight of 3 to 30 kDa.

3. The use according to claim 1, characterized in that: The preparation raw material is a milk protein culture medium with a milk protein content of 5%-20% (preferably milk protein from mammals such as cow, sheep, chicken, duck, shrimp, horse and camel), which is obtained through anaerobic fermentation of Lactobacillus bulgaricus, bacterial inactivation and ultrafiltration-nanofiltration purification process, wherein: the ultrafiltration molecular weight cut-off is 3-30kDa; the fermentation conditions are 30-60°C and 10-60h.

4. The use according to claim 1, wherein The preparation method of the postbiotic extract comprises the following steps: 1) preparing a culture medium with a milk protein content (preferably milk protein from mammals such as cow, sheep, chicken, duck, shrimp, horse and camel) of 5% to 20% (preferably 5-8%, more preferably 6-10%) as a fermentation medium, 2) inoculating Lactobacillus bulgaricus after sterilization; and fermenting at 30-60° C. (preferably 36-45° C., more preferably 38-42° C.) for 10-60 hours (preferably 15-25 hours, more preferably 20-22 hours); 3) After the fermentation is completed, the bacteria are sterilized to inactivate the bacteria; the fermentation broth is centrifuged to remove impurities and the supernatant is collected; 4) The supernatant is separated and purified by ultrafiltration and nanofiltration membranes, and the dried extract is the postbiotic extract.

5. The use according to claim 3 or 4, characterized in that: The centrifugation speed is 500-1000×g (preferably 600-900×g, more preferably 700-800×g), and the time is 20 min. collecting the fermentation supernatant; The fermentation supernatant is collected and diluted with water to 2-20 (preferably 5-15, more preferably 6-10) times the volume of the original fermentation supernatant, and then ultrafiltered, separated by an ultrafiltration membrane with a molecular weight cutoff of 3-30 kDa (preferably 6-20 kDa, more preferably 8-12 kDa), and the ultrafiltrate (i.e., the ultrafiltration membrane permeate) is collected; Nanofiltration is performed using a nanofiltration membrane with a molecular weight cutoff of ≤1 kDa (preferably 0.3-0.9 kDa, more preferably 0.6-0.8 kDa), and nanofiltration is performed to 0.5-5 (preferably 0.6-2, more preferably 0.8-1.5) times the volume of the fermentation supernatant, and the nanofiltration retentate (i.e., the retentate that has not passed through the nanofiltration membrane) is collected.

6. The use according to any one of claims 1 to 5, characterized in that: The improvement of skin aging is anti-UVB skin damage, i.e. anti-photoaging, including prevention and / or treatment of UVB (ultraviolet light) damage to the skin; The postbiotic extract can be used to prepare an anti-UVB skin damage preparation, which contains an effective amount of the postbiotic extract and a pharmaceutically / cosmetically acceptable carrier, and can achieve synergistic anti-aging effects of anti-inflammation, collagen repair, and promotion of epidermal cell proliferation.

7. A postbiotic extract preparation for improving skin aging, characterized in that: The preparation comprises an effective amount of a postbiotic extract and a pharmaceutically or cosmetically acceptable carrier, and can achieve synergistic anti-aging effects of anti-inflammation, collagen repair, and promotion of epidermal cell proliferation; The postbiotic extract is a postbiotic extract derived from Lactobacillus bulgaricus.

8. The preparation according to claim 7, wherein: The postbiotic extract in the preparation is prepared by fermenting lactobacillus bulgaricus as a fermentation strain through milk protein fermentation (fermentation culture in a fermentation medium containing milk protein (preferably milk protein from mammals such as cow, sheep, chicken, duck, shrimp, horse and camel), and contains active peptides with a molecular weight of 3 to 30 kDa; The preparation raw material is a milk protein culture medium with a milk protein content of 5%-20% by weight (preferably milk protein from mammals such as cow, sheep, chicken, duck, shrimp, horse and camel), which is obtained through anaerobic fermentation of Lactobacillus bulgaricus, bacterial inactivation and ultrafiltration-nanofiltration purification process, wherein: the ultrafiltration molecular weight cut-off is 3-30kDa (preferably 6-20kDa, more preferably 8-12kDa); the fermentation conditions are 30-60°C for 10-60h.

9. The preparation according to claim 7 or 8, characterized in that: The preparation method of the postbiotic extract comprises the following steps: 1) preparing a culture medium containing 5% to 20% (preferably 5-8%, more preferably 6-10%) milk protein (preferably milk protein from mammals such as cow, sheep, chicken, duck, shrimp, horse, and camel) by weight as a fermentation medium; 2) inoculating Lactobacillus bulgaricus after sterilization; and fermenting at 37° C. for 10 to 60 hours (preferably 15 to 25 hours, more preferably 20 to 22 hours); 3) After the fermentation is completed, the bacteria are sterilized to inactivate the bacteria; the fermentation broth is centrifuged to remove impurities and the supernatant is collected; 4) The supernatant is separated and purified by ultrafiltration and nanofiltration membranes, and the resulting extract after drying is the postbiotic extract; Centrifuge at a speed of 500-1000×g (preferably 600-900×g, more preferably 700-800×g) for 20 min, and collect the fermentation supernatant; The fermentation supernatant is collected and diluted with water to 2-20 (preferably 5-15, more preferably 6-10) times the volume of the original fermentation supernatant, and then ultrafiltered, separated by an ultrafiltration membrane with a molecular weight cutoff of 3-30 kDa (preferably 6-20 kDa, more preferably 8-12 kDa), and the ultrafiltrate (i.e., the ultrafiltration membrane permeate) is collected; Nanofiltration is performed using a nanofiltration membrane with a molecular weight cutoff of <1 kDa (preferably 0.3-0.9 kDa, more preferably 0.6-0.8 kDa), and nanofiltration is performed to 0.5-5 (preferably 0.6-2, more preferably 0.8-1.5) times the volume of the fermentation supernatant, and the nanofiltration retentate (i.e., the retentate that does not pass through the nanofiltration membrane) is collected.

10. The use according to claim 6 or the preparation according to claim 7, characterized in that: The preparation is a topical skin care dosage form, including but not limited to therapeutic gel, ointment, essence water, cream, essence, emulsion, mask or repair dressing.

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