Skin-derived infant streptococcus CCSM002 capable of improving skin health and metagen of infant streptococcus CCSM002

Abstract

The invention discloses a skin-sourced streptococcus infantis CCSM002 capable of improving skin health and a metagen of the streptococcus infantis CCSM002, and belongs to the field of microbial technologies and medicines. The streptococcus infantis CCSM002 provided by the invention has the capability of generating oleamide, D-erythrodisphingosine and (2-oxo-2, 3-dihydro-1H-indole-3-yl)-acetic acid, and the strain has a good effect of repairing a barrier function in the aspect of external use and a good application prospect, specifically, the activity of damaged HaCaT cells is improved in vitro, the activity of the damaged HaCaT cells is improved, the activity of the damaged HaCaT cells is improved, and the activity of the damaged HaCaT cells is improved. The expression of tissue kininolase is reduced, and the gene level expression of FLG, IVL, AQP3, ZO-1 and Occludin can be improved at the same time in the aspect of improving functional protein and structural protein after damage. Therefore, the streptococcus infantis CCSM002 has a huge application prospect in preparation of external daily chemical products or medicines for repairing the skin barrier.

Description

Technical Field

[0001] This invention relates to a skin-derived Streptococcus infantis strain CCSM002 that improves skin health and its post-biotic, belonging to the fields of microbial technology and pharmaceutical technology. Background Technology

[0002] The skin barrier is the body's first line of defense, playing a crucial protective role. It is mainly composed of the stratum corneum, the sebum film, and intercellular lipids. It not only prevents the invasion of harmful substances, pathogens, and allergens from the outside world but also prevents the loss of moisture and nutrients from the body. The integrity of the skin barrier directly affects the health of the skin. If the barrier function is impaired, the skin becomes dry and sensitive, prone to inflammation and infection, and may even lead to skin diseases such as eczema and psoriasis. Therefore, maintaining the integrity and health of the skin barrier is key to skincare, effectively resisting external aggressors and maintaining the skin's moisture balance and overall health.

[0003] While existing microbiome-related skin barrier repair technologies have shown some effectiveness, they still suffer from limitations such as poor stability of live bacteria, unclear active ingredients, insufficient understanding of mechanisms, and significant individual variability in responses, making it difficult to precisely control the repair effect. Although the skin microbiome can promote skin cell regeneration, inflammation regulation, and barrier structure reconstruction through the secretion of metabolites and signaling molecules when damaged, how to stably and standardizedly obtain these key active factors remains a technological bottleneck. This invention, through in-depth analysis of the action mechanisms of the skin microbiome and its key effector molecules, specifically overcomes the problems of unstable activity and unclear components in traditional technologies. It achieves more precise and efficient skin barrier repair with functional post-biotics with clearly defined targets and controllable activity, providing a new technological path and broad development space for the innovation of cosmetic formulations and functional raw materials. Summary of the Invention

[0004] This invention provides an infantile streptococcus ( Streptococcus infantis The application of CCSM002 and its post-genes in the preparation of products that repair the skin barrier.

[0005] This invention provides a strain of Streptococcus infantis (Streptococcus infantis) Streptococcus infantis The Streptococcus infantis strain described in CCSM002 is deposited at the Guangdong Provincial Center for Microbial Culture Collection, with accession number GDMCC No: 65086.

[0006] The Streptococcus infantis CCSM002 strain was derived from the skin of a healthy individual. The strain was sequenced and the obtained sequence was compared with the nucleic acid sequence in NCBI. The result showed that it was Streptococcus infantis, and it was named Streptococcus infantis CCSM002.

[0007] The colonies of *Streptococcus infantis* CCSM002 on BHI solid medium are raised, white, smooth, and round.

[0008] The present invention also provides a postbiotic prepared using the aforementioned Streptococcus infantis CCSM002.

[0009] In one embodiment, the metabiotic includes cell lysate, inactivated or dead cells, fermentation supernatant, or any of the above-mentioned powders prepared by drying. In one embodiment, the inactivated or dead cells are prepared by culturing the *Streptococcus infantis* CCSM002 in a culture medium for a period of time, collecting the bacterial cells in the cell culture medium, and obtaining inactivated bacterial cells after heat treatment. In one embodiment, the heat treatment conditions are: 65°C for 30 minutes.

[0010] In one embodiment, the method for preparing the bacterial lysate is as follows: the infantile streptococcus CCSM002 is cultured in a culture medium for a period of time, bacterial cells are collected, homogenized under high pressure, and the supernatant obtained by centrifugation is used to obtain the bacterial lysate.

[0011] In one embodiment, the fermentation supernatant is the supernatant obtained by culturing *Streptococcus infantis* CCSM002 in a culture medium for a period of time and then centrifuging it; optionally, the supernatant is further subjected to heat treatment to inactivate it.

[0012] The present invention also provides compositions containing the aforementioned Streptococcus infantis CCSM002 and / or its postbiotic.

[0013] In one embodiment, the composition includes, but is not limited to, pharmaceuticals or daily chemical products.

[0014] In one embodiment, the daily chemical products include skin care products or washing and care products.

[0015] In one embodiment, the composition further includes conventional excipients.

[0016] In one embodiment, the conventional excipients include one or more of fillers, flavoring agents, binders, disintegrants, lubricants, antacids, and nutritional fortifiers.

[0017] In one embodiment, the composition is a probiotic powder.

[0018] In one embodiment, the probiotic powder is a solid powder of probiotic prepared by drying the prepared liquid probiotic of Streptococcus infantis CCSM002.

[0019] In one embodiment, drying includes, but is not limited to, preparation by spray drying, vacuum freeze drying, fluidized bed drying, and vacuum drying.

[0020] The present invention also provides the use of the composition in the preparation of products that repair the skin barrier.

[0021] In one embodiment, the product includes at least one of the following functions: (1) Enhance the cell activity of skin keratinocytes (HaCaT) after sodium dodecyl sulfate (SDS) damage in vitro; (2) In vitro, the expression of filaggrin gene in keratinocytes (HaCaT) of the skin after sodium dodecyl sulfate (SDS) injury was increased; (3) In vitro, the expression of aquaporin gene in keratinocytes (HaCaT) of the skin after sodium dodecyl sulfate (SDS) injury was increased; (4) Increase the expression of inner lining protein gene in keratinocytes (HaCaT) after sodium dodecyl sulfate (SDS) damage in vitro; (5) Reduced tissue kininase gene expression in keratinocytes (HaCaT) after sodium dodecyl sulfate (SDS) damage in vitro. (6) In vitro, the expression of tight junction proteins (ZO-1, OCCLUDIN and CLDN) in skin keratinocytes (HaCaT) after sodium dodecyl sulfate (SDS) injury was increased.

[0022] In one implementation, the application method is local external application.

[0023] In one embodiment, the amount of *Streptococcus infantis* CCSM002 and the prepared postbiotic in the product is not less than 1 × 10⁻⁶ of the corresponding viable bacterial count. 7 CFU / mL.

[0024] In one embodiment, the product is a pharmaceutical product or a daily chemical product.

[0025] In one embodiment, the drug comprises the infantile streptococcus CCSM002, a drug carrier, and / or pharmaceutical excipients.

[0026] In one embodiment, the pharmaceutical excipient comprises excipients and additives.

[0027] In one embodiment, the pharmaceutical excipients include solvents, propellants, solubilizers, cosolvents, emulsifiers, colorants, binders, disintegrants, fillers, lubricants, wetting agents, osmotic pressure regulators, stabilizers, flow aids, flavoring agents, preservatives, suspending agents, coating materials, fragrances, anti-adhesion agents, integrators, penetration enhancers, pH adjusters, buffers, plasticizers, surfactants, foaming agents, defoamers, thickeners, encapsulating agents, humectants, absorbents, diluents, flocculants and anti-flocculation agents, filter aids, and release inhibitors.

[0028] In one embodiment, the daily chemical product contains Streptococcus infantis CCSM002, matrix raw materials, and / or conventional excipients.

[0029] In one embodiment, the daily chemical products include cosmetics, skin care products, or toiletries.

[0030] In one embodiment, the matrix raw materials include oil-based raw materials, wax-based raw materials, synthetic oil-based raw materials, powder-based raw materials, gel-based raw materials, coagulants, and surfactants.

[0031] In one embodiment, the conventional excipients include one or more of the following: moisturizers, whitening agents, flavoring agents, adhesives, lubricants, preservatives, film-forming agents, antioxidants, emulsifiers, and cosmetic nutritional additives.

[0032] The present invention also provides the use of the aforementioned Streptococcus infantis CCSM002 in the preparation of oleamide, D-erythrosine and / or (2-oxo-2,3-dihydro-1H-indol-3-yl)-acetic acid.

[0033] Beneficial effects: The infantile streptococcus of the present invention ( Streptococcus infantis CCSM002 has the ability to produce oleamide, D-erythrosine, and (2-oxo-2,3-dihydro-1H-indole-3-yl)-acetic acid. The metabiotics prepared from this strain have the ability to alleviate skin barrier damage and enhance the expression of related proteins when applied topically, specifically in the following ways: (1) Enhance the cell activity of skin keratinocytes (HaCaT) after sodium dodecyl sulfate (SDS) damage; (2) Regulate the mRNA expression of FLG, IVL, AQP3, KLK7 and tight junction proteins in skin keratinocytes (HaCaT) after sodium dodecyl sulfate (SDS) damage; Therefore, Streptococcus infantis ( Streptococcus infantis The post-biotic prepared by CCSM002 has great application potential in products that alleviate damage to the host skin barrier.

[0034] Preservation of biological materials Streptococcus infantis ( Streptococcus infantis CCSM002, taxonomically named Streptococcus infantis It was deposited on September 2, 2024, at the Guangdong Provincial Center for the Preservation of Microbial Cultures, with accession number GDMCCNo: 65086, located at 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou. Attached Figure Description

[0035] Figure 1 Evaluation of the effects of different concentrations of SDS on HaCaT cell damage; AC: fitted curves of cell viability at different incubation times; D: fitted data of the effect of different SDS concentrations on cell viability under 6h incubation conditions.

[0036] Figure 2 The effect of post-genetics on HaCaT cell proliferation.

[0037] Figure 3 The effect of post-genetic repair on HaCaT cell viability after SDS damage.

[0038] Figure 4 The effect of post-genetic repair of SDS damage on FLG mRNA expression in HaCaT.

[0039] Figure 5 The effect of post-genetic repair on IVL mRNA expression in HaCaT.

[0040] Figure 6 The effect of post-genetic repair of SDS damage on AQP3 mRNA expression in HaCaT.

[0041] Figure 7 The effect of post-genetic repair of SDS damage on KLK7 mRNA expression in HaCaT.

[0042] Figure 8 The effect of post-genetic repair of SDS-damaged tight junction mRNA expression in HaCaT.

[0043] Figure 9 LC-MS Graph of Oleamide Standard.

[0044] Figure 10 Liquid chromatography-mass spectrometry (LC-MS) plot of oleic acid amide content in fermentation supernatant sample.

[0045] Figure 11 Liquid chromatography-mass spectrometry (LC-MS) plot of oleic acid amide content in bacterial cell samples.

[0046] Figure 12 A comparison chart of retention times for oleamide standards, fermentation supernatant, and cell samples.

[0047] Figure 13 Liquid chromatography-mass spectrometry (LC-MS) of sphingosine standard.

[0048] Figure 14 Liquid chromatography-mass spectrometry (LC-MS) plot of sphingosine content in fermentation supernatant sample.

[0049] Figure 15 Liquid chromatography-mass spectrometry (LC-MS) plot of sphingosine content in bacterial samples.

[0050] Figure 16A comparison chart of retention times for sphingosine standards, fermentation supernatant, and cell samples.

[0051] Figure 17 LC-MS chromatogram of (2-oxo-2,3-dihydro-1H-indole-3-yl)-acetic acid standard.

[0052] Figure 18 Liquid chromatography-mass spectrometry (LC-MS) plot of (2-oxo-2,3-dihydro-1H-indole-3-yl)-acetic acid content in fermentation supernatant sample.

[0053] Figure 19 Liquid chromatography-mass spectrometry (LC-MS) plot of (2-oxo-2,3-dihydro-1H-indole-3-yl)-acetic acid content in bacterial samples.

[0054] Figure 20 A comparison chart of retention times for (2-oxo-2,3-dihydro-1H-indol-3-yl)-acetic acid, fermentation supernatant, and cell samples. Detailed Implementation

[0055] The present invention will be further described below with reference to specific embodiments.

[0056] The human keratinocytes (HaCaT) involved in the following examples were purchased from the Shanghai Cell Bank.

[0057] The culture media involved in the following examples are as follows: BHI solid medium: bovine brain extract: 4.0 g / L, bovine heart extract: 4.0 g / L, peptone: 5.0 g / L, casein peptone: 16.0 g / L, sodium chloride: 5.0 g / L, glucose: 2.0 g / L, disodium hydrogen phosphate: 2.5 g / L, and agar: 20 g / L, pH: 7.4±0.2.

[0058] BHI liquid culture medium: bovine brain extract: 4.0 g / L, bovine heart extract: 4.0 g / L, peptone: 5.0 g / L, casein peptone: 16.0 g / L, sodium chloride: 5.0 g / L, glucose: 2.0 g / L and disodium hydrogen phosphate: 2.5 g / L, pH: 7.4±0.2.

[0059] Modified BHI screening solid culture medium: bovine brain extract: 4.0 g / L, bovine heart extract: 4.0 g / L, peptone: 5.0 g / L, casein peptone: 16.0 g / L, sodium chloride: 5.0 g / L, glucose: 2.0 g / L, disodium hydrogen phosphate: 2.5 g / L, nicotinic acid: 0.3 g / L, neomycin: 0.4 g / L, and agar: 20 g / L.

[0060] Cell culture medium: 89% (v / v) DMEM medium + 10% (v / v) fetal bovine serum + 1% (v / v) 100× penicillin and streptomycin mixed solution (penicillin content 10000 U / mL, streptomycin concentration 10 mg / mL).

[0061] Example 1: Cell resuscitation and culture First, remove the frozen human keratinocyte cell line (HaCaT) from the -80℃ freezer, thaw it rapidly in a 37℃ water bath, then add 5 mL of cell culture medium and centrifuge at 1000 r / min for 3 min, discarding the supernatant. Resuspend the cells in 1 mL of cell culture medium and place them in a culture dish, then incubate in a 37℃ incubator containing 5% CO2. When the cells regain viability and reach 70%-80% confluence after 1-2 days, passage the cells.

[0062] Example 2: Isolation and screening of Streptococcus infantis The bacterial strain was screened from healthy human skin. A sterile swab was applied to the back of the hand three times with physiological saline, and then streaked directly onto a modified BHI screening solid medium. The culture was incubated at 37°C for 48 hours. Yellow, round colonies were picked and enriched in BHI liquid medium for 16 hours. The bacterial culture was then transferred to BHI solid plates for purification and incubation for 48 hours. Finally, single colonies were picked and transferred to BHI liquid medium for enrichment. The strain was preserved in 30% glycerol. The genome of the strain was extracted, and 16S rDNA was amplified and sequenced (performed by Suzhou Genewiz Biotechnology Co., Ltd.). The results, confirmed by NCBI sequence alignment, identified it as *Streptococcus infantis*, and it was named *Streptococcus infantis*. streptococcus infantarius CCSM002. The aforementioned Streptococcus infantis CCSM002 has been deposited at the Guangdong Provincial Center for Microbial Culture Collection.

[0063] Example 3: Preparation of postbiotics from Streptococcus infantis CCSM002 (1) Streptococcus infantis CCSM002 was streaked from the preservation tube and revived. It was cultured in a water-jacketed incubator at 37°C for 48 h on BHI solid medium to obtain single colonies. Single colonies were picked and inoculated into BHI liquid medium and cultured at 37°C for 12-18 h to obtain culture solution 1. Culture broth 1 was inoculated into BHI liquid medium at an inoculation rate of 2% (v / v) and cultured at 37°C for 12 h to obtain seed culture; The seed culture was inoculated into BHI liquid medium at 2% (v / v) for expansion culture, and cultured at 37℃ for 18h. The number of viable bacteria was recorded and bacterial culture a was obtained.

[0064] The bacterial culture a was centrifuged at 8000 r / min for 30 min, and the supernatant and bacterial sludge were collected. The supernatant was heat-treated (65℃, 30 min) and freeze-dried to obtain powder for later use, thus preparing the freeze-dried powder of the fermentation supernatant of Streptococcus infantis CCSM002 (denoted as CCSM002-Q). The bacterial sludge was resuspended in 75% of the original bacterial culture volume of double-distilled water, and the resuspended liquid was heat-treated (65℃, 30 min), and then homogenized under high pressure (1000 MPa, 10 times) using a high-pressure homogenizer. After homogenization, the supernatant was collected by centrifugation at 8000 r / min for 30 min to obtain the bacterial cell lysate (denoted as CCSM002-S).

[0065] The following were prepared by means of the above method: postbiotic of Streptococcus infantis CCSM002 (cell lysate CCSM002-S and lyophilized powder CCSM002-Q of fermentation supernatant).

[0066] Example 4: Establishment of an in vitro model of keratinocyte damage caused by sodium dodecyl sulfate (SDS) (HaCaT) (1) Take 100 μL of HaCaT cells in the logarithmic growth phase and use 1.2 × 10⁻⁶ ppm. 4 Cells were seeded at a concentration of 100 cells / well in 96-well plates, with the outermost ring filled with PBS solution to prevent edge effects. After culturing for 36 h to allow the cells to adhere, blank control, control and SDS treatment groups were set up. Control group: Contains only cell culture medium and does not contain HaCaT cells; Control group: Contains cell culture medium and HaCaT cells, but does not contain SDS; Treatment groups: containing cell culture medium, HaCaT cells, and SDS at final concentrations of 6.25 μg / mL, 12.5 μg / mL, 25 μg / mL, and 50 μg / mL, respectively.

[0067] (2) The above well plates were incubated in an incubator at a temperature of 37°C for 6 h, 12 h and 24 h respectively. After incubation, 10 μL of CCK8 solution was added to each well and incubated for 1.5 h to measure the absorbance (OD) at 450 nm.

[0068] Cell viability is calculated using the following formula: Cell viability (%) = (OD value of treatment group - OD value of blank group) / (OD value of control group - OD value of blank group) × 100%.

[0069] (3) The effect on cell activity after nonlinear regression fitting is as follows: Figure 1 As shown, compared with the control group (cell proliferation rate 100%), the cell viability of the model group treated with 15 μg / mL SDS for 6 h was 60.6%, and SDS modeling caused significant damage to HaCaT cells.

[0070] Example 5: Safety verification of postbiotics prepared from Streptococcus infantis CCSM002 on HaCaT cells (1) Take 100 μL of HaCaT cells in the logarithmic growth phase and use 1.2 × 10⁻⁶ ppm. 4 Cells per well were seeded at a concentration of 100 cells / well in 96-well plates, with the outermost ring filled with PBS solution to prevent edge effects. After culturing for 36 h to allow the cells to adhere, blank control, control and post-genetic treatment groups were set up. Control group: Contains only cell culture medium and does not contain HaCaT cells; Control group: Contains cell culture medium and HaCaT cells, but does not contain post-genetics; The metagenetic treatment group contained cell culture medium and HaCaT cells, as well as metagenes.

[0071] Preparation of the metabiotic solution: The metabiotic was resuspended in cell culture medium (the amount of resuspended metabiotic was the same as that fermented to a concentration of 1.0 × 10⁻⁶). 7 (The amount of metabiotic prepared from bacterial culture at CFU / ml was equivalent). In the metabiotic treatment group, the culture medium was replaced with 100 μL of metabiotic solution prepared from Streptococcus infantis CCSM002; in other groups, the culture medium was replaced with 100 μL of fresh culture medium.

[0072] (2) The above well plates were incubated in an incubator at 37°C for 24 h. After incubation, 10 μL of CCK8 solution was added to each well and incubated for 1.5 h. The absorbance (OD) at 450 nm was measured.

[0073] Cell viability is calculated using the following formula: Cell viability (%) = (OD value of treatment group - OD value of blank group) / (OD value of control group - OD value of blank group) × 100%.

[0074] Effects on cell viability, such as Figure 2 As shown, compared with the control group (cell proliferation rate 101.62%), the addition of postbiotics derived from Streptococcus infantis CCSM002 (CCSM002-Q and CCSM002-S) at an inactivated bacterial concentration of 1.0 × 10⁻⁶ cells significantly improved cell proliferation. 7 The cell proliferation rates at CFU / ml were 100.49% and 100.27%, respectively.

[0075] According to the ISO 10993-5:2009 toxicity classification evaluation method, cells with a viability greater than 70% can be considered non-toxic. The above results indicate that an inactivated bacterial concentration of 1.0 × 10⁻⁶ is appropriate. 7 HaCaT cells at a postbiotic concentration of CFU / ml showed high viability (over 95%). Considering their lack of cytotoxicity, an inactivated cell concentration of 1.0 × 10⁻⁶ was selected. 7CFU / ml is a suitable postbiotic concentration for subsequent cell experiments.

[0076] Example 6: Effect of postbiotic prepared from Streptococcus infantis CCSM002 on SDS-induced damage to HaCaT cells (1) Take 100 μL of HaCaT cells in the logarithmic growth phase and use 1.2 × 10⁻⁶ ppm. 4 A concentration of cells / well was seeded into 96-well plates, with the outermost ring filled with PBS solution to prevent edge effects. After culturing for 36 h to allow the cells to adhere, control group 1 was obtained. (2) Set up control group, model group and treatment group respectively for the well plates prepared in step (1), and then incubate them in an incubator at 37℃ for 6 h: Control group: After changing the medium of control group 1 in step (1), it contains cell culture medium and HaCaT cells, without post-genetic treatment and without SDS modeling agent; Model group: The control group 1 in step (1) was replaced with a cell culture medium containing 15 μg / mL SDS, containing the original HaCaT cells, without post-genetic treatment; the cell culture medium containing SDS modeling agent was prepared by mixing SDS evenly in the culture medium and passing it through a 0.22 μm aqueous filter membrane for sterilization, so that the final concentration of SDS in the cell culture medium was 15 μg / mL. Postgenetic treatment group: The treatment method is the same as that of the model group.

[0077] (3) After incubation, discard the original culture medium and wash once with PBS. Add the corresponding metagenic sample to the metagenic treatment group, and add an equal amount of cell culture medium to the control group and the model group. Incubate again for 24 hours.

[0078] In the metabiotic treatment group, the metabiotics were resuspended in cell culture medium to obtain a metabiotic solution (the amount of resuspended metabiotics was the same as that fermented to a concentration of 1.0 × 10⁻⁶). 7 The amount of postbiotic prepared from bacterial culture of CFU / ml is equivalent to that of bacteria. 100 μL of postbiotic solution prepared from Streptococcus infantis CCSM002 was added to each solution.

[0079] (4) Add 10 μL of CCK8 solution to each well and incubate for 1.5 h to measure the absorbance (OD) at 450 nm.

[0080] Cell viability was calculated using the following formulas: Model group cell viability (%) = (Model group OD value - Blank group OD value) / (Control group OD value - Blank group OD value) × 100%; Treatment group cell viability (%) = (Treatment group OD value - Blank group OD value) / (Control group OD value - Blank group OD value) × 100%.

[0081] The results of the effect of post-genetic agents on SDS-induced HaCaT cell damage are as follows: Figure 3 Compared with the control group (100% cell viability), the cell viability of the model group was 66.76%, indicating that SDS modeling caused damage to HaCaT cells.

[0082] After adding CCSM002-S and CCSM002-Q to the treatment groups, the cell viability was 69.36% and 84.52%, respectively. Compared with the model group (66.76%), CCSM002-Q could increase the viability of HaCaT cells by 1.27 times, indicating that the metagenetic agent (fermentation supernatant) of Streptococcus infantis CCSM002 can effectively alleviate the damage to HaCaT cells caused by SDS.

[0083] Example 7: Effect of postbiotics prepared from Streptococcus infantis CCSM002 on FLG gene expression in HaCaT cells damaged by SDS. (1) HaCaT cells were injected at a concentration of 1.8 × 10⁻⁶. 4 One cell per well was seeded into a 6-well plate and cultured for 36 hours until the cells adhered.

[0084] (2) Set up control group, model group and treatment group respectively for the well plates prepared in step (1), and then incubate them in an incubator at 37℃ for 6 h: Control group: After changing the medium of control group 1 in step (1), it contains cell culture medium and HaCaT cells, without post-genetic treatment and without SDS modeling agent; Model group: The control group 1 in step (1) was replaced with a cell culture medium containing 15 μg / mL SDS, containing the original HaCaT cells, without post-genetic treatment; the cell culture medium containing SDS modeling agent was prepared by mixing SDS evenly in the culture medium and passing it through a 0.22 μm aqueous filter membrane for sterilization, so that the final concentration of SDS in the cell culture medium was 15 μg / mL. Postgenetic treatment group: The treatment method is the same as that of the model group.

[0085] (3) After incubation, discard the original culture medium and wash once with PBS. Add the corresponding metagenic sample to the metagenic treatment group, and add an equal amount of cell culture medium to the control group and the model group. Incubate again for 24 hours.

[0086] In the metabiotic treatment group, the metabiotics were resuspended in cell culture medium to obtain a metabiotic solution (the amount of resuspended metabiotics was the same as that fermented to a concentration of 1.0 × 10⁻⁶). 7 The amount of postbiotic prepared from bacterial culture of CFU / ml is equivalent to that of bacteria. 2 mL of postbiotic solution prepared from Streptococcus infantis CCSM002 (CCSM002-Q) was added to each solution.

[0087] (4) After incubation, discard the culture supernatant, wash each well three times rapidly with PBS, add 1 mL of cell lysis buffer to each well, repeatedly pipette, extract RNA from the cell lysis buffer, and reverse transcribe it into cDNA using an RT-PCR reverse transcription kit. Detect gene expression in HaCaT cells using real-time quantitative PCR. -△△Ct Formula calculation FLG The mRNA expression levels were measured, with GAPDH as the internal reference. The primers are described in Table 1 below, and the results are as follows: Figure 4 As shown.

[0088] Table 1 Primer Sequences

[0089] Filamentin (FLG) plays a crucial role in the skin barrier function. It is an important molecule connecting keratin fibers in the stratum corneum, forming a robust physical barrier on the outermost layer of the epidermis by helping keratin fibers aggregate in an orderly manner, preventing moisture loss and blocking the invasion of external allergens. FLG deficiency is closely associated with various skin diseases, such as eczema (atopic dermatitis) and ichthyosis. In these diseases, FLG deficiency or absence weakens the skin barrier function, making the skin more sensitive to external stimuli. Therefore, reducing SDS-induced damage to keratinocytes by increasing FLG gene expression has become a therapeutic target. FLG expression results are obtained from… Figure 4 It can be seen that, with the control group's FLG mRNA expression level as 1, the expression level in the model group decreased to 0.63 after SDS intervention. The post-biotic prepared by Streptococcus infantis CCSM002 (fermentation supernatant) significantly upregulated the FLG mRNA expression level to 1.57.

[0090] Example 8: Effect of postbiotic preparation of Streptococcus infantis CCSM002 on IVL gene expression in HaCaT cells damaged by SDS. For specific implementation details, refer to Example 7. After incubation, discard the culture supernatant, wash each well three times rapidly with PBS, add 1 mL of cell lysis buffer to each well, repeatedly pipette, extract RNA from the cell lysis buffer, and reverse transcribe it into cDNA using an RT-PCR reverse transcription kit. Detect gene expression in HaCaT cells using real-time quantitative PCR. -△△Ct Formula calculation IVL The mRNA expression levels were measured, with GAPDH as the internal reference. The primers were described in Table 1, and the results are as follows: Figure 5 As shown.

[0091] In keratinocytes, inner lamina protein (IVL) cross-links with laminarin via transglutaminase catalysis, forming an insoluble keratinized capsule that constitutes the unique stratum corneum barrier structure of the epidermis. Simultaneously, it interacts with other keratinocyte differentiation proteins such as filaggrin (FLG) to jointly maintain the structure and function of the skin barrier. IVL expression results are derived from… Figure 5 It can be seen that, with the expression level of IVL mRNA in the control group as 1, the expression level in the model group decreased to 0.46 after SDS intervention, and the fermentation supernatant of Streptococcus infantis CCSM002 could increase the expression level of IVL to 1.15.

[0092] Example 9: Effect of postbiotic preparation from Streptococcus infantis CCSM002 on AQP3 gene expression in HaCaT cells damaged by SDS. For specific implementation details, refer to Example 7. After incubation, discard the culture supernatant, wash each well three times rapidly with PBS, add 1 mL of cell lysis buffer to each well, repeatedly pipette, extract RNA from the cell lysis buffer, and reverse transcribe it into cDNA using an RT-PCR reverse transcription kit. Detect gene expression in HaCaT cells using real-time quantitative PCR. -△△Ct Formula calculation AQP3 The mRNA expression levels were measured, with GAPDH as the internal reference. The primers were described in Table 1, and the results are as follows: Figure 6 As shown.

[0093] Aquaporins (AQPs) play a crucial role in skin barrier function, especially AQP3. AQPs regulate the transport and distribution of water in the skin, maintaining skin hydration and elasticity by controlling the water content within the stratum corneum. AQP3 not only promotes water transport but also regulates glycerol transport, playing a vital role in maintaining skin suppleness and barrier function. The expression of AQP3 is determined by… Figure 6 It can be seen that, with the mRNA expression level of AQP3 in the control group as 1, the expression level in the model group decreased to 0.52 after SDS intervention, and the expression level of AQP3 was restored to 1.34 after being repaired by fermentation supernatant of Streptococcus infantis CCSM002.

[0094] Example 10: Effect of postbiotic preparation of Streptococcus infantis CCSM002 on KLK7 gene expression in HaCaT cells damaged by SDS. For specific implementation details, refer to Example 7. After incubation, discard the culture supernatant, wash each well three times rapidly with PBS, add 1 mL of cell lysis buffer to each well, repeatedly pipette, extract RNA from the cell lysis buffer, and reverse transcribe it into cDNA using an RT-PCR reverse transcription kit. Detect gene expression in HaCaT cells using real-time quantitative PCR. -△△CtFormula calculation KLK7 The mRNA expression levels were measured, with GAPDH as the internal reference. The primers were described in Table 1, and the results are as follows: Figure 7 As shown.

[0095] Tissue kallikrein 7 (KLK7) is a serine protease that plays a crucial role in the formation and maintenance of the skin barrier. KLK7 is primarily expressed in the stratum corneum of the skin, participating in the degradation of intercellular junction proteins (such as keratinocyte adhesion proteins). Excessive KLK7 activity can lead to excessive flaking, weakened skin barrier function, increased moisture loss, and increased sensitivity to harmful external substances; therefore, it is important to control the expression of this enzyme to a low level. KLK7 expression is determined by… Figure 7 It can be seen that the mRNA expression level of KLK7 in the control group was about 1, while the expression level in the model group increased to 1.75 after SDS intervention. After repair by fermentation supernatant of Streptococcus infantis CCSM002, the expression level of AQP3 was downregulated to 0.73.

[0096] Example 11: Effect of post-genetic agents prepared from Streptococcus infantis CCSM002 on the expression of tight junction-related protein genes in HaCaT cells damaged by SDS. For specific implementation details, refer to Example 7. After incubation, discard the culture supernatant, wash each well three times rapidly with PBS, add 1 mL of cell lysis buffer to each well, repeatedly pipette, extract RNA from the cell lysis buffer, and reverse transcribe it into cDNA using an RT-PCR reverse transcription kit. Detect gene expression in HaCaT cells using real-time quantitative PCR. -△△Ct Formula calculation ZO-1 and Occludin The mRNA expression levels were measured, with GAPDH as the internal reference. The primers were described in Table 1, and the results are as follows: Figure 8 As shown.

[0097] Tight junction proteins are essential components for maintaining skin barrier function. Located between keratinocytes, they form sealed junctions that prevent the permeation of external substances and water through the intercellular spaces, thus maintaining the integrity of the skin barrier. Zona occlusion protein (ZO-1) and Occludin are core components of tight junctions and important structural proteins of the skin barrier. They play a crucial role in regulating the transport of substances through the intercellular spaces, maintaining the selective permeability of the skin barrier. Furthermore, these proteins not only participate in physical cell-cell connections but also function in intracellular signal transduction, influencing cell proliferation, differentiation, and migration. The expression of ZO-1 and Occludin is controlled by… Figure 8As shown, the mRNA expression of these two proteins in the model group decreased to 0.47 and 0.34, respectively. However, after repair by fermentation supernatant of Streptococcus infantis CCSM002, the expression of these three proteins could be upregulated to different degrees, up to 1.53 and 1.63, respectively.

[0098] Example 12: Effective substance analysis of fermentation supernatant of Streptococcus infantis CCSM002 1. Substance identification based on non-targeted metabolomics: Sample preparation and detection before CCSM002 fermentation supernatant metabolomics analysis: (1) After culturing Streptococcus infantis CCSM002 for 12 h, take 1 mL of bacterial solution (live count of 1 x 10⁻⁶). 7 CFU / mL), 4℃, 10000 g Centrifuge for 5 minutes and collect the supernatant. (2) Transfer 100 μL of supernatant into a 1.5 mL centrifuge tube; (3) Add 400 μL of methanol:acetonitrile = (1:1, v / v) (pre-cooled at -20℃ in advance) to precipitate the protein; (4) Vortex for 30 s, followed by ice bath ultrasound for 10 min; (5) Place the sample in a -20℃ refrigerator for 1 h to increase the protein precipitation rate (secondary precipitation removes protein). (6) Centrifuge at 15000 rpm for 15 min at 4℃. (7) Take the supernatant and concentrate it under vacuum; (8) Redissolve by adding 100 μL of acetonitrile:water (1:1) and vortexing for 30 s; (9) Centrifuge at 15,000 rpm for 15 min at 4℃, take the supernatant, transfer the appropriate volume into a vial for instrument testing; (10) For polar metabolites, this project used a Vanquish (Thermo Fisher Scientific) ultra-high performance liquid chromatograph (UPLC) with a Waters ACQUITY UPLC BEH Amide (2.1 mm × 50 mm, 1.7 μm) column for chromatographic separation of the target compounds. Phase A of the HPLC was aqueous, containing 25 mmol / L ammonium acetate and 25 mmol / L ammonia, and phase B was acetonitrile. Sample tray temperature: 4℃, injection volume: 2 μL. The Orbitrap Exploris 120 mass spectrometer was able to acquire primary and secondary mass spectrometry data under the control of the control software (Xcalibur, version 4.4, Thermo). Detailed parameters are as follows: Sheath gasflow rate: 50 Arb, Aux gas flow rate: 15 Arb, Capillary temperature: 320℃, Full ms resolution: 60000, MS / MS resolution: 15000, Collision energy: SNCE 20 / 30 / 40, Spray voltage: 3.8 kV (positive) or -3.4 kV (negative).

[0099] 2. Sample preparation and detection before CCSM002 bacterial cell structure and metabolomics analysis: After culturing *Streptococcus infantis* CCSM002 for 12 hours as described in step 1, take 1 mL of the bacterial culture and incubate at 4°C for 10,000 minutes. g Centrifuge for 5 minutes to collect the bacterial sludge; (1) Wash once with pre-cooled 0.9% physiological saline to remove residual culture medium; (2) After centrifuging to remove physiological saline, 1.5 mL centrifuge tubes containing bacterial sludge are placed into liquid nitrogen for quenching to stop metabolic activity; (3) Remove the sample from the liquid nitrogen and add 500 μL of methanol:acetonitrile = (1:1, v / v) (pre-cooled at -20℃ in advance); (4) After vortexing for 30 seconds, the cells were repeatedly frozen and thawed three times with liquid nitrogen to lyse the cells and release intracellular metabolites. (5) Place the sample in a -20℃ refrigerator for 1 h to increase the protein precipitation rate (secondary precipitation removes protein). (6) Centrifuge at 15,000 rpm for 15 min at 4℃; (7) Take the supernatant and evaporate it to dryness using a rotary evaporator; (8) Redissolve by adding 100 μL of acetonitrile:water (1:1) and vortexing for 30 s; (9) Centrifuge at 15,000 rpm for 15 min at 4℃, take the supernatant, transfer an appropriate volume into a vial for instrument testing; (10) For polar metabolites, this project used a Vanquish (Thermo Fisher Scientific) ultra-high performance liquid chromatograph (UPLC) with a Waters ACQUITY UPLC BEH Amide (2.1 mm × 50 mm, 1.7 μm) column for chromatographic separation of the target compounds. Phase A of the HPLC was aqueous, containing 25 mmol / L ammonium acetate and 25 mmol / L ammonia, and phase B was acetonitrile. Sample tray temperature: 4℃, injection volume: 2 μL. The Orbitrap Exploris 120 mass spectrometer was able to acquire primary and secondary mass spectrometry data under the control of the control software (Xcalibur, version 4.4, Thermo). Detailed parameters are as follows: Sheath gasflow rate: 50 Arb, Aux gas flow rate: 15 Arb, Capillary temperature: 320℃, Full ms resolution: 60000, MS / MS resolution: 15000, Collision energy: SNCE 20 / 30 / 40, Spray voltage: 3.8 kV (positive) or -3.4 kV (negative).

[0100] 3. Analysis of active ingredients in CCSM002: The raw data was converted into mzXML format using ProteoWizard software, and then metabolite identification was performed using a collaboratively developed R package. The database used was BiotreeDB (V3.0), and then visualization analysis was performed using a self-developed R package.

[0101] Literature review was conducted to collect the chemical formulas, molecular formulas, and molecular weights of potential effective substances in CCSM002. Based on non-target metabolomics, and according to the P-value < 0.05 and the differences compared with the pre-fermentation state, as well as research on skin barrier repair, potential effective substances were screened. The substances are shown in Table 2.

[0102] Table 2 Potential active ingredients related to skin barrier in CCSM002

[0103] 4. Detection of the content of active ingredients Based on the results of non-targeted metabolomics, the following substances were detected in the sample after targeted quantitative detection: (1) Oleamide like Figures 9-12 As shown, this substance was present in both the fermentation supernatant and the bacterial cells, at concentrations of 1.7 ppm and 4.92 ppm, respectively. Oleamide is a class of endogenous fatty amide molecules, which has recently been considered a potential active ingredient for regulating skin barrier homeostasis. In vitro studies in primary human keratinocyte and immune cell models have confirmed that oleamide can significantly inhibit the production of inflammatory factors such as TNF-α and IL-1β, thereby reducing the damage to the skin barrier caused by the inflammatory microenvironment. Simultaneously, oleamide can exert immunomodulatory and homeostatic effects by regulating the PPAR signaling pathway and CB2 receptor.

[0104] In terms of barrier repair, oleamides have been shown to promote the production of key barrier lipids in keratinocytes, including ceramides, which helps restore and optimize the lamellar structure of the stratum corneum lipids, enhancing the integrity and stability of the sebum film. By improving the skin's resistance to external stimuli (such as dryness, microbial invasion, and inflammatory factors), oleamides demonstrate significant application potential in skin health maintenance and barrier function repair.

[0105] (2) D-erythrosine disphingosine like Figures 13-16 As shown, this substance was present in both the fermentation supernatant and the bacterial cells, with concentrations of 0.96 ppm and 5.22 ppm, respectively. Sphingosine, a key molecule in the skin's surface lipid metabolism network, is an important component in maintaining barrier homeostasis and microecological balance. In vitro studies in a primary human keratinocyte model have confirmed that sphingosine not only significantly inhibits the proliferation of various opportunistic skin pathogens (such as Staphylococcus aureus and Corynebacterium spp.), but also reduces the activation of excessive inflammatory signals by regulating the TLR2 / TLR4 receptor pathway, thereby reducing the release of downstream inflammatory mediators of NF-κB and restoring cellular microenvironmental homeostasis. Furthermore, sphingosine promotes the synthesis of intrinsic lipids (including ceramides and their precursor sphingomyelin) within keratinocytes, enhances the activity of the sphingomyelin-ceramide metabolic axis, and further improves the integrity and functionality of the stratum corneum structure. Through multiple mechanisms of antibacterial, anti-inflammatory, and lipid homeostasis regulation, sphingosine demonstrates significant physiological regulatory potential in improving the skin barrier and maintaining a healthy microecology.

[0106] (3) (2-oxo-2,3-dihydro-1H-indol-3-yl)-acetic acid like Figures 17-20As shown, this substance was present in both the fermentation supernatant and the bacterial cells, with concentrations of 21.89 ppb and 13.13 ppb, respectively. (2-oxo-2,3-dihydro-1H-indol-3-yl)-acetic acid, as a structurally stable indolone derivative, possesses potential anti-inflammatory and antioxidant capabilities, providing multiple supports for maintaining skin homeostasis. Previous studies have shown that indolone molecules can alleviate oxidative stress damage induced by exogenous stimuli such as ultraviolet radiation and pollution by regulating the redox state of keratinocytes and reducing excessive ROS production. Furthermore, these molecules can inhibit inflammation-related NF-κB and MAPK signaling pathways, reduce the expression of inflammatory factors such as TNF-α and IL-6, and restore the skin to a more stable immune environment.

[0107] In terms of barrier function, (2-oxo-2,3-dihydro-1H-indol-3-yl)-acetic acid is expected to promote keratinocyte differentiation and the expression of barrier-related proteins (such as FLG, LOR, and IVL), thereby enhancing the integrity of the stratum corneum. Its structural properties may also enhance the activity of the skin's inherent antioxidant system (such as SOD and GSH-Px), helping cells resist environmental stress. Overall, this molecule possesses the potential to regulate inflammation, improve oxidative stress, and promote stratum corneum homeostasis, making it a potential functional candidate for skin repair and barrier support ingredients.

[0108] Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Any person skilled in the art can make various modifications and alterations without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be determined by the claims.

Claims

1. Streptococcus infantis ( Streptococcus infantis CCSM002 was deposited at the Guangdong Provincial Center for Microbial Culture Collection on September 2, 2024, with accession number GDMCC No: 65086.

2. The postbiotic prepared using the *Streptococcus infantis* CCSM002 described in claim 1, characterized in that, The metabiotic includes the fermentation supernatant and / or cell lysate of Streptococcus infantis CCSM002 as described in claim 1.

3. The method for preparing the postgenetic agent according to claim 2, characterized in that, Including (a) or (b): (a) The infantile streptococcus CCSM002 of claim 1 is cultured to the logarithmic growth phase, the fermentation supernatant is collected, and the supernatant is inactivated by heat treatment to obtain the metagene; (b) The infantile streptococcus CCSM002 of claim 1 is cultured to the logarithmic growth phase, the bacterial cells are collected, inactivated by heat treatment, and then homogenized under high pressure. The supernatant is collected to obtain the metagene.

4. A composition containing the Streptococcus infantis CCSM002 of claim 1 and / or its postbiotic.

5. The composition according to claim 4, characterized in that, The composition includes pharmaceuticals or daily chemical products, including skin care products or personal care products.

6. The composition according to claim 5, characterized in that, The composition further includes conventional excipients, which include one or more of fillers, flavoring agents, binders, disintegrants, lubricants, antacids, and nutritional fortifiers.

7. The composition according to any one of claims 4 to 6, characterized in that, The dosage forms of the composition include powders, tablets, blocks, liquid formulations, ointments, films, or gels.

8. The use of the infantile streptococcus CCSM002 and / or its postbiotics as described in claim 1 in the preparation of products for repairing the skin barrier.

9. The application as described in claim 8, characterized in that, The products include pharmaceuticals or daily chemical products; the repair of the skin barrier includes repairing skin keratinocytes and / or regulating the level of barrier-related genes.

10. The use of the *Streptococcus infantis* CCSM002 of claim 1 in the preparation of oleamide, D-erythrosine and / or (2-oxo-2,3-dihydro-1H-indol-3-yl)-acetic acid.

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