Probiotic prepared from paracasei ccfm1294 having improved skin cell ha content

Abstract

The application discloses a postbiotic prepared from paracasei CCFM1294 and having the function of improving the HA content of skin cells, and belongs to the technical field of microorganisms. The postbiotic prepared from paracasei CCFM1294 has the function of promoting the synthesis of HA of a host: (1) significantly up-regulating the UGDH mRNA expression of skin cells; (2) significantly up-regulating the HAS1 mRNA expression of skin cells; (3) significantly up-regulating the HAS2 mRNA expression of skin cells; (4) significantly up-regulating the HAS3 mRNA expression of skin cells; (5) increasing the HA content of skin cells; therefore, the postbiotic prepared from paracasei CCFM1294 has the function of promoting the synthesis of HA of a host and preventing and / or repairing the symptoms, such as dry skin and aging, and has a great application prospect.

Description

Technical Field

[0001] This invention relates to a metabiotic prepared from Lactobacillus paracasei CCFM1294 that improves HA content in skin cells, belonging to the field of microbial technology. Background Technology

[0002] Hyaluronic acid (HA) is the most important moisturizing substance in skin tissue. The skin's HA content is highest during adolescence, and gradually decreases with age. The resulting signs of aging, such as dryness, roughness, wrinkles, loss of elasticity, and lack of hydration, are all related to this decrease in HA content.

[0003] Currently, it has been reported that epidermal growth factor, transforming growth factor β, retinoic acid, N-methylserine, cytokines, and estradiol derivatives can enhance HA synthesis in cells (Journal of Investigative Dermatology.120(6),1038-1044, Journal of Biological Chemistry, 276(23), 20428–20435, BiochemicalJournal, 404, 327–336, Circulation Research, 94(5), 592–600, Journal of Biological Chemistry, 285(32), 24639–24645.).

[0004] CN111902149A discloses a method for promoting hyaluronic acid (HA) synthesis, which involves using a polysaccharide derivative or its salt to generate cell contact with hyaluronic acid, thereby promoting the synthesis of hyaluronic acid in the cells.

[0005] CN 113081878A discloses a composition for promoting skin HA synthesis, the composition comprising 0.01-5.0 parts by weight of L lactic acid.

[0006] The safety of the above-mentioned substances that increase HA content still needs to be further evaluated through clinical trials. This invention uses postbiotic substances prepared by probiotics to specifically increase the HA content of skin cells, innovatively linking the microbial field with HA synthesis.

[0007] Hyaluronic acid (HA) is a linear glycosaminoglycan formed by the alternating linkage of two precursors, UDP-GlcA and UDP-GlcNAc, under the action of hyaluronic acid synthase (HAS). It has significant effects on improving skin problems caused by UV exposure, such as skin inflammation, lipid peroxidation, and apoptosis. The synthesis of HA precursors originates from G6P, a product of glycolysis. UDP-GlcA is synthesized from G1P under the mediation of UDP-glucose dehydrogenase (UGDH), while UDP-GlcNAc is synthesized from F6P via the hexosamine biosynthesis pathway. Two important factors affecting HA synthesis are the activity of hyaluronic acid synthase (HAS) and the supply of the HA precursors UDP-GlcA and UDP-GlcNAc. In mammals, three isoenzymes, HAS1, HAS2, and HAS3, can all participate in HA synthesis. In the HA synthesis pathway, UGDH is the only rate-limiting enzyme mediating UDP-GlcA synthesis. Changes in its expression can affect HA levels by influencing UDP-GlcA accumulation. Studies have shown that upregulating the expression of UGDH mRNA, HAS1 mRNA, HAS2 mRNA, and HAS3 mRNA during HA synthesis can promote HA synthesis, thereby increasing intracellular HA content. Therefore, using probiotic-prepared metabiotics to increase HA levels in skin cells and improve skin health by regulating the expression of UGDH mRNA, HAS1 mRNA, HAS2 mRNA, and HAS3 mRNA has practical application significance. Summary of the Invention

[0008] This invention provides a method that can be produced by Lactobacillus paracasei ( Lacticaseibacillus paracasei This is a post-biotic prepared by [organism name] that can increase the HA content of skin cells.

[0009] This invention provides a strain of Lactobacillus paracasei ( Lacticaseibacillus paracasei CCFM1294, the Lactobacillus paracasei ( Lacticaseibacillus paracasei It was deposited on November 13, 2022, at the Guangdong Provincial Center for the Preservation of Microbial Cultures, with accession number GDMCC No: 62972, located at Building 59, No. 100, Xianlie Middle Road, Guangzhou.

[0010] The Lactobacillus paracasei ( Lacticaseibacillus paracasei CCFM1294 was isolated from the feces of healthy human beings. After sequencing analysis, the sequence obtained was compared with the nucleic acid sequence in NCBI. After the comparison result, it was identified as Lactobacillus paracasei.

[0011] The Lactobacillus paracasei ( Lacticaseibacillus paracasei The colonies of CCFM1294 on MRS solid medium are round, white, and smooth.

[0012] The Lactobacillus paracasei ( Lacticaseibacillus paracasei CCFM1294 is a Gram-positive, facultative anaerobic bacterium that thrives in warm temperatures. Its optimal growth temperature is 35-40℃ and its optimal growth pH is 6.0-7.0.

[0013] The present invention provides a composition comprising the above-mentioned *Lactobacillus paracasei* CCFM1294 and / or a metabiotic prepared from the above-mentioned *Lactobacillus paracasei* CCFM1294.

[0014] In one embodiment of the present invention, the metabiotic includes dead cells, fermentation supernatant, cell lysate and / or fermentation broth.

[0015] In one embodiment of the present invention, the fermentation broth is prepared by inoculating the above-mentioned Lactobacillus paracasei CCFM1294 into a fermentation medium and culturing it to obtain a bacterial solution, followed by heat treatment to obtain the fermentation broth.

[0016] In one embodiment of the present invention, the heat treatment conditions are 60~70°C for 25~35 min.

[0017] In one embodiment of the present invention, the method for preparing the cell lysate is to homogenize the fermentation broth prepared above under high pressure and centrifuge it to obtain the cell lysate.

[0018] In one embodiment of the present invention, the dead cells are inactivated bacterial cells obtained by centrifuging the above-mentioned fermentation broth to obtain bacterial sludge precipitate, and then heat-treating or freeze-drying them.

[0019] In one embodiment of the present invention, the post-generic agent can be dried into powder or used directly by various drying methods such as vacuum drying, spray drying, vacuum freeze drying, and fluidized bed drying.

[0020] This invention provides the use of the aforementioned Lactobacillus paracasei CCFM1294 or the aforementioned composition in the preparation of pharmaceuticals for preventing and / or repairing dry skin and delaying aging.

[0021] This invention provides the application of the metabiotic prepared from Lactobacillus paracasei CCFM1294 in the preparation of cosmetics for preventing and / or repairing dry skin and delaying aging.

[0022] This invention provides the application of the metabiotic prepared from Lactobacillus paracasei CCFM1294 in the preparation of skin care products for preventing and / or repairing dry skin and delaying aging.

[0023] In one embodiment of the present invention, the product includes cosmetics, skin care products, and pharmaceuticals.

[0024] In one embodiment of the present invention, the cosmetic comprises the above-described composition, matrix raw materials and / or conventional excipients.

[0025] In one embodiment of the present invention, 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.

[0026] In one embodiment of the present invention, 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.

[0027] In one embodiment of the present invention, the skin care product includes the above-described composition, basic raw materials and / or conventional excipients.

[0028] In one embodiment of the present invention, the basic raw materials include hydrocarbons, waxes, and oils.

[0029] In one embodiment of the present invention, the hydrocarbons include liquid paraffin, solid paraffin, microcrystalline wax, ceresin, and petrolatum.

[0030] In one embodiment of the present invention, the waxes include palm wax, candelilla wax, jojoba wax, wood wax, lanolin, and beeswax.

[0031] In one embodiment of the present invention, the oil raw materials include olive oil, coconut oil, castor oil, cottonseed oil, rice bran oil, and tea seed oil.

[0032] In one embodiment of the present invention, the conventional excipients include solvents, softeners, penetrants, stabilizers, chelating agents, thickeners, and dispersants.

[0033] In one embodiment of the present invention, the pharmaceutical product comprises a composition, a drug carrier, and / or pharmaceutical excipients.

[0034] In one embodiment of the present invention, the drug carrier comprises microcapsules, microspheres, nanoparticles, and liposomes.

[0035] In one embodiment of the present invention, the pharmaceutical excipient comprises excipients and additives.

[0036] In one embodiment of the present invention, 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.

[0037] In one embodiment of the present invention, the additives comprise microcrystalline cellulose, hydroxypropyl methylcellulose, and refined lecithin.

[0038] In one embodiment of the present invention, the dosage form of the medicine includes capsules, granules, mixtures, tablets or tinctures.

[0039] The present invention also provides a pharmaceutical product containing the above-mentioned Lactobacillus paracasei CCFM1294 or the composition thereof; the product has the effect of preventing and / or repairing symptoms related to dry skin and aging.

[0040] In one embodiment of the invention, symptoms associated with dry skin include skin becoming inelastic, rough, and developing fine lines.

[0041] In one embodiment of the present invention, symptoms related to skin aging include increased skin sensitivity to temperature, sagging, irregular pigmentation, significantly increased wrinkles, and decreased skin resistance.

[0042] In one embodiment of the present invention, the product includes at least one of the following functions:

[0043] (1) Significantly upregulated the expression level of UGDH mRNA in skin cells;

[0044] (2) Significantly upregulated the expression level of HAS1 mRNA in skin cells;

[0045] (3) Significantly upregulated the expression level of HAS2 mRNA in skin cells;

[0046] (4) Significantly upregulated the expression level of HAS3 mRNA in skin cells;

[0047] (5) Significantly increases the HA content of skin cells.

[0048] The present invention also provides a cosmetic containing a metabiotic prepared from Lactobacillus paracasei CCFM1294.

[0049] The present invention also provides a skin care product containing a postbiotic prepared from Lactobacillus paracasei CCFM1294.

[0050] In one embodiment of the present invention, the product includes cosmetics, skin care products, and pharmaceuticals.

[0051] In one embodiment of the present invention, the cosmetic comprises the above-described composition, matrix raw materials and / or conventional excipients.

[0052] In one embodiment of the present invention, 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.

[0053] In one embodiment of the present invention, 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.

[0054] In one embodiment of the present invention, the skin care product includes the above-described composition, basic raw materials and / or conventional excipients.

[0055] In one embodiment of the present invention, the basic raw materials include hydrocarbons, waxes, and oils.

[0056] In one embodiment of the present invention, the hydrocarbons include liquid paraffin, solid paraffin, microcrystalline wax, ceresin, and petrolatum.

[0057] In one embodiment of the present invention, the waxes include palm wax, candelilla wax, jojoba wax, wood wax, lanolin, and beeswax.

[0058] In one embodiment of the present invention, the oil raw materials include animal fats and vegetable oils.

[0059] In one embodiment of the present invention, the animal fats include mink oil, egg yolk oil, lanolin, and lecithin.

[0060] In one embodiment of the present invention, the vegetable oils include olive oil, coconut oil, castor oil, cottonseed oil, rice bran oil, and tea seed oil.

[0061] In one embodiment of the present invention, the conventional excipients include solvents, softeners, penetrants, stabilizers, chelating agents, thickeners, and dispersants.

[0062] In one embodiment of the present invention, the pharmaceutical product comprises a composition, a drug carrier, and / or pharmaceutical excipients.

[0063] In one embodiment of the present invention, the drug carrier comprises microcapsules, microspheres, nanoparticles, and liposomes.

[0064] In one embodiment of the present invention, the pharmaceutical excipient comprises excipients and additives.

[0065] In one embodiment of the present invention, 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.

[0066] In one embodiment of the present invention, the additives comprise microcrystalline cellulose, hydroxypropyl methylcellulose, and refined lecithin.

[0067] In one embodiment of the present invention, the dosage form of the medicine includes capsules, granules, mixtures, tablets or tinctures.

[0068] The present invention also provides the use of the above-mentioned Lactobacillus paracasei CCFM1294 or the composition thereof in the preparation of products that increase the hyaluronic acid content in cells.

[0069] Beneficial effects

[0070] This invention screened and obtained a strain of Lactobacillus paracasei ( Lacticaseibacillus paracasei CCFM1294, this is Lactobacillus paracasei ( Lacticaseibacillus paracasei The post-biotic prepared with CCFM1294 has the effect of repairing symptoms related to dry skin and aging, specifically manifested in:

[0071] (1) Significantly upregulated the expression of UGDH mRNA in skin cells;

[0072] (2) Significantly upregulated the expression of HAS1 mRNA in skin cells;

[0073] (3) Significantly upregulated the expression of HAS2 mRNA in skin cells;

[0074] (4) Significantly upregulated the expression of HAS3 mRNA in skin cells;

[0075] (5) Significantly increases the HA content in skin cells;

[0076] Therefore, Lactobacillus paracasei ( Lacticaseibacillus paracasei The post-biotic prepared by CCFM1294 has great application potential in the preparation of products for preventing and / or repairing dry and aging skin, and even preventing and / or alleviating skin problems related to aging and metabolic disorders.

[0077] Preservation of biological materials

[0078] A strain of Lactobacillus paracasei ( Lacticaseibacillus paracasei CCFM1294, taxonomically named Lacticaseibacillus paracasei It was deposited on November 13, 2022, at the Guangdong Provincial Center for Microbial Culture Collection, with accession number GDMCC No: 62972, located at Building 59, No. 100 Xianlie Middle Road, Guangzhou. Attached Figure Description

[0079] Figure 1 Effects of different epigenetics on the expression of UGDH mRNA in skin cells.

[0080] Figure 2 Effects of different epigenetics on HAS1 mRNA expression in skin cells.

[0081] Figure 3 Effects of different epigenetics on HAS2 mRNA expression in skin cells.

[0082] Figure 4 Effects of different epigenetics on HAS3 mRNA expression in skin cells.

[0083] Figure 5 The effect of different post-genes on the HA content of skin cells.

[0084] "*" indicates a statistically significant difference from the Model group (P<0.05), "**" indicates a statistically significant difference from the Model group (P<0.01), and "***" indicates an extremely statistically significant difference from the Model group (P<0.001). Detailed Implementation

[0085] The human immortalized keratinocytes (HaCaT) involved in the following examples were purchased from the Shanghai Cell Bank.

[0086] The *Lactobacillus paracasei* FXJWS3M2 postbiotic and *Lactobacillus plantarum* 23 postbiotic involved in the following examples were obtained from the laboratory of the Food Biotechnology Center of Jiangnan University.

[0087] The present invention will be further described below with reference to specific embodiments.

[0088] The culture media involved in the following examples are as follows:

[0089] MRS liquid culture medium: yeast extract 5.0 g / L, beef extract 10.0 g / L, peptone 10.0 g / L, glucose 20.0 g / L, anhydrous sodium acetate 2.0 g / L, diammonium citrate 2.0 g / L, dipotassium hydrogen phosphate 2.6 g / L, manganese sulfate monohydrate 0.25 g / L, magnesium sulfate heptahydrate 0.5 g / L, and Tween-80 1 mL / L, pH 6.2~6.4.

[0090] MRS solid culture medium: yeast extract 5.0 g / L, beef extract 10.0 g / L, peptone 10.0 g / L, glucose 20.0 g / L, anhydrous sodium acetate 2.0 g / L, diammonium citrate 2.0 g / L, dipotassium hydrogen phosphate 2.6 g / L, manganese sulfate monohydrate 0.25 g / L, magnesium sulfate heptahydrate 0.5 g / L, Tween-80 1 mL / L, and agar 20.0 g / L, pH 6.2~6.4.

[0091] 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).

[0092] Example 1: Screening and identification of Lactobacillus paracasei

[0093] The specific steps are as follows:

[0094] 1. Screening

[0095] The samples were derived from feces of healthy individuals. After pretreatment, the samples were stored in 30% glycerol at -80°C. After thawing, the samples were mixed and 0.5 mL was added to 4.5 mL of physiological saline. The samples were then serially diluted with physiological saline containing 9 g / L. The appropriate serial dilutions were plated onto MRS solid medium and incubated at 37°C for 48 h. Typical colonies of *Lactobacillus paracasei* were picked and streaked onto MRS solid medium for purification. Single colonies were then transferred to MRS liquid medium for enrichment and preserved in 30% glycerol to obtain *Lactobacillus paracasei* CCFM1294. The typical colonies of *Lactobacillus paracasei* are round, milky white, smooth, and raised.

[0096] 2. Identification

[0097] The genome of strain CCFM1294 was extracted, and the 16S rDNA of strain CCFM1294 was amplified and sequenced (performed by Suzhou Genewiz Biotechnology Co., Ltd.). The nucleotide sequence of the amplified 16S rDNA of CCFM1294 was compared with the nucleic acid sequence in NCBI, and the results showed that the strain was Lactobacillus paracasei, and it was named Lactobacillus paracasei. Lacticaseibacillus paracasei (CCFM1294)

[0098] Example 2: Cell resuscitation and culture

[0099] First, remove the cryopreserved human immortalized keratinocytes (HaCaT), quickly thaw them in a 37°C water bath, then centrifuge at 1000 r / min for 3 min, discard the supernatant, add an appropriate volume of cell culture medium to resuspend the cells, place them in a culture dish, and incubate them in a 37°C incubator containing 5% CO2. When the cells grow for 1-2 days and reach 70%-80% confluence, passage the cells.

[0100] Example 3: Preparation of postbiotics from Lactobacillus paracasei CCFM1294

[0101] (1) Use MRS solid medium to culture in a water-jacketed constant temperature incubator at 37℃ for 24~48 h to obtain single colonies; pick single colonies and inoculate them into MRS liquid medium, and culture at 37℃ for 12~18 h to obtain culture solution 1;

[0102] Culture medium 1 was inoculated into MRS liquid medium at an inoculation rate of 2% (v / v) and cultured at 37°C for 12 h to obtain seed culture;

[0103] The seed culture was inoculated at 3-5% (v / v) into MRS liquid medium for expansion, and cultured at 37℃ for 18-24 h to obtain the bacterial culture with a concentration of 4.2 × 10⁻⁶. 10 CFU / ml.

[0104] The bacterial culture was heat-treated (65 °C, 30 min), and then homogenized under high pressure (800~1200 MPa, 3 times) to obtain bacterial cell lysate. The lysate was then freeze-dried to obtain post-liferogen freeze-dried powder for later use, thus preparing post-liferogen of Lactobacillus paracasei CCFM1294.

[0105] (2) Lactobacillus paracasei FXJWS3M2 and Lactobacillus plantarum 23 postbiotics were prepared according to the method in step (1).

[0106] Example 4: Effect of postbiotic prepared from Lactobacillus paracasei CCFM1294 on UGDH mRNA in HaCaT cells

[0107] The specific steps are as follows:

[0108] (1) HaCaT cells were injected at a concentration of 2.5 × 10⁻⁶. 6 Cells were seeded at 12-well plates at a density of 10 cells / ml and cultured overnight until cell attachment. The old culture medium was discarded, and the cells were washed three times with PBS. A control group and a treatment group were set up.

[0109] The control group consisted of groups that did not receive the post-genetic agent.

[0110] The treatment group consisted of metabiotics resuspended in cell culture medium (the amount of resuspended metabiotics was the same as that fermented to a concentration of 1.0 × 10⁻⁶). 7 The amount of metabiotics prepared from bacterial culture at CFU / ml is equivalent.

[0111] Add 2 ml of the Lactobacillus paracasei CCFM1294 postgenetic, Lactobacillus paracasei FXJWS3M2 postgenetic, and Lactobacillus plantarum 23 postgenetic prepared in Example 3 to well plates containing HaCaT cells respectively.

[0112] Each group was incubated at 37°C for 4 hours, with three replicates for each sample.

[0113] (2) After the metagenics and cells were co-cultured, the culture supernatant was discarded, each well was washed three times with PBS, 1 mL of cell lysis buffer was added to each well, and the cells were repeatedly pipetted. RNA was extracted from the cell lysis buffer and reverse transcribed into cDNA using an RT-PCR reverse transcription kit. The expression of genes in HaCaT cells was detected by real-time quantitative PCR. -△△Ct The formula calculates the expression level of UGDH mRNA, with GAPDH as the internal reference. The primers are described in Table 1 below, and the results are as follows: Figure 1 As shown.

[0114] Table 1: Primer sequences

[0115]

[0116] The results show that, by Figure 1 It can be seen that, with the relative expression level of the control group (Control) as 1, the relative expression levels of UGDH mRNA of Lactobacillus paracasei CCFM1294 postgenerant, Lactobacillus paracasei FXJWS3M2 postgenerant, and Lactobacillus plantarum 23 postgenerant were 1.94, 1.21, and 1.35, respectively. The Lactobacillus paracasei CCFM1294 postgenerant group significantly promoted the expression of UGDH mRNA (94% higher than the control group), while the Lactobacillus paracasei FXJWS3M2 postgenerant only increased it by about 21% compared with the control group.

[0117] Cellular experiments showed that the post-genetic group of Lactobacillus paracasei CCFM1294 has the ability to upregulate UGDH mRNA expression.

[0118] Example 5: Effects of post-biotics prepared from *Lactobacillus paracasei* CCFM1294 on HAS1 mRNA, HAS2 mRNA, and HAS3 mRNA in HaCaT cells

[0119] The specific steps are as follows:

[0120] (1) HaCaT cells were injected at a concentration of 2.5 × 10⁻⁶. 6 Cells were seeded at a density of 10 cells / ml in 12-well plates and cultured overnight until cell attachment was achieved. The old culture medium was discarded, and the cells were washed three times with PBS. A control group and a treatment group were set up.

[0121] The control group consisted of groups that did not receive the post-genetic agent.

[0122] The treatment group consisted of metabiotics resuspended in cell culture medium (the amount of resuspended metabiotics was the same as that fermented to a concentration of 1.0 × 10⁻⁶). 7 The amount of metabiotics prepared from bacterial culture at CFU / ml is equivalent.

[0123] Add 2 ml of the Lactobacillus paracasei CCFM1294 postgenetic, Lactobacillus paracasei FXJWS3M2 postgenetic, and Lactobacillus plantarum 23 postgenetic prepared in Example 3 to well plates containing HaCaT cells respectively.

[0124] Each group was incubated at 37°C for 4 hours, with three replicates for each sample.

[0125] (2) After the metagenics and cells were co-cultured, the culture supernatant was discarded, each well was washed three times with PBS, 1 mL of cell lysis buffer was added to each well, and the cells were repeatedly pipetted. RNA was extracted from the cell lysis buffer and reverse transcribed into cDNA using an RT-PCR reverse transcription kit. The expression of genes in HaCaT cells was detected by real-time quantitative PCR. -△△Ct The formula calculates the expression levels of HAS1 mRNA, HAS2 mRNA, and HAS3 mRNA, with GAPDH as the internal reference. The primers are described in Table 2 below, and the results are as follows: Figures 2 - 4 As shown.

[0126] Table 2: Primer sequences

[0127]

[0128] The results showed that the expression levels of HAS1 mRNA, HAS2 mRNA, and HAS3 mRNA in the control group were 1.

[0129] The relative expression levels of HAS1 mRNA in the metagenic groups prepared from *Lactobacillus paracasei* CCFM1294, *Lactobacillus paracasei* FXJWS3M2, and *Lactobacillus plantarum* 23 were 1.68, 0.33, and 0.91, respectively; the relative expression levels of HAS2 mRNA were 1.44, 0.68, and 1.01, respectively; and the relative expression levels of HAS3 mRNA were 1.49, 0.74, and 1.24, respectively.

[0130] This shows that Lactobacillus paracasei CCFM1294 significantly promoted the expression of HAS1 mRNA (68% higher than the control group), HAS2 mRNA (44% higher than the control group), and HAS3 mRNA (49% higher than the control group).

[0131] Cellular experiments showed that the post-genetic group prepared from Lactobacillus paracasei CCFM1294 has the ability to upregulate the expression of HA synthase.

[0132] Example 6: Effect of metabiotic prepared from Lactobacillus paracasei CCFM1294 on HA content in HaCaT cells

[0133] The specific steps are as follows:

[0134] (1) HaCaT cells were injected at a concentration of 2.5 × 10⁻⁶. 6 Cells were seeded at a density of 10 cells / ml in 12-well plates and cultured overnight until cell attachment was achieved. The old culture medium was discarded, and the cells were washed three times with PBS. A control group and a treatment group were set up.

[0135] The control group consisted of groups that did not receive the post-genetic agent.

[0136] The treatment group used cell culture medium to resuspend the metabiotics (the amount of resuspended metabiotics was the same as that fermented to a concentration of 1.0 × 10⁻⁶). 7 The amount of metabiotics prepared from bacterial culture at CFU / ml is equivalent.

[0137] Add 2 ml of postbiotics prepared from Lactobacillus paracasei CCFM1294, Lactobacillus paracasei FXJWS3M2, and Lactobacillus plantarum 23 to well plates containing HaCaT cells respectively, and incubate for 4 h. Each sample is in triplicate.

[0138] (2) After the co-culture of metagenin and cells is complete, discard the culture supernatant, wash each well three times quickly with PBS, add 1 mL of cell lysis buffer to each well, repeatedly pipette, and aspirate the cell lysis buffer to detect the HA content of skin cells using ELISA. The results are as follows: Figure 5 As shown.

[0139] The results show that, by Figure 5It can be seen that the HA content of the control group was 29.61 µg / l, the HA content of the post-genetic group prepared by Lactobacillus paracasei CCFM1294 was 46.79 µg / l, the HA content of the post-genetic group prepared by Lactobacillus paracasei FXJWS3M2 was 31.39 µg / l, and the HA content of the post-genetic group prepared by Lactobacillus plantarum 23 was 33.60 µg / l.

[0140] Compared with the control group and other metagener groups, the metagener prepared from Lactobacillus paracasei CCFM1294 significantly increased the HA content of skin cells (upregulated by 58% compared with the control group), while the other metagener groups showed the highest increase in HA content of skin cells compared with the control group, up by 13.48%.

[0141] Cellular experiments showed that the metabiotic prepared from Lactobacillus paracasei CCFM1294 promoted HA synthesis by upregulating the expression of precursor enzymes UGDH and HA synthase in the HA synthesis process, thereby increasing the HA content in skin cells.

[0142] Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Anyone 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. A strain of Lactobacillus paracasei ( Lacticaseibacillus paracasei CCFM1294 was deposited at the Guangdong Provincial Center for Microbial Culture Collection on November 13, 2022, with accession number GDMCC No: 62972, and the deposit address is Building 59, No. 100, Xianlie Middle Road, Guangzhou.

2. A composition, characterized in that, The composition comprises *Lactobacillus paracasei* CCFM1294 as described in claim 1 and / or a metagenetic agent prepared from *Lactobacillus paracasei* CCFM1294 as described in claim 1, wherein the metagenetic agent comprises dead cells and cell lysates.

3. A method for preparing postbiotics from *Lactobacillus paracasei* CCFM1294, characterized in that, The method involves inoculating the *Lactobacillus paracasei* CCFM1294 of claim 1 into a fermentation medium to obtain a bacterial solution, and then subjecting the bacterial solution to heat treatment and homogenization lysis to obtain a metabiotic.

4. The use of the metabiotic prepared from Lactobacillus paracasei CCFM1294 as described in claim 1 in the preparation of cosmetics for preventing and / or repairing dry skin and delaying aging.

5. The use of the post-biotic prepared from Lactobacillus paracasei CCFM1294 as described in claim 1 in the preparation of skin care products for preventing and / or repairing dry skin and delaying aging.

6. A cosmetic product, characterized in that, The cosmetic contains a metabiotic prepared from Lactobacillus paracasei CCFM1294 as described in claim 1.

7. A skincare product, characterized in that, The skincare product contains a metabiotic prepared from Lactobacillus paracasei CCFM1294 as described in claim 1.

8. The use of Lactobacillus paracasei CCFM1294 of claim 1 or the composition of claim 2 in the preparation of products that increase the hyaluronic acid (HA) content in cells.

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