CN116083315B - A strain of Lactobacillus plantarum CCFM1296 with the ability to regulate host HA content and its postbiotics - Google Patents

Abstract

The present invention discloses a strain of plant lactobacillus CCFM1296 and its postbiotics capable of regulating the host HA content, and belongs to the fields of microbial technology and pharmaceutical technology. The plant lactobacillus CCFM1296 provided by the present invention and the postbiotics prepared therefrom have the function of regulating the host HA content, including: (1) promoting the expression of HA synthase in intestinal cells and inhibiting the expression of HA degrading enzymes; (2) promoting the expression of HA synthase in skin cells; (3) up-regulating the HA level in the skin of aging mice; (4) up-regulating the HA content in the joints of aging mice; (5) up-regulating the brain HA level of aging mice; (6) up-regulating the eyeball HA level of aging mice. The plant lactobacillus CCFM1296 provided by the present invention and the postbiotics prepared therefrom can be used to prepare products for preventing and/or treating skin and joint-related diseases caused by HA loss, and have great application prospects.

Description

A strain of Lactobacillus plantarum CCFM1296 with the ability to regulate host HA content and its postbiotics

Technical Field

The invention relates to a strain of Lactobacillus plantarum CCFM1296 capable of regulating host HA content and a postbiotic thereof, and belongs to the technical field of microorganisms and the technical field of medicine.

Background Art

Hyaluronic acid (HA), also known as hyaluronic acid, is an acidic mucopolysaccharide composed of repeating disaccharide units of D-glucuronic acid and N-acetylglucosamine. It was isolated from cow eyes by Meyer, a professor of ophthalmology at Columbia University in the United States, in 1934.

HA is widely present in connective tissues of animals and humans, with high levels in vitreous body, skin, cartilage and synovial fluid. HA also plays an important role in the development and function of the brain and heart. The role of HA in the body is reflected in lubricating joints and eyeballs, regulating the permeability of blood vessel walls, regulating the diffusion of water and electrolytes, slowing down keratin differentiation, and removing free radicals.

The synthesis of HA comes from the product of glycolysis, glucose-6-phosphate (G6P). G6P is converted into glucose-1-phosphate (G1P) and fructose-6-phosphate (F6P) under the catalysis of corresponding enzymes. UDP-glucuronic acid (UDP-GlcA) is synthesized from G1P as a substrate under the action of a series of enzymes. The conversion of UDP-glucose (UDP-G) to UDP-GlcA catalyzed by UDP-glucose dehydrogenase (UGDH) is a rate-limiting step. Regulating the gene expression of UGDH can affect the content of the final product HA. UDP-N-acetylglucosamine (UDP-GlcNAc) is synthesized from F6P as a substrate through the hexosamine biosynthesis pathway (HBP). The finally synthesized UDP-GlcA and UDP-GlcNAc are alternately connected by HA synthase (HAS1-3) on the cytoplasmic membrane through β-1,3 and β-1,4 glycosidic bonds to form linear glycosaminoglycans with multiple repeating disaccharide units. HA synthase is crucial to the synthesis of HA, and any regulation of the expression changes of the three isozymes will cause changes in the content of HA. In the degradation of HA, HA is mainly circulated and metabolized through HYAL1, HYAL2, and HYBID. HYAL1 and HYAL2 are the most important enzymes for degrading HA in somatic cells. Inhibiting their expression can reduce the loss of HA. HYBID is an HA degradation enzyme independent of HYAL1 and HYAL2, and is involved in the metabolism of HA in the skin, joints, and brain. Therefore, by regulating the changes in the enzyme expression of UGDH, HAS1-3, HYAL1, HYAL2, and HYBID, it can promote the synthesis of HA and inhibit HA degradation, thereby achieving the purpose of maintaining the body's HA content. However, because HYAL1 and HYAL2 are necessary enzymes to ensure the body's HA turnover, inhibiting their activity will cause local accumulation of HA, leading to edema and obstruction of material transport. Therefore, the changes in UGDH, HAS1-3, and HYBID mRNA expression are used as targets to screen postbiotics with potential to regulate the host's HA content in cells.

Summary of the invention

The technical problem to be solved by the present invention is to provide a Lactobacillus planturum capable of regulating the HA content of a host and a postbiotic prepared therefrom.

The present invention provides a strain of Lactobacillus plantarum CCFM1296 (Lactiplantibacillus plantarum), which was deposited in Guangdong Provincial Microbiological Culture Collection Center on November 13, 2022, with a deposit number of GDMCC No. 62974, and a deposit address of Building 59, No. 100 Xianlie Middle Road, Guangzhou.

The Lactobacillus planturum CCFM1296 is isolated from healthy human feces and has the following characteristics:

The cells of Lactiplantibacillus planturum CCFM1296 appear as slightly irregular, round-ended, non-motile Campylobacter under a microscope, and the colonies inoculated on MRS medium and cultured for 48 hours are generally milky white, smooth and convex, and circular with a diameter of 0.5-2 mm.

The Lactobacillus planturum CCFM1296 is a Gram-positive bacterium, facultative anaerobic, thermophilic, with an optimum growth temperature of 35-40° C. and an optimum growth pH of 6.0-7.0.

The invention provides a composition, which contains the Lactobacillus plantarum CCFM1296 or a postbiotic prepared from the Lactobacillus plantarum CCFM1296.

In one embodiment, the postbiotics include dead cells, fermentation supernatant, bacterial lysate and/or fermentation broth.

In one embodiment, the method for preparing the postbiotics is to inoculate the above-mentioned Lactobacillus plantarum CCFM1296 into a fermentation medium to obtain a bacterial liquid, perform heat treatment and lysis, and obtain the postbiotics.

In one embodiment, the heat treatment is performed at 60-70° C. for 25-35 min.

In one embodiment, the method for preparing bacterial lysate is to homogenize the heat-treated fermentation broth under high pressure and centrifuge to obtain the bacterial lysate.

In one embodiment, the dead cells are bacterial sludge precipitates obtained by centrifuging the fermentation broth, and inactivated bacterial cell precipitates obtained by heat treatment or freeze drying.

In one embodiment, the fermentation supernatant is the supernatant obtained by centrifuging the fermentation broth.

In one embodiment, the postbiotics can be dried into powder or used directly by various drying methods such as vacuum drying, spray drying, vacuum freeze drying, fluidized bed drying, etc.

The present invention provides the use of the above-mentioned Lactobacillus planturum CCFM1296 in preparing a product for regulating the HA content of a host.

In one embodiment, the regulation of host HA content mainly includes HA levels in tissues such as skin, joints, and brain.

In one embodiment, the application method includes but is not limited to topical application or oral administration.

The present invention provides the use of the composition in the preparation of a drug for preventing and/or treating skin and joint related diseases caused by HA loss

In one embodiment, the skin-related diseases caused by the loss of HA include but are not limited to dry skin, atopic dermatitis, psoriasis, and skin aging.

In one embodiment, the joint-related diseases caused by HA loss include but are not limited to joint stiffness, knee pain, cartilage lesions, osteoarthritis, cartilage defects, cartilage damage or meniscus damage.

The invention also provides food, health products, medicine or cosmetics containing the Lactobacillus plantarum CCFM1296 postbiotics.

In one embodiment, the food comprises the above composition and conventional auxiliary materials.

In one embodiment, the conventional auxiliary materials include one or more of fillers, flavoring agents, binders, disintegrants, lubricants, antacids, and nutritional enhancers.

In one embodiment, the health care product comprises the above composition and conventional auxiliary materials.

In one embodiment, the conventional auxiliary materials include one or more of fillers, flavoring agents, binders, disintegrants, lubricants, antacids, and nutritional enhancers.

In one embodiment, the drug comprises the above composition, a drug carrier and/or a pharmaceutical excipient.

In one embodiment, the pharmaceutical excipient comprises an excipient and an additive.

In one embodiment, the pharmaceutical excipients include solvents, propellants, solubilizers, cosolvents, emulsifiers, colorants, binders, disintegrants, fillers, lubricants, wetting agents, osmotic pressure regulators, stabilizers, glidants, flavoring agents, preservatives, suspending agents, coating materials, fragrances, anti-adhesives, integrities, penetration enhancers, pH regulators, buffers, plasticizers, surfactants, foaming agents, defoamers, thickeners, inclusion agents, humectants, absorbents, diluents, flocculants and deflocculating agents, filter aids and release retardants.

In one embodiment, the cosmetic comprises the above composition, matrix raw materials and/or conventional auxiliary materials.

In one embodiment, the matrix raw materials include oil raw materials, wax raw materials, synthetic oil raw materials, powdery raw materials, colloid raw materials, coagulants, and surfactants.

In one embodiment, the conventional auxiliary materials include one or more of moisturizers, whitening agents, flavoring agents, adhesives, lubricants, preservatives, film agents, antioxidants, emulsifiers, and cosmetic nutritional additives.

The present invention provides a product for regulating the HA content of a host, wherein the product comprises the above-mentioned Lactobacillus planturum CCFM1296 and/or the above-mentioned composition.

In one embodiment, the product comprises at least one of the following effects:

(1) Promote the expression of HA synthase and inhibit the expression of HA degradation enzyme in intestinal cells (HT29);

(2) Promote the expression of HA synthase in skin cells (HaCaT);

(3) Significantly upregulated HA levels in the skin of aged mice by promoting the expression of HA synthase and inhibiting the expression of HA degradase;

(4) Significantly upregulates the HA content in the joints of aged mice by promoting the expression of HA synthase;

(5) It significantly upregulated the brain HA level of aged mice by promoting the expression of HA synthase and inhibiting the expression of HA degradation enzymes;

(6) Significantly up-regulated the eye HA level of aged mice by promoting the expression of HA synthase;

In one embodiment, in the product, the dosage of the postbiotics prepared by Lactobacillus planturum CCFM1296 is not less than 60 mg/kg body weight.

In one embodiment, the product comprises food, health care product, medicine, and cosmetics.

In one embodiment, the food comprises the above composition and conventional auxiliary materials.

In one embodiment, the conventional auxiliary materials include one or more of fillers, flavoring agents, binders, disintegrants, lubricants, antacids, and nutritional enhancers.

In one embodiment, the health care product comprises the above composition and conventional auxiliary materials.

In one embodiment, the conventional auxiliary materials include one or more of fillers, flavoring agents, binders, disintegrants, lubricants, antacids, and nutritional enhancers.

In one embodiment, the drug comprises a composition, a pharmaceutical carrier and/or a pharmaceutical excipient.

In one embodiment, the drug carrier comprises microcapsules, microspheres, nanoparticles and liposomes.

In one embodiment, the pharmaceutical excipient comprises an excipient and an additive.

In one embodiment, the pharmaceutical excipients include solvents, propellants, solubilizers, cosolvents, emulsifiers, colorants, binders, disintegrants, fillers, lubricants, wetting agents, osmotic pressure regulators, stabilizers, glidants, flavoring agents, preservatives, suspending agents, coating materials, fragrances, anti-adhesives, integrities, penetration enhancers, pH regulators, buffers, plasticizers, surfactants, foaming agents, defoamers, thickeners, inclusion agents, humectants, absorbents, diluents, flocculants and deflocculating agents, filter aids and release retardants.

In one embodiment, the additional agent comprises microcrystalline cellulose, hydroxypropyl methylcellulose and refined lecithin.

In one embodiment, the dosage form of the drug comprises granules, capsules, tablets, pills or oral liquids.

In one embodiment, the cosmetic comprises the above composition, matrix raw materials and/or conventional auxiliary materials.

In one embodiment, the matrix raw materials include oil raw materials, wax raw materials, synthetic oil raw materials, powdery raw materials, colloid raw materials, coagulants, and surfactants.

In one embodiment, the conventional auxiliary materials include one or more of moisturizers, whitening agents, flavoring agents, adhesives, lubricants, preservatives, film agents, antioxidants, emulsifiers, and cosmetic nutritional additives.

Beneficial effects:

The present invention screened and obtained a strain of Lactiplantibacillus planturum CCFM1296. The postbiotics prepared by the strain of Lactiplantibacillus planturum CCFM1296 have the ability to regulate the HA content of the host, which is specifically embodied in:

(1) Promote the expression of HA synthase and inhibit the expression of HA degradation enzyme in intestinal cells (HT29);

(2) Promote the expression of HA synthase in skin cells (HaCaT);

(3) Significantly upregulated HA levels in the skin of aged mice by promoting the expression of HA synthase and inhibiting the expression of HA degradase;

(4) Significantly upregulates the HA content in the joints of aged mice by promoting the expression of HA synthase;

(5) It significantly upregulated the brain HA level of aged mice by promoting the expression of HA synthase and inhibiting the expression of HA degradation enzymes;

(6) Significantly up-regulated the eye HA level of aged mice by promoting the expression of HA synthase;

Therefore, postbiotics prepared by Lactobacillus planturum CCFM1296 have great application prospects in the preparation of products that regulate host HA content.

Biomaterial Deposit

A strain of Lactobacillus planturum CCFM1296, taxonomically named Lactiplantibacillus plantarum, was deposited in the Guangdong Provincial Microbiological Culture Collection Center on November 13, 2022, with the deposit number GDMCC No.62974, and the deposit address is Building 59, No. 100 Xianlie Middle Road, Guangzhou.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1: Effect of postbiotic CCFM1296 on HT29 cell proliferation.

Figure 2: Effect of postbiotic CCFM1296 on the expression of UGDH mRNA, HAS2 mRNA, and HYBID mRNA in HT29 cells.

Figure 3: Effect of postbiotic CCFM1296 on HaCaT cell proliferation.

Figure 4: Effect of postbiotic CCFM1296 on the expression of UGDH mRNA, HAS2 mRNA, and HAS3 mRNA in HaCaT cells.

Figure 5: Effects of Lactobacillus plantarum CCFM1296 and postbiotics on the HA content and the expression of HAS2 mRNA, HYBID mRNA, HYAL1 mRNA, and HYAL2 mRNA in mouse skin.

Figure 6: Effects of Lactobacillus plantarum CCFM1296 and postbiotics on HA content, UGDH mRNA, HAS2 mRNA, and HAS2 mRNA in mouse joints.

Figure 7: Effects of Lactobacillus plantarum CCFM1296 and postbiotics on HA content, HAS2 mRNA and HYBID mRNA in mouse brain.

Figure 8: Effects of Lactobacillus plantarum CCFM1296 and postbiotics on HA content and HAS1 mRNA, HAS2 mRNA, and HAS3 mRNA in mouse eyes.

“*” indicates statistical difference compared with the Model group (P<0.05), “**” indicates significant statistical difference compared with the Model group (P<0.01), and “***” indicates extremely significant statistical difference compared with the Model group (P<0.001).

DETAILED DESCRIPTION

The present invention will be further described below in conjunction with specific embodiments.

The BALB/c mice involved in the following examples were purchased from Weitong Lihua Co., Ltd. The strains of Lactobacillus plantarum 23 and Lactobacillus paracasei FXJWS3M2 were self-screened strains from the laboratory of the Food Biology Center of Jiangnan University;

The culture medium involved in the following examples is as follows:

MRS liquid culture medium: yeast powder 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 hydrogen 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.

MRS solid culture medium: yeast powder 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 hydrogen 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.

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

Example 1: Screening and identification of Lactobacillus plantarum

1. Screening

The sample comes from healthy human feces. After pretreatment, the sample is stored in a -80°C refrigerator in 30% glycerol. After being taken out and thawed, it is mixed and 0.5 mL of the sample is pipetted and added to 4.5 mL of normal saline, and gradient dilution is performed with 9 g/L normal saline. The appropriate gradient dilution liquid is selected and spread on MRS solid culture medium, and cultured at 37°C for 48 hours. The typical colony of Lactobacillus plantarum is picked and streaked on MRS solid culture medium for purification, and a single colony is picked and transferred to MRS liquid culture medium for bacterial enrichment, and preserved in 30% glycerol to obtain Lactobacillus plantarum CCFM1296; wherein, the typical colony of Lactobacillus plantarum is round, milky white, and smooth and convex.

2. Identification

The genome of strain CCFM1296 was extracted, and the 16S rDNA of strain CCFM1296 was amplified and sequenced (by Suzhou Jinweizhi Biotechnology Co., Ltd., and the nucleotide sequence amplified from the 16S rDNA of CCFM1296 was compared with the nucleic acid sequence in NCBI, and the results showed that the strain was Lactobacillus plantarum, and was named Lactobacillus planturum CCFM1296.

Example 2: Cell recovery and culture

First, take out the frozen human colon cancer cell line (HT29) and human immortalized keratinocytes (HaCaT), quickly thaw them in a 37°C water bath, then centrifuge at 1000r/min for 3min, discard the supernatant, add an appropriate volume of cell culture medium to resuspend the cells, place them in a culture dish, and culture them in a 37°C incubator containing 5% _CO2 . When the cells grow to 70% to 80% fusion, cell passage is performed.

Example 3: Preparation of postbiotics by Lactobacillus plantarum CCFM1296

MRS solid culture medium is cultured in a water-insulated constant temperature incubator at 37°C for 24-48 hours to obtain a single colony; a single colony is picked and inoculated into an MRS liquid culture medium, and cultured at 37°C for 12-18 hours to obtain a culture solution 1; the culture solution 1 is inoculated into an MRS liquid culture medium at an inoculation amount of 2% (v/v), and cultured at 37°C for 12 hours to obtain a seed solution; the seed solution is inoculated into an MRS liquid culture medium at 3% (v/v) for expansion culture, and cultured at 37°C for 18-24 hours to obtain a bacterial solution with a concentration of 8.1×10 ¹⁰ CFU/ml.

The bacterial solution was heat treated at 65°C for 30 min, and then high-pressure homogenized (800-1200 MPa, 3 times) to obtain bacterial lysate, which was freeze-dried to obtain postbiotic freeze-dried powder for later use.

Example 4: Effect of postbiotics prepared by Lactobacillus plantarum CCFM1296 on HT29 cell proliferation

The preparation method of Lactobacillus plantarum CCFM1296 postbiotics or Lactobacillus plantarum 23 postbiotics is the same as that in Example 3.

100 μl of HT29 cells in the logarithmic growth phase were inoculated into a 96-well plate at a concentration of 5×10 ⁵ cells/well, and the outermost circle was filled with PBS solution to prevent edge effect. After culturing for 24 h to allow them to adhere to the wall, blank group, control group, and treatment group were set up.

Blank group: containing only cell culture medium without HT29 cells;

Control group: contained both cell culture medium and HT29 cells without postbiotics;

Treatment group: Resuspend the postbiotics in cell culture medium (the amount of the postbiotics after resuspending is equivalent to the amount of the postbiotics prepared by fermenting the bacterial solution to 1.0×10 ⁷ CFU/ml), add 100μl of Lactobacillus plantarum CCFM1296 postbiotics or Lactobacillus plantarum 23 postbiotics respectively, incubate in an incubator at 37°C for 4h, add 10μl of CCK8 solution to each well after incubation for 2h, and measure the absorbance (OD) at 450nm. Calculate the cell proliferation rate according to the following formula: Cell proliferation rate (%) = (OD value of treatment group - OD value of blank group) / (OD value of control group - OD value of blank group) × 100%.

The effect on cell proliferation is shown in Figure 1. Compared with the control group (cell proliferation rate is 100%), the cell proliferation rates of adding Lactobacillus plantarum CCFM1296 postbiotics or Lactobacillus plantarum 23 postbiotics are 126% and 101%, respectively, and there is no inhibitory effect on the proliferation of HT29 cells. It can be selected as an appropriate postbiotic concentration for subsequent cell experiments.

Example 5: Effects of postbiotics prepared by Lactobacillus plantarum CCFM1296 on the expression of UGDH mRNA, HAS2 mRNA and HYBID mRNA in HT29 cells

The preparation methods of Lactobacillus plantarum CCFM1296 postbiotics and Lactobacillus plantarum 23 postbiotics are the same as those in Example 3.

HT29 cells were seeded at 2.5×10 ⁶ cells/ml on a 12-well plate and cultured overnight until the cells adhered to the plate. The old culture medium was discarded and the cells were rinsed three times with PBS to set up a control group and a treatment group.

Control group: contained both cell culture medium and HT29 cells without postbiotics;

Treatment group: resuspend the postbiotics in cell culture medium (the amount of the resuspended postbiotics is equivalent to the amount of the postbiotics prepared by fermenting the bacterial solution to 1.0×10 ⁷ CFU/ml), and add 2 ml of Lactobacillus plantarum CCFM1296 postbiotics and Lactobacillus plantarum 23 postbiotics to 12-well plates, and culture at 37°C for 4 hours, with three replicates for each sample.

After the co-culture of postbiotics and cells was completed, the culture supernatant was discarded, each well was quickly washed 3 times with PBS, 1 mL of cell lysate was added to each well, and the cells were repeatedly pipetted and the cell lysate was aspirated to extract RNA, and the RT-PCR reverse transcription kit was used to reverse transcribe it into cDNA. The expression of genes in HT29 cells was detected by real-time fluorescence quantitative method, and the 2- ^△△Ct formula was used to calculate the expression of UGDHmRNA, HAS2 mRNA and HYBID mRNA, where the internal reference was GAPDH, and the primers were shown in Table 1.

Table 1 Primers

Oligo Name sequence describe F-qPCR-hUGDH CTTGCCCAGAGAATAAGCAG UDP-glucose dehydrogenase (UGDH) R-qPCR-hUGDH CAAATTCAGAACATCCTTTTGGA UDP-glucose dehydrogenase (UGDH) F-qPCR-hHAS2 TCCTGGATCTCATTCCTCAGC Hyaluronan synthase 2 (HAS2) R-qPCR-hHAS2 TGCACTGAACACACCCAAAATA Hyaluronan synthase 2 (HAS2) F-qPCR-hHYBID TCTTTGGGCCACTGCTTCTTCACG Hyaluronan Depolymerization Protein (HYBID) R-qPCR-hHYBID GTCTTGCCTGGGCTTGGGGATGTA Hyaluronan Depolymerization Protein (HYBID)

As can be seen from Figure 2, taking the expression levels of UGDH mRNA, HAS2 mRNA and HYBID mRNA in the control group as 1, the relative expression levels of UGDH mRNA, HAS2 mRNA and HYBID mRNA in the postbiotic group prepared by Lactobacillus plantarum CCFM1296 were 2.77, 4.40 and 0.21, respectively, while the expression levels of the postbiotic group prepared by Lactobacillus plantarum 23 were 0.63, 1.13 and 0.57, respectively. The above results indicate that the postbiotics prepared by Lactobacillus plantarum CCFM1296 significantly promoted the expression of UGDH mRNA and HAS2 mRNA in HT29 cells, and also significantly inhibited the expression of HYBID mRNA.

UGDH and HAS2 are both key enzymes in the HA synthesis process. By regulating the expression of UGDH mRNA, the content of UDP-GlcA can be controlled. UDP-GlcA is a precursor substance in the HA synthesis process, which can affect the HA content. Under the action of HAS2, the two precursors of HA, UDP-GlcA and UDP-GlcNAc, are alternately connected to form linear glycosaminoglycans through β-1,3 and β-1,4 glycosidic bonds on the cell membrane. By regulating the expression of HAS2 mRNA, the level of HA in the cell can be controlled. In addition, HYBID is a key enzyme for the degradation of large molecular HA. Studies have reported that inhibiting its expression can prevent the loss of HA.

It can be seen from this that the postbiotics prepared by Lactobacillus plantarum CCFM1296 have the potential to affect the HA content of the host by regulating the expression of UGDHmRNA and HAS2 mRNA in the intestine to affect the synthesis of HA or regulating the expression of HYBID mRNA to affect the degradation of HA.

Example 6: Effect of postbiotics prepared by Lactobacillus plantarum CCFM1296 on HaCaT cell proliferation

The preparation method of Lactobacillus plantarum CCFM1296 postbiotics and Lactobacillus paracasei FXJWS3M2 postbiotics is the same as that in Example 3.

100 μl of HaCaT cells in the logarithmic growth phase were inoculated into a 96-well plate at a concentration of 5×10 ⁵ cells/well, and the outermost circle was filled with PBS solution to prevent edge effect. After culturing for 24 h to allow them to adhere to the wall, blank group, control group, and treatment group were set up.

Blank group: containing only cell culture medium without HaCaT cells;

Control group: contained both cell culture medium and HaCaT cells without postbiotics;

Treatment group: Resuspend the postbiotics in cell culture medium (the amount of postbiotics after resuspending is equivalent to the amount of postbiotics prepared by fermenting to 1.0×10 ⁷ CFU/ml bacterial solution), add 100μl of Lactobacillus plantarum CCFM1296 postbiotics and Lactobacillus paracasei FXJWS3M2 postbiotics to 96-well plates, and incubate in an incubator at 37°C for 4h. After the incubation, add 10μl of CCK8 solution to each well and incubate for 2h to measure the absorbance (OD) at 450nm. Calculate the cell proliferation rate according to the following formula: Cell proliferation rate (%) = (OD value of treatment group - OD value of blank group) / (OD value of control group - OD value of blank group) × 100%.

The effect of postbiotics on cell proliferation is shown in Figure 3. Compared with the control group (cell proliferation rate is 99%), the cell proliferation rates of postbiotics prepared by adding Lactobacillus plantarum CCFM1296 and Lactobacillus paracasei FXJWS3M2 were 135% and 116%, respectively, which had no inhibitory effect on the proliferation of HaCaT cells. The appropriate postbiotic concentration can be selected for subsequent cell experiments. Example 7: Effect of postbiotics prepared by Lactobacillus plantarum CCFM1296 on UGDH mRNA, HAS2 mRNA and HAS3 mRNA in HaCaT cells

The preparation method of Lactobacillus plantarum CCFM1296 postbiotics and Lactobacillus paracasei FXJWS3M2 postbiotics is the same as that in Example 3.

HaCaT cells were seeded at 2.5×10 ⁶ cells/ml on a 12-well plate and cultured overnight until the cells adhered to the plate. The old culture medium was discarded and the cells were rinsed three times with PBS to set up a control group and a treatment group.

Control group: contained both cell culture medium and HaCaT cells without postbiotics;

Treatment group: resuspend the postbiotics in cell culture medium (the amount of the resuspended postbiotics is equivalent to the amount of the postbiotics prepared by fermenting the bacterial solution to 1.0×10 ⁷ CFU/ml), and add 2 ml of Lactobacillus plantarum CCFM1296 postbiotics and Lactobacillus paracasei FXJWS3M2 postbiotics to 12-well plates, and culture them in an incubator at 37°C for 4 hours, with three replicates for each sample.

After the co-culture of postbiotics and cells was completed, the culture supernatant was discarded, each well was quickly washed 3 times with PBS, 1 mL of cell lysate was added to each well, and the cells were repeatedly pipetted and the cell lysate was aspirated to extract RNA, which was reversely transcribed into cDNA using an RT-PCR reverse transcription kit. The expression of genes in HaCaT cells was detected by real-time fluorescence quantitative method, and the expression levels of UGDH mRNA, HAS2 mRNA and HAS3 mRNA were calculated using the 2- ^△△Ct formula, where the internal reference was GAPDH and the primers were shown in Table 2.

Table 2 Primers

Oligo Name sequence describe F-qPCR-hUGDH CTTGCCCAGAGAATAAGCAG UDP-glucose dehydrogenase (UGDH) R-qPCR-hUGDH CAAATTCAGAACATCCTTTTGGA UDP-glucose dehydrogenase (UGDH) F-qPCR-hHAS2 TCCTGGATCTCATTCCTCAGC Hyaluronan synthase 2 (HAS2) R-qPCR-hHAS2 TGCACTGAACACACCCAAAATA Hyaluronan synthase 2 (HAS2) F-qPCR-hHAS3 GAGATGTCCAGATCCTCAACAA Hyaluronan synthase 3 (HAS3) R-qPCR-hHAS3 CCCACTAATACACTGCACAC Hyaluronan synthase 3 (HAS3)

As shown in Figure 4, the expression levels of UGDH mRNA, HAS2 mRNA and HAS3 mRNA in the control group were 1, the expression levels of UGDH mRNA, HAS2 mRNA and HAS3 mRNA in the postbiotic group prepared by Lactobacillus plantarum CCFM1296 were 2.28, 1.99 and 2.10 respectively, while the expression levels of Lactobacillus paracasei FXJWS3M2 were 1.21, 0.68 and 0.74 respectively. From the above results, it can be seen that the postbiotics prepared by Lactobacillus plantarum CCFM1296 significantly promoted the expression of UGDH mRNA, HAS2 mRNA and HAS3 mRNA in HaCaT cells. Among them, UGDH is the rate-limiting enzyme for synthesizing UDP-GlcA, UDP-GlcA is the precursor substance in the HA synthesis process, HAS2 and HAS3 are important HA synthases, and their expression changes directly affect the molecular weight and content of HA. It can be seen from this that the postbiotics prepared by Lactobacillus plantarum CCFM1296 have the potential to increase the synthesis of HA in skin cells by regulating the expression of UGDH mRNA, HAS2 mRNA, and HAS3 mRNA, thereby increasing the host HA level.

Example 8: Regulation of HA content in the skin of aged mice by postbiotics prepared by Lactobacillus plantarum CCFM1296.

The preparation method of live bacteria of Lactobacillus plantarum CCFM1296 is the same as that of Example 3, except that the bacterial liquid is centrifuged and then the bacterial sludge is collected without heat treatment and high pressure homogenization. The preparation method of live bacteria of Lactobacillus plantarum FXJWS3M2 and Lactobacillus plantarum 23 is the same as that of Lactobacillus plantarum CCFM1296.

The preparation method of Lactobacillus plantarum FXJWS3M2 postbiotics and Lactobacillus plantarum 23 postbiotics is the same as that in Example 3.

45 healthy male BALB/c mice aged 8 weeks were randomly divided into 9 cages, with 5 mice in each cage. The 9 cages were divided into: 2 cages for the model group (Model) and 1 cage for each of the other groups. The groups were divided into the following groups:

Blank group (Control): Use normal saline as control;

CCFM1296-L group: live Lactobacillus plantarum CCFM1296 was used at a dose of 5 × 10 ⁹ CFU/kg mouse body weight;

CCFM1296-D group: Lactobacillus plantarum CCFM1296 postbiotics were used at a dose of 500 mg/kg mouse body weight;

23-L group: Lactobacillus plantarum 23 live bacteria were used at a dose of 5 × 10 ⁹ CFU/kg mouse body weight;

Group 23-D: Lactobacillus plantarum 23 postbiotics, dosed at 500 mg/kg mouse body weight;

3M2-L group: live Lactobacillus plantarum FXJWS3M2 was used at a dose of 5×10 ⁹ CFU/kg mouse body weight;

3M2-D group: Lactobacillus plantarum FXJWS3M2 postbiotics were used at a dose of 500 mg/kg mouse body weight;

Among them, each postbiotic is a postbiotic prepared by fermenting the corresponding live bacteria to a bacterial liquid with an equal bacterial volume.

The experiment lasted for 9 weeks: After the mice adapted for one week, D-galactose was injected subcutaneously in all groups except the blank group. From the second week, the mice in each intervention group were gavaged with 0.2 mL/mouse/day of freeze-dried powder of the corresponding strain or freeze-dried powder of postbiotics prepared from the strain (dissolved in normal saline at the corresponding dose). The blank group and the model group were gavaged with the same amount of normal saline as a control until the end of the experiment. All groups had free access to water and food.

After the experiment, the mice were killed, and the skin tissue on the back of the mice was cut and homogenized at a weight-to-volume ratio of 1:10 with PBS. The HA content of the skin was detected by ELISA HA kit, and the gene expression levels of the key enzymes UGDH, HAS1-3, HYBID, and HYAL1-2 in the HA synthesis process of skin tissue were detected. The internal reference was mouse GAPDH, and the sequence is shown in Table 3.

Table 3 Primers

Oligo Name sequence describe F-qPCR-hHAS2 TGTGAGAGGTTTCTATGTGTCCT Hyaluronan synthase 2 (HAS2) R-qPCR-hHAS2 ACCGTACAGTCCAAATGAGAAGT Hyaluronan synthase 2 (HAS2) F-qPCR-hHYBID TGATGGGAGTCGAGGTCAC Hyaluronan Depolymerization Protein (HYBID) R-qPCR-hHYBID GAGCACTATGGAATTGTCAGGG Hyaluronan Depolymerization Protein (HYBID) F-qPCR-hHYAL1 ACCTGCTTCGCATCTCTACTC Hyaluronidase 1 (HYAL1) R-qPCR-hHYAL1 GGTTGGATACCACGGAACCTC Hyaluronidase 1 (HYAL1) F-qPCR-hHYAL2 AGATGTTTACCGACAGTCTTCAC Hyaluronidase 2 (HYAL2) R-qPCR-hHYAL2 TGACGTAACGGAGAGTGTTCAA Hyaluronidase 2 (HYAL2)

As can be seen from Figure 5, compared with the HA content of 7.89 ng/mg in the blank group, the HA content of the model group (6.24 ng/mg) was significantly reduced, the HA content after intervention with live bacteria of Lactobacillus plantarum CCFM1296 was 7.00 ng/mg skin tissue, the HA content of the postbiotic intervention group prepared by Lactobacillus plantarum CCFM1296 was 6.64 ng/mg, the HA contents of the live bacteria group of Lactobacillus plantarum 23 and the postbiotic group were 6.08 ng/mg and 6.09 ng/mg skin tissue, respectively; the HA contents of the live bacteria group of Lactobacillus paracasei FXJWS3M2 and the postbiotic group were 6.07 ng/mg and 6.28 ng/mg skin tissue, respectively. From the above results, it can be seen that the HA content of live bacteria of Lactobacillus plantarum CCFM1296 and postbiotics was significantly increased compared with the model group.

Further detection of the expression of HA synthase and degradation enzyme genes in the groups with increased HA content revealed that, taking the expression of HAS2mRNA, HYBID mRNA, HYAL1 mRNA, and HYAL2 mRNA in the model group as 1, the expression of HAS2 mRNA, HYBID mRNA, HYAL1 mRNA, and HYAL2 mRNA in the live bacteria group of Lactobacillus plantarum CCFM1296 were 7.47, 0.69, 1.37, and 1.09, respectively; the expression of HAS2 mRNA, HYBID mRNA, HYAL1 mRNA, and HYAL2 mRNA in the postbiotic group prepared by Lactobacillus plantarum CCFM1296 were 3.65, 0.67, 0.63, and 0.45, respectively; the expression of HAS2 mRNA, HYBID mRNA, HYAL1 mRNA, and HYAL2 mRNA in the postbiotic group prepared by Lactobacillus plantarum 23 were 3.84, 0.89, 0.91, and 1.10, respectively. The expression levels of mRNA were 0.99, 1.00, 1.27 and 1.58 respectively; the expression levels of postbiotics prepared by Lactobacillus paracasei FXJWS3M2 were 1.33, 1.06, 1.20 and 1.28 respectively. From the above results, it can be seen that compared with the model group, oral administration of live Lactobacillus plantarum CCFM1296 and postbiotics prepared significantly promoted the expression of HAS2 mRNA in skin tissue, and inhibited the expression of HYBID mRNA, HYAL1 mRNA and HYAL2 mRNA.

The results of cell experiments and animal experiments showed that the postbiotics prepared by Lactobacillus plantarum CCFM1296, which can promote the expression of HAS2 mRNA and inhibit HYBID mRNA in HT29 cells and HaCaT cells, inhibited the loss of HA in aging mice in oral animal experiments, indicating that the postbiotics prepared by Lactobacillus plantarum CCFM1296 can maintain the host's HA level by upregulating the expression of HAS2 mRNA and downregulating HYBID mRNA.

Example 9: Regulation of HA content in joints of aged mice by postbiotics prepared by Lactobacillus plantarum CCFM1296.

The preparation method of live bacteria of Lactobacillus plantarum CCFM1296 is the same as that of Example 3, except that the bacterial liquid is centrifuged and then the bacterial sludge is collected without heat treatment and high pressure homogenization. The preparation method of live bacteria of Lactobacillus plantarum FXJWS3M2 and Lactobacillus plantarum 23 is the same as that of Lactobacillus plantarum CCFM1296.

The preparation method of Lactobacillus plantarum FXJWS3M2 postbiotics and Lactobacillus plantarum 23 postbiotics is the same as that in Example 3.

45 healthy male BALB/c mice aged 8 weeks were randomly divided into 9 cages, with 5 mice in each cage. The 9 cages were as follows: except for the model group (Model) with 2 cages, the other groups had 1 cage each, and were divided into groups according to the following settings:

Blank group (Control): Use normal saline as control;

CCFM1296-L group: live Lactobacillus plantarum CCFM1296 was used at a dose of 5 × 10 ⁹ CFU/kg mouse body weight;

CCFM1296-D group: Lactobacillus plantarum CCFM1296 postbiotics were used at a dose of 500 mg/kg mouse body weight;

23-L group: Lactobacillus plantarum 23 live bacteria were used at a dose of 5 × 10 ⁹ CFU/kg mouse body weight;

Group 23-D: Lactobacillus plantarum 23 postbiotic group, the dose was 500 mg/kg mouse body weight);

3M2-L group: live Lactobacillus plantarum FXJWS3M2 was used at a dose of 5×10 ⁹ CFU/kg mouse body weight;

Group 3M2-D: The postbiotic group using Lactobacillus plantarum FXJWS3M2 was administered at a dose of 500 mg/kg mouse body weight.

Among them, each postbiotic is a postbiotic prepared by fermenting the corresponding live bacteria to a bacterial liquid with an equal bacterial volume.

The experiment lasted for 9 weeks: After the mice adapted for one week, D-galactose was injected subcutaneously in all groups except the blank group. From the second week, the mice in each intervention group were gavaged with 0.2 mL/mouse/day of freeze-dried powder of the corresponding strain or freeze-dried powder of postbiotics prepared from the strain (dissolved in normal saline at the corresponding dose). The blank group and the model group were gavaged with the same amount of normal saline as a control until the end of the experiment. All groups had free access to water and food.

After the experiment, the mice were killed, and the joint tissues were collected and homogenized at a weight-to-volume ratio of 1:6 with PBS. The HA content in the joints was detected by ELISA HA kit, and the gene expression levels of the key enzymes UGDH, HAS1-3, HYBID, and HYAL1-2 in the HA synthesis process of the joint tissues were detected. The internal reference was mouse GAPDH, and the primers were shown in Table 4.

Table 4 Primers

Oligo Name sequence describe F-qPCR-hUGDH AGTAGTCGAATCCTGTCGAGG UDP-glucose dehydrogenase (UGDH) R-qPCR-hUGDH CTCCGTGACAATTTTGTACCCA UDP-glucose dehydrogenase (UGDH) F-qPCR-hHAS1 GGCGAGCACTCACGATCATC Hyaluronan synthase 1 (HAS1) R-qPCR-hHAS1 AGGAGTCCATAGCGATCTGAAG Hyaluronan synthase 1 (HAS1) F-qPCR-hHAS2 TGTGAGAGGTTTCTATGTGTCCT Hyaluronan synthase 2 (HAS2) R-qPCR-hHAS2 ACCGTACAGTCCAAATGAGAAGT Hyaluronan synthase 2 (HAS2)

As can be seen from Figure 6, compared with the HA content of 0.91 ng/mg in the blank group, the HA content in the model group (0.70 ng/mg) was significantly reduced. The HA content after intervention with live bacteria of Lactobacillus plantarum CCFM1296 was 0.83 ng/mg of joint tissue, the HA content in the postbiotic intervention group of Lactobacillus plantarum CCFM1296 was 0.86 ng/mg, the HA contents of the live bacteria group of Lactobacillus plantarum 23 and the postbiotic group were 0.73 ng/mg and 0.67 ng/mg of joint tissue, respectively; the HA contents of the live bacteria group of Lactobacillus paracasei FXJWS3M2 and the postbiotic group were 0.76 ng/mg and 0.77 ng/mg of skin tissue, respectively. From the above results, it can be seen that the HA content of live bacteria of Lactobacillus plantarum CCFM1296 and postbiotics was significantly increased compared with that of the model group.

Further detection of HA synthase and degradation enzyme gene expression in the HA elevated group revealed that, taking the expression of UGDHmRNA, HAS1 mRNA, and HAS2 mRNA in the model group as 1, the expression of UGDH mRNA, HAS1 mRNA, and HAS2 mRNA in the live bacteria group of Lactobacillus plantarum CCFM1296 were 2.90, 2.55, and 4.73, respectively; the expression of the postbiotic group prepared by Lactobacillus plantarum CCFM1296 were 2.01, 1.58, and 5.34, respectively; the expression of UGDH mRNA, HAS1 mRNA, and HAS2 mRNA in the postbiotic group prepared by Lactobacillus plantarum 23 were 0.67, 0.99, and 0.91, respectively; the expression of the postbiotics prepared by Lactobacillus paracasei FXJWS3M2 were 0.89, 0.92, and 1.10, respectively. The above results show that compared with the model group, the live bacteria of Lactobacillus plantarum CCFM1296 and the prepared postbiotic group significantly promoted the expression of UGDH in joint tissues. The expression of mRNA, HAS1 mRNA and HAS2 mRNA.

Through the comprehensive results of cell experiments and animal experiments, it can be seen that the postbiotics obtained in HT29 cells and HaCaT cells can promote the expression of UGDH mRNA and HAS2 mRNA. The postbiotics prepared by Lactobacillus plantarum CCFM1296 inhibited the loss of HA in aging mice in oral animal experiments, indicating that the postbiotics prepared by Lactobacillus plantarum CCFM1296 can maintain the host's HA level by up-regulating the expression of UGDH mRNA and HAS2 mRNA and promoting the HA synthesis process.

Example 10: Regulation of HA content in the brain of aged mice by postbiotics prepared by Lactobacillus plantarum CCFM1296.

The preparation method of live bacteria of Lactobacillus plantarum CCFM1296 is the same as that of Example 3, except that the bacterial liquid is centrifuged and then the bacterial sludge is collected without heat treatment and high pressure homogenization. The preparation method of live bacteria of Lactobacillus plantarum FXJWS3M2 and Lactobacillus plantarum 23 is the same as that of Lactobacillus plantarum CCFM1296.

The preparation method of Lactobacillus plantarum FXJWS3M2 postbiotics and Lactobacillus plantarum 23 postbiotics is the same as that in Example 3.

45 healthy male BALB/c mice aged 8 weeks were randomly divided into 9 cages, with 5 mice in each cage. The 9 cages were as follows: except for the model group (Model) with 2 cages, the other groups had 1 cage each, and were divided into groups according to the following settings:

Blank group (Control); physiological saline was used as the control;

CCFM1296-L group: live Lactobacillus plantarum CCFM1296 was used at a dose of 5 × 10 ⁹ CFU/kg mouse body weight;

CCFM1296-D group: Lactobacillus plantarum CCFM1296 postbiotics were used at a dose of 500 mg/kg mouse body weight;

23-L group: Lactobacillus plantarum 23 live bacteria were used at a dose of 5 × 10 ⁹ CFU/kg mouse body weight;

Group 23-D: Lactobacillus plantarum 23 postbiotics, dosed at 500 mg/kg mouse body weight;

3M2-L group: plant FXJWS3M2 live bacteria were used at a dose of 5×10 ⁹ CFU/kg mouse body weight;

3M2-D group: plant FXJWS3M2 postbiotic was used at a dose of 500 mg/kg mouse body weight.

Among them, each postbiotic is a postbiotic prepared by fermenting the corresponding live bacteria to a bacterial liquid with an equal bacterial volume.

The experiment lasted for 9 weeks: After the mice adapted for one week, D-galactose was injected subcutaneously in all groups except the blank group. From the second week, the mice in each intervention group were gavaged with 0.2 mL/mouse/day of freeze-dried powder of the corresponding strain or freeze-dried powder of postbiotics prepared from the strain (dissolved in normal saline at the corresponding dose). The blank group and the model group were gavaged with the same amount of normal saline as a control until the end of the experiment. All groups had free access to water and food.

After the experiment, the mice were killed, and the brain tissue was cut and homogenized with PBS at a weight-to-volume ratio of 1:3. The HA content in the brain was detected by ELISA HA kit, and the gene expression levels of key enzymes UGDH, HAS1-3, HYBID, and HYAL1-2 in the HA synthesis process of brain tissue were detected. The internal reference was mouse GAPDH, and the sequence is shown in Table 5.

Table 5 Primers

Oligo Name sequence describe F-qPCR-hHAS2 TGTGAGAGGTTTCTATGTGTCCT Hyaluronan synthase 2 (HAS2) R-qPCR-hHAS2 ACCGTACAGTCCAAATGAGAAGT Hyaluronan synthase 2 (HAS2) F-qPCR-hHYBID TGATGGGAGTCGAGGTCAC Hyaluronan Depolymerization Protein (HYBID) R-qPCR-hHYBID GAGCACTATGGAATTGTCAGGG Hyaluronan Depolymerization Protein (HYBID)

As can be seen from Figure 7, compared with the HA content of 0.61 ng/mg in the blank group, the HA content of the model group (0.47 ng/mg) was significantly reduced, the HA content after intervention with live bacteria of Lactobacillus plantarum CCFM1296 was 0.46 ng/mg brain tissue, the HA content of the postbiotic intervention group prepared by Lactobacillus plantarum CCFM1296 was 0.54 ng/mg, and the HA content of the live bacteria group of Lactobacillus plantarum 23 and the postbiotic group was 0.49 ng/mg brain tissue; the HA contents of the live bacteria group of Lactobacillus paracasei FXJWS3M2 and the postbiotic group were 0.49 ng/mg and 0.47 ng/mg brain tissue, respectively. From the above results, it can be seen that the HA content of the postbiotics prepared by Lactobacillus plantarum CCFM1296 was significantly increased compared with that of the model group.

Further detection of HA synthase and degradation enzyme gene expression in the HA elevated group revealed that, taking the expression levels of HAS2mRNA and HYBID mRNA in the model group as 1, the expression levels of HAS2 mRNA and HYBID mRNA in the live bacteria group of Lactobacillus plantarum CCFM1296 were 0.90 and 0.93, respectively; the expression levels of the postbiotic group prepared by Lactobacillus plantarum CCFM1296 were 2.70 and 0.70, respectively; the expression levels of HAS2 mRNA and HYBID mRNA in the postbiotic group prepared by Lactobacillus plantarum 23 were 0.77 and 1.12, respectively; the expression levels of the postbiotics prepared by Lactobacillus paracasei FXJWS3M2 were 1.02 and 0.91, respectively. From the above results, it can be seen that compared with the model group, oral administration of postbiotics prepared by Lactobacillus plantarum CCFM1296 significantly promoted the expression of HAS2mRNA in brain tissue and inhibited the expression of HYBID mRNA.

Through the combined results of cell experiments and animal experiments, it can be seen that the postbiotics prepared by Lactobacillus plantarum CCFM1296, which can promote the expression of HAS2 mRNA and down-regulate the expression of HYBID mRNA in HT29 and HaCaT cells, inhibited the loss of brain HA in aging mice in oral animal experiments, indicating that the postbiotics prepared by Lactobacillus plantarum CCFM1296 can regulate the host's HA level by up-regulating the expression of HAS2 mRNA and down-regulating the expression of HYBID mRNA.

Example 11: Regulation of HA content in eyes of aged mice by postbiotics prepared by Lactobacillus plantarum CCFM1296.

45 healthy male BALB/c mice aged 8 weeks were randomly divided into 9 cages with 5 mice in each cage. The grouping was the same as in Example 10.

The experiment lasted for 9 weeks: After the mice adapted for one week, D-galactose was injected subcutaneously in all groups except the blank group. From the second week, the mice in each intervention group were gavaged with 0.2 mL/mouse/day of freeze-dried powder of the corresponding strain or freeze-dried powder of postbiotics prepared from the strain (dissolved in normal saline at the corresponding dose). The blank group and the model group were gavaged with the same amount of normal saline as a control until the end of the experiment. All groups had free access to water and food.

After the experiment, the mice were killed, and the eyeballs were removed and homogenized with PBS at a weight-to-volume ratio of 1:5. The HA content in the brain was detected by ELISA HA kit, and the gene expression levels of key enzymes UGDH, HAS1-3, HYBID, and HYAL1-2 in the HA synthesis process of brain tissue were detected. The internal reference was mouse GAPDH, and the sequence is shown in Table 6.

Table 6

Oligo Name sequence describe F-qPCR-hHAS2 TGTGAGAGGTTTCTATGTGTCCT Hyaluronan synthase 2 (HAS2) R-qPCR-hHAS2 ACCGTACAGTCCAAATGAGAAGT Hyaluronan synthase 2 (HAS2) F-qPCR-hHAS3 CCTGGAGCACCGTCGAATG Hyaluronan synthase 3 (HAS3) R-qPCR-hHAS3 CCTTGAGGTTTGGAAAGGCAA Hyaluronan synthase 3 (HAS3)

As can be seen from Figure 8, compared with the HA content of 4.23 ng/mg in the blank group, the HA content of the model group (3.05 ng/mg) was significantly reduced. The HA content after intervention with live bacteria of Lactobacillus plantarum CCFM1296 was 3.38 ng/mg of eye tissue, the HA content of the postbiotic intervention group prepared by Lactobacillus plantarum CCFM1296 was 3.44 ng/mg, the HA contents of the live bacteria group of Lactobacillus plantarum 23 and the postbiotic group were 3.05 ng/mg and 3.17 ng/mg of eye tissue, respectively; the HA contents of the live bacteria group of Lactobacillus paracasei FXJWS3M2 and the postbiotic group were 3.02 ng/mg and 2.86 ng/mg of eye tissue, respectively. From the above results, it can be seen that the HA content of the postbiotics prepared by Lactobacillus plantarum CCFM1296 was significantly increased compared with that of the model group.

Further detection of HA synthase and degradase gene expression in the HA elevated group revealed that, taking the expression levels of HAS1mRNA, HAS2 mRNA and HAS3 mRNA in the model group as 1, the expression levels of HAS1 mRNA, HAS2 mRNA and HAS3 mRNA in the live bacteria group of Lactobacillus plantarum CCFM1296 were 1.06, 2.03 and 1.14, respectively; the expression levels of the postbiotic group prepared by Lactobacillus plantarum CCFM1296 were 2.10, 2.04 and 1.63, respectively; the expression levels of HAS1 mRNA, HAS2 mRNA and HAS3 mRNA in the postbiotic group prepared by Lactobacillus plantarum 23 were 0.50, 0.56 and 0.55, respectively; the expression levels of the postbiotics prepared by Lactobacillus paracasei FXJWS3M2 were 0.64, 0.83 and 0.81, respectively. From the above results, it can be seen that compared with the model group, oral administration of live Lactobacillus plantarum CCFM1296 significantly promoted the expression of HAS2 mRNA in ocular tissue, while administration of postbiotics prepared by Lactobacillus plantarum CCFM1296 promoted the expression of HAS1-3 mRNA in ocular tissue.

Through the combined results of cell experiments and animal experiments, it can be seen that the postbiotics prepared by Lactobacillus plantarum CCFM1296, which can promote the expression of HAS2-3 mRNA in HT29 and HaCaT cells, inhibited the loss of brain HA in aging mice in oral animal experiments, indicating that the postbiotics prepared by Lactobacillus plantarum CCFM1296 can regulate the host's HA level by upregulating the expression of HAS2 mRNA and HAS3 mRNA.

Although the present invention has been disclosed as above in the form of a preferred embodiment, it is not intended to limit the present invention. Anyone familiar with this technology can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be based on the definition of the claims.

Claims (6)

1. Lactiplantibacillus plantarum CCFM1296 was deposited in Guangdong Provincial Microbiological Culture Collection Center on November 13, 2022, with the deposit number GDMCC No.62974. 2. A composition containing the plant lactobacillus CCFM1296 according to claim 1. 3. A postbiotic prepared by using the plant lactobacillus CCFM1296 according to claim 1, characterized in that the postbiotic is dead cells and/or bacterial lysate of plant lactobacillus CCFM1296. 4. The postbiotic according to claim 3 is characterized in that the preparation method comprises: inoculating the plant lactobacillus CCFM1296 according to claim 1 into a fermentation medium for cultivation to obtain a bacterial liquid, and then heat-treating and cracking the bacterial liquid to obtain the postbiotic. 5. Food containing the postbiotics according to claim 3 or 4. 6. A health product, medicine or cosmetic containing the postbiotic according to claim 3 or 4.