Lactobacillus paracasei ccfm1355 and its postbiotic having anti-glycation and anti-aging functions

Abstract

The application discloses a strain of paracasei CCFM1355 with anti-glycation and anti-aging functions and a postbiotic of the strain, and belongs to the technical field of microorganisms and the technical field of medicines.The strain has been preserved in the Guangdong Microbial Culture Collection Center on October 25, 2023, and the preservation number is GDMCC No: 63925.The strain provided in the application can reduce the content of in-vitro fluorescent AGE, prevent the decrease in the activity of HSF caused by acetone aldehyde, and abnormal expression of DDOST mRNA, MMP-9 mRNA, EGR2 mRNA and NFKB1 mRNA after HSF is damaged in high-glucose culture.In addition, the content of fluorescent AGE and inflammatory markers in serum and skin can be reduced, the water content in the matrix layer, the elasticity of the skin and the content of collagen can be improved, and the AGE-RAGE-mediated glycation damage can be reduced.

Description

TECHNICAL FIELD

[0001] The present application relates to a strain of Paracaseolyticum CCFM1355 with anti-glycation and anti-aging functions and its postbiotic, and belongs to the field of microbial technology and medical technology. BACKGROUND

[0002] Non-enzymatic glycosylation occurs widely in the human body, and its mechanism is that the carbonyl group on the reducing sugar reacts with the free amino group on the macromolecule such as protein, lipid or nucleic acid through non-enzymatic condensation reaction, i.e. non-enzymatic modification of proteins by sugars. Advanced glycation end products (AGEs) are stable covalent adducts generated by glycation reaction, which react with corresponding receptors or proteins in the body. With the growth of age, AGEs also slowly accumulate in the human body, and are involved in the pathological changes of various diseases such as diabetes, inflammation, neurodegenerative diseases and cardiovascular diseases.

[0003] Skin is the largest organ of the human body and the boundary between the organism and the environment. Therefore, the skin is not only affected by the internal aging process, but also by various external pressures leading to significant changes in skin structure, not only affecting its youthful appearance, but also affecting its various physiological functions. In recent years, the role of AGEs in skin aging has been discussed more and more. CN116650579A discloses the application of Camellia oleifera Abel extract in removing freckles, whitening and anti-glycation, determines that the Camellia oleifera Abel extract inhibits the activity of tyrosinase, inhibits the synthesis of melanin, has anti-glycation effect, and determines the role of the Camellia oleifera Abel extract in preventing and treating diseases related to melanin and AGEs, such as freckles, yellow spots, dull skin and the like, and expands its application in pharmaceuticals and daily chemical products.

[0004] In order to develop more efficient and comprehensive anti-glycation products, four anti-glycation strategies can be proposed according to the occurrence and development of glycation damage, and potential effective biological raw materials can be screened based on these paths: (1) reducing the generation of AGE; (2) hindering the combination of AGE-RAGE (AGE receptor); (3) destroying the cross-linking of AGE and protein; (4) inhibiting the signal activation after AGE-RAGE combination. At present, the most studied is to compete with the intermediate products of glycation reaction or limit the generation of intermediate reaction to reduce the generation and accumulation of AGE. For example, patent CN110506106A discloses that Lactobacillus paracasei KF00816 can reduce the content of glycosylation end product, but its effect on reducing the content of AGE is still limited, and simply limiting the generation of AGE is not enough, because its receptor RAGE-AGE specific combination will quickly trigger other damage in multiple pathways downstream, so reducing the combination of the two is one of the key targets to be solved. Therefore, it has very important scientific significance and practical value to find a probiotic strain that can more efficiently degrade AGE and further inhibit the occurrence of AGE-induced glycation damage by inhibiting the above glycation damage pathways. SUMMARY

[0005] To solve the above technical problems, the present application provides a Lactobacillus paracasei (Lactobacillus paracasei) Lacticaseibacillus paracasei ) CCFM1355 probiotic in the preparation of anti-glycation and anti-aging products.

[0006] The present application provides a Lactobacillus paracasei (Lactobacillus paracasei) Lacticaseibacillus paracasei ) CCFM1355, which was deposited with the Guangdong Microbial Culture Collection Center on October 25, 2023, and the deposit number is GDMCC NO: 63925.

[0007] The 16S rDNA sequence of the Lactobacillus paracasei (Lactobacillus paracasei) Lacticaseibacillus paracasei ) CCFM1355 is shown as SEQ ID NO. 1.

[0008] The present application also provides a Lactobacillus paracasei CCFM1355 probiotic, which comprises a culture, a dry matter, an inactivated matter and / or a broken matter of Lactobacillus paracasei CCFM1355.

[0009] In one embodiment, the culture is a culture supernatant obtained by culturing Lactobacillus paracasei CCFM1355 in a fermentation medium; preferably, the Lactobacillus paracasei CCFM1355 is inoculated in a fermentation medium for culture, a culture liquid is obtained, and the obtained culture supernatant is collected.

[0010] In an embodiment, the dry matter is a powder prepared from Paracaseicoccus caseicofaciens CCFM1355 by a drying process, which can be thermal drying, spray drying, vacuum freeze drying, fluidized bed drying or vacuum drying.

[0011] In an embodiment, the inactivated matter is inactivated cells of Paracaseicoccus caseicofaciens CCFM1355 obtained by an inactivation process, which can be heat treatment or freeze-drying treatment; preferably, the Paracaseicoccus caseicofaciens CCFM1355 is inoculated in a fermentation medium, the bacterial slurry is collected, and the inactivated bacterial cells are obtained after heat treatment at 60-65°C for 25-35 min.

[0012] In an embodiment, the broken matter is broken matter of Paracaseicoccus caseicofaciens CCFM1355 obtained by breaking; preferably, the Paracaseicoccus caseicofaciens CCFM1355 is inoculated in a fermentation medium, the bacterial slurry is collected, and the bacterial slurry is resuspended in a fermentation medium at a volume of 50-80%, and the bacterial slurry is obtained by high-pressure homogenization.

[0013] In an embodiment, the bio-organic matter can be dried into powder by various drying methods such as vacuum drying, spray drying, vacuum freeze drying, fluidized bed drying, or directly used.

[0014] In an embodiment, the concentration of Paracaseicoccus caseicofaciens CCFM1355 used in the preparation of the bio-organic matter is not less than 5.0 x 10 7 CFU / mL.

[0015] The present application also provides a product containing the above-mentioned Paracaseicoccus caseicofaciens CCFM1355 and / or bio-organic matter of Paracaseicoccus caseicofaciens CCFM1355.

[0016] In an embodiment, the product comprises food, health care products, pharmaceuticals or cosmetics.

[0017] In an embodiment, the food comprises the above-mentioned composition and conventional adjuvants.

[0018] In an embodiment, the conventional adjuvants comprise one or more of fillers, flavoring agents, binders, disintegrants, lubricants, antacids, and nutritional fortifiers.

[0019] In an embodiment, the health care products comprise the above-mentioned composition and conventional adjuvants.

[0020] In an embodiment, the conventional adjuvants comprise one or more of fillers, flavoring agents, binders, disintegrants, lubricants, antacids, and nutritional fortifiers.

[0021] In one embodiment, the pharmaceutical product comprises the above composition, a pharmaceutical carrier, and / or a pharmaceutical excipient.

[0022] In one embodiment, the pharmaceutical excipient comprises an excipient and an additional agent.

[0023] In one embodiment, the pharmaceutical excipient comprises at least one of a solvent, a propellant, a solubilizer, a co-solvent, an emulsifier, a coloring agent, a binding agent, a disintegrating agent, a filler, a lubricant, a wetting agent, an osmotic pressure adjusting agent, a stabilizer, a glidant, a flavoring agent, a preservative, a suspending agent, a coating material, a fragrance, an anti-adhesion agent, an integrating agent, a penetration enhancer, a pH adjusting agent, a buffer, a plasticizer, a surfactant, a foaming agent, an antifoaming agent, a thickening agent, an inclusion agent, a humectant, an absorbent, a diluent, a flocculating agent and a deflocculating agent, a filter aid, or a release retardant.

[0024] In one embodiment of the present application, the cosmetic product comprises the above composition, a base material, and / or a conventional excipient.

[0025] In one embodiment of the present application, the base material comprises an oil-based material, a wax-based material, a synthetic oil-based material, a powder-based material, a gum-based material, a gelling agent, and a surfactant.

[0026] In one embodiment of the present application, the conventional excipient comprises one or more of a humectant, a whitening agent, a flavoring agent, a binding agent, a lubricant, a preservative, a film-forming agent, an antioxidant, an emulsifier, and a cosmetic nutrient additive.

[0027] In one embodiment, the product is a fermentation product of the Paracaseicoccus casei CCFM1355 after fermentation in a medium containing peanut skin.

[0028] In one embodiment, the formulation of the medium containing peanut skin comprises 1-10 g / L of peanut skin extract, 1-5 g / L of glucose, 1-10 g / L of yeast powder, and 2-6 g / L of calcium carbonate; preferably, 5 g / L of peanut skin extract, 4 g / L of glucose, 5 g / L of yeast powder, and 4 g / L of calcium carbonate.

[0029] The present application also provides a method for improving the anti-glycation effect of peanut skin, or preventing and / or delaying the aging effect, the method comprising mixing a medium containing peanut skin with a microorganism containing the Paracaseicoccus casei CCFM1355, and fermenting.

[0030] In one embodiment, the formula of the peanut skin-containing medium comprises: peanut skin extract 1-10 g / L, 1-5 g / L glucose, 1-10 g / L yeast powder, 2-6 g / L calcium carbonate; preferably, peanut skin extract 5 g / L, 4 g / L glucose, 5 g / L yeast powder, 4 g / L calcium carbonate.

[0031] In one embodiment, the inoculation amount of the Paracaseicoccus casei mixed is 1-5% (v / v), preferably 2% (v / v).

[0032] In one embodiment, the fermentation conditions are: fermentation temperature 25-40℃, fermentation time 10-30 h; preferably, fermentation temperature 37℃, fermentation time 18-24 h.

[0033] In one embodiment, the anti-glycation or the prevention and / or delay of aging of the peanut skin is improved by at least one of the following aspects:

[0034] (1) in vitro inhibition of fluorescent AGE generation;

[0035] (2) alleviation of the decrease in HSF cell viability caused by methylglyoxal;

[0036] (3) reduction of blood AGE levels;

[0037] (4) enhancement of the expression level of the skin RAGE competitive receptor DDOST gene;

[0038] (5) enhancement of the elasticity of aged skin;

[0039] (6) enhancement of the expression level of the skin Cu / Zn-SOD gene.

[0040] The present application also provides a fermentation composition obtained by fermentation according to the above method.

[0041] The present application also provides the use of the above-mentioned Paracaseicoccus casei CCFM1355, or the Paracaseicoccus casei CCFM1355 probiotic, or the fermentation composition in the preparation of anti-glycation or anti-aging products.

[0042] In one embodiment, the glycation includes the accumulation of glycation aging markers AGE in blood and / or other tissues and organs.

[0043] In one embodiment, the aging includes skin aging.

[0044] In one embodiment, the skin aging includes skin dryness, reduced elasticity, relaxation, wrinkle formation, oxidative damage or collagen loss.

[0045] In one embodiment, the application comprises at least one of the following 1) - 9) effects:

[0046] 1) inhibiting the generation of fluorescent AGE in vitro;

[0047] 2) alleviating the damage or functional decline of skin fibroblasts (HSF) caused by an intermediate of AGE formation (methylglyoxal);

[0048] 3) reducing the NFKB1 gene expression level or the MMP-9 gene expression level of skin fibroblasts under high-sugar treatment; or increasing the DDOST gene expression level or the EGR2 gene expression level of skin fibroblasts under high-sugar treatment;

[0049] 4) reducing the AGE level in the skin or blood;

[0050] 5) reducing the TNF-α or IL-6 level in the skin or blood;

[0051] 6) increasing the water content of the stratum corneum of aged skin or the skin elasticity performance;

[0052] 7) increasing the COL3A1 gene expression level of aged skin, or increasing the content of collagen type III of aged skin, or reducing the MMP-9 gene expression level of aged skin;

[0053] 8) reducing the expression of the RAGE gene of the skin, or increasing the expression of the DDOST gene of the RAGE competitive receptor;

[0054] 9) reducing the NFKB1 gene expression level of the skin, or increasing the Cu / Zn-SOD gene expression level.

[0055] In one embodiment, the dose of the metagenomic prepared by the Paracaseiclovarcasei CCFM1355 in the product is not less than 10 mg / kg of body weight.

[0056] Beneficial effects:

[0057] The Paracaseiclovarcasei CCFM1355 of the present application and the metagenomic prepared by the same have the ability to alleviate the glycation damage of the host and reduce the corresponding aging problems, which is embodied in: Lacticaseibacillus paracasei (1) inhibiting the generation of fluorescent AGE in a fructose-bovine serum albumin system in vitro;

[0058] (2) preventing the decrease of cell viability in a skin fibroblast (HSF) damage model caused by methylglyoxal;

[0059]

[0060] ​(3) Prevent abnormal expression of DDOST mRNA, MMP-9 mRNA, EGR2 mRNA and NFKB1 mRNA in skin fibroblasts (HSF) after glucose damage under high glucose culture;

[0061] (4) Reduce the AGE content in the serum and skin of aging individuals;

[0062] (5) Reduce the levels of inflammatory markers IL-6 and TNF-α in the serum and skin of aging individuals;

[0063] (6) Reduce the decrease in the moisture content of the stratum corneum of the back skin in aging individuals caused by glycation damage;

[0064] (7) To alleviate the decreased elasticity of the back skin in aging individuals caused by glycation damage;

[0065] (8) Reduce the decrease in type III collagen content in the back skin of aging individuals caused by glycation damage;

[0066] (9) Relieves the aggravated glycation damage to the back skin mediated by AGE-RAGE in aging individuals;

[0067] (10) Alleviate collagen synthesis, degradation and inflammatory response caused by glycation damage in aging individuals.

[0068] In addition, Lactobacillus paracasei ( Lacticaseibacillus paracasei CCFM1355 fermentation of peanut skins can enhance the following properties of peanut skins:

[0069] (1) Ability to inhibit the formation of fluorescent AGEs in vitro;

[0070] (2) The ability to prevent the decline in HSF cell viability caused by acetone aldehyde;

[0071] (3) Inhibits the ability of serum AGE production;

[0072] (4) The ability to inhibit AGE-RAGE binding and alleviate skin glycation damage;

[0073] (5) Improves skin elasticity;

[0074] (6) Enhance the ability of peanut skin to relieve oxidative stress after skin glycation.

[0075] Therefore, Lactobacillus paracasei ( Lacticaseibacillus paracasei CCFM1355 and its prepared metabiotics have great application potential in products that alleviate glycation damage to the host and reduce corresponding aging.

[0076] Preservation of biological materials

[0077] Paracaseicoccus casei subsp. casei (CCFM1355, taxonomic name Lacticaseibacillus paracasei ) was deposited in the Guangdong Microbial Culture Collection Center on October 25, 2023, and the deposit number is GDMCC No: 63925, and the deposit address is No. 59, Building, Guangzhou, Guangdong Province. Lacticaseibacillus paracasei BRIEF DESCRIPTION OF DRAWINGS

[0078] Figure 1 : Effects of different postbiotics on the inhibition of fluorescence AGE generation in the fructose-bovine serum albumin system in vitro.

[0079] Figure 2 : Effects of different postbiotics on HSF cell proliferation.

[0080] Figure 3 : Effects of different postbiotics on HSF cell viability under the action of methylglyoxal, an intermediate of glycation reaction.

[0081] Figure 4 : Effects of different postbiotics on the expression of anti-glycation related genes (DDOST mRNA, MMP-9 mRNA, EGR2 mRNA, NFKB1 mRNA) in HSF cells under high glucose culture.

[0082] Figure 5 : Flowchart of mouse experiment.

[0083] Figure 6 : Effects of Paracaseicoccus casei subsp. casei and its CCFM1355 prepared postbiotics on the content of AGE in the blood and skin tissue of mice.

[0084] Figure 7 : Effects of Paracaseicoccus casei subsp. casei and its CCFM1355 prepared postbiotics on the content of inflammatory markers TNF-α and IL-6 in the serum and skin of aging mice.

[0085] Figure 8 : Effects of Paracaseicoccus casei subsp. casei and its CCFM1355 prepared postbiotics on the water content of the stratum corneum and the elasticity of the skin.

[0086] Figure 9 : Effects of Paracaseicoccus casei subsp. casei and its CCFM1355 prepared postbiotics on the synthesis, degradation and content of collagen type III in the skin.

[0087] Figure 10 : Effects of Paracaseicoccus casei subsp. casei and its CCFM1355 prepared postbiotics on the inhibition of AGE-RAGE binding to alleviate glycation damage of the skin.

[0088] Figure 11 ​: The effect of Paracaseicoccus variabilis and its CCFM1355 prepared postbiotic on inhibiting downstream sugar injury inflammatory pathway and oxidative stress pathway.

[0089] Figure 12 : The effect of Paracaseicoccus variabilis CCFM1355 fermented peanut skin on improving the in vitro anti-glycation ability of peanut skin.

[0090] Figure 13 : The effect of Paracaseicoccus variabilis CCFM1355 fermented peanut skin on improving the oral inhibition of AGE accumulation in serum and subsequent receptor binding of peanut skin.

[0091] Figure 14 : The effect of Paracaseicoccus variabilis CCFM1355 fermented peanut skin on improving the oral relief of glycation-induced skin elasticity and oxidative stress of peanut skin.

[0092] " " indicates a statistically significant difference from the Model group (P < 0.05), " " indicates a statistically significant difference from the Model group (P < 0.01), " " indicates a statistically significant difference from the Model group (P < 0.001), and " " indicates a statistically significant difference from the Model group (P < 0.0001). DETAILED DESCRIPTION

[0093] The application will be further described below in conjunction with specific examples.

[0094] The human skin fibroblasts (HSF) involved in the following examples were purchased from Kunming Cell Bank.

[0095] The BALB / c mice involved in the following examples were purchased from Vivotecnia.

[0096] The Paracaseicoccus variabilis CCFM1355 and Paracaseicoccus variabilis FQHXN96L1 involved in the following examples were obtained from the Food Biotechnology Center of Jiangnan University.

[0097] The peanut skin extract was purchased from Shaanxi Shengheng Biological Technology Co., Ltd., batch number: SH20220328.

[0098] The culture medium involved in the following examples is as follows:

[0099] MRS liquid medium: yeast powder 5.0 g / L, beef extract 10.0 g / L, peptone 10.0 g / L, glucose 20.0 g / L, sodium acetate anhydrous 2.0 g / L, citric acid biamine 2.0 g / L, potassium phosphate dibasic 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.

[0100] MRS solid medium: yeast powder 5.0 g / L, beef extract 10.0 g / L, peptone 10.0 g / L, glucose 20.0 g / L, sodium acetate anhydrous 2.0 g / L, citric acid biamine 2.0 g / L, potassium phosphate dibasic 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.

[0101] MRS simplified liquid medium: glucose 8 g / L, yeast powder 5 g / L, calcium carbonate 6 g / L, sodium acetate anhydrous 2 g / L, citric acid biamine 2 g / L, potassium phosphate dibasic 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.

[0102] Peanut skin fermentation medium: peanut skin extract 5 g / L, 4 g / L glucose, 5 g / L yeast powder, 4 g / L calcium carbonate, pH adjusted to 6.8~7.2, sterilized at 115 ℃ for 20 min to prepare a peanut skin fermentation medium (abbreviated as hsp).

[0103] 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 in the mixed solution).

[0104] Example 1: Cell resuscitation and culture

[0105] First, the cryopreserved human skin fibroblast strain (HSF) was taken out, quickly melted in a 37℃ water bath, then centrifuged at 1000 r / min for 3 min, the supernatant was discarded, and the cells were resuspended in an appropriate volume of cell culture medium and placed in a culture dish, then placed in a 37℃ incubator containing 5% CO2 for culture. When the cells grew to 70%~80% confluence after 1~2 d, the cells were subcultured.

[0106] Example 2: Screening of Paracasei CCFM1355 and preparation of postbiotic

[0107] (1) Screening of Paracaseicilvus paracasei CCFM1355

[0108] The sample was derived from healthy human feces, and after pretreatment, the sample was stored in 20% glycerol at -80°C in a refrigerator. After being taken out and thawed, the sample was mixed and 0.5 mL of the sample was taken and added to 4.5 mL of normal saline. Gradient dilution was performed with normal saline, and the appropriate gradient dilution liquid was coated on the MRS solid culture medium and cultured at 37°C for 48 h. Typical colonies of Paracaseicilvus paracasei were picked and streaked on MRS solid culture medium, and single colonies were picked and transferred to MRS liquid medium for enrichment. The strain was obtained, and the genome of the strain was extracted for 16S rDNA amplification and sequencing (performed by Suzhou Jinyuzhi Biological Technology Co., Ltd.). The 16S rDNA sequencing result is shown as SEQ ID No. 1. The result was determined as Paracaseicilvus paracasei by NCBI sequence alignment, and named as Paracaseicilvus paracasei CCFM1355.

[0109] (2) Preparation of Paracaseicilvus paracasei CCFM1355 and Paracaseicilvus paracasei FQHXN96L1 probiotics

[0110] 1) Paracaseicilvus paracasei CCFM1355 was streaked from the bacteria preservation tube and cultured on MRS solid medium in a 37°C water-jacketed incubator for 24-48 h to obtain single colonies. The single colonies were inoculated into MRS liquid medium and cultured at 37°C for 12-18 h to obtain culture liquid 1;

[0111] Culture liquid 1 was inoculated into MRS liquid medium at a 2% (v / v) inoculation amount and cultured at 37°C for 12 h to obtain seed liquid;

[0112] The seed liquid was inoculated into MRS liquid medium and MRS simplified liquid medium at 2-5% (v / v) respectively for expansion culture, and cultured at 37°C for 18-24 h. The concentrations of the two bacterial liquids were adjusted to the same level to obtain bacterial liquid a (MRS liquid medium) and bacterial liquid b (MRS simplified liquid medium).

[0113] The supernatant obtained by centrifuging bacterial liquid b at 8000 r / min for 30 min was heat-treated (65°C, 30 min), and then freeze-dried to obtain a powder for standby. Paracaseicilvus paracasei CCFM1355 fermentation supernatant (denoted as CCFM1355_M) was prepared.

[0114] The bacteria slurry obtained by centrifuging the bacteria liquid a at 8000 r / min for 30 min was resuspended with double distilled water at a volume of 75% of the original bacteria liquid. The resuspended liquid was heat treated (65 ℃, 30 min) and then subjected to high pressure homogenization (1000-1200 MPa, 10P times) in a high pressure homogenizer to obtain a bacteria lysate (denoted as CCFM1355_Z). The postbiotic freeze-dried powder was obtained by freeze-drying and was ready for use.

[0115] The preparation method of the live bacteria of Lactobacillus paracasei CCFM1355 was the same as that of the postbiotic, except that the bacteria slurry obtained by centrifuging the bacteria liquid b at 8000 r / min for 30 min was resuspended with a freeze-drying protective agent at a ratio of 1 g:2 mL and then directly freeze-dried to obtain the live bacteria powder of Lactobacillus paracasei CCFM1355, denoted as CCFM1355.

[0116] The postbiotic of Lactobacillus paracasei CCFM1355 (bacteria lysate CCFM1355_Z and fermentation supernatant CCFM1355_M) was prepared by the above-mentioned means.

[0117] 2) The postbiotic of Lactobacillus paracasei FQHXN96L1 (bacteria lysate FQHXN96L1_Z and fermentation supernatant FQHXN96L1_M) was prepared according to the method of step 1).

[0118] Example 3: Preparation of peanut skin fermentation broth by Lactobacillus paracasei CCFM1355 fermentation of peanut skin

[0119] The bacteria liquid of Lactobacillus paracasei CCFM1355 was streaked on MRS solid medium with an inoculation loop and cultured at 37 ℃ for 48 h. A single colony was taken to MRS liquid medium and cultured at 37 ℃ under aerobic conditions for 18 h. After mixing, the bacteria liquid was inoculated into new MRS liquid medium at an inoculation amount of 2% (v / v) and cultured, and this operation was repeated for 3 times to obtain the activated bacteria liquid.

[0120] The obtained activated bacteria liquid was inoculated into peanut skin fermentation medium (hsp) at an inoculation amount of 2% (v / v) and cultured at 37 ℃ with 200 rpm shaking for 72 h. The fermentation broth was taken at 72 h, the supernatant was obtained by centrifuging the bacteria liquid at 8000 r / min for 30 min, and then the supernatant was heat treated (65 ℃, 30 min) and freeze-dried to obtain a powder for standby. The peanut skin fermentation supernatant of Lactobacillus paracasei CCFM1355 (denoted as CCFM1355_H) was prepared.

[0121] Example 4: Effect of postbiotic prepared by Lactobacillus paracasei CCFM1355 on inhibition of fluorescence AGE generation in fructose-bovine serum albumin system in vitro

[0122] Bovine serum albumin and d-(+)-fructose were dissolved in 0.1 M phosphate buffer (pH 7.4) as solvent, so that the final mixture had a concentration of 10 mg / mL bovine serum albumin and 0.5 M (i.e. 90 mg / mL) d-(+)-fructose. After mixing well by magnetic stirring, the fructose-bovine serum albumin glycation system was obtained by filtration through a 0.22 μm water filter membrane under sterile conditions.

[0123] Experimental group: different metaplasma samples were added to the fructose-bovine serum albumin glycation system and incubated at 37 °C for 7 days. The volume ratio of the metaplasma sample solution to the fructose-bovine serum albumin glycation solution was 1:1. The incubated samples were used to detect the formation of fluorescent AGEs after 7 days of incubation. The metaplasma samples included each group of metaplasma prepared according to the method in Example 2: metaplasma prepared from Paracasei CCFM1355 (CCFM1355_Z and CCFM1355_M), metaplasma prepared from Paracasei FQHXN96L1 (FQHXN96L1_Z and FQHXN96L1_M).

[0124] Control group: an equal volume of PBS solution was added to the fructose-bovine serum albumin glycation solution, and incubated at 37 °C for 7 days. The formation of fluorescent AGEs was detected after 7 days of incubation.

[0125] After incubation, the fluorescent AGEs in the fructose-modified BSA were detected using an excitation wavelength of 370 nm and an emission wavelength of 440 nm. The percentage of inhibition of the generation of fluorescent AGEs was calculated as 1 minus the difference in fluorescence intensity between the sample and the control (BSA+ / fructose+).

[0126] The effect of the inhibition rate of the generation of fluorescent AGEs in the fructose-bovine serum albumin system in vitro is shown in Table 1. Figure 1 As shown in Table 1, the AGE inhibition rates of CCFM1355_Z and CCFM1355_M were 51.60% and 29.91%, respectively, which were significantly better than those of other Paracasei (the AGE inhibition rates of FQHXN96L1_Z and FQHXN96L1_M were 16.74% and 14.12%, respectively).

[0127] Example 5: Effect of metaplasma prepared from Paracasei CCFM1355 on the proliferation of HSF cells

[0128] The specific steps are as follows:

[0129] (1) 100 μL of HSF cells in the logarithmic growth phase were taken and diluted to a concentration of 3×10 4The cells were inoculated in 96-well plates at a concentration of 1 cell / well, with the outermost circle filled with PBS solution to prevent edge effects, and cultured for 24 h to allow them to adhere to the wall. After that, blank, control and metatonic treatment groups were set up.

[0130] The blank group contained only cell culture medium without HSF cells.

[0131] The control group contained cell culture medium and HSF cells without metatonic.

[0132] The metatonic treatment group contained cell culture medium with different metatonic obtained from Example 2 and HSF cells.

[0133] Preparation of cell culture medium containing metatonic: resuspend the metatonic in cell culture medium (the amount of resuspended metatonic is equivalent to the amount of metatonic prepared from bacteria solution with a concentration of 5.0 x 10 7 CFU / mL), to obtain cell culture medium containing metatonic.

[0134] (2) Incubate the above-mentioned hole plates in an incubator at a temperature of 37°C for 24 h. After incubation, add 10 μL of CCK8 solution to each well and incubate for 2 h to measure the absorbance at 450 nm (OD).

[0135] Calculate the cell viability according to the following formula: cell viability (%) = (treatment group OD value - blank group OD value) / (control group OD value - blank group OD value) x 100%.

[0136] The effect of metatonic on cell proliferation is shown in Figure 2 Compared with the control group (cell proliferation rate 100%), the cell proliferation rates of the metatonic from Paracasei CCFM1355 (CCFM1355_M and CCFM1355_Z), Paracasei FQHXN96L1 (FQHXN96L1_M and FQHXN96L1_Z) were 104.78%, 107.45%, 97.22% and 98.50%, respectively.

[0137] According to the toxicity grading evaluation method in ISO 10993-5:2009, if the cell viability is greater than 70%, it can be considered non-toxic. The above results show that the HSF cell viability under the above-mentioned metatonic concentration is higher than 90%, and considering its non-cytotoxicity, the inactivated bacteria concentration of 5.0 x 10 7 CFU / mL is a suitable metatonic concentration for subsequent cell experiments.

[0138] Example 6: Effect of metatonic prepared from Paracasei CCFM1355 on prevention of glycation damage to HSF cells caused by methylglyoxal

[0139] The specific steps are as follows:

[0140] (1) Take 100 μL of HSF cells in the logarithmic growth phase at a concentration of 3×10 4 cells / well to a 96-well plate, fill the outermost circle with PBS solution to prevent edge effects, and culture for 24 h until the cells adhere, then set up a blank group, a control group 1, and a treatment group 1;

[0141] The blank group contains only cell culture medium without HSF cells.

[0142] The control group 1 contains cell culture medium and HSF cells without metachronism.

[0143] The treatment group 1 contains cell culture medium with different metachronisms obtained in Example 2 and HSF cells.

[0144] Preparation of cell culture medium containing metachronism: resuspend the metachronism in cell culture medium (the amount of resuspended metachronism is equivalent to the amount of metachronism prepared from bacteria fermented to a concentration of 5.0×10 7 CFU / mL), and obtain cell culture medium containing metachronism.

[0145] (2) Incubate the above-mentioned plates in an incubator at a temperature of 37℃ for 24 h, discard the old medium of the control group and the model group after incubation, rinse with PBS for 3 times, and set up a control group, a model group, and a treatment group:

[0146] The control group is the control group 1 after changing the liquid, containing cell culture medium and HSF cells, without metachronism treatment, and without methylglyoxal modeling agent.

[0147] The model group is the control group 1 changed into cell culture medium containing methylglyoxal modeling agent, containing original HSF cells, without metachronism treatment,

[0148] The cell culture medium containing methylglyoxal modeling agent is prepared by mixing methylglyoxal uniformly in ordinary cell culture medium and sterilizing it through a 0.22 μm water system filter membrane, and the final concentration of methylglyoxal in the cell culture medium is 400 μmol / L.

[0149] The treatment group is the treatment group 1 changed into cell culture medium containing methylglyoxal modeling agent, containing original HSF cells.

[0150] (3) Incubate the above-mentioned plates in an incubator at a temperature of 37℃ for 24 h, add 10 μL of CCK8 solution to each well for 2 h of incubation, and measure the absorbance value (OD) at 450 nm.

[0151] Cell viability was calculated according to the following formula: Model group cell viability (%) = (model group OD value-blank group OD value) / (control group OD value-blank group OD value) x 100%; treatment group cell viability (%) = (treatment group OD value-blank group OD value) / (control group OD value-blank group OD value) x 100%.

[0152] The results of the effect of preventing acetol from damaging HSF cells are as follows Figure 3 Compared with the control group (cell viability 100%), the cell viability of the model group was 54.93%, and acetol modeling caused significant damage to HSF cells.

[0153] The cell viability of the treatment group after adding CCFM1355_M and CCFM1355_Z was 64.71% and 61.36%, respectively, among which CCFM1355_Z significantly improved the HSF cell viability compared with the model group, indicating that the probiotic of Paracasei CCFM1355 can effectively prevent the glycation damage of HSF cells caused by acetol;

[0154] After the treatment group of the comparative example Paracasei FQHXN96L1 probiotic (FQHXN96L1_M and FQHXN96L1_Z) was treated, the HSF cell viability was 43.96% and 47.63%, respectively, that is, the probiotic of Paracasei FQHXN96L1 does not have the ability to prevent acetol from damaging HSF cells.

[0155] Example 7: Effect of probiotic prepared by Paracasei CCFM1355 on prevention of expression of MMP-9 mRNA and EGR2 mRNA in HSF cells under high glucose culture.

[0156] The specific steps are as follows:

[0157] (1) HSF cells were inoculated in a 6-well plate at 1x10 5 cells / mL, and the cells were cultured overnight to adhere. The old culture medium was discarded, and PBS was used to rinse 3 times, and control group 1 and treatment group 1 were set;

[0158] The control group 1 is a group without probiotic;

[0159] The treatment group 1 is divided into: using probiotic to treat cells, the treatment method is: resuspending the probiotic (CCFM1355_Z or FQHXN96L1_Z) in the cell culture medium, and replacing the original cell culture medium with the cell culture medium containing the probiotic.

[0160] (2) The above-mentioned hole plate is incubated in an incubator at a temperature of 37°C for 24 hours. After incubation, the old culture medium of the control group and the modeling agent group is discarded, and the PBS is washed for 3 times. The control group, the model group and the treatment group are set:

[0161] The control group is that the control group 1 is replaced with the original HSF cell without postbiotic treatment, and 2 mL of ordinary cell culture medium is added.

[0162] The model group is that the control group 1 is replaced with the original HSF cell without postbiotic treatment.

[0163] The treatment group is that the control group 1 is replaced with the original HSF cell without postbiotic treatment.

[0164] (3) The above-mentioned hole plate is incubated in an incubator at a temperature of 37°C for 24 hours. The culture supernatant is discarded, and each hole is rapidly washed with PBS for 3 times. 1 mL of cell lysis solution is added to each hole, and repeated blowing and beating is performed. The cell lysis solution is extracted to extract RNA, and RT-PCR reverse transcription kit is used for reverse transcription to cDNA. The expression of genes in HSF cells is detected by real-time fluorescent quantitative method, and 2 -△△Ct The expression of DDOST mRNA, MMP-9 mRNA, EGR2 mRNA and NFKB1 mRNA is calculated by the formula, in which the internal reference is β-actin. The primers are described in Table 1, and the results are shown in Figure 4 .

[0165] Table 1: Primer sequence

[0166]

[0167] AGE receptor-1 (DDOST, also known as AGER1) is a protein with strong AGE-specific binding ability, which has been proved to directly accelerate the absorption and clearance of AGE, prevent the increase of cell AGE-RAGE-mediated active oxygen and pro-inflammatory cytokines, and inhibit RAGE signal by competing with AGE. Glycation injury can cause the expression of DDOST mRNA to decrease, and aggravate AGE-RAGE binding. Therefore, the target relieves AGE-RAGE injury and strengthens the anti-glycation function of DDOST by increasing the expression of DDOST mRNA. The results are shown in Figure 4As can be seen, the expression of DDOST mRNA in the control group is about 1, and the expression of DDOST mRNA in the model group after intervention of high-glucose medium is reduced to 0.58; the postbiotic prepared from Paracasei CCFM1355 (CCFM1355_Z) significantly increases the expression of DDOST mRNA in HSF cells to 1.51 (increased by 159.7% compared with the model group); and the expression of DDOST mRNA after treatment with the postbiotic of FQHXN96L1 Paracasei (FQHXN96L1_Z) is only 0.73, which does not show a significant up-regulation effect on the expression of DDOST mRNA compared with the model group.

[0168] Matrix metalloproteinase 9 (MMP-9) is a kind of enzyme belonging to the zinc-metalloproteinase family, which is an enzyme mainly degrading type IV collagen and elastin, and is involved in the degradation of extracellular matrix in normal physiological processes and pathological processes; under high-glucose conditions, the expression of MMP-9 is increased, which causes the proliferation of skin fibroblasts to slow down, the activity to decrease, and the migration and collagen secretion capacity to decrease. Figure 4 As can be seen, the expression of MMP-9 mRNA in the control group is about 1, and the expression in the model group after intervention of high-glucose medium is increased to 2.16; the postbiotic prepared from Paracasei CCFM1355 (CCFM1355_Z) significantly reduces the expression of MMP-9 mRNA in HSF cells to 0.82 (decreased by 62.1% compared with the model group); and the expression of MMP-9 mRNA after treatment with the postbiotic of FQHXN96L1 Paracasei (FQHXN96L1_Z) is 1.61, which does not show a significant down-regulation effect on the expression of MMP-9 mRNA compared with the model group.

[0169] Early growth response protein 2 (EGR2) is induced by T cell receptors and is essential for inducing T cell anergy, and is involved in the regulation of inflammatory pathways; under high-glucose conditions, the expression of EGR2 is reduced, which inhibits suppressor of cytokine signaling 1 (Socs1) and increases pro-inflammatory genes. The expression of EGR2 mRNA in the control group is about 1, and the expression after intervention of high-glucose medium is reduced to 0.75; the postbiotic prepared from Paracasei CCFM1355 (CCFM1355_Z) significantly increases the expression of EGR2 mRNA in HSF cells to 1.74 (increased by 125.9% compared with the model group); and the expression of EGR2 mRNA after treatment with the postbiotics of other Paracasei (FQHXN96L1_Z) is about 1.09, 0.68, and 1.02, respectively, which does not show a significant up-regulation effect on the expression of EGR2 mRNA compared with the model group.

[0170] Nuclear factor kappa-B (NF-κB) is a group of important transcription factors involved in various biological processes of cells, including cell proliferation and inflammation; NFKB1 is one of the members, NFKB1 encodes p50 protein, under hyperglycemic conditions, p50 expression increases and exacerbates the formation of NF-κB complex in which it participates, increasing the pro-inflammatory potential; by controlling the expression amount of NFKB1 mRNA under the interference of glycation reaction, stable and not abnormally high, it can also reduce the exacerbation of downstream inflammatory response caused by glycation loss. Figure 4 It can be seen that the expression amount of NFKB1 mRNA in the control group is about 1, and the expression amount of the model group after high glucose medium intervention rises to 1.77; the metagenomic (CCFM1355_Z) prepared by Paracasei CCFM1355 significantly reduces the expression amount of NFKB1 mRNA in HSF cells to 0.78 (decreased by 55.7% compared with the model group); and the metagenomic (FQHXN96L1_Z) treated by Paracasei CCFM1355 has an expression amount of NFKB1 mRNA of about 1.10, which does not show a significant down-regulation effect on NFKB1 mRNA expression compared with the model group.

[0171] From the experimental results, it can be seen that the metagenomic (bacterial lysate) prepared by Paracasei CCFM1355 can down-regulate the expression of MMP-9 mRNA and NFKB1 mRNA, up-regulate the expression of DDOST mRNA and EGR2 mRNA, and prevent the sugar damage to HSF cells caused by high glucose culture conditions.

[0172] Example 8: Effect of Paracasei CCFM1355 and the metagenomic prepared therefrom on the AGE level in the blood and skin of aging mice

[0173] The preparation method of the metagenomic (CCFM1355_M and CCFM1355_Z) of Paracasei CCFM1355 involved in the following examples is the same as that of Example 2, but CCFM1355_Z in this example specifically refers to the bacterial slurry obtained by centrifuging the bacterial liquid a obtained in Example 2 at 8000 r / min for 30 min, and the bacterial slurry is resuspended in physiological saline at 75% of the original bacterial liquid volume. The resuspended liquid is heat treated (65°C, 30 min), and the dead bacterial cells (denoted as CCFM1355_Z) are obtained without high-pressure homogenization, and the metagenomic freeze-dried powder is obtained by freeze-drying for standby.

[0174] The specific steps are as follows:

[0175] (1) Take 25 healthy 8-week-old male BALB / c mice, and randomly divide them into 5 cages, 5 mice in each cage. The 5 cages are respectively: 1 cage for the blank group (Control), 1 cage for the model group (Model), 1 cage for the CCFM1355 live bacteria group, 1 cage for the CCFM1355_Z group, and 1 cage for the CCFM1355_M group, which are respectively:

[0176] The blank group (Control): physiological saline is used as a control;

[0177] The model group (Model): physiological saline is used as a control;

[0178] The CCFM1355 live bacteria group: intragastrically administer live bacteria of the Paracasei CCFM1355, with a dose of 5×10 9 CFU / kg of mouse body weight;

[0179] The CCFM1355_Z group: intragastrically administer the Paracasei CCFM1355 postbiotic (dead bacteria), with a dose of 500 mg / kg of mouse body weight;

[0180] The CCFM1355_M group: intragastrically administer the fermentation supernatant of the Paracasei CCFM1355, with a dose of 500 mg / kg of mouse body weight;

[0181] Among the above groups, the dead bacteria or the fermentation supernatant is: the dead bacteria or the fermentation supernatant prepared from the bacteria liquid obtained after fermentation of the bacteria with an equal amount of bacteria as 5×10 9 CFU / kg of mouse body weight of live bacteria.

[0182] The experiment lasts for 7 weeks: after one week of adaptation, the mice in the blank group are subcutaneously injected with D-galactose (1000 mg / kg) at a dose of 0.1 mL / mouse / day. From the second week, the CCFM1355 live bacteria group, the CCFM1355_Z group, and the CCFM1355_M group are intragastrically administered with the corresponding strain of freeze-dried powder or postbiotic freeze-dried powder (dead bacteria or fermentation supernatant) prepared from the strain at the corresponding dose dissolved in physiological saline at a dose of 0.1 mL / mouse / day. The blank group and the model group are intragastrically administered with an equal amount of physiological saline as a control, until the end of the experiment. All groups are allowed to drink and eat freely, and the experimental process is as shown in Figure 5 .

[0183] After the end of the experiment, the mice were sacrificed and the eyeball blood was taken. After standing for 40 min, the blood supernatant was centrifuged at 3000 r / min for 20 min for ELISA detection; the back skin tissue was cut and ground to prepare a homogenate at a weight-volume ratio of 1:10 with PBS, centrifuged at 3000 r / min for 20 min, and the skin supernatant was taken to detect the AGE content in the mouse serum and skin by an ELISA kit, and the results are shown in Figure 6

[0184] (1) Serum AGE content: compared with the control group (262.70 ng / L), the AGE content in the serum of the model group was significantly increased to 406.22 ng / L. After oral administration of Paracaseiclostridium CCFM1355 and its prepared metaplasma, the content of AGE, a marker of glycation and aging, in the serum of mice was reduced; the CCFM1355 live bacteria group, the CCFM1355_Z group and the CCFM1355_M group reduced the AGE content in the serum to 180.06 ng / mL, 333.56 ng / L and 218.58 ng / L, respectively, which was 55.7%, 17.9% and 46.2% lower than the model group, respectively. That is, oral administration of Paracaseiclostridium CCFM1355 and its prepared metaplasma (lysate and supernatant) can reduce the content of AGE in the serum of aging mice and reduce the accumulation of glycation loss.

[0185] (2) Skin AGE content: compared with the control group (336.76 ng / L), the AGE content in the serum of the model group was significantly increased to 463.21 ng / L. Compared with the model group, oral administration of Paracaseiclostridium CCFM1355 and its prepared metaplasma significantly reduced the content of AGE, a marker of glycation and aging, in the skin of mice. The CCFM1355 group, the CCFM1355_Z group and the CCFM1355_M group reduced the AGE content in the skin to 340.51 ng / L, 435.85 ng / L and 376.37 ng / L, respectively, which was 26.5%, 5.9% and 18.7% lower than the model group, respectively. Oral administration of Paracaseiclostridium CCFM1355 and its prepared metaplasma (lysate and supernatant) can reduce the content of AGE in the skin of aging mice and reduce the accumulation of glycation loss.

[0186] The above results show that Paracaseiclostridium CCFM1355 and its prepared metaplasma have the ability to alleviate the accumulation of AGE content in the serum and skin of aging mice, and the improvement is obvious compared with the model group.

[0187] Example 9: Effect of Paracaseiclostridium CCFM1355 and its prepared metaplasma on the content of inflammatory markers in the serum and skin of aging mice

[0188] ​The animal experiment design and gavage group in the following examples are the same as those in Example 8. The TNF-α and IL-6 contents in the serum and skin of the aging mice were detected using a Nanjing Sunbioga Elisa kit, and the results are shown in Table 2. Figure 7

[0189] (1) Serum TNF-α content: Compared with the control group (297.24 ng / L), the TNF-α content in the serum of the model group significantly increased to 382.15 ng / L. Oral administration of Paracaseicoccus casei CCFM1355 and the metaplast prepared from Paracaseicoccus casei CCFM1355 significantly reduced the TNF-α content in the serum of the mice. The CCFM1355_Z group, the CCFM1355_M group and the CCFM1355 live bacteria group made the TNF-α content in the serum reach 335.82 ng / L, 375.48 ng / L and 335.83 ng / L, respectively, which corresponded to a decrease of 12.1%, 1.7% and 12.1% in the TNF-α content in the serum of the model group, respectively.

[0190] (2) Serum IL-6 content: Compared with the control group (80.20 ng / L), the IL-6 content in the serum of the model group significantly increased to 126.79 ng / L. Oral administration of Paracaseicoccus casei CCFM1355 and the metaplast prepared from Paracaseicoccus casei CCFM1355 significantly reduced the IL-6 content in the serum of the mice. The CCFM1355_Z group, the CCFM1355_M group and the CCFM1355 live bacteria group made the IL-6 content in the serum reach 106.30 ng / L, 115.29 ng / L and 114.28 ng / L, respectively, among which the CCFM1355_Z group reduced the IL-6 content in the serum of the model group the most, by 16.2% compared with the model group.

[0191] (3) Skin TNF-α content: Compared with the control group (351.25 ng / L), the TNF-α content in the skin of the model group significantly increased to 486.73 ng / L. Oral administration of Paracaseicoccus casei CCFM1355 and the metaplast prepared from Paracaseicoccus casei CCFM1355 significantly reduced the TNF-α content in the skin of the mice. The CCFM1355_Z group, the CCFM1355_M group and the CCFM1355 live bacteria group made the TNF-α content in the skin reach 389.71 ng / L, 347.50 ng / L and 396.98 ng / L (decreased by 19.9%, 28.6% and 18.4% compared with the model group, respectively).

[0192] ​(4) Skin IL-6 content: Compared with the control group (80.20 ng / L), the IL-6 content in the skin of the model group increased significantly to 126.79 ng / L. Oral administration of Lactobacillus paracasei CCFM1355 and its prepared postbiotic significantly reduced the IL-6 content in mouse skin. CCFM1355 live bacteria, CCFM1355_Z group and CCFM1355_M group reduced the serum IL-6 content to 88.28 ng / L, 96.12 ng / L and 86.59 ng / L, respectively. Among them, CCFM1355_M reduced the inflammatory factor IL-6 content in the model group the most (19.8% lower than the model group), CCFM1355_Z reduced it by 11.0% compared with the model group, and CCFM1355 reduced it by 18.3% compared with the model group.

[0193] Based on the results of relevant biochemical indicators in animal serum, it can be concluded that Lactobacillus paracasei CCFM1355 and its prepared metabiotic can reduce the levels of inflammatory factors IL-6 and TNF-α in the serum and skin of aging mice, thereby alleviating inflammation and combating the overall aging of the host.

[0194] Example 10: Effects of Lactobacillus paracasei CCFM1355 and its prepared metabiotics on the stratum corneum moisture content and skin elasticity of aging mice.

[0195] The animal experimental design and gavage groups involved in the following examples are the same as in Example 8. At the end of the experiment, the skin moisture content of the stratum corneum on the back of each mouse and the elasticity of the skin on the back of the mouse were measured using a skin moisture meter (equipped with a Corneometer CM825 stratum corneum moisture measurement probe) from CK GmbH, Germany. The results are as follows: Figure 8 As shown.

[0196] (1) The moisture content of the stratum corneum is from Figure 8 It can be seen that, compared with the blank group (66.78%), the water content of the model group was significantly reduced to 47.95%. The water content of the cuticle in the *Lactobacillus paracasei* CCFM13555_Z group (67.23%) was 40.2% higher than that in the model group. The water content of the *Lactobacillus paracasei* CCFM1355_M group (57.33%) was 19.6% higher than that in the model group. The water content of the *Lactobacillus paracasei* CCFM1355 group (53.33%) was 11.2% higher than that in the model group. In other words, the experimental results show that the postbiotic prepared from *Lactobacillus paracasei* CCFM1355, especially its dead bacterial cells (*Lactobacillus paracasei* CCFM1355_Z), can increase the water content of the back of aged mice after glycation damage.

[0197] During the aging process, the concentration of AGE gradually increases, crosslinks with surrounding long-lived proteins, or enhances the expression of metalloproteinases through the AGE-RAGE pathway, leading to loose skin structure and decreased water retention capacity of the stratum corneum. Exogenous supplementation of postbiotics with anti-glycation function can alleviate skin water loss during the aging process. Postbiotics prepared from Paracaseicoliformis CCFM1355 can prevent skin dryness during the aging process. Among them, postbiotics prepared from Paracaseicoliformis CCFM1355 (CCFM1355_Z) has more significant effect on alleviating the decrease of water content in the stratum corneum caused by glycation damage than pure fermentation supernatant CCFM1355_M group.

[0198] (2) The skin elasticity performance R2 is calculated by Figure 8 It can be seen that compared with the blank group (49.77%), the skin elasticity performance of the model group is significantly reduced to 30.16%, the skin elasticity of the Paracaseicoliformis CCFM1355_Z group (72.93%) is increased by 141.8% compared with the model group, the skin elasticity of the CCFM1355_M group (79.00%) is increased by 161.9% compared with the model group, and the skin elasticity of the CCFM1355 group (75.75%) is increased by 151.2% compared with the model group.

[0199] It can be seen from the experimental results that the postbiotics prepared from Paracaseicoliformis CCFM1355 significantly increases the skin elasticity performance of the back of the aging mouse. During the aging process, the concentration of AGE gradually increases, the synthesis of collagen decreases after being affected by AGE and its receptor interaction, and the function of the original structural protein is damaged, resulting in decreased elasticity of the cell skeleton and cell space. Exogenous supplementation of anti-glycation functional products can alleviate the decrease of skin elasticity during the aging process. Therefore, intragastrically administering postbiotics prepared from Paracaseicoliformis CCFM1355 significantly increases the skin elasticity performance of the back of the aging mouse.

[0200] Example 11: Effect of Paracaseicoliformis CCFM1355 and postbiotics prepared therefrom on the synthesis and content of type 3 collagen in the skin of glycation-damaged aging mice

[0201] The animal experiment design and intragastric administration groups in the following examples are the same as those in Example 8. The content of type 3 collagen in the skin of the mouse was detected by using a Nanjing Senbeiga Elisa kit.

[0202] The skin RNA was extracted by Trizol method, and the key target gene expression in the synthesis and degradation process of type 3 collagen in the skin was detected by reverse transcription into cDNA. The primers of COL3A1 mRNA and MMP-9 mRNA are described in Table 2 below.

[0203] Table 2: Primer sequences

[0204]

[0205] The content of collagen III in the back skin of mice was determined as shown in the table below. Figure 9 The content of collagen III in the back skin of mice was determined as shown in the table below.

[0206] The expression of COL3A1 mRNA was detected. Figure 9 The results showed that the expression of COL3A1 mRNA was significantly up-regulated by Paracaseitol CCFM1355 and the probiotics CCFM1355_Z and CCFM1355_M, and the relative expression was 1.30, 1.61 and 0.90, respectively (increased by 38.78% and 70.9% compared with the model group 0.94), which could alleviate the abnormal decrease of collagen III caused by glycation loss and maintain the normal function of collagen.

[0207] The expression of MMP-9 mRNA was detected. Figure 9 The results showed that the expression of MMP-9 mRNA was significantly down-regulated by Paracaseitol CCFM1355 and the probiotics CCFM1355_Z and CCFM1355_M, and the relative expression was 0.66, 2.95 and 0.68, respectively (decreased by 82.0%, 19.88% and 81.6% compared with the model group 3.69), which could alleviate the abnormal increase of MMP-9 caused by glycation loss and maintain the normal function of protein.

[0208] Example 12: Effect of Paracaseitol CCFM1355 and the probiotics prepared therefrom on relieving multi-angle glycation damage of skin

[0209] The animal experiment design, gavage group, and RNA extraction and detection in the following examples are the same as those in Example 11. The primers for the key genes DDOST, RAGE, NFKB1, and Cu / Zn-SOD in the skin of aged mice are described in Table 3 below. The results of the gene expression affected by inhibiting AGE-RAGE binding to alleviate the effects of skin glycation damage are shown in Figure 10 ; and the effects of inhibiting the downstream sugar damage inflammation pathway and oxidative stress pathway are shown in Figure 11 .

[0210] Table 3: Primer sequences

[0211]

[0212] (1) Effects of Paracaseicoccus casei CCFM1355 and the postbiotic prepared therefrom on inhibiting AGE-RAGE binding to alleviate skin glycation damage

[0213] As can be seen by detecting the expression of RAGE mRNA, Paracaseicoccus casei CCFM1355 and the postbiotic prepared therefrom CCFM1355_Z can significantly down-regulate the expression of RAGE mRNA to 1.00 and 0.70 (66.1% and 76.3% lower than the model group 2.95, respectively). In addition, the expression of DDOST mRNA is increased to 1.86 and 0.68 in the CCFM1355_Z group and the CCFM1355_M group (232.1% and 21.4% higher than the model group 0.56), but the expression of DDOST mRNA in the CCFM1355 group is only 0.36.

[0214] That is, oral administration of the postbiotic prepared from Paracaseicoccus casei CCFM1355 can inhibit AGE-RAGE binding through two pathways, down-regulating the expression of RAGE mRNA and up-regulating the expression of the competitive receptor DDOST mRNA of RAGE, while oral administration of Paracaseicoccus casei CCFM1355 can only down-regulate the expression of RAGE mRNA to alleviate AGE-RAGE binding; however, both oral administration of Paracaseicoccus casei CCFM1355 and the postbiotic prepared therefrom CCFM1355_Z can achieve the effect of alleviating skin glycation damage.

[0215] (2) Effects of Paracaseicoccus casei CCFM1355 and the postbiotic prepared therefrom on inhibiting the downstream sugar damage inflammation pathway and oxidative stress pathway

[0216] Detection of NFKB1 mRNA expression revealed that *Lactobacillus paracasei* CCFM1355 and its prepared metagenic derivative CCFM1355_Z downregulated NFKB1 mRNA expression, resulting in relative expression levels of 1.08 and 0.49, respectively (reduced by 61.1% and 82.4% compared to the model group's 2.77). Simultaneously, Cu / Zn-SOD mRNA expression increased to 1.21 and 2.20 in the oral CCFM1355 and CCFM1355_Z groups, respectively (increased by 148.1% and 353.8% compared to the model group's 0.49). This indicates that oral administration of *Lactobacillus paracasei* CCFM1355 and its prepared metagenic derivative (lysate) can alleviate subsequent glycation damage in the skin by inhibiting the effects of downstream glycation-induced inflammation and oxidative stress pathways.

[0217] Example 13: Effect of Lactobacillus paracasei CCFM1355 fermentation of peanut skin on enhancing the anti-saccharification ability of peanut skin applied externally.

[0218] The preparation of peanut skin fermentation culture medium (hsp) and peanut skin fermentation supernatant of Lactobacillus paracasei CCFM1355 (CCFM1355_H) is as shown in Example 3; the construction of the in vitro fructose-bovine serum albumin system and the generation and detection of fluorescent AGE are as shown in Example 4; the detection of the prevention of glycosylation damage caused by acetone aldehyde to HSF cells is as shown in Example 6.

[0219] (1) Fermentation of peanut skin supernatant by Lactobacillus paracasei CCFM1355 enhances the ability of peanut skin to inhibit the formation of fluorescent AGEs in vitro.

[0220] Peanut skin extract mainly contains flavonoids such as proanthocyanidins, resveratrol, and quercetin. Oligomeric proanthocyanidins possess bioactivities including free radical scavenging, antioxidant activity, cardiovascular protection, anti-inflammation, and tumor inhibition. However, the content of anti-glycation active substances in peanut skin is low, and their bioavailability is not high. To better utilize the anti-glycation properties of peanut skin, *Lactobacillus paracasei* CCFM1355 was used to ferment peanut skin to prepare *Lactobacillus paracasei* CCFM1355 fermented peanut skin supernatant (CCFM1355_H). The effect of *Lactobacillus paracasei* CCFM1355 on the anti-glycation ability of peanut skin was verified using a peanut skin fermentation culture medium of 100 μg / mL and the fermented peanut skin supernatant concentration.

[0221] Depend on Figure 12 It can be seen that the original inhibition rate of the fluorescent AGE formation of the fructose-bovine serum albumin system by peanut skin was 61.70%, while the inhibition rate of the supernatant after fermentation with Lactobacillus paracasei CCFM1355 reached 71.18%, which was 15.4% higher than that of the unfermented group, and the ability to inhibit AGE formation was significantly increased.

[0222] (2) The ability of L. paracasei CCFM1355 fermented peanut skin supernatant to improve the HSF cell viability decline caused by methylglyoxal in vitro

[0223] As can be seen from Figure 12 , the HSF cell viability after peanut skin was 56.16% (2.2% higher than the model group of 54.93%), but after fermentation by L. paracasei CCFM1355, the HSF cell viability after CCFM1355_H was 77.55% (41.2% higher than the model group of 54.93%). The anti-glycation effect of peanut skin was significantly improved by L. paracasei CCFM1355.

[0224] Example 14: Effect of L. paracasei CCFM1355 fermented peanut skin on improving the oral inhibition of AGE accumulation in serum and subsequent receptor binding by peanut skin

[0225] The construction of the aging mouse experiment is as shown in Example 8, the R2 detection of the elasticity of the mouse back skin is as shown in Example 10, and the method for detecting the DDOST mRNA of the skin is as shown in Example 11. The preparation of peanut skin fermentation broth (hsp) and L. paracasei CCFM1355 fermented peanut skin supernatant (CCFM1355_H) is as shown in Example 3; the peanut skin synthetic preparation is administered by gavage (live bacteria L. paracasei CCFM1355 and peanut skin fermentation broth (hsp), denoted as CCFM1355+hsp.

[0226] As can be seen from Figure 13 , although peanut skin itself can reduce the AGE content in the serum of aging mice to 246.95 ng / L (control group 262.70 ng / L, model group 406.22 ng / L), which is significantly reduced by 39.2% compared with the model group; but the effect of peanut skin fermented by L. paracasei CCFM1355 is better, and the AGE content is 174.61 ng / L, which is reduced by 57.0% compared with the model group; the effect of the peanut skin synthetic preparation group is not as good as the other two groups, which can only reduce the model group by 35.5% (AGE content 262.08 ng / L).

[0227] The expression of DDOST mRNA in the hsp group administered by gavage was increased to 0.71 (29.94% higher than the model group of 0.56), but after the participation of L. paracasei CCFM1355, the expression of skin DDOST mRNA after oral administration of CCFM1355_H and CCFM1355+hsp was 6.21 and 3.78, respectively (1040.4% and 594.9% higher than the model group). L. paracasei CCFM1355 improves the ability of peanut skin to inhibit AGE-RAGE binding to alleviate skin glycation damage through fermentation.

[0228] Example 15: Effects of Lactobacillus paracasei CCFM1355 fermented peanut skin on improving skin elasticity and oxidative stress induced by oral administration of peanut skin to alleviate glycation.

[0229] The experimental setup for aging mice is as shown in Example 8, and the method for detecting Cu / Zn-SOD mRNA in the skin is as shown in Example 12. The preparation of peanut skin fermentation culture medium (hsp) and peanut skin fermentation supernatant of Lactobacillus paracasei CCFM1355 (CCFM1355_H) is as shown in Example 3; the group that was gavaged with the peanut skin synthetic preparation (live Lactobacillus paracasei CCFM1355 and peanut skin fermentation culture medium (hsp) is denoted as CCFM1355+hsp).

[0230] Depend on Figure 14 It was found that although peanut skin itself could increase the elasticity R2 of the back skin of aging mice to 63.98% (74.90% in the control group and 53.70% in the model group), a significant increase of 19.1% compared with the model group; the peanut skin fermentation supernatant (CCFM1355_H) fermented with Lactobacillus paracasei CCFM1355 had a better effect, with the skin elasticity R2 reaching 66.18% (an increase of 23.2%); the effect of the peanut skin synthetic preparation group was also improved, with the elasticity reaching 71.15% (an increase of 32.5% compared with the model).

[0231] Depend on Figure 14 It was found that the expression of Cu / Zn-SOD mRNA in the gavage hsp group was increased to 0.67 (36.7% higher than the model group's 0.49). However, after the involvement of *Lactobacillus paracasei* CCFM1355, specifically after oral administration of CCFM1355_H and CCFM1355+hsp, the expression levels of Cu / Zn-SOD mRNA in the skin were 1.78 and 2.18, respectively (263.3% and 344.9% higher than the model group). *Lactobacillus paracasei* CCFM1355 enhances the ability of peanut skin to alleviate post-glycation oxidative stress in the skin through fermentation.

[0232] 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 CCFM1355 was deposited at the Guangdong Provincial Center for Microbial Culture Collection on October 25, 2023, with accession number GDMCC NO: 63925.

2. The postbiotic prepared using the P. parapsilum CCFM 1355 of claim 1, characterized by, The postbiotic comprises a culture of Lacticaseibacillus paracasei CCFM1355 or a dry matter of Lacticaseibacillus paracasei CCFM1355.

3. A product comprising Lacticaseibacillus paracasei CCFM1355 of claim 1 and / or the postbiotic of claim 2.

4. The product of claim 3, wherein, The product comprises a food, a nutraceutical or a pharmaceutical product.

5. The product of claim 4, wherein, The product is a fermentate of the Lacticaseibacillus paracasei CCFM1355 after fermentation in a medium containing peanut skins.

6. Use of Lacticaseibacillus paracasei CCFM1355 of claim 1 or the postbiotic of claim 2 for the preparation of a pharmaceutical product against glycation or for the prevention and / or delay of aging.

7. Use according to claim 6, characterized in that, The glycation comprises the accumulation of the glycation aging marker AGE in the blood or in the skin.

8. Use according to claim 6, characterized in that, The aging comprises skin aging.

9. Use according to claim 8, characterized in that, The skin aging comprises dryness, loss of elasticity, laxity, wrinkle production, oxidative damage or loss of collagen.

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