Lactobacillus acidophilus ccfm1356 with anti-glycation and anti-aging functions and postbiotic

Abstract

The application discloses a strain of lactobacillus acidophilus CCFM1356 with anti-glycation and anti-aging functions and a postbiotic thereof, and belongs to the technical fields of microorganisms and medicines. The lactobacillus acidophilus CCFM1356 is preserved in the Guangdong Microbial Culture Collection Center on October 25, 2023, and the preservation number is GDMCC NO: 63926. The paracasei lactobacillus provided by the application can reduce the content of in-vitro fluorescent AGE, the decrease in the activity of HSF, the abnormal mRNA expression of BAX and NFKB1 genes after HSF is damaged. Moreover, the content of fluorescent AGE and inflammatory markers in serum and skin can be reduced, the water content in the matrix layer, the skin elasticity and the collagen content can be improved, and the AGE-RAGE-mediated glycation damage can be reduced. In addition, the bioavailability of peanut skin can be further improved.

Description

TECHNICAL FIELD

[0001] The present application relates to a strain of Lactobacillus acidophilus CCFM1356 with anti-sugar and anti-aging effects and a postbiotic thereof, and belongs to the technical fields of microorganisms and medicine. BACKGROUND

[0002] Extensive glycation occurs in human body. The essence of non-enzymatic glycation (NEG) is the non-enzymatic condensation reaction between the carbonyl group on the reducing sugar and the free amino group on the macromolecules such as proteins, lipids or nucleic acids, i.e. the non-enzymatic modification of proteins by sugars. The stable covalent adducts generated by glycation are collectively referred to as advanced glycation end products (AGEs), which react with corresponding receptors or proteins in vivo, causing numerous harmful physiological effects, including directly inducing intracellular proteins, changing the biochemical properties of specific protein binding sites and the normal recognition function of protein molecules, cross-linking long-lived proteins to change their mechanical properties and cause tissue hardening, etc. In addition, glycation damage also accelerates the aging process and exacerbates the occurrence of various complications.

[0003] Glycation damage proposes four anti-sugar strategies, and potential effective biological raw materials are screened based on these paths: (1) reducing the generation of AGEs; (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. In the study of microbial anti-glycation, the main focus is on reducing the generation of AGEs. For example, patent CN110506106A discloses that Lactobacillus acidophilus sk2 can reduce the content of non-fluorescent glycation end products in vitro, but on the one hand, its effect on reducing the content of AGEs is still limited, and on the other hand, it does not verify the effect of reducing the content of AGEs in vivo. In addition, reducing the generation of AGEs is only one of the above four paths, and the prior art does not study the anti-glycation effect of microorganisms on the above multiple paths. Therefore, it is of great scientific significance and practical value to find a probiotic strain that can more efficiently degrade AGEs and further inhibit the occurrence of AGE-induced glycation damage by inhibiting the above glycation damage paths. SUMMARY

[0004] To solve the above technical problems, the present application provides a Lactobacillus acidophilus (Lactobacillus acidophilus) CCFM1356 and a postbiotic thereof. Lactobacillus acidophilus The present application provides a Lactobacillus acidophilus (Lactobacillus acidophilus) CCFM1356 and a postbiotic thereof.

[0005] The present application provides a Lactobacillus acidophilus (Lactobacillus acidophilus) CCFM1356 and a postbiotic thereof. Lactobacillus acidophilus) Lactobacillus acidophilus CCFM1356, which was preserved in Guangdong Microbial Culture Collection Center on October 26, 2023, and the preservation number is GDMCC NO: 63926.

[0006] The 16S rDNA sequence of the Lactobacillus acidophilus CCFM1356 is shown as SEQ ID NO. 1. Lactobacillus acidophilus The 16S rDNA sequence of the Lactobacillus acidophilus CCFM1356 is shown as SEQ ID NO. 1.

[0007] Lactobacillus acidophilus The 16S rDNA sequence of the Lactobacillus acidophilus CCFM1356 is shown as SEQ ID NO. 1.

[0008] The 16S rDNA sequence of the Lactobacillus acidophilus CCFM1356 is shown as SEQ ID NO. 1. Lactobacillus acidophilus The 16S rDNA sequence of the Lactobacillus acidophilus CCFM1356 is shown as SEQ ID NO. 1.

[0009] The present application also provides a Lactobacillus acidophilus CCFM1356 probiotic, which comprises a culture, a dry product, an inactivated product and / or a broken product of the Lactobacillus acidophilus CCFM1356.

[0010] In an embodiment, the culture is a culture supernatant obtained by culturing the Lactobacillus acidophilus CCFM1356 in a fermentation medium; preferably, the Lactobacillus acidophilus CCFM1356 is inoculated in a fermentation medium for culture, a culture solution is obtained, and the obtained culture supernatant is collected.

[0011] In an embodiment, the dry product is a powder prepared by drying the Lactobacillus acidophilus CCFM1356, and the drying treatment can be thermal drying, spray drying, vacuum freeze drying, fluidized bed drying or vacuum drying.

[0012] In an embodiment, the inactivated product is an inactivated cell obtained by inactivating the Lactobacillus acidophilus CCFM1356, and the inactivation treatment can be heat treatment or freeze-drying treatment; preferably, the Lactobacillus acidophilus CCFM1356 is inoculated in a fermentation medium for culture, bacterial slurry is collected, and the inactivated bacterial cell is obtained after heat treatment at 60-65℃ for 25-35 min.

[0013] ​In one embodiment, the broken substance is a broken substance of Lactobacillus acidophilus CCFM1356; preferably, the Lactobacillus acidophilus CCFM1356 is inoculated into a fermentation medium, cultured, the bacterial slurry is collected, resuspended in the fermentation medium to a volume of 50-80%, and homogenized under high pressure to obtain the bacterial broken substance.

[0014] In one embodiment, the postbiotic can be dried into powder by various drying methods such as vacuum drying, spray drying, vacuum freeze drying, fluidized bed drying, etc., or used directly.

[0015] In one embodiment, the concentration of the Lactobacillus acidophilus CCFM1356 used in the preparation of the postbiotic is not less than 5.0 x 10 7 CFU / mL.

[0016] The present application also provides a product containing the above-mentioned Lactobacillus acidophilus CCFM1356 and / or Lactobacillus acidophilus CCFM1356 postbiotic.

[0017] In one embodiment, the conventional adjuvant includes one or more of a filler, a flavoring agent, a binder, a disintegrant, a lubricant, an antacid, and a nutritional fortifier.

[0018] In one embodiment, the health product includes the above-mentioned composition and conventional adjuvant,

[0019] In one embodiment, the conventional adjuvant includes one or more of a filler, a flavoring agent, a binder, a disintegrant, a lubricant, an antacid, and a nutritional fortifier.

[0020] In one embodiment, the pharmaceutical product contains the above-mentioned composition, a pharmaceutical carrier and / or a pharmaceutical adjuvant.

[0021] In one embodiment, the pharmaceutical adjuvant contains an excipient and an additional agent.

[0022] In one embodiment, the pharmaceutical adjuvant contains at least one of a solvent, a propellant, a solubilizer, a co-solvent, an emulsifier, a coloring agent, a binder, a disintegrant, a filler, a lubricant, a wetting agent, an osmotic pressure regulator, a stabilizer, a flow aid, a flavoring agent, a preservative, a suspending agent, a coating material, an aromatic agent, an anti-adhesion agent, an integration agent, a penetration enhancer, a pH regulator, 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.

[0023] In one embodiment of the present application, the cosmetic product contains the above-mentioned composition, a base raw material, and / or a conventional adjuvant.

[0024] In one embodiment of the present application, the matrix material includes oil and fat material, wax material, synthetic oil and fat material, powder material, gum material, coagulant, surfactant.

[0025] In one embodiment of the present application, the conventional adjuvant includes one or more of moisturizing agent, whitening agent, flavoring agent, binding agent, lubricant, preservative, film agent, antioxidant, emulsifying agent, and cosmetic nutrient additive.

[0026] In one embodiment, the product is a fermentation product of Lactobacillus acidophilus CCFM1356 fermented in a peanut skin-containing medium.

[0027] In one embodiment, the formulation of the peanut skin-containing medium includes: 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.

[0028] The present application also provides a method for improving the anti-glycation of peanut skin, or preventing and / or delaying the aging effect, which comprises mixing the peanut skin-containing medium with the microorganism of Lactobacillus acidophilus CCFM1356 and fermenting. Lactobacillus acidophilus )CCFM1356.

[0029] In one embodiment, the formulation of the peanut skin-containing medium includes: 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.

[0030] In one embodiment, the inoculation amount of Lactobacillus acidophilus CCFM1356 is 1-5% (v / v), preferably 2% (v / v).

[0031] 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.

[0032] In one embodiment, the improvement of the anti-glycation of peanut skin, and / or the delay of aging includes the improvement of at least one of the following aspects of peanut skin:

[0033] (1) inhibiting the generation of in vitro fluorescent AGE;

[0034] (2) reducing the AGE level in the blood and / or skin of mice;

[0035] (3) enhancing the elasticity performance of aging skin;

[0036] (4) down-regulating the expression level of MMP-2 gene in aged skin;

[0037] (5) increasing the content of collagen type 3 in aged skin, and / or the expression level of COL3A1 gene.

[0038] The present application also provides a fermentation composition obtained by fermentation of the above method.

[0039] The present application also provides use of the above Lactobacillus acidophilus CCFM1356, or Lactobacillus acidophilus CCFM1356 probiotic, or fermentation composition in preparation of a product for resisting glycation, or preventing and / or delaying aging.

[0040] In one embodiment, the glycation includes accumulation of glycation aging marker AGE in blood and / or other tissue organs.

[0041] In one embodiment, the aging includes skin aging.

[0042] In one embodiment, the skin aging includes skin dryness, decreased elasticity, relaxation, wrinkle generation, oxidative damage or collagen loss.

[0043] In one embodiment, the use includes at least one of the following 1) - 8):

[0044] 1) inhibiting generation of fluorescent AGE in vitro;

[0045] 2) relieving damage and functional decline of skin fibroblasts (HSF) caused by an intermediate of AGE formation (methylglyoxal);

[0046] 3) reducing the expression level of NFKB1 or BAX gene of skin fibroblasts of an individual under high sugar treatment;

[0047] 4) reducing the AGE level in skin or blood of an individual;

[0048] 5) reducing the TNF-α or IL-6 level in skin or blood of an individual;

[0049] 6) increasing the water content of stratum corneum of aged individuals or skin elasticity performance;

[0050] 7) increasing the expression level of collagen type III synthesis enzyme COL3A1 gene of aged individuals, or increasing the content of collagen type III in skin of aged individuals;

[0051] 8) reducing the expression of RAGE gene or MMP-2 gene of skin, or increasing the expression of DDOST gene.

[0052] In an embodiment, the product contains the probiotic Lactobacillus acidophilus CCFM1356 in a dose of not less than 10 mg / kg body weight.

[0053] Beneficial effects

[0054] The probiotic Lactobacillus acidophilus CCFM1356 of the present application has the ability to relieve the host from glycation damage and reduce the corresponding aging problems, which is manifested in: Lactobacillus acidophilus

[0055] (1) Inhibition of the generation of fluorescent AGE in the fructose-bovine serum albumin system in vitro;

[0056] (2) Prevention of the decrease in cell viability in the skin fibroblast (HSF) damage model caused by methylglyoxal;

[0057] (3) Prevention of the abnormal expression of mRNA of the apoptosis factor BAX and the downstream cascade reaction related NFKB1 mRNA of skin fibroblasts (HSF) under high glucose culture;

[0058] (4) Reduction of the content of AGE in the serum and skin of aging individuals;

[0059] (5) Reduction of the content of inflammatory markers IL-6 and TNF-α in the serum and skin of aging individuals;

[0060] (6) Reduction of the decrease in the water content of the stratum corneum of the back skin of aging individuals caused by glycation damage;

[0061] (7) Relief of the decrease in the elasticity and tightness of the back skin of aging individuals caused by glycation damage;

[0062] (8) Reduction of the decrease in the content of collagen type III in the back skin of aging individuals caused by glycation damage;

[0063] (9) Relief of the aggravation of glycation damage in the back skin of aging individuals mediated by AGE-RAGE combination;

[0064] (10) Relief of the collagen synthesis, degradation and inflammatory response triggered by glycation damage in aging individuals.

[0065] In addition, the Paracaseicola casei CCFM1356 fermented peanut skin can improve the following abilities of peanut skin: Lacticaseibacillus paracasei

[0066] (1) Inhibition of the generation of fluorescent AGE in vitro;

[0067] (2) Inhibition of the generation of serum and skin AGE;

[0068] (3) Improvement of the elasticity of the skin;​​

[0069] (4) Alleviate the weakening of protein function caused by glucose damage during aging.

[0070] Therefore, Lactobacillus acidophilus ( Lactobacillus acidophilus The metabiotic prepared by CCFM1356 has great application potential in products that alleviate glycation damage to the host and reduce corresponding aging.

[0071] Preservation of biological materials

[0072] A strain of Lactobacillus acidophilus ( Lactobacillus acidophilus CCFM1356, taxonomically named Lactobacillus acidophilus It was deposited on October 25, 2023, at the Guangdong Provincial Center for the Preservation of Microbial Cultures, with accession number GDMCC No:63926, located at Building 59, No. 100 Xianlie Middle Road, Guangzhou. Attached Figure Description

[0073] Figure 1 Effects of different post-genetics on the inhibition of fluorescent AGE formation in the in vitro fructose-bovine serum albumin system.

[0074] Figure 2 The effects of different epigenetics on HSF cell proliferation.

[0075] Figure 3 The effect of different post-genetic agents on the activity of acetone aldehyde, an intermediate in the glycation reaction, on HSF cell viability.

[0076] Figure 4 The effects of different metabiotics on the expression of BAX mRNA and NFKB1 mRNA in HSF cells under high glucose culture.

[0077] Figure 5 Flowchart of mouse experiments.

[0078] Figure 6 Effects of Lactobacillus acidophilus CCFM1356 and its prepared postbiotic on AGE content in mouse blood and skin tissue.

[0079] Figure 7 Effects of Lactobacillus acidophilus CCFM1356 and its prepared postbiotic on the levels of inflammatory markers IL-6 and TNF-α in the serum and skin of aging mice.

[0080] Figure 8 Effects of Lactobacillus acidophilus CCFM1356 and its prepared metabiotic on the moisture content of the stratum corneum and the elastic properties of the skin.

[0081] Figure 9: Effects of Lactobacillus acidophilus CCFM1356 and its prepared postbiotics on the synthesis and content of skin type III collagen.

[0082] Figure 10 : Effects of Lactobacillus acidophilus CCFM1356 and its prepared postbiotics on the expression of different pathways to relieve skin glycation damage (DDOST mRNA, RAGE mRNA, MMP2 mRNA).

[0083] Figure 11 : Effects of Lactobacillus acidophilus CCFM1356 fermented peanut skin on improving the in vitro anti-glycation ability of peanut skin.

[0084] Figure 12 : Effects of Lactobacillus acidophilus CCFM1356 fermented peanut skin on improving the oral anti-AGE accumulation ability of peanut skin.

[0085] Figure 13 : Effects of Lactobacillus acidophilus CCFM1356 fermented peanut skin on improving the oral ability of peanut skin to relieve glycation-induced abnormal protein degradation and oxidative stress.

[0086] “*” indicates a statistically significant difference from the Model group (P < 0.05), “**” indicates a significantly statistically significant difference from the Model group (P < 0.01), “***” indicates a very significantly statistically significant difference from the Model group (P < 0.001), and “****” indicates a very significantly statistically significant difference from the Model group (P < 0.0001). DETAILED DESCRIPTION

[0087] The application will be further described below in conjunction with specific examples.

[0088] The human skin fibroblasts (HSF) involved in the following examples were purchased from the Kunming Cell Bank.

[0089] The BALB / c mice involved in the following examples were purchased from Vivotecnia.

[0090] The Lactobacillus acidophilus CCFM1356, Lactobacillus acidophilus JCM1132, Lactobacillus acidophilus FFJND7L5, and Lactobacillus acidophilus FSHXBX32L130 involved in the following examples were obtained from the Food Biotechnology Center of Jiangnan University.

[0091] The peanut skin extract was purchased from Shaanxi Shengheng Biotechnology Co., Ltd., batch number: SH20220328.

[0092] The culture media involved in the following examples are as follows:

[0093] 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, anhydrous sodium acetate 2.0 g / L, citric acid diamine 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.

[0094] 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, anhydrous sodium acetate 2.0 g / L, citric acid diamine 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.

[0095] MRS simplified liquid medium: glucose 8 g / L, yeast powder 5 g / L, calcium carbonate 6 g / L, anhydrous sodium acetate 2 g / L, citric acid diamine 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.

[0096] 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).

[0097] 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).

[0098] Example 1: Cell resuscitation and culture

[0099] 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 of recovery and growth, the cells were subcultured.

[0100] Example 2: Screening of Lactobacillus acidophilus CCFM1356 and preparation of postbiotic

[0101] (1) Screening of Lactobacillus acidophilus CCFM1356

[0102] 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 thawing, 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 was coated on the MRS solid culture medium and incubated at 37°C for 48 h. Typical colonies of Lactobacillus acidophilus were picked and streaked on MRS solid culture medium, and single colonies were transferred to MRS liquid medium for enrichment. The strain was obtained by 30% glycerol preservation. The strain genome was extracted for 16S rDNA amplification and sequencing (performed by Suzhou Jinyuzhi Biological Technology Co., Ltd.). The 16S rDNA sequencing result is shown in SEQ ID No. 1. The result was determined as Lactobacillus acidophilus by NCBI sequence alignment, and was named Lactobacillus acidophilus CCFM1356.

[0103] (2) Preparation of postbiotics of Lactobacillus acidophilus CCFM1356, Lactobacillus acidophilus JCM1132 and Lactobacillus acidophilus FFJND7L5

[0104] 1) Lactobacillus acidophilus CCFM1356 was streaked from the 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 incubated at 37°C for 12-18 h to obtain culture solution 1;

[0105] Culture solution 1 was inoculated into MRS liquid medium at a 2% (v / v) inoculation amount and incubated at 37°C for 12 h to obtain seed solution;

[0106] The seed solution was inoculated into MRS liquid medium and MRS (simplified) liquid medium at 2-5% (v / v) respectively for expansion, and incubated at 37°C for 18-24 h. The concentrations of the two bacterial solutions were adjusted to the same level to obtain bacterial solution a (MRS liquid medium) and bacterial solution b (MRS simplified liquid medium).

[0107] The supernatant obtained by centrifugation of bacterial solution 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. Lactobacillus acidophilus CCFM1356 fermentation supernatant (denoted as CCFM1356_M) was prepared.

[0108] The bacterial slurry obtained by centrifuging the bacterial liquid a at 8000 r / min for 30 min was resuspended with double-distilled water at a volume of 75% of the original bacterial 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 bacterial lysate (denoted as CCFM1356_Z). The postbiotic freeze-dried powder was obtained by freeze-drying and was ready for use.

[0109] The preparation method of Lactobacillus acidophilus CCFM1356 live bacteria was the same as that of the postbiotic, except that the bacterial slurry obtained by centrifuging the bacterial liquid b at 8000 r / min for 30 min was resuspended with a freeze-drying protectant at a ratio of 1 g:2 mL and then directly freeze-dried to obtain Lactobacillus acidophilus CCFM1356 live bacteria powder, denoted as CCFM1356.

[0110] The postbiotic of Lactobacillus acidophilus CCFM1356 (bacterial lysate CCFM1356_Z and fermentation supernatant CCFM1356_M) was prepared by the above-mentioned means.

[0111] 2) The postbiotic of Lactobacillus acidophilus JCM1132 (bacterial lysate JCM1132_Z and fermentation supernatant JCM1132_M), the postbiotic of Lactobacillus acidophilus FFJND7L5 (bacterial lysate FFJND7L5_Z and fermentation supernatant FFJND7L5_M) were prepared according to the method of step (1).

[0112] Example 3: Preparation of peanut skin fermentation broth by Lactobacillus acidophilus CCFM1356 fermentation of peanut skin

[0113] The bacterial liquid of Lactobacillus acidophilus CCFM1356 was streaked on MRS solid medium with a inoculating loop, and cultured at 37 ℃ for 48 h. A single colony was taken and inoculated in MRS liquid medium, and cultured at 37 ℃ for 18 h. After mixing, the bacterial liquid was inoculated in new MRS liquid medium at a inoculation amount of 2% (v / v) and cultured, and the operation was repeated for 3 times to obtain the activated bacterial liquid.

[0114] The obtained activated bacterial liquid was inoculated in peanut skin fermentation medium (hsp) at a 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, and the supernatant obtained by centrifuging the bacterial liquid at 8000 r / min for 30 min was heat-treated (65 ℃, 30 min) and freeze-dried to obtain a powder for standby. The peanut skin fermentation supernatant of Lactobacillus acidophilus CCFM1356 (denoted as CCFM1356_H) was prepared.

[0115] Example 4: Effect of postbiotics prepared from Lactobacillus acidophilus CCFM1356 on inhibition of fluorescence AGE formation in an in vitro fructose-BSA system

[0116] An in vitro fructose-BSA system was prepared by dissolving BSA and d-(+)-fructose in 0.1 M phosphate buffer (pH 7.4) to a final concentration of 10 mg / mL BSA and 0.5 M (i.e. 90 mg / mL) d-(+)-fructose, and mixing well by magnetic stirring. The reaction solution was passed through a 0.22 μm water filter to obtain a sterile fructose-BSA glycation system before adding the sample for incubation.

[0117] The experimental group was incubated at 37 °C for 7 days with different postbiotic samples added to the fructose-BSA glycation system, with the volume ratio of postbiotic sample solution to fructose-BSA glycation solution being 1:1. The incubated samples were used to detect the formation of fluorescence AGEs after 7 days of incubation. The postbiotic samples included each group of postbiotics prepared according to the method of Example 2: postbiotics prepared from Lactobacillus acidophilus CCFM1356 (CCFM1356_Z and CCFM1356_M), postbiotics prepared from Lactobacillus acidophilus JCM1132 (JCM1132_Z and JCM1132_M), and postbiotics prepared from Lactobacillus acidophilus FFJND7L5 (FFJND7L5_Z and FFJND7L5_M).

[0118] The control group was incubated at 37 °C for 7 days with an equal volume of PBS solution added to the fructose-BSA glycation solution, and the formation of fluorescence AGEs was detected after 7 days of incubation.

[0119] After incubation, the fluorescence AGEs in the fructose-modified BSA were detected using an excitation wavelength of 370 nm and an emission wavelength of 440 nm, and the percentage of inhibition of fluorescence AGE formation was calculated as 1 minus the difference in fluorescence intensity between the sample and the control (BSA+ / fructose+).

[0120] The effect of the inhibition rate of fluorescence AGE formation in the in vitro fructose-BSA system is shown in Table 1. Figure 1 As shown in Table 1, the AGE inhibition rates of CCFM1356_Z and CCFM1356_M were 19.19% and 20.68%, respectively, while the AGE inhibition rates of JCM1132_M and JCM1132_Z were only 17.82% and 14.11%, respectively, and the AGE inhibition rates of FFJND7L5_M and FFJND7L5_Z were 16.06% and 0.63%, respectively.

[0121] Example 5: Effect of postbiotics prepared from Lactobacillus acidophilus CCFM1356 on HSF cell proliferation

[0122] The specific steps are as follows:

[0123] (1) 100 μL of HSF cells in the logarithmic growth phase were inoculated in a 96-well plate at a concentration of 3×10 4 cells / well, the outermost circle was filled with PBS solution to prevent edge effects, and the cells were cultured for 24 h to adhere to the wall. Then, a blank group, a control group, and a metatron treatment group were set up;

[0124] The blank group only contained cell culture medium without HSF cells.

[0125] The control group contained cell culture medium and HSF cells without metatron.

[0126] The metatron treatment group contained cell culture medium with different metatrons obtained in Example 2 and HSF cells.

[0127] Preparation of cell culture medium containing metatron: the metatron was resuspended in cell culture medium (the amount of resuspended metatron was equivalent to the amount of metatron prepared from bacteria fermented to a concentration of 5.0×10 7 CFU / mL), and the cell culture medium containing metatron was obtained.

[0128] (2) The above-mentioned hole plates were incubated at a temperature of 37 ℃ for 24 h, and then 10 μL of CCK8 solution was added to each hole for 2 h of incubation to measure the absorbance value (OD) at 450 nm.

[0129] The cell viability was calculated according to the following formula: cell viability (%) = (treatment group OD value-blank group OD value) / (control group OD value-blank group OD value)×100%.

[0130] The effect of metatron on cell proliferation is shown in Figure 2 Compared with the control group (cell proliferation rate 101.11%), the cell proliferation rates of Lactobacillus acidophilus CCFM1356 metatron (CCFM1356_M and CCFM1356_Z), Lactobacillus acidophilus JCM1132 metatron (JCM1132_M and JCM1132_Z), Lactobacillus acidophilus FFJND7L5 metatron (FFJND7L5_M and FFJND7L5_Z), and Lactobacillus acidophilus FSHXBX32L130 metatron (FSHXBX32L130_M and FSHXBX32L130_Z) were 101.11%, 102.22%, 105.30%, 98.50%, 102.58%, 99.99%, 97.42%, and 107.73%, respectively.

[0131] According to the toxicity grading evaluation method in ISO 10993-5:2009, if the cell viability is greater than 70%, it can be considered as non-toxic. The above results show that the HSF cell viability under the above postbiotic concentration is higher than 90%, and considering its non-cytotoxicity, the inactivated bacterial body concentration can be selected as 5.0×10 7 CFU / mL is a suitable postbiotic concentration for subsequent cell experiments.

[0132] Example 6: Effect of postbiotic prepared by Lactobacillus acidophilus CCFM1356 on prevention of glycation damage to HSF cells caused by methylglyoxal

[0133] The specific steps are as follows:

[0134] (1) Take 100 μL of HSF cells in logarithmic growth phase at a concentration of 3×10 4 cells / well in 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;

[0135] The blank group only contains cell culture medium without HSF cells.

[0136] The control group 1 contains cell culture medium and HSF cells without postbiotic.

[0137] The treatment group 1 contains different postbiotic cell culture medium and HSF cells obtained in Example 2.

[0138] Preparation of postbiotic-containing cell culture medium: resuspend the postbiotic in cell culture medium (the amount of resuspended postbiotic is equivalent to the amount of postbiotic prepared from bacteria liquid fermented to a concentration of 5.0×10 7 CFU / mL), and obtain postbiotic-containing cell culture medium.

[0139] (2) Incubate the above-mentioned hole plates at a temperature of 37℃ for 24 h, and after incubation, discard the old culture medium of the control group and the modeling agent group, rinse with PBS for 3 times, and set up a control group, a model group, and a treatment group:

[0140] The control group is the control group 1 after liquid change, which contains cell culture medium and HSF cells without methylglyoxal modeling agent.

[0141] The model group is the control group 1 after liquid change into cell culture medium containing methylglyoxal modeling agent, containing original HSF cells,

[0142] The cell culture medium containing methylglyoxal modeling agent is that methylglyoxal is uniformly mixed in ordinary cell culture medium and sterilized by 0.22 μm water system filter membrane, and the final concentration of methylglyoxal in the cell culture medium is 400 μmol / L.

[0143] The treatment group is that the treatment group 1 is changed into a cell culture medium containing methylglyoxal modeling agent, and the original HSF cells are contained.

[0144] (3) The above well plates were respectively incubated in an incubator at a temperature of 37°C for 24 h, 10 μL of CCK8 solution was added to each well after the incubation was completed, and the absorbance value (OD) at 450 nm was measured after 2 h of incubation.

[0145] The 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%.

[0146] The results of the effect of preventing methylglyoxal from causing damage to HSF cells are as follows: Figure 3 Compared with the control group (cell viability 100.08%), the cell viability of the model group was 57.22%, and methylglyoxal modeling caused significant damage to HSF cells.

[0147] The cell viabilities of the treatment groups after adding CCFM1356_M and CCFM1356_Z were 81.29% and 93.18% respectively, and among them, CCFM1356_Z significantly improved the HSF cell viability compared with the model group, indicating that the probiotics of Lactobacillus acidophilus CCFM1356 can effectively prevent the glycation damage of methylglyoxal to HSF cells;

[0148] After the treatment of the probiotics of Lactobacillus acidophilus JCM1132 (JCM1132_M and JCM1132_Z) and the probiotics of Lactobacillus acidophilus FFJND7L5 (FFJND7L5_M and FFJND7L5_Z) in other treatment groups, the HSF cell viabilities were 47.66%, 37.63%, 39.42% and 45.25% respectively, that is, the probiotics of other Lactobacillus acidophilus do not have the ability to prevent the damage of methylglyoxal to HSF cells.

[0149] Example 7: Effect of probiotics prepared by Lactobacillus acidophilus CCFM1356 on preventing the expression of BAX mRNA and NFKB1 mRNA in HSF cells under high glucose culture.

[0150] The specific steps are as follows:

[0151] (1) HSF cells were inoculated on a 6-well plate at a concentration of 1 x 10 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 the control group 1 and the treatment group 1 were set;

[0152] Control group 1 is a group without adding probiotics;

[0153] The processing group 1 is grouped as: Lactobacillus acidophilus CCFM1356 lysate (CCFM1356_Z), Lactobacillus acidophilus JCM1132 lysate (JCM1132_Z), Lactobacillus acidophilus FFJND7L5 lysate (FFJND7L5_Z) are resuspended in cell culture medium, standby (the amount of probiotics prepared by resuspending the amount of bacteria liquid fermented to a concentration of 5.0×10 7 CFU / mL is equivalent).

[0154] Respectively, 2 mL of Lactobacillus acidophilus CCFM1356 lysate (CCFM1356_Z), Lactobacillus acidophilus JCM1132 lysate (JCM1132_Z), Lactobacillus acidophilus FFJND7L5 lysate (FFJND7L5_Z) resuspended in cell culture medium are added to 6-well plates, and cultured for 24 h, with three parallel samples for each sample.

[0155] (2) The above hole plate is incubated in an incubator at 37°C for 24 h, and after incubation, the old culture medium of the control group and the modeling agent group is discarded, and the PBS is washed 3 times, and the control group, the model group and the processing group are set:

[0156] The control group is that the control group 1 is replaced with 2 mL of ordinary cell culture medium after the original HSF cells are added;

[0157] The model group is that the control group 1 is replaced with cell culture medium containing 35 mmol / L glucose, containing the original HSF cells;

[0158] The processing group is that the processing group 1 is replaced with cell culture medium containing 35 mmol / L glucose, containing the original HSF cells.

[0159] (3) The above hole plate is incubated in an incubator at 37 ℃ for 24 h, and the culture supernatant is discarded, and each hole is washed with PBS for 3 times, and 1 mL of cell lysate is added to each hole, and repeated blowing and beating, and the cell lysate is extracted to extract RNA, and the RT-PCR reverse transcription kit is used to reverse transcribe into cDNA, and the expression of genes in HSF cells is detected by real-time fluorescent quantitative method, and 2 -△△Ct The expression of BAX mRNA and NFKB1 mRNA is calculated by the formula, wherein the internal reference is β-actin, the primers are described in Table 1, and the results are shown in Figure 4 .

[0160] Table 1: Primer sequence

[0161]

[0162] The results show that,Figure 4 It can be seen that the expression amount of BAX mRNA in the control group is about 1, and the expression amount of the model group after high-glucose medium intervention rises to 4.33; the metronidazole Lactobacillus CCFM1356 prepared postbiotic (CCFM1356_Z) significantly reduces the expression amount of BAX mRNA in HSF cells to 0.64; and the expression amount of BAX mRNA after treatment of other metronidazole Lactobacillus postbiotics JCM1132_Z, FFJND7L5_Z) is about 0.85, 2.42, and the down-regulation effect of BAX mRNA expression caused by modeling is not as prominent as CCFM1356_Z. In addition, the expression amount of NFKB1 in the control group is about 1, and the expression amount of the model group after high-glucose medium intervention rises to 2.17; the metronidazole Lactobacillus CCFM1356 prepared postbiotic (CCFM1356_Z) significantly reduces the expression amount of NFKB1 mRNA in HSF cells to 0.75; and the expression amount of NFKB1 mRNA after treatment of other metronidazole Lactobacillus postbiotics JCM1132_Z, FFJND7L5_Z) is about 1.70, 1.82, and compared with the model group, they do not show a down-regulation effect on NFKB1 mRNA expression.

[0163] BAX is a water-soluble related protein homologous to BCL-2, which is an apoptosis-promoting gene in the BCL-2 gene family, and overexpression of the BAX gene can antagonize the protective effect of BCL-2 and make cells tend to die; under high-glucose conditions, the expression of the BAX gene increases, causing the activity of skin fibroblasts to decrease and cell apoptosis to intensify. 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, and NFKB1 encodes p50 protein. Under high-glucose conditions, the expression of p50 increases and intensifies the formation of the NF-κB complex in which it is involved, increasing the pro-inflammatory potential.

[0164] Therefore, the metronidazole Lactobacillus CCFM1356 prepared postbiotic (bacterial lysate) reduces the expression of BAX mRNA and NFKB1 mRNA after high-glucose culture, preventing sugar damage to HSF cells caused by high-glucose culture conditions.

[0165] Example 8: Effect of metronidazole Lactobacillus CCFM1356 and postbiotics prepared therefrom on the AGE level in the blood and skin of aging mice

[0166] The preparation method of the probiotics (CCFM1356_M and CCFM1356_Z) of Lactobacillus acidophilus CCFM1356 involved in the following examples is the same as that of Example 2, except that CCFM1356_Z in this example refers to the bacterial slurry obtained by centrifugation of 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 volume of the original bacterial liquid. The resuspended liquid is heat treated (65°C, 30 min), and the dead bacterial cells CCFM1356_Z are obtained without high-pressure homogenization. The probiotic freeze-dried powder is obtained by freeze-drying for standby use.

[0167] The specific steps are as follows:

[0168] (1) Take 25 healthy 8-week-old male BALB / c mice and randomly divide them into 5 cages, 5 mice per cage. The 5 cages are respectively: 1 cage of Control group, 1 cage of Model group, 1 cage of CCFM1356 live bacteria group, 1 cage of CCFM1356_Z group, and 1 cage of CCFM1356_M group.

[0169] Control group (Control): physiological saline is used as a control;

[0170] Model group (Model): physiological saline is used as a control;

[0171] CCFM1356 group: Lactobacillus acidophilus CCFM1356 live bacteria are administered by gavage, with a dose of 5×10 9 CFU / kg of mouse body weight;

[0172] CCFM1356_Z group: Lactobacillus acidophilus CCFM1356 probiotics (dead bacterial cells) are administered by gavage, with a dose of 500 mg / kg of mouse body weight;

[0173] CCFM1356_M group: Lactobacillus acidophilus CCFM1356 fermentation supernatant is administered by gavage, with a dose of 500 mg / kg of mouse body weight;

[0174] Among the above groups, the dead bacterial cells or fermentation supernatant are obtained by fermenting the bacterial cells in an amount equivalent to 5×10 9 CFU / kg of mouse body weight of live bacteria.

[0175] The experiment lasted for 7 weeks: after one week of adaptation, the mice in the groups except the blank group were injected subcutaneously with D-galactose (1000 mg / kg) at a dose of 0.1 mL per mouse per day. From the second week, the mice in the intervention groups were administered with the corresponding strain of freeze-dried powder or postbiotic freeze-dried powder (dead bacteria or fermentation supernatant) prepared from the strain at a corresponding dose dissolved in normal saline at a dose of 0.1 mL per mouse per day. The blank group and the model group were administered with an equal amount of normal saline as a control until the end of the experiment. All groups were allowed to drink and eat freely, and the experimental procedure is shown in Figure 5 .

[0176] After 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-to-volume ratio of 1:10 with PBS. The homogenate was centrifuged at 3000 r / min for 20 min, and the skin supernatant was collected for ELISA detection.

[0177] The AGE content in the serum and skin of the mice was detected by an ELISA kit, and the results are shown in Figure 6 .

[0178] (1) Serum AGE content: compared with the control group (262.70 ng / L), the AGE content in the serum of the model group significantly increased to 409.97 ng / L. Oral administration of postbiotics prepared from Lactobacillus acidophilus CCFM1356 significantly reduced the content of the glycation aging marker AGE in the serum of the mice. The CCFM1356_Z group, the CCFM1356_M group, and the CCFM1356 group reduced the AGE content in the serum to 263.03 ng / L, 271.89 ng / L, and 290.38 ng / L, respectively.

[0179] (2) Skin AGE content: compared with the control group (336.76 ng / L), the AGE content in the serum of the model group significantly increased to 463.21 ng / L. Oral administration of postbiotics prepared from Lactobacillus acidophilus CCFM1356 significantly reduced the content of the glycation aging marker AGE in the skin of the mice. The CCFM1356_Z group, the CCFM1356_M group, and the CCFM1356 group reduced the AGE content in the skin to 412.97 ng / L, 406.33 ng / L, and 350.13 ng / L, respectively.

[0180] The above results show that Lactobacillus acidophilus CCFM1356 live bacteria and postbiotics prepared from Lactobacillus acidophilus CCFM1356 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.

[0181] Example 9: Effect of Lactobacillus acidophilus CCFM1356 and its prepared postbiotics on the content of inflammatory markers in serum and skin of aging mice

[0182] The animal experiment design and gavage groups involved in the following examples are the same as Example 8. The content of TNF-α and IL-6 in serum and skin of aging mice was detected using Elisa kit of Nanjing Senbeiga Company as shown in Figure 7 .

[0183] (1) Serum TNF-α content: compared with the control group (297.24 ng / L), the content of TNF-α in the serum of the model group increased significantly to 382.86 ng / L, and oral administration of Lactobacillus acidophilus CCFM1356 live bacteria and postbiotics prepared from Lactobacillus acidophilus CCFM1356 significantly reduced the content of TNF-α in the serum of mice. The content of TNF-α in the serum of CCFM1356_Z group, CCFM1356_M group and CCFM1356 live bacteria group reached 337.77 ng / L, 355.66 ng / L and 394.92 ng / L respectively, and CCFM1356_Z significantly reduced the content of inflammatory factor TNF-α in the model group by 11.8%.

[0184] (2) Serum IL-6 content: compared with the control group (80.20 ng / L), the content of IL-6 in the serum of the model group increased significantly to 129.01 ng / L, and oral administration of Lactobacillus acidophilus CCFM1356 live bacteria and postbiotics prepared from Lactobacillus acidophilus CCFM1356 significantly reduced the content of IL-6 in the serum of mice. The content of IL-6 in the serum of CCFM1356_Z group, CCFM1356_M group and CCFM1356 live bacteria group was 117.07 ng / L, 85.81 ng / L and 123.20 ng / L respectively, and CCFM1356_M reduced the content of inflammatory factor IL-6 in the model group the most (33.5% lower than the model group).

[0185] (3) Skin TNF-α content: compared with the control group (351.25 ng / L), the content of TNF-α in the skin of the model group increased significantly to 486.77 ng / L, and oral administration of Lactobacillus acidophilus CCFM1356 live bacteria and postbiotics prepared from Lactobacillus acidophilus CCFM1356 significantly reduced the content of TNF-α in the skin of mice. The content of TNF-α in the skin of CCFM1356_Z group, CCFM1356_M group and CCFM1356 live bacteria group reached 407.55 ng / L, 442.29 ng / L and 377.50 ng / L respectively.

[0186] (4) Skin IL-6 content: Compared with the control group (74.78 ng / L), the IL-6 content in the skin of the model group increased significantly to 106.26 ng / L. Oral administration of live Lactobacillus acidophilus CCFM1356 and the postbiotic prepared from Lactobacillus acidophilus CCFM1356 significantly reduced the IL-6 content in the skin of mice. The CCFM1356_Z group, CCFM1356_M group and CCFM1356 live bacteria group reduced the serum IL-6 content to 96.79 ng / L, 92.03 ng / L and 109.48 ng / L, respectively. Among them, CCFM1356_M reduced the inflammatory factor IL-6 content in the model group the most (reduced by 13.39% compared with the model group).

[0187] Based on the results of relevant biochemical indicators in animal serum, it can be concluded that live Lactobacillus acidophilus CCFM1356 and the postbiotic prepared from Lactobacillus acidophilus CCFM1356 can reduce the content 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.

[0188] Example 10: Effects of Lactobacillus acidophilus CCFM1356 and its prepared metabiotic on the stratum corneum moisture content and skin elasticity of aging mice.

[0189] 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.

[0190] (1) The moisture content of the stratum corneum is from Figure 8 It can be seen that, compared with the blank group (66.85%), the water content of the model group was significantly reduced to 48.08%. The water content of the stratum corneum in the Lactobacillus acidophilus CCFM13565_Z group (67.19%) was 39.7% higher than that in the model group. The water content of the Lactobacillus acidophilus CCFM1356_M group (65.30%) was about 35.8% higher than that in the model group. The water content of the Lactobacillus acidophilus CCFM1356 group (62.13%) was 29.2% higher than that in the model group. In other words, the experimental results show that Lactobacillus acidophilus CCFM1356 and its prepared metabiotics, especially its dead bacterial cells (Lactobacillus acidophilus CCFM1356_Z), can increase the water content of the back of aged mice after glycation damage.

[0191] In 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 holding 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 Lactobacillus acidophilus CCFM1356 can prevent skin dryness during the aging process. Among them, postbiotics prepared from Lactobacillus acidophilus CCFM1356 (CCFM1356_Z) has more significant effect on alleviating the decrease of water content in the stratum corneum caused by glycation damage than pure fermentation supernatant CCFM1356_M group.

[0192] (2) The skin elasticity performance R2 is calculated by Figure 8 It can be seen that compared with the blank group (75.15%), the skin elasticity performance of the model group is significantly reduced to 53.45%, and the skin elasticity of the Lactobacillus acidophilus CCFM1356_Z group (79.43%) is increased by 48.6% compared with the model group, and the skin elasticity of the CCFM1356_M group (75.63%) is increased by 41.5% compared with the model group.

[0193] It can be seen from the experimental results that Lactobacillus acidophilus CCFM1356 and postbiotics prepared therefrom significantly increase the skin elasticity performance of the back of the aging mouse. In 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, gavage of Lactobacillus acidophilus CCFM1356 and postbiotics prepared therefrom (especially dead bacteria CCFM1356_Z) significantly increases the skin elasticity performance of the back of the aging mouse.

[0194] Example 11: Effect of Lactobacillus acidophilus CCFM1356 and postbiotics prepared therefrom on the synthesis and content of type 3 collagen in the skin of glycation-damaged aging mice

[0195] The animal experiment design and gavage groups in the following examples are the same as those in Example 8. The content of type 3 collagen in the skin of mice was detected using a Nanjing Senbeiga Elisa kit.

[0196] The skin RNA was extracted by Trizol method, and the key target gene expression in the synthesis process of type 3 collagen in the skin was detected by reverse transcription into cDNA. The primer of COL3A1 is described in Table 2 below.

[0197] Table 2: Primer sequence

[0198]

[0199] The content of collagen III in the back skin of mice was determined as shown in Figure 9 The content of collagen III in the model group was significantly reduced to 6.07 μg / L compared with the blank group (7.17 μg / L), the content of collagen III in the L. acidophilus CCFM1356_Z group (7.87 μg / L) was increased by 29.7% compared with the model group, the content of collagen III in the L. acidophilus CCFM1356_M group (8.87 μg / L) was increased by 46.1% compared with the model group, and the content of collagen III in the L. acidophilus CCFM1356 live bacteria group (7.93 μg / L) was increased by 30.6% compared with the model group. The L. acidophilus CCFM1356 and the metapiont prepared therefrom significantly increased the content of collagen III in the back skin of the aging mice.

[0200] The expression of COL3A1 mRNA was detected Figure 9 ), the metapiont CCFM1356_Z prepared from the L. acidophilus CCFM1356 significantly up-regulated the expression of the collagen III synthesis enzyme COL3A1 mRNA, and the relative expression amount was 1.70 (increased by 295% compared with 0.43 of the model group), and the expression amount of COL3A1 mRNA in the L. acidophilus CCFM1356 live bacteria group was 0.84 (increased by 95.3% compared with 0.43 of the model group). The L. acidophilus CCFM1356 and the metapiont CCFM1356_Z prepared therefrom can alleviate the abnormal decrease of collagen III synthesis caused by glycation damage and maintain the normal function of collagen.

[0201] Example 12: Effect of the L. acidophilus CCFM1356 and the metapiont prepared therefrom on the expression of key target genes in the skin of sugar-damaged aging mice

[0202] The animal experiment design, gavage group, RNA extraction and detection in the following examples were the same as in Example 11, and the primers of the key genes DDOST, RAGE and NFKB1 in the skin of the aging mice were described in Table 3, and the gene expression results were shown in Figure 10 .

[0203] Table 3: Primer sequences

[0204]

[0205] (1) AGE receptor RAGE and DDOST: Lactobacillus acidophilus CCFM1356 and its prepared probiotics can significantly down-regulate the expression of AGE receptor RAGE mRNA, and the relative expression amount after intragastric administration of CCFM1356 live bacteria group and CCFM1356_Z group is 0.45 and 0.36 (decreased by 84.7% and 87.9% respectively compared with the model group 2.95). CCFM1356_Z group can also up-regulate the expression of RAGE competitive receptor GAGE1, that is, DDOST mRNA to 2.11 (increased by 276.8% compared with the model group 0.56), thereby hindering AGE-RAGE binding from two aspects.

[0206] (2) MMP-2: The expression amount of MMP-2 mRNA in the back skin of mice is up-regulated from 1.00 in the control group to 2.72 in the model group due to modeling, but after intragastric administration of Lactobacillus acidophilus CCFM1356 and its prepared probiotics, the relative expression amount of MMP-2 mRNA in CCFM1356 group and CCFM1356_Z group is 0.37 and 0.93, respectively, which hinders the abnormal proteolysis on the skin with aggravated glycation damage.

[0207] Lactobacillus acidophilus CCFM1356 and its prepared probiotics can hinder AGE-RAGE binding to reduce the continuous progress of glycation downstream reactions, and target anti-glycation and anti-aging, and can alleviate the weakening of protein function caused by sugar damage in the aging process by down-regulating the expression of MMP-2 mRNA.

[0208] Example 13: Effect of Lactobacillus acidophilus CCFM1356 fermented peanut skin on improving the anti-glycation ability of peanut skin for external use

[0209] The preparation of peanut skin fermentation culture solution (hsp) and Lactobacillus acidophilus CCFM1356 fermented peanut skin supernatant (CCFM1356_H) is shown in Example 3; the construction of in vitro fructose-bovine serum albumin system and the detection of fluorescent AGE are shown in Example 4.

[0210] Peanut skin extract mainly contains flavonoids such as proanthocyanidins, resveratrol, quercetin, etc., and oligomeric proanthocyanidins have biological activities such as free radical scavenging, antioxidant, heart and brain blood vessel protection, anti-inflammatory, tumor inhibition, etc. However, the content of anti-glycation effective substances in peanut skin is low, and the bioavailability is not high. In order to better exert the anti-glycation function and characteristics of peanut skin, Lactobacillus acidophilus CCFM1356 is used to ferment peanut skin to prepare Lactobacillus acidophilus CCFM1356 fermented peanut skin supernatant (CCFM1356_H), and the effects of Lactobacillus acidophilus CCFM1356 on the anti-glycation ability of peanut skin are verified at a concentration of 100 μg / mL of peanut skin fermentation culture solution and fermented peanut skin supernatant.

[0211] From the above experiments, it can be seen that Lactobacillus acidophilus CCFM1356 and its prepared probiotics can inhibit the expression of AGE receptor RAGE mRNA, up-regulate the expression of RAGE competitive receptor GAGE1, and inhibit the expression of MMP-2 mRNA, thereby hindering the binding of AGE-RAGE and inhibiting the abnormal proteolysis on the skin with aggravated glycation damage. Figure 11It can be seen that the original inhibition rate of the fluorescent AGE formation of the fructose-bovine serum albumin system was 59.68%, while the inhibition rate of the supernatant after fermentation with Lactobacillus acidophilus CCFM1356 reached 73.13%, which was 22.5% higher than that of the unfermented group, and the ability to inhibit AGE formation was significantly increased.

[0212] Example 14: Effect of Lactobacillus acidophilus CCFM1356 fermentation of peanut skin on enhancing the inhibition of AGE accumulation by oral administration of peanut skin.

[0213] The methods for constructing experiments on aging mice and detecting serum and skin AGEs are as described in Example 8. Peanut skin gavage group (hsp), peanut skin supernatant fermented with Lactobacillus acidophilus CCFM1356 group (denoted as CCFM1356_H) and peanut skin synthetic preparation gavage group (live Lactobacillus acidophilus CCFM1356 and peanut skin fermentation culture medium (hsp), denoted as CCFM1356+hsp).

[0214] Depend on Figure 12 It was found that although peanut skin itself could 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), a significant reduction of 39.2% compared with the model group; the peanut skin fermentation broth treated with Lactobacillus acidophilus CCFM1356 was even more effective, with the AGE content reaching 244.32 ng / L, a reduction of 39.9% compared with the model group; the effect of the peanut skin synthetic preparation group was not as good as the other two groups, and it could only reduce the AGE content by 36.0% compared with the model group.

[0215] In addition to reducing the levels of glycation end products (AGEs) in serum, the accumulation of AGEs in the skin was also alleviated, such as... Figure 12 As shown, peanut skin itself reduced the AGE content in the skin of aging mice to 376.40 ng / L (336.76 ng / L in the control group and 456.72 ng / L in the model group), a significant reduction of 17.6% compared with the model group. The AGE content reduction effect of peanut skin fermentation broth with Lactobacillus acidophilus CCFM1356 was slightly improved compared with the unfermented broth, with the AGE content reduced to 326.17 ng / L, a reduction of 28.6% compared with the model group. In addition, the AGE content in the skin of the synthetic preparation group was reduced to 393.99 ng / L after gavage (a reduction of 13.7% compared with the model group).

[0216] The above experimental results, combined with in vitro experimental results, demonstrate that Lactobacillus acidophilus CCFM1356 fermentation of peanut skins not only enhances the anti-glycation function of unfermented peanut skins in inhibiting AGE production in vitro, but also exerts a synergistic effect in vivo after oral administration. That is, Lactobacillus acidophilus CCFM1356 further enhances the anti-glycation ability of peanut skins.

[0217] Example 15: Effect of Lactobacillus acidophilus CCFM1356 fermented peanut skin on improving peanut skin oral administration to alleviate the damage of glycation to skin

[0218] The skin elasticity of mice was detected as described in Example 10, and the expression of key enzymes and the collagen content of the skin of mice were tested as described in Examples 12 and 11, and the results are shown in Figure 13 .

[0219] The skin elasticity R2 is one of the important indicators for evaluating the elasticity of the skin of aging mice. The R2 of the model group mice was only 53.7% (74.90% for the control group), and the skin elasticity of the aging mice was restored to different degrees after intragastrically administering the fermented culture solution of peanut skin (hsp), the supernatant of Lactobacillus acidophilus CCFM1356 fermented peanut skin (CCFM1356_H), and the synthetic preparation of Lactobacillus acidophilus CCFM1356 plus peanut skin (CCFM1356+hsp). The R2 values were 63.98%, 67.38%, and 74.53%, respectively, i.e., the fermented peanut skin of Lactobacillus acidophilus CCFM1356 and the synthetic preparation both improved the ability of unfermented peanut skin to alleviate the elasticity of the skin of aging mice.

[0220] The expression of MMP-2 mRNA in the skin of the control group was about 1, and the expression of MMP-2 mRNA in the skin of the mice after intragastrically administering the fermented culture solution of peanut skin (hsp) was 0.80, which was down-regulated by 71.1% (2.77 for the model group) compared with the model group. However, through the fermentation effect of Lactobacillus acidophilus CCFM1356, the expression of MMP-2 mRNA after intragastrically administering the supernatant of Lactobacillus acidophilus CCFM1356 fermented peanut skin (CCFM1356_H) and the synthetic preparation of Lactobacillus acidophilus CCFM1356 plus peanut skin (CCFM1356+hsp) was 0.229 and 0.459, respectively (down-regulated by 91.7% and 83.4% compared with the model group), i.e., Lactobacillus acidophilus CCFM1356 could improve the functional properties of peanut skin in reducing the expression of MMP-2 mRNA after glycation damage.

[0221] After the occurrence of sugar damage, the collagen type 3 content of the skin of aging mice was Figure 13As shown, compared with the blank group (7.17 μg / L), the collagen type 3 content of the model group was reduced to 6.09 μg / L, the collagen type 3 content of the mouse skin was increased to 7.43 μg / L (22% higher than the model) after intragastrically administering the peanut skin fermentation broth (hsp), while the collagen type 3 content of the mouse skin was 7.41 μg / L and 7.88 μg / L respectively after intragastrically administering the Lactobacillus acidophilus CCFM1356 fermented peanut skin supernatant (CCFM1356_H) and the synthetic preparation of Lactobacillus acidophilus CCFM1356 plus peanut skin (CCFM1356+hsp), that is, the synthetic preparation group improved the ability of peanut skin to maintain the collagen content of the skin under the relief of sugar damage.

[0222] The relative expression amount of the collagen type III synthesis enzyme COL3A1 mRNA of the mouse skin was 0.87 (0.50 of the model group) after intragastrically administering the peanut skin fermentation broth (hsp), and the expression amount of COL3A1 mRNA of the mouse skin was 0.83 and 2.05 respectively after intragastrically administering the Lactobacillus acidophilus CCFM1356 fermented peanut skin supernatant (CCFM1356_H) and the synthetic preparation of Lactobacillus acidophilus CCFM1356 plus peanut skin (CCFM1356+hsp), that is, the synthetic preparation group significantly improved the ability of peanut skin to relieve the expression of collagen type III synthesis enzyme under the relief of sugar damage.

[0223] Through the above results, it is shown that the Lactobacillus acidophilus CCFM13565 fermented peanut skin supernatant (or synthetic preparation) can improve the ability of peanut skin to relieve the abnormal protein hydrolysis of the mouse under the relief of aging by improving the ability of peanut skin to relieve the skin elasticity of the mouse under the relief of aging and the synthesis and content of collagen type III of the mouse skin.

[0224] Although the present application has been disclosed with the above preferred embodiments, it is not intended to limit the present application, and any person skilled in the art can make various modifications and modifications without departing from the spirit and scope of the present application, therefore the protection scope of the present application should be defined by the claims.

Claims

1. A strain of Lactobacillus acidophilus ( Lactobacillus acidophilus CCFM1356 was deposited at the Guangdong Provincial Center for Microbial Culture Collection on October 25, 2023, with accession number GDMCC NO: 63926.

2. The metabiotic prepared using Lactobacillus acidophilus CCFM1356 according to claim 1, characterized in that, The metabiotic includes cultures and / or freeze-dried forms of Lactobacillus acidophilus CCFM1356.

3. A drug containing Lactobacillus acidophilus CCFM1356 as described in claim 1, and / or the metabiotic as described in claim 2.

4. The drug according to claim 3, characterized in that, The drug is a fermentation product of Lactobacillus acidophilus CCFM1356 fermented in a culture medium containing peanut skins.

5. The use of Lactobacillus acidophilus CCFM1356 as described in claim 1, or the metabiotic as described in claim 2, in the preparation of medicaments for anti-glycation, or prevention and / or delay of aging.

6. The application according to claim 5, characterized in that, The glycation mentioned includes the accumulation of AGEs (glycated aging markers) in the blood or skin.

7. The application according to claim 6, characterized in that, The aging process includes skin aging.

8. The application according to claim 7, characterized in that, Skin aging includes dry skin, reduced elasticity, sagging, wrinkles, oxidative damage, or collagen loss.

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