Novel combined lactobacillus salivarius as well as anti-aging composition and application thereof

Abstract

The invention provides novel combined lactobacillus salivarius, which is classified and named as Ligilobacillus salivarius, is preserved in Guangdong Microbial Culture Collection Center, and has a preservation number of GDMCC No: 65184. The invention further provides an anti-aging composition containing the saliva combined lactobacillus. The anti-aging composition comprises an oral composition and / or an external composition. Meanwhile, the invention further provides application of the saliva combined lactobacillus or the anti-aging composition in preparation of anti-aging and anti-oxidation products. The saliva combined lactobacillus disclosed by the invention is separated from excrement of healthy adults, and can effectively metabolize lactic acid and short-chain fatty acid, promote intestinal flora balance, enhance the antioxidant capacity of a body and improve the antioxidant defense and immune regulation functions of the body, thereby delaying senescence.

Description

Technical Field

[0001] The present invention relates to the fields of microorganisms and biomedicine, and particularly to a novel Ligilactobacillus salivarius and its anti-aging composition and application. Background Art

[0002] Aging is an inevitable physiological process of organisms, and the accompanying oxidative stress is one of the important mechanisms of aging and various age-related diseases. Oxidative stress refers to cell damage caused by the accumulation of free radicals (such as superoxide anions, hydroxyl radicals, hydrogen peroxide, etc.) in cells. These free radicals can damage cell membranes, proteins, and DNA, leading to cell function decline and death, and ultimately accelerating the aging process. With the occurrence of aging, the body's antioxidant defense system gradually weakens, and the antioxidant capacity decreases, thus further exacerbating the accumulation of free radicals. Therefore, developing new antioxidants and anti-aging substances, especially products that can delay aging by reducing the effect of oxidative stress, has become an important direction in anti-aging research in recent years.

[0003] Oxidative damage is considered to be one of the fundamental causes of organism aging and various degenerative diseases. To counteract oxidative stress, antioxidants such as vitamin C, vitamin E, selenium, and polyphenols are widely used in dietary supplements to scavenge excessive free radicals and reduce oxidative damage. However, existing antioxidant nutritional supplements often contain only one or a few components, with a single mechanism of action, and it is difficult to comprehensively cope with the complex aging process.

[0004] There is a close interaction between the gut microbiota and host health. Research shows that the gut microbiota plays an important role in regulating the immune system, maintaining metabolic balance, and antioxidant defense. Lactobacilli, especially Ligilactobacillus salivarius, as a common probiotic, show unique potential in improving the gut microecology, regulating the immune system, and antioxidant. It is reported that L. salivarius strains exhibit strong antioxidant activity and antibacterial properties, and can scavenge free radicals and increase the level of antioxidant enzymes in the host. In addition, L. salivarius can produce a variety of metabolites (such as organic acids, exopolysaccharides, and bacteriocin, etc.), and these active products have biological functions such as anti-inflammatory, antioxidant, and immunomodulatory. These characteristics make it have great potential in promoting human health. However, in the prior art, the research and products applying probiotics to the anti-aging field are relatively few. Especially the application of L. salivarius in anti-aging has not been fully reported and applied.

[0005] In view of this, the present invention attempts to screen a suitable Ligilactobacillus salivarius and use it as the core functional strain for application in the anti-aging field. Summary of the Invention

[0006] The technical problem to be solved by the present invention is to provide a novel Ligilactobacillus salivarius, its anti-aging composition and application. The Ligilactobacillus salivarius is isolated from the feces of healthy adults, can effectively metabolize lactic acid and short-chain fatty acids, promote the balance of intestinal flora, enhance the antioxidant capacity of the body, improve the antioxidant defense and immune regulation functions of the body, and thus delay aging.

[0007] The present invention adopts the following technical solutions to solve the above technical problems:

[0008] A novel Ligilactobacillus salivarius, classified and named Ligilactobacillus salivarius, was deposited in the Guangdong Provincial Microbial Culture Collection Center on September 23, 2024 and was shown to be viable. The deposit number is GDMCC No: 65184, and the deposit address is the 5th floor of Building 59, No. 100 Compound, Xianlie Middle Road, Guangzhou.

[0009] As one of the preferred embodiments of the present invention, the full-length sequence of the 16S rRNA gene of the Ligilactobacillus salivarius is as shown in SEQ ID NO.1.

[0010] An anti-aging composition containing the above novel Ligilactobacillus salivarius.

[0011] As one of the preferred embodiments of the present invention, it includes an oral composition and / or a topical composition. When used orally, the Ligilactobacillus salivarius exerts its effects by improving the intestinal flora, promoting the production of short-chain fatty acids, regulating the immune response, etc. When used topically, it can directly act on the skin to promote the antioxidant capacity of the skin and slow down the external manifestations of aging.

[0012] As one of the preferred embodiments of the present invention, the oral composition comprises the following components: Ligilactobacillus salivarius fermentation broth 10^6 - 10^8 CFU / mL, vitamin C 100 - 200 g / L, vitamin E 20 - 50 g / L, selenium 1 - 5 g / L, zinc 5 - 20 g / L, green tea extract 20 - 50 g / L, excipients 80 - 120 g / L.

[0013] As one of the preferred embodiments of the present invention, the external composition comprises the following components: Lactobacillus salivarius fermentation broth 10^6 - 10^8 CFU / mL, vitamin C 10 - 50 g / L, vitamin E 5 - 20 g / L, green tea extract 10 - 30 g / L, glutathione 1 - 5 g / L, glycerol 30 - 80 g / L, sodium hyaluronate 1 - 10 g / L, panthenol 5 - 20 g / L, triglyceride 20 - 50 g / L, amino acid surfactant 10 - 30 g / L, phenoxyethanol 5 - 10 g / L.

[0014] Use of the above-mentioned novel Lactobacillus salivarius or anti-aging composition in the preparation of anti-aging and antioxidant products.

[0015] As one of the preferred embodiments of the present invention, the Lactobacillus salivarius or anti-aging composition is used for preventing or slowing down functional degradation caused by aging, including but not limited to skin aging, cardiovascular aging, immune system decline, etc.

[0016] As one of the preferred embodiments of the present invention, the anti-aging and antioxidant products include but not limited to drugs, skin care products, etc.

[0017] The advantages of the present invention compared with the prior art are as follows:

[0018] In the present invention, a novel Lactobacillus salivarius strain with a high DPPH free radical scavenging rate and the ability to significantly increase the antioxidant enzyme activity of the host was isolated from the feces of healthy adults, and it was used as the core functional strain in the anti-aging composition. This strain plays a core role in the composition: on the one hand, it has the activities of scavenging reactive oxygen species and activating antioxidant enzymes, which can directly reduce the oxidative stress level in the body; on the other hand, after colonizing in the intestine, it can also improve the intestinal microecological balance, produce anti-inflammatory factors, and reduce chronic inflammation; moreover, this strain can also enhance the function of the body's natural antioxidant system, such as up-regulating the expression activities of enzymes such as superoxide dismutase (SOD) and peroxidase (GPx) in host cells; at the same time, its metabolites (such as lactic acid, exopolysaccharides, short-chain fatty acids, etc.) can regulate cell signaling pathways, such as activating the Keap1-Nrf2 antioxidant pathway to enhance the antioxidant gene expression of cells; in addition, this strain has a two-way regulatory effect on the immune system: promoting the beneficial Th1-type immune response, increasing the production of anti-inflammatory cytokines (such as IL-10), and inhibiting the pro-inflammatory Th2-type response and the overexpression of related cytokines; through this immune regulation, the composition of the present invention can reduce chronic inflammation related to aging (inflammaging), which helps to maintain tissue function.

[0019] In summary, the anti-aging composition of the present invention has multiple mechanisms of action and can be widely applied to prevent or slow down the functional degradation caused by aging. Compared with the existing anti-aging products with a single ingredient, this composition shows more significant effects and synergistic advantages in scavenging free radicals, protecting cells from oxidative damage, and enhancing immune function. The present invention also relates to the application of this composition in the preparation of anti-aging and antioxidant products, which has good application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 It is a morphological characteristic diagram of the aFMT-301 strain in Example 1 of the present invention. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0021] The embodiments of the present invention will be described in detail below. These embodiments are implemented on the premise of the technical solution of the present invention, and detailed implementation manners and specific operation processes are given. However, the protection scope of the present invention is not limited to the following embodiments. At the same time, unless otherwise specified, the reagents, methods, and equipment used in the present invention are conventional reagents, methods, and equipment in the technical field.

[0022] Example 1: Screening and isolation of the aFMT-301 strain:

[0023] A new strain was isolated from the feces of healthy adults. The colony morphology is as Figure 1 shown. After preliminary identification and 16S rRNA gene sequencing (SEQ ID NO.1), it was confirmed to be Ligilactobacillus salivarius. This strain can effectively metabolize lactic acid and short-chain fatty acids, promote the balance of the intestinal flora, and enhance the antioxidant capacity of the body.

[0024] Based on the identification results, this strain was named Ligilactobacillus salivarius aFMT-301 strain, and it was preserved and shown to be viable in the Guangdong Provincial Microbial Culture Collection Center on September 23, 2024. The preservation number is GDMCC No: 65184, and the preservation address is the 5th floor of Building 59, No. 100 Yard, Xianlie Middle Road, Guangzhou.

[0025] Example 2: Fermentation culture of the aFMT-301 strain:

[0026] Use a modified medium for culture and adopt anaerobic fermentation to ensure the growth of the strain and obtain the corresponding fermentation broth.

[0027] Specific culture method:

[0028] (1) Under aseptic conditions, inoculate the aFMT-301 strain into the modified MRS medium and culture it at 37°C for 12 h to obtain a logarithmic-phase seed solution. The formula of the modified MRS medium is: glucose 2%, peptone 1%, yeast extract 0.5%, beef extract 1%, sodium acetate 0.5%, potassium dihydrogen phosphate 0.2%, magnesium sulfate 0.01%, manganese sulfate 0.005%, Tween-80 0.1%, and the initial pH is adjusted to 6.5.

[0029] (2) Take the seed solution and inoculate it into a fermenter (or conical flask) at an inoculation amount of 5%, and carry out anaerobic fermentation at 37°C for 18 - 24 h. Keep gentle stirring during the fermentation process and do not need to aerate (lactic acid bacteria grow anaerobically or microaerophilically). When the fermentation reaches the end point, the pH of the medium drops to about 4.0 - 4.5, and the viable bacteria count reaches 10^6 - 10^8 CFU / mL.

[0030] (3) After the fermentation is completed, collect the fermentation broth. The fermentation broth is not only rich in viable aFMT-301 bacteria but also contains its metabolites (such as lactic acid, short-chain fatty acids, antioxidant enzymes, etc.), which are used as anti-aging active ingredients.

[0031] Example 3. Anti-aging composition (oral):

[0032] The anti-aging composition of this example includes the following components: Lactobacillus salivarius aFMT-301 fermentation broth (Example 2) 10^8 CFU / mL, vitamin C 150 g / L, vitamin E 35 g / L, selenium 3 g / L, zinc 12 g / L, green tea extract 35 g / L, excipients (maltodextrin, glucose) 100 g / L.

[0033] Among them, Lactobacillus salivarius aFMT-301 fermentation broth: plays a role by improving the intestinal flora, promoting the production of short-chain fatty acids, regulating immune responses, etc.

[0034] Vitamin C (ascorbic acid): A widely used antioxidant with a significant free radical scavenging effect.

[0035] Vitamin E (tocopherol): A fat-soluble antioxidant that can protect cell membranes from oxidative damage and cooperate with water-soluble antioxidants to enhance the combined antioxidant effect.

[0036] Selenium and zinc: Are cofactors of important antioxidant enzymes (such as superoxide dismutase, glutathione peroxidase), and can improve the antioxidant capacity of the body.

[0037] Green tea extract (catechins): Catechins in green tea have a strong antioxidant effect, can protect the skin from damage by the external environment, and fight against aging.

[0038] Excipients (maltodextrin, glucose): As carriers in oral form.

[0039] Preparation method:

[0040] ① Mixing and dissolution: Mix the Lactobacillus salivarius aFMT - 301 fermentation broth, antioxidant components (vitamin C, vitamin E), selenium, zinc, and excipients (maltodextrin, glucose, etc.) to ensure uniform dispersion of all components.

[0041] ② Concentration and drying: After ultrafiltration and concentration of the obtained mixed solution, perform freeze - drying to prepare a dry powder.

[0042] ③ Final product: Fill the prepared dry powder into enteric - coated capsules, seal and store to maintain good antioxidant activity.

[0043] Administration method: Continuously take orally for more than 3 months to enhance the antioxidant capacity in the body and delay the aging process.

[0044] Example 4, Anti - aging composition (oral):

[0045] The anti - aging composition of this example is basically the same as that of Example 3. The main difference is that it includes the following specific composition: Lactobacillus salivarius aFMT - 301 fermentation broth (Example 2) 10^6 CFU / mL, vitamin C 100 g / L, vitamin E 20 g / L, selenium 1 g / L, zinc 5 g / L, green tea extract 20 g / L, excipients (maltodextrin, glucose) 80 g / L.

[0046] Example 5, Anti - aging composition (oral):

[0047] The anti - aging composition of this example is basically the same as that of Example 3. The main difference is that it includes the following specific composition: Lactobacillus salivarius aFMT - 301 fermentation broth (Example 2) 10^7 CFU / mL, vitamin C 200 g / L, vitamin E 50 g / L, selenium 5 g / L, zinc 20 g / L, green tea extract 50 g / L, excipients (maltodextrin, glucose) 120 g / L.

[0048] Example 6, Anti - aging composition (topical):

[0049] The anti - aging composition of this example includes the following composition: Lactobacillus salivarius fermentation broth 10^8 CFU / mL, vitamin C 20 g / L, vitamin E 10 g / L, green tea extract 20 g / L, glutathione 2 g / L, glycerol 50 g / L, sodium hyaluronate 5 g / L, panthenol (vitamin B5) 10 g / L, triglyceride 30 g / L, amino acid surfactant 20 g / L, phenoxyethanol 8 g / L, pH 5.0.

[0050] Among them, the fermentation broth of Lactobacillus salivarius aFMT-301: directly acts on the skin, promotes the antioxidant capacity of the skin, and slows down the external manifestations of aging.

[0051] Vitamin C (ascorbic acid): a widely used antioxidant with a significant free radical scavenging effect.

[0052] Vitamin E (tocopherol): a fat-soluble antioxidant that can protect cell membranes from oxidative damage and alleviate oxidative damage caused by ultraviolet irradiation.

[0053] Green tea extract (catechins): Catechins in green tea have a powerful antioxidant effect, can protect the skin from damage by the external environment, and fight aging.

[0054] Glutathione: has a broad-spectrum antioxidant capacity, can scavenge excess free radicals in the body, reduce the retention of free radicals, and prevent damage to the skin; at the same time, after scavenging free radicals, it can also promote cell regeneration and increase cell vitality, making the skin elastic and improving the survival ability of cells.

[0055] Glycerol: a commonly used moisturizer that can form a protective film on the skin surface to prevent water loss and keep the skin moist.

[0056] Sodium hyaluronate: a powerful moisturizing ingredient that helps attract and lock in moisture, enhancing the elasticity and moisture of the skin.

[0057] Panthenol (vitamin B5): has good moisturizing and soothing effects and can enhance the skin's barrier function.

[0058] Triglyceride: provides a fat-soluble component for the emulsifier, enhancing the stability and spreadability of the formulation.

[0059] Amino acid surfactant: used for emulsifying and stabilizing the formulation, improving the texture and absorbency of the product.

[0060] Phenoxyethanol: mainly used as a preservative and antibacterial agent, which can inhibit the growth of bacteria in skin care products and prevent product deterioration.

[0061] Preparation method:

[0062] ① Emulsification and mixing: For external products (such as creams, masks, etc.), the components of the oil phase and water phase need to be emulsified. Mix the oily components with the aqueous components and form a stable emulsion under high-speed stirring. Subsequently, after adding the fermentation broth of Lactobacillus salivarius aFMT-301 and antioxidants (vitamin C, vitamin E, etc.), continue to stir to ensure uniform dispersion.

[0063] ② Adjust pH and viscosity: Use citric acid to adjust the pH value of the product and use a thickening agent to adjust the viscosity of the product.

[0064] ③Final product: The prepared composition is filled into bottles, sealed and packaged. External products should be stored in a suitable environment to avoid degradation of the active ingredients.

[0065] Administration method: Act directly on skin cells through facial care, anti-aging masks, etc. to improve the skin's antioxidant defense.

[0066] Example 7, Anti-aging composition (for external use):

[0067] The anti-aging composition of this example is basically the same as that of Example 6. The main differences are as follows: The specific composition includes Lactobacillus salivarius fermentation broth 10^6 CFU / mL, vitamin C 10 g / L, vitamin E 5 g / L, green tea extract 10 g / L, glutathione 1 g / L, glycerol 30 g / L, sodium hyaluronate 1 g / L, panthenol (vitamin B5) 5 g / L, triglyceride 20 g / L, amino acid surfactant 10 g / L, phenoxyethanol 5 g / L, pH 4.5.

[0068] Example 8, Anti-aging composition (for external use):

[0069] The anti-aging composition of this example is basically the same as that of Example 6. The main differences are as follows: The specific composition includes Lactobacillus salivarius fermentation broth 10^7 CFU / mL, vitamin C 50 g / L, vitamin E 20 g / L, green tea extract 30 g / L, glutathione 5 g / L, glycerol 80 g / L, sodium hyaluronate 10 g / L, panthenol (vitamin B5) 20 g / L, triglyceride 50 g / L, amino acid surfactant 30 g / L, phenoxyethanol 10 g / L, pH 6.0.

[0070] Experimental example

[0071] This experimental example is used to evaluate the anti-aging and antioxidant effects of the Lactobacillus salivarius aFMT-301 of the present invention.

[0072] I. Determination of antioxidant capacity

[0073] The antioxidant activity was determined using the DPPH free radical scavenging method, ABTS free radical scavenging method, etc.

[0074] 1. Determination by DPPH free radical scavenging method

[0075] The DPPH free radical scavenging method is commonly used to evaluate the ability of antioxidant substances. By measuring the scavenging rate of the sample on DPPH free radicals, its antioxidant ability can be indirectly calculated.

[0076] Specific steps:

[0077] The aFMT-301 fermentation broth obtained in Example 2 (at different concentrations of 10^6, 10^7, 10^8 CFU / mL) was added to the DPPH solution (24.4 mg of DPPH powder dissolved in 100 mL of ethanol), and the final concentration of DPPH was 0.1 mM.

[0078] 10 μL of the aFMT-301 fermentation broth was mixed with 190 μL of the DPPH solution. After incubation at room temperature for 30 min, the absorbance was measured using a spectrophotometer (wavelength 517 nm), and the scavenging rate of DPPH radicals was calculated.

[0079] Control group: 10 μL of sterile medium was mixed with 190 μL of the DPPH solution.

[0080] Calculation formula:

[0081]

[0082] The results are shown in Table 1.

[0083] Table 1. Experimental results of aFMT-301 fermentation broth at different concentrations in the DPPH radical scavenging method

[0084] Fermentation broth concentration (CFU / mL) Absorbance (OD 517nm) Clearance rate (%) Control group 0.950 0％ 10^6CFU / mL 0.880 7.37％ 10^7CFU / mL 0.750 21.05％ 10^8CFU / mL 0.620 34.74％

[0085] 2. ABTS radical scavenging method

[0086] The ABTS radical scavenging method was used to further verify the antioxidant capacity.

[0087] The ABTS radical solution was mixed with the aFMT-301 fermentation broth at different concentrations, incubated at 37 °C for 30 min, the absorbance was measured using a spectrophotometer (λ = 734 nm), and the scavenging rate was calculated.

[0088] Specific steps:

[0089] 7.4 mg of ABTS powder was dissolved in 1 mL of deionized water to obtain a concentration of 20 mM of ABTS. Stir well to ensure complete dissolution of ABTS to obtain a 20 mM ABTS stock solution.

[0090] The ABTS stock solution was mixed with a 0.2 mM hydrogen peroxide solution. Usually, the ratio of ABTS to hydrogen peroxide is 1:1 to activate the generation of ABTS radicals. The solution after the activation reaction should be incubated in the dark for 5 min until a dark green or blue ABTS radical is formed.

[0091] The activated ABTS solution is usually used at a concentration of 0.1 mM. When used, the absorbance of the ABTS radical solution is usually measured at 734 nm.

[0092] Mix the ABTS radical solution with the sample to be tested (aFMT-301 fermentation broth). The mixing ratio is: 190 μL of the ABTS radical solution and 10 μL of the fermentation broth at different concentrations. Incubate the sample solution and the ABTS radical solution at 37 °C for 30 min to ensure a complete reaction. Measure the absorbance (A_sample) of the mixed solution at a wavelength of 734 nm using a spectrophotometer.

[0093] Control group: Use the same ABTS solution and solvent (without sample) to measure the absorbance (A_control).

[0094] Calculation formula:

[0095]

[0096] The results are shown in Table 2.

[0097] Table 2. Experimental results of aFMT-301 fermentation broth at different concentrations in the ABTS radical scavenging method

[0098] Fermentation broth concentration (CFU / mL) Absorbance (OD 734nm) Clearance rate (%) Control group 0.950 0％ 10^6CFU / mL 0.900 5.26％ 10^7CFU / mL 0.750 21.05％ 10^8CFU / mL 0.500 47.37％

[0099] II. Cell viability assessment

[0100] Use the CCK-8 method to evaluate the effects of aFMT-301 fermentation broth at different concentrations on cell proliferation and survival rate.

[0101] Select the commonly used HFF-1 human skin fibroblasts as the research object to evaluate the effects of the fermentation broth at different concentrations on cell viability and verify its potential promoting or inhibitory effects on cell proliferation and survival rate.

[0102] 1. Cell line selection and culture

[0103] Cell line: Select HFF-1 human skin fibroblasts (Human Foreskin Fibroblasts-1), which is commonly used in skin repair and aging research.

[0104] Culture medium: Use DMEM (high-glucose medium), supplemented with 10% fetal bovine serum (FBS) and 1% penicillin / streptomycin, and the culture conditions are 37 °C and 5% CO2.

[0105] Seed cells: Seed the cells at 1×10^4 cells / well into a 96-well plate and culture until about 70%-80% confluent.

[0106] 2. Experimental group design

[0107] Experimental group: Cells were treated with aFMT-301 fermentation broth at different concentrations, namely 10^6 CFU / mL, 10^7 CFU / mL, and 10^8 CFU / mL, for 24 h, 48 h, and 72 h, with a total of 3 time points set.

[0108] Control group: Cells were treated with sterile liquid or culture medium as a reference group.

[0109] 3. Cell senescence assessment

[0110] Detection of cell aging markers:

[0111] Senescent cells were detected by β-galactosidase staining to evaluate the number of senescent cells.

[0112] The level of ROS (reactive oxygen species) was detected. The intracellular ROS level was detected by the fluorescent dye DCFH-DA to evaluate the oxidative stress in senescent cells.

[0113] Cell viability was evaluated by the CCK-8 method: The cell viability of different treatment groups at 24 h, 48 h, and 72 h was determined by the CCK-8 method to evaluate the effect of anti-aging components on maintaining cell activity.

[0114] 4. Results

[0115] The results are shown in Table 3.

[0116] Table 3. Results of the effects of AFMT-301 fermentation broth at different concentrations on cell proliferation and viability

[0117]

[0118] 5. Results analysis

[0119] Cell viability:

[0120] The cell viability of the control group was 90% after 72 h, indicating that under normal culture conditions, the cells experienced mild senescence and metabolic stress, resulting in a decrease in cell viability.

[0121] The cell viabilities of the 10^6 CFU / mL fermentation broth group at 24 h, 48 h, and 72 h were 96%, 94%, and 92% respectively, showing good cell activity retention ability, indicating that low-concentration AFMT-301 can effectively inhibit senescence-related cell death.

[0122] The cell viabilities of the 10^7 CFU / mL fermentation broth group at 24 h, 48 h, and 72 h were 92%, 89%, and 85% respectively. Although there was a certain decrease, compared with the control group, it still maintained relatively high cell activity.

[0123] The cell survival rate of the 10^8 CFU / mL fermentation broth group was 80% after 72 h, which was relatively low. This might be because the high-concentration fermentation broth had a slight inhibitory effect on the cell microenvironment.

[0124] Senescence marker (β-galactosidase):

[0125] The proportion of senescent cells in the control group was 35%, showing obvious signs of senescence, which was in line with the law of natural senescence of cells over time.

[0126] The proportion of senescent cells in the 10^6 CFU / mL fermentation broth group was 25%, indicating that the low concentration of AFMT-301 had a significant inhibitory effect on cell senescence.

[0127] The proportion of senescent cells in the 10^7 CFU / mL fermentation broth group was further reduced to 18%, showing a better anti-aging effect of the medium-concentration treatment.

[0128] The proportion of senescent cells in the 10^8 CFU / mL fermentation broth group was 12%. Although the high-concentration treatment had the strongest anti-aging effect, the high concentration might also have a slight inhibitory effect on cells, resulting in a relatively low survival rate.

[0129] ROS level:

[0130] The ROS level of the control group was 100%, indicating that the untreated cells experienced a certain degree of oxidative stress and had a high ROS level.

[0131] The ROS level of the 10^6 CFU / mL fermentation broth group was 80%, indicating that the low concentration of AFMT-301 could significantly reduce the intracellular ROS level and alleviate oxidative damage.

[0132] The ROS level of the 10^7 CFU / mL fermentation broth group was 70%, further verifying the advantage of the medium-concentration treatment group in antioxidant capacity.

[0133] The ROS level of the 10^8 CFU / mL fermentation broth group was 60%, and the antioxidant effect was the most obvious, indicating that the high concentration of AFMT-301 effectively reduced oxidative damage but had a certain impact on cell survival.

[0134] 6. Conclusion

[0135] The AFMT-301 fermentation broth of the present invention can effectively slow down cell senescence at different concentrations. Especially at low concentration (10^6 CFU / mL) and medium concentration (10^7 CFU / mL), it can significantly improve cell survival rate, reduce the proportion of senescent cells, and lower the ROS level.

[0136] Although the high-concentration fermentation broth (10^8 CFU / mL) exhibits the strongest anti-aging and antioxidant effects, it has a certain inhibitory effect on cell viability. Therefore, the concentration range needs to be optimized in practical applications.

[0137] AFMT-301 has good anti-aging effects and antioxidant capacity, and can effectively protect cells from aging and oxidative stress, providing a theoretical basis for its application in anti-aging products.

[0138] III. Immune function assessment

[0139] By evaluating the effects of aFMT-301 fermentation broth on the immune function of mice, especially by detecting the antioxidant enzyme activities (such as superoxide dismutase SOD and catalase CAT) in the sera of mice, its antioxidant capacity was evaluated.

[0140] 1. Experimental animals

[0141] Experimental animals: Healthy male C57BL / 6 mice, weighing about 20 - 25 g, 8 - 10 weeks old.

[0142] Grouping:

[0143] Experimental group: Intragastric administration of aFMT-301 fermentation broth every day, with concentrations of 10^6 / 10^7 / 10^8 CFU / mL, and the treatment lasted for 6 weeks.

[0144] Control group: Oral administration of an equal volume of sterile normal saline every day.

[0145] 2. Experimental design

[0146] Treatment for the experimental group:

[0147] Mice in the experimental group were orally administered aFMT-301 fermentation broth at 10^6 / 10^7 / 10^8 CFU / mL every day through a gavage tube.

[0148] After 6 weeks of treatment, mouse serum samples were collected for detection of antioxidant enzyme activities.

[0149] 3. Detection of serum antioxidant enzyme activities

[0150] Blood collection: After the experiment, blood was collected from the orbital vein and serum was separated.

[0151] Determination of enzyme activities:

[0152] Determination of superoxide dismutase (SOD) activity: Using an SOD kit, the activity of SOD in mouse serum was determined according to the instructions.

[0153] Determination of catalase (CAT) activity: Using a CAT kit, the activity of CAT in serum was determined according to the instructions.

[0154] Immunoglobulin detection:

[0155] The levels of immunoglobulins (such as IgG, IgA, IgM) in serum were detected by enzyme-linked immunosorbent assay (ELISA) to evaluate the regulatory ability of the immune system.

[0156] 4. Experimental procedures

[0157] Determination of SOD activity: The activity of SOD in mouse serum was determined using an SOD kit and operated according to the kit instructions.

[0158] Determination of CAT activity: The activity of CAT in mouse serum was determined using a CAT kit, and the absorbance value was recorded.

[0159] Determination of immunoglobulin levels: The levels of IgG, IgA, and IgM in serum were detected using an ELISA kit to evaluate immune function.

[0160] Statistical analysis: Statistical methods were used to analyze the effects of fermentation broth at different concentrations on immune function and antioxidant enzyme activity.

[0161] 5. Experimental results

[0162] The results are shown in Table 4.

[0163] Table 4. Detection results of enzyme activities and immunoglobulins in each group

[0164]

[0165]

[0166] 6. Result analysis

[0167] Antioxidant enzyme activity:

[0168] Control group: The SOD activity was 3.2 U / mL and the CAT activity was 4.5 U / mL, which represented the antioxidant enzyme activity of mice under normal conditions.

[0169] 10^6 CFU / mL fermentation broth group: The SOD activity was 3.8 U / mL and the CAT activity was 5.2 U / mL, indicating that low-concentration aFMT-301 enhanced the antioxidant ability of mice to a certain extent.

[0170] 10^7 CFU / mL fermentation broth group: The SOD activity was 4.5 U / mL and the CAT activity was 5.9 U / mL, showing a significant enhancing effect of medium-concentration fermentation broth on antioxidant enzymes in mice.

[0171] The 10^8 CFU / mL fermentation broth group: The SOD activity was 5.2 U / mL and the CAT activity was 6.5 U / mL, showing the best effect of high-concentration aFMT-301 in enhancing the antioxidant enzyme activity.

[0172] Immunoglobulin levels (IgG, IgA, IgM):

[0173] Control group: IgG was 220 ng / mL, IgA was 130 ng / mL, and IgM was 110 ng / mL, showing the immune function level of normal mice.

[0174] The 10^6 CFU / mL fermentation broth group: IgG was 240 ng / mL, IgA was 160 ng / mL, and IgM was 130 ng / mL, indicating that low-concentration aFMT-301 has a mild enhancing effect on the immune system.

[0175] The 10^7 CFU / mL fermentation broth group: IgG was 260 ng / mL, IgA was 180 ng / mL, and IgM was 150 ng / mL, indicating that medium-concentration aFMT-301 has a significant promoting effect on the levels of immunoglobulins.

[0176] The 10^8 CFU / mL fermentation broth group: IgG was 280 ng / mL, IgA was 210 ng / mL, and IgM was 180 ng / mL, showing the strongest immune-enhancing effect, indicating that high-concentration aFMT-301 significantly improved the immune function of mice.

[0177] 7. Conclusions

[0178] The aFMT-301 fermentation broth has a significant effect on the antioxidant enzyme activity and immune function of mice at different concentrations, and shows a concentration-dependent manner.

[0179] Low concentration (10^6 CFU / mL) can moderately enhance the antioxidant capacity and immune function of mice.

[0180] Medium concentration (10^7 CFU / mL) can significantly increase the antioxidant enzyme activity and immunoglobulin levels.

[0181] High concentration (10^8 CFU / mL) shows the strongest antioxidant effect and immune-enhancing effect, and can significantly increase the levels of SOD, CAT, IgG, IgA, and IgM.

[0182] Optimal concentration:

[0183] The aFMT-301 fermentation broth at a concentration of 10^8 CFU / mL shows the best performance in enhancing immune function and antioxidant effect, but it is also necessary to pay attention to optimizing the concentration in actual applications to avoid potential adverse effects on the health of mice caused by high concentrations.

[0184] IV. Detection of Aging-Related Markers

[0185] The effect of aFMT-301 on the expression levels of aging-related markers (such as p16 and p53) was detected by Western blot to evaluate its anti-aging effect. Among them, p16 and p53 are important marker proteins related to cellular senescence and are commonly used in aging research.

[0186] 1. Experimental Materials and Methods

[0187] Cell line: After inoculating HFF-1 cells, they were treated with aFMT-301 fermentation broth at different concentrations (10^6, 10^7, 10^8 CFU / mL) for 48 h.

[0188] 2. Experimental Methods and Procedures

[0189] (1) Experimental Cells

[0190] Cell line selection: Human Foreskin Fibroblasts-1 (HFF-1) were used as experimental cells and inoculated in 6-well plates at a cell density of 1×10^5 cells / well.

[0191] Culture medium: Standard cell culture medium (DMEM high-glucose medium supplemented with 10% fetal bovine serum and 1% penicillin / streptomycin) was used.

[0192] (2) Cell Treatment

[0193] Experimental group: HFF-1 cells were treated with aFMT-301 fermentation broth at different concentrations (10^6, 10^7, 10^8 CFU / mL).

[0194] Control group: Cells were treated with a sterile solution or culture medium.

[0195] (3) Western Blot Analysis

[0196] Western blot was used to detect the expression of aging-related markers p16 and p53 in cells.

[0197] ① Cell Treatment

[0198] Cultivation time: The treatment time was 48 h to ensure sufficient time for cells to respond to the treatment with aFMT-301.

[0199] ② Cell Lysis and Protein Extraction

[0200] The treated cells were washed twice with PBS.

[0201] Lyse cells using RIPA lysis buffer (containing protease inhibitors) to extract total cellular proteins.

[0202] Measure the protein concentration using a BCA protein quantification kit and adjust the sample concentrations to be the same.

[0203] ③ SDS-PAGE electrophoresis

[0204] Load the protein sample (20 μg) onto a 12% polyacrylamide gel. Perform SDS-PAGE electrophoresis to separate the proteins.

[0205] ④ Blotting and blocking

[0206] Transfer the proteins from the gel to a PVDF membrane for 1 h (100 V). Block the membrane with 5% non-fat milk at room temperature for 1 h.

[0207] ⑤ Antibody incubation

[0208] Primary antibody incubation: Incubate p16 antibody (1:1000) and p53 antibody (1:1000) overnight at 4°C.

[0209] Secondary antibody incubation: Use an HRP-conjugated secondary antibody (1:5000) and incubate for 1 h at room temperature.

[0210] ⑥ Chemiluminescence detection

[0211] Detect the protein bands on the membrane using an ECL chemiluminescence kit and acquire images using an imaging system.

[0212] (4) Experimental results

[0213] Table 5 shows the effects of different concentrations of aFMT-301 fermentation broth treatment on the expression levels of p16 and p53 in HFF-1 cells. Quantitatively detect the band intensity of the proteins by Western blot.

[0214] Table 5. Results of p16 and p53 expression levels under different concentrations of aFMT-301 fermentation broth treatment

[0215]

[0216] (5) Conclusion

[0217] By detecting the expression levels of senescence marker proteins p16 and p53 in HFF-1 cells treated with aFMT-301 by Western blot, we found that as the concentration of the aFMT-301 fermentation broth increased, the expression of both p16 and p53 showed a downward trend. Especially in the high-concentration group at 10^8 CFU / mL, the expression levels of p16 and p53 proteins were significantly reduced, indicating that aFMT-301 can effectively inhibit the expression of senescence-related markers and has potential anti-aging effects.

[0218] In summary, the salivary combined Lactobacillus aFMT-301 (Ligilactobacillus salivarius) of the present invention has the following remarkable effects:

[0219] Enhanced antioxidant capacity: aFMT-301 can significantly enhance the antioxidant capacity of the body, increase the activities of antioxidant enzymes such as superoxide dismutase (SOD) and catalase (CAT), effectively scavenge free radicals in the body, and reduce the damage caused by oxidative stress. Experimental data show that aFMT-301 can effectively increase the activities of antioxidant enzymes in serum at different concentrations, thereby reducing cell damage caused by free radicals.

[0220] Delay the aging process: Through Western blot analysis, aFMT-301 significantly reduced the expression of senescence markers p16 and p53. This indicates that the strain has the effect of inhibiting cell senescence-related proteins, can delay the aging process by reducing the levels of senescence markers, and enhance cell viability and survival ability.

[0221] Promote cell proliferation: The experimental results by the CCK-8 method show that low concentrations of aFMT-301 can promote cell proliferation and increase cell survival rate. The strain has a positive effect on cell proliferation at low concentrations, which helps to improve the cell growth environment and promote cell activity.

[0222] Safety and low toxicity: As a probiotic, aFMT-301 has low toxicity and can play anti-aging and antioxidant roles without damaging the normal physiological functions of the body. The fermentation broths of the bacteria at different concentrations showed good safety in cell proliferation and immune function regulation, and no obvious side effects were observed.

[0223] Broad application prospects: The effects of aFMT-301 of the present invention in anti-aging, antioxidant, immune regulation, etc. indicate that it has broad application prospects in multiple fields such as food, health products, drugs, and cosmetics. It can be used as a natural and effective anti-aging ingredient, providing a new solution for delaying aging and enhancing immune function.

[0224] The above are only the preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.

Claims

1. A novel salivary lactobacillus, characterized in that: The strain was classified and named as Ligilactobacillus salivarius and deposited in Guangdong Microbiological Culture Collection Center with the deposit number GDMCC No: 65184.

2. The novel salivary lactobacillus according to claim 1, characterized in that: The full-length sequence of the 16S rRNA gene of the salivary lactobacillus is shown in SEQ ID NO.

1.

3. An anti-aging composition containing the novel salivary lactobacillus according to claim 1 or 2.

4. The anti-aging composition according to claim 3, characterized in that It includes oral compositions and / or external compositions.

5. The anti-aging composition according to claim 4, characterized in that The oral composition comprises the following components: 10^6-10^8 CFU / mL of saliva combined with lactobacillus fermentation liquid, 100-200 g / L of vitamin C, 20-50 g / L of vitamin E, 1-5 g / L of selenium, 5-20 g / L of zinc, 20-50 g / L of green tea extract, and 80-120 g / L of auxiliary materials.

6. The anti-aging composition according to claim 4, characterized in that The topical composition comprises the following components: 10^6-10^8 CFU / mL of saliva combined with lactobacillus fermentation liquid, 10-50g / L of vitamin C, 5-20g / L of vitamin E, 10-30g / L of green tea extract, 1-5g / L of glutathione, 30-80g / L of glycerol, 1-10g / L of sodium hyaluronate, 5-20g / L of panthenol, 20-50g / L of triglyceride, 10-30g / L of amino acid surfactant, and 5-10g / L of phenoxyethanol.

7. Use of the novel salivary lactobacillus as claimed in any one of claims 1 to 2, or the anti-aging composition as claimed in any one of claims 3 to 6, in the preparation of anti-aging and antioxidant products.

8. The use according to claim 7, characterized in that: The saliva combined with lactobacillus or anti-aging composition is used for preventing or slowing down functional degradation caused by aging.

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