Lactobacillus sakei strain with anti-sugar / anti-aging efficacy, composition and application thereof

Abstract

The present application relates to the field of microorganisms, in particular to a lactobacillus sakei strain, a composition comprising the strain and application thereof. Latilactobacillus sakei The lactobacillus sakei (LeSK-508) of the present application is preserved in the China General Microbiological Culture Collection Center, and the preservation number is CGMCC No. 38652, and the preservation date is May 12, 2026. The lactobacillus sakei strain of the present application has high safety, strong anti-glycosylation activity, excellent antioxidant performance, and has important theoretical value and application prospect in the development of new microbial preparations with anti-glycosylation and anti-aging dual functions.

Description

Technical Field

[0001] This invention relates to the field of microbiology, and more specifically, to a sake-growing Lactobacillus strain with anti-glycation / anti-aging effects, its composition, and its applications. Background Technology

[0002] Skin aging is the most direct manifestation of bodily aging on the skin's surface and the most easily perceived aspect of aging in human tissues and organs. Modern skin biology research has clearly established that the skin aging process is driven by both endogenous physiological aging and exogenous environmental damage. These two factors overlap and amplify each other, ultimately manifesting as a series of irreversible skin problems such as sagging skin, deepening wrinkles, decreased elasticity, dullness and roughness, weakened skin barrier, and inflammatory sensitivity. Among these, glycation, as one of the core mechanisms of endogenous aging, is widely recognized by the academic community as the "silent killer of skin aging." Its damage to skin structure and function persists throughout the entire life cycle and intensifies with age, changes in diet, abnormal metabolic state, and increased oxidative stress levels, becoming a key bottleneck restricting the effectiveness of anti-aging products.

[0003] Glycation is essentially a spontaneous reaction between reducing sugars (such as glucose and fructose) and the free amino groups of biomolecules such as proteins, lipids, and nucleic acids under non-enzymatic conditions. This reaction involves intermediate stages such as Schiff base and Amadori rearrangement, ultimately producing irreversible and stable products—advanced glycation end products (AGEs). In skin tissue, collagen, elastin, fibronectin, and other core components of the extracellular matrix are the primary targets of glycation. When these structural proteins are continuously modified by glycation, abnormal cross-linking, folding errors, and loss of function occur, leading to the collapse of the dermal support structure, decreased resilience, and loss of elasticity, directly causing skin laxity and wrinkles. Simultaneously, the continuous accumulation of AGEs in the skin further activates their receptor RAGE, initiating downstream oxidative stress and inflammatory signaling pathways. This promotes the excessive generation of reactive oxygen species (ROS) and the overexpression of matrix metalloproteinases (MMPs), thereby accelerating collagen degradation, damaging the skin barrier, and exacerbating chronic low-grade inflammation, forming a vicious cycle of "glycation—oxidation—inflammation—aging." In addition, AGEs can also lead to skin pigment metabolism disorders, resulting in a cumulative effect of photoaging symptoms such as dullness, yellowing, and age spots, which significantly reduces the appearance of the skin.

[0004] With the upgrading of public health awareness and skincare needs, anti-glycation and anti-aging have become core research and development directions in the food, health product, and cosmetic fields. Currently, the functional ingredients used in commercially available anti-glycation and anti-aging products mainly fall into three categories: First, chemically synthesized ingredients, such as aminoguanidine and ALT-711, while exhibiting strong AGEs inhibition capabilities in vitro, raise concerns about cell safety, in vivo metabolic risks, and skin irritation, making long-term use difficult. Second, plant extracts, such as resveratrol, proanthocyanidins, oleuropein, and tea polyphenols, possess certain antioxidant and anti-glycation activities, but suffer from drawbacks such as complex composition, poor stability, low bioavailability, unclear target sites, and inconsistent effects. Third, traditional vitamins, such as vitamin C, vitamin E, and niacinamide, primarily function as antioxidants, but have limited ability to block existing AGEs and glycation pathways. Overall, existing ingredients generally suffer from drawbacks such as difficulty in balancing safety and efficacy, single mechanism of action, insufficient targeting, and weak in vivo synergistic effects, failing to meet consumers' demands for safe, gentle, highly effective, and long-term tolerable anti-glycation and anti-aging products.

[0005] In recent years, microbial resources have become a new hotspot in the research field of anti-glycation and anti-aging due to their advantages such as natural safety, mild effects, abundant metabolites, and industrial production capabilities. Probiotics and their fermentation products and post-biotics can block or alleviate glycation damage from the source through multiple pathways, including scavenging free radicals, inhibiting glycation reactions, reducing AGEs accumulation, regulating oxidative stress, inhibiting inflammatory factors, protecting skin cells, and promoting collagen synthesis, thereby achieving systemic anti-aging effects. However, the number of probiotic strains with clear anti-glycation functions is currently limited, and research on their mechanisms of action is not yet in-depth. Most strains are still only at the stage of in vitro antioxidant screening, lacking target validation at the cellular and molecular levels, and standardized and industrially scalable functional strains and formulation technologies have not yet been formed. Therefore, screening for specialized functional probiotics with high safety, strong anti-glycation activity, excellent antioxidant performance, and clear mechanisms of action, and developing novel microbial preparations with dual effects of anti-glycation and anti-aging, to fill the market gap for natural and safe anti-glycation raw materials, has important theoretical value and application prospects for promoting technological upgrading and product innovation in the food, health product, and cosmetic industries. Summary of the Invention

[0006] In view of the above-mentioned problems existing in the prior art, the present invention proposes the following technical solutions.

[0007] In a first aspect, the present invention provides a *Lactobacillus spp.* (sake-growing lactobacillus) Latilactobacillus sakei The *Lactobacillus thuringiensis* strain described is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 38652 and deposit date of May 12, 2026.

[0008] In a second aspect, the present invention provides a composition comprising at least one of the *Lactobacillus sacchariformis* strains or their culture supernatants from the first aspect of the present invention.

[0009] In a third aspect, the present invention provides the use of the *Lactobacillus spp.* strain of sake from the first aspect of the present invention, or the composition from the second aspect of the present invention, in at least one of the following aspects: a) Anti-glycation; b) Antioxidant; c) Maintain collagen homeostasis; d) Anti-aging.

[0010] In a fourth aspect, the present invention provides the use of the *Lactobacillus sacchariflorus* strain of the first aspect of the present invention, or the composition of the second aspect of the present invention, in the preparation of a medicament for preventing or alleviating chronic skin damage or thinning caused by abnormal glucose metabolism in the body.

[0011] The beneficial effects of the present invention include at least one or more of the following: This invention provides a novel *Lactobacillus saccharidus* strain that exerts anti-glycation, antioxidant, collagen homeostasis-maintaining, and / or anti-aging effects through a multi-target mechanism: First, this strain exhibits significant DPPH free radical scavenging and anti-lipid peroxidation capabilities; second, it significantly inhibits the formation of advanced glycation end products (AGEs) in vivo, downregulates RAGE receptor expression, and blocks glycation damage pathways; finally, it activates the skin's antioxidant system (increasing SOD) and maintains collagen homeostasis by upregulating COL3A1 and downregulating MMP9. This *Lactobacillus saccharidus* strain exhibits high safety, strong anti-glycation activity, and excellent antioxidant performance, possessing significant theoretical value and application prospects in the development of novel microbial preparations with both anti-glycation and anti-aging effects. Attached Figure Description

[0012] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0013] Figure 1 The in vitro antioxidant and anti-glycosylation activity evaluation of the *Lactobacillus sacchariformis* strain from the present invention is shown.

[0014] Figure 2The effects of the *Lactobacillus sakei* strain of this invention on the levels of AGEs and CML in a HaCaT cell model of MGO injury are illustrated. Specifically, (A) shows the effect of *Lactobacillus sakei* strain LeSK-508 on the AGEs level in the cell injury model; (B) shows the effect of *Lactobacillus sakei* strain LeSK-508 on the CML level in the cell injury model; and (C) shows the effect of *Lactobacillus sakei* strain LeSK-508 on the ROS level in the cell injury model.

[0015] Figure 3 This invention demonstrates the effects of the *Lactobacillus sakei* strain from this invention on the improvement of skin physiological state in D-galactose-induced aging mice and on AGE content in blood and skin. Specifically, (A) shows the effect of gavage administration of *Lactobacillus sakei* strain LeSK-508 on the stratum corneum moisture content of aging mouse skin; (B) shows the effect of gavage administration of *Lactobacillus sakei* strain LeSK-508 on the skin elasticity of aging mice; (C) shows the effect of gavage administration of *Lactobacillus sakei* strain LeSK-508 on the AGE content in the blood of aging mice; and (D) shows the effect of gavage administration of *Lactobacillus sakei* strain LeSK-508 on the AGE content in the skin tissue of aging mice. n=8, *p< 0.05, **p< 0.01, ***p< 0.001.

[0016] Figure 4 This invention illustrates the molecular mechanism by which the *Lactobacillus sakei* strain improves D-galactose-induced aging in mice. Specifically, (A) shows the effect of gavage administration of *Lactobacillus sakei* strain LeSK-508 on inhibiting AGE-RAGE binding and alleviating skin glycation damage; (B) shows the effect of gavage administration of *Lactobacillus sakei* strain LeSK-508 on superoxide dismutase (SOD); and (CD) show the effect of gavage administration of *Lactobacillus sakei* strain LeSK-508 on the synthesis and degradation of type III collagen in the skin. n=8, *p< 0.05, **p< 0.01, ***p< 0.001. Detailed Implementation

[0017] The present invention will now be described in detail. It should be understood that the following description is merely illustrative and is not intended to limit the scope of the invention; the scope of protection of the invention is defined by the appended claims. Furthermore, those skilled in the art will understand that modifications can be made to the technical solutions of the present invention without departing from its spirit and intent. Unless otherwise specified, the technical means used in the embodiments are conventional means well known to those skilled in the art.

[0018] Furthermore, unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. It should be noted that the terminology used in this specification is for the purpose of describing particular embodiments only and is not intended to limit the invention. The foregoing summary section and the following detailed description are for illustrative purposes only and are not intended to limit the invention in any way.

[0019] In cases where numerical ranges are provided, such as concentration ranges, percentage ranges, or ratio ranges, it should be understood that, unless the context explicitly specifies otherwise, all intermediate values ​​between the upper and lower limits of the range, up to one-tenth of the lower limit unit, and any other values ​​or intermediate values ​​within the range are included in the subject matter. The upper and lower limits of these smaller ranges may be independently included in the smaller ranges, and such embodiments are also included in the subject matter, limited by any specific excluded limit values ​​within the range. Where the range includes one or two limit values, the range excluding any one or both of those included limit values ​​is also included in the subject matter.

[0020] In the context of this invention, many embodiments use the expressions "comprising," "including," or "basically / mainly composed of." The expressions "comprising," "including," or "basically / mainly composed of" are generally understood as open-ended expressions, indicating that they include not only the elements, components, parts, or method steps specifically listed after the expression, but also other elements, components, parts, or method steps. However, in this document, the expressions "comprising," "including," or "basically / mainly composed of" can also be understood as closed-ended expressions in certain cases, indicating that they only include the elements, components, parts, or method steps specifically listed after the expression, and do not include any other elements, components, parts, or method steps. In this case, the expression is equivalent to the expression "composed of."

[0021] As described above, this invention provides a *Lactobacillus saccharidus* strain from sake brewing, a composition comprising this strain, and its applications. Through multiple studies, the inventors discovered that the *Lactobacillus saccharidus* strain of this invention not only possesses significant DPPH free radical scavenging ability and anti-lipid peroxidation ability, but also significantly inhibits the formation of advanced glycation end products (AGEs) in vivo, downregulates RAGE expression, blocks glycation damage pathways, activates the skin's antioxidant system, and maintains collagen homeostasis by upregulating COL3A1 and downregulating MMP9. Thus, this invention was completed.

[0022] Therefore, in a first aspect, the present invention provides a *Lactobacillus spp.* (sake-growing lactobacillus) Latilactobacillus sakeiThe *Lactobacillus sacchariflorus* strain described herein is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 38652 and deposit date of May 12, 2026. This strain was isolated by the inventors from fecal samples in a live bacteria bank and is also named LeSK-508 in this invention.

[0023] In one embodiment, the 16S rDNA sequence of the *Lactobacillus sacchariformis* strain of the present invention is shown in SEQ ID NO.1.

[0024] The inventors have confirmed through research that the Lactobacillus strain widely distributed in sake in this invention is: 1) It has significant antioxidant activity, effectively scavenging DPPH free radicals, resisting lipid peroxidation, and effectively scavenging excess reactive oxygen species (ROS) in cells. 2) It has strong anti-glycosylation activity, which can effectively inhibit glycosylation reaction and significantly reduce the generation and accumulation of advanced glycosylation products; 3) It has the activity of maintaining collagen homeostasis, and can promote collagen synthesis while inhibiting its degradation.

[0025] Furthermore, the inventors conducted a more detailed study on the relevant molecular mechanisms and discovered that the *Lactobacillus saccharidus* strain of the present invention can block the inflammatory cascade response triggered by advanced glycation end products by downregulating RAGE expression, strongly activate the skin's antioxidant defense system by increasing the expression of superoxide dismutase (SOD), and maintain collagen homeostasis by upregulating COL3A1 and downregulating MMP9 expression.

[0026] The above-mentioned functions of the *Lactobacillus spp.* strain from sake of this invention demonstrate its important theoretical value and application prospects in the fields of anti-glycation, anti-oxidation, maintaining collagen homeostasis, and / or anti-aging.

[0027] In a second aspect, the present invention provides a composition comprising at least one of the *Lactobacillus sacchariformis* strains or their culture supernatants from the first aspect of the present invention.

[0028] In a preferred embodiment, the *Lactobacillus sacchariformis* strain can be either live or inactivated.

[0029] In a more preferred embodiment, the *Lactobacillus sacchariformis* strain is in live form.

[0030] In this document, the term "live bacteria" refers to active bacteria with intact cellular structure and maintaining normal metabolic activity. The terms "inactivated bacteria" and "dead bacteria" are used interchangeably, referring to bacteria that have lost their growth and metabolic capacity after being treated by physical or chemical methods; their cells may still retain an intact morphology (or be partially ruptured) but they are no longer active. "Physical methods" include heat inactivation, ultraviolet irradiation, etc., and "chemical methods" include treatment with formaldehyde, ethanol, etc., and this invention does not impose any particular limitations on these methods. As an example, inactivated bacteria can be obtained by subjecting live bacteria to high-temperature treatment (e.g., treatment at 70°C for 30 min).

[0031] In this article, the terms “culture supernatant”, “fermentation supernatant” or “supernatant” are used interchangeably to refer to the liquid portion obtained after centrifuging or filtering to remove all bacterial cells from the bacterial culture. In this article, it may also refer to the metabolic products of the bacterial strain.

[0032] In one embodiment, the culture supernatant of the *Lactobacillus sacchariformis* strain refers to the supernatant obtained by separating the strain after inoculating it into liquid MRS medium and culturing it to the stationary phase (e.g., 24-48 hours). This separation can be performed using methods known in the art, such as centrifugation. In one specific embodiment, the culture supernatant is obtained by centrifuging the cultured bacterial solution at 13,000 rpm and 4°C for 5-15 min. The culture supernatant can be further filtered through a 0.22 μm filter membrane to completely remove residual bacteria.

[0033] In one embodiment, the composition further comprises a cosmetically, edible, or pharmaceutically acceptable carrier or excipient.

[0034] Suitable carriers may include, for example, liposomes, microcapsules, polymer micelles, etc.

[0035] Suitable excipients may include, for example, solubilizers, preservatives, fillers, excipients, emulsifiers, pH adjusters, antioxidants, colorants, etc.

[0036] It is understood that those skilled in the art can select appropriate carriers or excipients based on specific dosage form requirements.

[0037] In one embodiment, the composition is administered orally or topically.

[0038] In one embodiment, the composition is formulated as a solution, tablet, lozenge, powder, granule, capsule, gel, cream, emulsion, spray, or patch.

[0039] In one embodiment, the composition is formulated as a functional food, dietary supplement, pharmaceutical preparation, cosmetic, or skin care preparation.

[0040] In one embodiment, the functional food or dietary supplement is an anti-glycation, anti-oxidation, and / or anti-aging functional food or dietary supplement. The functional food or dietary supplement may be a product form common in the art, such as ready-to-eat probiotic powder, probiotic compressed candy, probiotic oral liquid, probiotic capsules, or probiotic dairy products.

[0041] In a third aspect, the present invention provides the use of the *Lactobacillus spp.* strain of sake from the first aspect of the present invention, or the composition from the second aspect of the present invention, in at least one of the following aspects: a) Anti-glycation; b) Antioxidant; c) Maintain collagen homeostasis; d) Anti-aging.

[0042] According to the present invention, the anti-glycation can be achieved by inhibiting glycosylation reactions, reducing the generation and accumulation of advanced glycation end products and / or downregulating RAGE expression to inhibit inflammatory factors, thereby blocking the glycation damage pathway.

[0043] According to the present invention, the antioxidant capacity can be improved by the *Lactobacillus saccharis* strain of sake of the present invention scavenging free radicals, resisting lipid peroxidation and / or increasing the expression of superoxide dismutase (SOD) to enhance ROS scavenging ability.

[0044] According to the present invention, maintaining collagen homeostasis can be achieved by upregulating the expression of COL3A1 and downregulating the expression of MMP9 by the *Lactobacillus sacchariformis* strain of the present invention.

[0045] According to the present invention, the anti-aging effect can be achieved through the synergistic effect of the *Lactobacillus saccharidus* strain of sake of the present invention in resisting glycation, anti-oxidation and / or maintaining collagen homeostasis.

[0046] In one embodiment, the use includes improving dull, lackluster, or rough skin caused by skin glycation or lack of sleep, and / or improving skin elasticity loss, sagging, or wrinkles caused by collagen glycation.

[0047] In one specific implementation, the use includes increasing skin moisture content and restoring or increasing skin elasticity.

[0048] In a fourth aspect, the present invention provides the use of the *Lactobacillus sacchariflorus* strain of the first aspect of the present invention, or the composition of the second aspect of the present invention, in the preparation of a medicament for preventing or alleviating chronic skin damage or thinning caused by abnormal glucose metabolism in the body.

[0049] As described above, the *Lactobacillus sacchariformis* strain of sake of the present invention has significant anti-glycation / antioxidant effects. Therefore, it is understood that it may also help prevent or alleviate chronic skin damage or thinning caused by abnormal sugar metabolism in the body.

[0050] The embodiments of the present invention will be described in detail below with reference to examples. Those skilled in the art will understand that the following examples are merely illustrative and should not be considered as limiting the scope of the present invention. Where specific techniques or conditions are not specified in the examples, they are performed according to the techniques or conditions described in the literature in the art or according to the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be obtained commercially.

[0051] Example 1: Isolation and sequencing identification of Lactobacillus sacchariformis strain LeSK-508 in sake 1.1 Isolation of Lactobacillus sacchariformis strain LeSK-508 from sake The *Lactobacillus sacchariformis* strain of this invention was isolated from fecal samples of healthy adults in the Beijing area. The subjects had not taken antibiotics or had a history of probiotic use prior to collection. The specific steps are as follows: Fecal samples were collected from healthy adults, diluted, and spread onto MRS solid agar plates (10 g peptone, 10 g beef extract, 5 g yeast extract, 2 g diammonium citrate, 20 g glucose, 1 mL Tween-80, 5 g sodium acetate, 2 g dipotassium hydrogen phosphate, 0.58 g magnesium sulfate, 0.25 g manganese sulfate, 18 g agar, and 1000 mL distilled water). The plates were anaerobic at 37°C for 24 h, and different single colonies were isolated from the plates. Different single colonies were picked using a sterile inoculation loop and streaked onto new MRS solid agar plates for purification. The plates were then anaerobic at 37°C for 24 h to obtain purified single colonies, which were then frozen at -80°C for later use.

[0052] 1.2 Culture and identification of Lactobacillus sacchariformis strain LeSK-508 in sake ① Strains culture and identification The test strain, frozen at -80℃, was streaked in MRS solid medium (10 g peptone, 10 g beef extract, 5 g yeast extract, 2 g diammonium citrate, 20 g glucose, 1 mL Tween 80, 5 g sodium acetate, 2 g dipotassium hydrogen phosphate, 0.58 g magnesium sulfate, 0.25 g manganese sulfate, 18 g agar, 1000 mL distilled water) and cultured anaerobically at 37℃ for 24 h. Single colonies were then picked and cultured in liquid MRS medium (10 g peptone, 10 g beef extract, 5 g yeast extract, 2 g diammonium citrate, 20 g glucose, 1 mL Tween 80, 5 g sodium acetate, 2 g dipotassium hydrogen phosphate, 0.58 g magnesium sulfate, 0.25 g manganese sulfate, 1000 mL distilled water) and cultured anaerobically at 37℃ for 24 h until the stationary phase to obtain the activated working bacterial suspension. A portion of the bacterial culture to be tested was centrifuged at 13000 rpm and 4℃ for 5 min to obtain bacterial cell precipitate, which was then sent to a sequencing company for sequencing. The 16S rDNA deoxyribonucleic acid sequence of the strain is shown in SEQ ID NO: 1:

[0053] Based on the 16S rDNA sequence of this strain in NCBI's BLAST results, this bacterium belongs to the *Lactobacillus sakei* species. Therefore, the applicant named it *Lactobacillus sakei* (…). Latilactobacillus sakei LeSK-508, this strain was deposited on May 12, 2026 at the China General Microbiological Culture Collection Center, located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC No: 38652.

[0054] ②Preparation of bacterial strain samples After centrifuging the above working bacterial solution at 13000 rpm and 4℃ for 10 min, collect the supernatant, which is the culture supernatant. Store it at 4℃ for subsequent experiments.

[0055] Resuspending the bacterial pellet in physiological saline yields a live bacterial suspension containing viable cells. Adjust the suspension to the appropriate concentration required for animal experiments (e.g., 10⁻⁶). 9 (CFU / mL).

[0056] By treating the live bacterial suspension at 70°C for 30 minutes, an inactivated bacterial solution containing the inactivated Lactobacillus sacchariformis LeSK-508 strain can be obtained.

[0057] Example 2: In vitro antioxidant capacity assessment of Lactobacillus sacchariformis strain LeSK-508 in sake This embodiment is used to evaluate the DPPH free radical scavenging ability and lipid peroxidation resistance of the *Lactobacillus saccharidus* strain of the present invention. These two abilities are of great guiding significance for the development of products using this strain.

[0058] 2.1 Evaluation of the in vitro DPPH clearance rate of Lactobacillus sacchariformis LeSK-508 in sake The experiments were conducted using the metabolites obtained according to Example 2 of the present invention. The experimental setup included sample groups and sample control groups, blank groups and blank control groups, and positive groups and positive control groups. Specifically, 30 μL of fermentation supernatant (sample group and sample control group) or 30 μL of anhydrous ethanol (blank group and blank control group) or 0.1 mg / mL vitamin C (sample group and sample control group) was added to a 1.5 mL PE tube, followed by 120 μL of DPPH solution (sample group, blank group, and positive group) or anhydrous ethanol (sample control group, blank control group, and positive control group). All groups were shaken and mixed for 1 min, then incubated at room temperature in the dark for 30 min. After centrifugation, 40 μL of the supernatant from each group was transferred to a 96-well plate, and the absorbance was measured at 517 nm. The DPPH radical scavenging rate of the samples was calculated using the following formula: Sample DPPH radical scavenging rate (%) = [1 - (OD sample group / OD positive group - OD sample control group / OD positive control group) / (OD blank group - OD blank control group)] × 100 (%).

[0059] 2.2 Evaluation of the in vitro anti-lipid peroxidation capacity of Lactobacillus sacchariflorus LeSK-508 in sake The experiments were conducted using the metabolites (i.e., culture supernatant) obtained according to Example 1. Sample groups and sample control groups, blank groups and blank control groups, and positive groups and positive control groups were set up as needed. In 1.5 mL PE tubes, 30 μL of PBS (pH 7.2), 100 μL of linoleic acid emulsion, and 100 μL of 1% FeSO4 were added sequentially. Then, 30 μL of fermentation supernatant sample (sample group and sample control group), 30 μL of PBS solution (blank group and blank control group), or 30 μL of butylated hydroxyanisole (BHA) solution with a final concentration of 0.05 mg / mL (positive group and positive control group) were added. After shaking, the tubes were incubated in a 37°C water bath for 90 min. After incubation, remove the sample and vortex to mix. Add 20 μL of 4% trichloroacetic acid (TCA), followed by 200 μL of 0.8% thiobarbituric acid (TBA) (sample group, blank group, and positive group) or aqueous solution (sample control group, blank control group, and positive control group). Mix well and incubate at 100°C for 30 min, then cool in cold water. After cooling, centrifuge at 5000 r / min for 5 min and collect the supernatant. (Note: The blank group has more precipitate; be careful to avoid aspirating precipitate when collecting the supernatant, as this will affect the absorbance value. If the precipitate is slightly suspended, increase the centrifugation speed to 10000 r / min and centrifuge again.) Then, take 50 μL of the supernatant and perform a four-well replicate in a 96-well plate to measure its absorbance (OD) at a wavelength of 532 nm. The anti-lipid peroxidation rate of the sample is calculated using the following formula: Anti-lipid peroxidation rate of sample (%) = [(OD sample group / OD positive group - OD sample control group / OD positive control group) / (OD blank group - OD blank control group)] × 100 (%).

[0060] like Figure 1 As shown in the left figure (A), the DPPH scavenging rate of the positive group (Vc) was approximately 74%, while the scavenging rate of the *Lactobacillus spp.* supernatant group of this invention was approximately 63%, indicating that this strain has a significant DPPH free radical scavenging ability. Figure 1 As shown in Figure A (right), the lipid peroxidation inhibition rate of the positive group was approximately 80%. The supernatant of *Lactobacillus thuringiensis* from sake brewing also exhibited significant anti-lipid peroxidation activity, with a lipid peroxidation inhibition rate of approximately 55%, demonstrating that the metabolites of this strain can effectively block the lipid peroxidation chain reaction. These results indicate that this strain possesses significant DPPH free radical scavenging ability and anti-lipid peroxidation ability, exhibiting excellent antioxidant activity.

[0061] Example 3: Evaluation of the in vitro anti-glycation effect of Lactobacillus saccharidus LeSK-508 in sake Experiments were conducted using the culture supernatant samples of the strain obtained according to Example 1. Sample groups and control groups, blank groups and control groups, positive groups and positive control groups were set up as needed. 1.5 mol / L fructose and 60 mg / mL bovine serum albumin (BSA) solutions were prepared using 0.2 mol / L, pH 7.4 phosphate buffer. In a 1.5 mL test tube with a screw cap, first add 210 μL of BSA solution, 210 μL of fructose solution (sample group, blank group, and positive group), or PBS solution (sample control group, blank control group, and positive control group). Then add 70 μL of fermentation supernatant (sample group and sample control group), or 70 μL of 100 μg / mL aminoguanidine (AG) working solution (0.01 g of AG powder dissolved in 1 mL of pure water as AG stock solution, diluted 1:100 to prepare AG working solution) (positive group and positive control group), or 70 μL of PBS solution (blank group and blank control group). Shake to mix well, seal the centrifuge tube with sealing film, and react at 50℃ for 24 h. After incubation, add each mixed solution to a 96-well black plate, 70 μL per well, setting up 6 parallel wells. Measure the fluorescence intensity (A) of all reaction solutions using an F-7000 fluorescence spectrophotometer at an excitation wavelength of 370 nm and an emission wavelength of 440 nm. The AGE inhibition rate of the sample is calculated using the following formula: Sample AGE inhibition rate (%) = [1 - (A sample group / A positive group - A sample control group / A positive control group) / (A blank group - A blank control group)] × 100 (%).

[0062] like Figure 1 As shown in Figure B, in the fructose-BSA-induced in vitro glycosylation model, the *Lactobacillus sakebi* strain LeSK-508 of this invention exhibited an inhibitory effect on the formation of advanced glycation end products (AGEs). The figure shows that the positive group showed the highest inhibition rate of aminoguanidine (64.6%), while the glycosylation inhibition rate of the *Lactobacillus sakebi* supernatant was 49.7%, demonstrating strong anti-glycosylation activity. These data confirm that the *Lactobacillus sakebi* strain of this invention can effectively inhibit non-enzymatic protein glycosylation reactions and reduce AGE formation, indicating that this strain has great application potential in preventing skin yellowing and aging caused by glycation.

[0063] Example 4: Effects of Lactobacillus sacchariflorus LeSK-508 on AGE and CML levels in a MGO-damaged HaCaT cell model According to experimental requirements, the following groups were set up: blank group, which was untreated HaCaT cells; model group, which was cells that underwent glucose damage induced by methylglyoxal (MGO) without any other treatment; Lactobacillus sakei group, which was cells that underwent glucose damage induced by MGO and then treated with the culture supernatant of Lactobacillus sakei LeSK-508 of the present invention; positive group, which was cells that underwent glucose damage induced by MGO and then treated with aminoguanidine (AG).

[0064] The glucose-induced injury model was established through the following steps: HaCaT immortalized human keratinocytes (purchased from Pronosai, catalog number: CL-0090) in logarithmic growth phase were harvested and subjected to a glucose-induced injury at a concentration of 2.5 × 10⁻⁶ cells / mL. 5 Inoculate the cells at a density of 100 cells / mL into 12-well plates, and then add methylglyoxal (MGO) to a final concentration of 800 μM to induce glycogen damage for 24 hours. For each treatment group, after the induced damage is completed, remove the saccharification medium and add fresh medium containing 10% Lactobacillus sacchariformis LeSK-508 fermentation supernatant or 500 μg / mL aminoguanidine (AG) and continue culturing for 24 hours.

[0065] AGE and Na-(carboxymethyl)lysine (CML) detection: Cells were collected and the levels of advanced glycosylation products (AGE) and Na-(carboxymethyl)lysine (CML) in cells were detected using an ELISA kit (purchased from Nanjing Jiancheng Biotechnology Co., Ltd., catalog number: H250-1-1).

[0066] ROS assay: Cell pellets from each group were collected, resuspended in serum-free medium, and incubated at 37°C for 20 minutes in the dark with the DCFH-DA fluorescent probe (10 μM) (purchased from MCE, catalog number: HY-D0940). Cells were washed three times with serum-free medium to thoroughly remove any probes that had not entered the cells. Fluorescence intensity was measured using a fluorescence microplate reader. The fluorescence intensity value represents the relative level of intracellular reactive oxygen species (ROS).

[0067] The results are as follows Figure 2 As shown, the levels of AGE, CML, and ROS in the MGO-induced model group cells were all significantly increased, indicating that the glucose damage and oxidative stress model was successfully constructed. Specifically, the AGE content detection results ( Figure 2 A) shows that, compared with the model group, the *Lactobacillus sacchariformis* group significantly reduced the intracellular AGE content, which decreased to approximately 1.26 μg / mg, while the model group reached as high as 8.0 μg / mg. Furthermore, the inhibitory effect of the *Lactobacillus sacchariformis* group was significantly better than that of the positive aminoguanidine (AG) treatment group (4.29 μg / mg).

[0068] CML is a major and stable biomarker for AGEs. CML content detection results ( Figure 2B) showed that the intracellular CML content in the model group surged to 71.64 ng / mg, while the *Lactobacillus shochuensis* group significantly inhibited CML production, reducing its content to 11.60 ng / mg, which was significantly lower than that in the model group and the positive group (28.33 ng / mg).

[0069] ROS level detection results ( Figure 2 C) showed that the intracellular ROS fluorescence intensity in the model group surged to 295.9 RFU (relative fluorescence intensity), indicating a severe state of oxidative stress. The *Lactobacillus shochuensis* group significantly cleared excess ROS from the cells, with the fluorescence intensity decreasing to 107.6 RFU, close to the level of the blank group (89.5 RFU), and significantly lower than the positive AG treatment group (171.2 RFU).

[0070] The above experimental results indicate that *Lactobacillus saccharidus* LeSK-508 can not only significantly reduce the accumulation of AGEs and their marker product CML by inhibiting glycosylation, but also effectively scavenge excess reactive oxygen species (ROS) induced by MGO. This demonstrates that this strain possesses a dual synergistic protective mechanism of "anti-glycation-antioxidation," which can alleviate glycosylation damage by blocking oxidative stress pathways, thereby exerting remarkable anti-aging and cell protection effects at the cellular level.

[0071] Example 5: Evaluation of the effect of Lactobacillus sacchariflorus LeSK-508 on the physiological function of skin in D-galactose-induced aging mice This embodiment establishes a D-galactose-induced subacute aging model in mice to evaluate the anti-aging and anti-glycation effects of Lactobacillus sacchariflorus LeSK-508 in vivo.

[0072] Healthy male BALB / c mice (weighing 20-22 g) were selected and, after one week of acclimatization, randomly divided into a control group, a model group, a LeSK-508 live bacteria intervention group, and a LeSK-508 dead bacteria (inactivated bacteria) intervention group, with 8 mice in each group. Except for the control group, which received daily subcutaneous injections of saline, all other groups received daily subcutaneous injections of D-galactose (1000 mg / kg) at a dose of 0.1 mL / mouse / day to establish a subacute aging model. Simultaneously with model establishment, the control and model groups were administered the same volume of saline via gavage daily. For the *Lactobacillus cereus* LeSK-508 live bacteria intervention group, *Lactobacillus cereus* LeSK-508 live bacteria suspension (dose of 1...) was administered via gavage. 10 9(CFU / mL / mouse / day); For the *Lactobacillus cereus* LeSK-508 inactivated bacteria intervention group: *Lactobacillus cereus* LeSK-508 inactivated bacteria solution was administered by gavage (same bacterial volume as the live bacteria group, treated at 65℃ for 30 min), and the intervention was continued for 6 weeks. At the end of the experiment, the stratum corneum water content and skin elasticity of the back of each mouse were measured using a skin moisture meter (equipped with a Corneometer CM825 stratum corneum moisture measurement probe) from Courage+Khazaka electronic GmbH (CK), Germany.

[0073] After a 6-week experiment, mice were euthanized, and blood was collected from their eyeballs. The blood was allowed to stand for 40 minutes, then centrifuged at 3000 rpm for 20 minutes, and the supernatant was collected for analysis. Skin tissue from the back was excised, and PBS was added at a 1:10 weight-to-volume ratio. The tissue was homogenized, centrifuged at 3000 rpm for 20 minutes, and the skin supernatant was collected for analysis. The AGE (advanced glycation end products) content in mouse serum and skin supernatant was detected using a corresponding human advanced glycation end products ELISA kit.

[0074] Experimental results are as follows Figure 3 As shown, intervention with *Lactobacillus sacchariformis* strain LeSK-508 significantly improved the skin physiological state of aging mice. Specifically, the results of the stratum corneum moisture content among skin physiological indicators ( Figure 3 A) showed that the moisture content of the stratum corneum in the skin of mice in the model group decreased significantly to 39.0%, while that in the live *Lactobacillus sacchariformis* LeSK-508 intervention group significantly increased to 61.5% (P < 0.001). Although the effect of the dead *Lactobacillus sacchariformis* LeSK-508 intervention group was not as good as the live group, it still showed a significantly better stratum corneum moisture content than the model group (49.3%), indicating that this strain has significant moisturizing and repairing functions. Skin elasticity results ( Figure 3 B) shows that the R2 value of skin elasticity in the model group mice decreased to 27.6%, while the R2 value of the live Lactobacillus sacchariformis LeSK-508 intervention group significantly increased to 74.6%, and the R2 value of the dead Lactobacillus sacchariformis LeSK-508 intervention group also significantly increased to 59.9%, indicating that this strain can effectively improve skin laxity and increase skin elasticity.

[0075] like Figure 3 As shown in C and 3D, the AGE levels in the serum and skin tissue of mice in the D-galactose model group were significantly increased, indicating an increased level of glycosylation in vivo. Compared with the model group, the *Lactobacillus cereus* LeSK-508 live bacteria intervention group showed a significant anti-glycation effect. Serum AGE levels (…) Figure 3(C) showed that the serum AGE content in the model group mice was approximately 375.6 ng / L. In contrast, the serum AGE content in the live *Lactobacillus cereus* LeSK-508 intervention group was significantly reduced to 145.2 ng / L, and the serum AGE content in the dead *Lactobacillus cereus* LeSK-508 intervention group was 167.2 ng / L, all approaching the normal range. Skin AGE content results ( Figure 3 D) showed that the AGE content in the skin tissue of the model group mice surged to about 414.5 ng / L. In contrast, the live Lactobacillus sakebi LeSK-508 intervention group significantly inhibited it to about 273.9 ng / L, and the dead Lactobacillus sakebi LeSK-508 intervention group had a level of 333.3 ng / L, indicating that this strain also has the ability to block glycosylation in skin tissue.

[0076] The experimental data above confirm that the Lactobacillus sacchariformis LeSK-508 strain can effectively reduce the level of systemic glycosylation in aging mice and significantly reduce the accumulation of AGEs in skin tissue through oral administration. This is an important material basis for its anti-aging effects.

[0077] Example 6: Effects of Lactobacillus saccharidonis LeSK-508 on the molecular mechanisms of aging mice All samples involved in this embodiment were derived from Example 5. After the experiment in Example 5, mice were euthanized, skin tissue was collected, skin RNA was extracted using the Trizol method, and reverse transcribed into cDNA. Using GAPDH as an internal reference gene, RT-qPCR technology was used to detect the gene expression of skin RAGE gene, SOD superoxide dismutase, and key target sites in the synthesis and degradation of type 3 collagen.

[0078] like Figure 4 As shown, intervention with *Lactobacillus spp.* LeSK-508 significantly improved the skin condition of aging mice and regulated the expression of related genes. Specifically, RAGE expression results ( Figure 4 A) showed that RAGE expression was significantly increased (4.2-fold) in the model group, while it was significantly downregulated (0.5-fold) in the *Lactobacillus cereus* LeSK-508 live bacteria intervention group compared to the control group (P < 0.001), indicating that it can block the AGE-induced inflammatory cascade by downregulating RAGE expression. Antioxidant capacity results ( Figure 4 (B) The results showed that the expression level of superoxide dismutase (SOD) in the model group decreased by 0.5-fold, while it significantly increased by 2.3-fold in the live bacteria intervention group compared with the control group (P < 0.001), and by 1.1-fold in the dead bacteria group, indicating that live bacteria can more effectively activate the skin's antioxidant defense system. Collagen synthesis results ( Figure 4C) showed that the mRNA expression level of type III collagen (COL3A1) in the *Lactobacillus thuringiensis* LeSK-508 intervention group was significantly upregulated by approximately 1.4 times compared to the control group, suggesting that it can improve elasticity by promoting collagen synthesis. Matrix metalloproteinases (MMPs) Figure 4 D) The results showed that the expression level of MMP9 in the model group increased by 4.9 times compared with the blank group, while the expression level of Lactobacillus sacchariflorus LeSK-508 live bacteria in the intervention group was significantly downregulated by 0.6 times compared with the blank group, indicating that it can inhibit collagen degradation by downregulating the expression of matrix metalloproteinases.

[0079] The above experimental results indicate that the *Lactobacillus sacchariformis* strain LeSK-508 exerts its anti-aging effects through a multi-target mechanism: on the one hand, it can significantly inhibit the production of AGEs in vivo, downregulate RAGE expression, and block the glycation damage pathway; on the other hand, it can activate the skin's antioxidant system (increase SOD) and maintain collagen homeostasis by upregulating COL3A1 and downregulating MMP9. Furthermore, the overall efficacy of live bacteria is significantly better than that of dead bacteria, suggesting that its active metabolites or live colonization may play a key role in the anti-aging process.

[0080] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A type of widely distributed lactobacillus in sake ( Latilactobacillus sakei The *Lactobacillus sacchariflorus* strain is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 38652 and accession date of May 12, 2026.

2. A composition wherein, The composition comprises at least one of the *Lactobacillus sacchariformis* strains according to claim 1 or 2, or their culture supernatant; preferably, the *Lactobacillus sacchariformis* strain is in live or inactivated form, more preferably in live form.

3. The composition according to claim 2, wherein, The composition also includes a cosmetically, edible, or pharmaceutically acceptable carrier or excipient.

4. The composition according to claim 2 or 3, wherein, The composition is administered orally or topically.

5. The composition according to any one of claims 2-4, wherein, The composition is formulated as a solution, tablet, lozenge, powder, granule, capsule, gel, cream, emulsion, spray, or patch.

6. The composition according to any one of claims 2-5, wherein, The composition is formulated as a functional food, dietary supplement, pharmaceutical preparation, cosmetic, or skin care preparation.

7. The composition according to claim 6, wherein, The functional food or dietary supplement is an anti-glycation, anti-oxidation and / or anti-aging functional food or dietary supplement; preferably, the functional food or dietary supplement is an instant probiotic powder, probiotic compressed candy, probiotic oral liquid, probiotic capsule, or probiotic dairy product.

8. Use of the *Lactobacillus thuringiensis* strain from sake according to claim 1, or the composition according to any one of claims 2-7, in at least one of the following aspects: a) Anti-glycation; b) Antioxidant; c) Maintain collagen homeostasis; d) Anti-aging.

9. The use according to claim 8, wherein, The uses include improving dull, lackluster, or rough skin caused by skin glycation or lack of sleep, and / or improving skin elasticity loss, sagging, or wrinkles caused by collagen glycation.

10. Use of the *Lactobacillus sacchariflorus* strain according to claim 1, or the composition according to any one of claims 2-7, in the preparation of a medicament for the prevention or relief of chronic skin damage or thinning caused by abnormal glucose metabolism in the body.

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