Application of stenotrophomonas maltophilia metabolite and product of stenotrophomonas maltophilia metabolite

Abstract

The invention discloses application of a stenotrophomonas maltophilia metabolite and a product of the stenotrophomonas maltophilia metabolite. The invention identifies a skin symbiotic bacterium, namely stenotrophomonas maltophilia, and provides an application of a metabolite of the stenotrophomonas maltophilia in anti-aging. The metabolite can regulate the balance of oxidized glutathione and glutathione in cells, improve the oxidation-reduction state in the cells, and regulate the internal oxidation-reduction balance and damage repair from the cellular level. The anti-aging scheme can fundamentally adjust the essential process of aging, and is high in effectiveness and good in persistence. The bacterial strain is a skin symbiotic bacterium, the metabolite of the bacterial strain is naturally compatible with the skin microecology, the adverse reaction can be reduced, the safety is good, and the affinity is high. The anti-aging effect of the stenotrophomonas maltophilia metabolite provided by the invention is verified by multi-dimensional experiments with quantitative indexes, and the stenotrophomonas maltophilia metabolite has strong scientificity and high reliability. The application scheme also provides another thought for developing more safe, effective and lasting anti-aging products, and the application potential is huge.

Description

Technical Field

[0001] The present invention relates to the field of microbial technology, and in particular to an application of a metabolite of Stenotrophomonas maltophilia and a product thereof. Background Art

[0002] Aging is an inevitable physiological process that occurs in cells and tissues under the influence of long-term physiological and environmental factors. During the aging process, oxidative stress and the accumulation of free radicals are one of the important reasons for the decline of cell function and tissue damage. Glutathione (GSH), also known as reduced glutathione, is an important antioxidant molecule in cells. It removes oxides by reacting with free radicals, while oxidized glutathione (GSSG) is its oxidized product. The accumulation of GSSG aggravates oxidative damage and further promotes aging. The intracellular GSH / GSSG ratio is an important marker of the cell's antioxidant capacity.

[0003] In the related art, most anti-aging products rely on exogenous antioxidant ingredients, such as traditional antioxidant ingredients such as vitamin C, vitamin E, tea polyphenols, and coenzyme Q10. These ingredients can neutralize free radicals on the surface of the skin by external application, prevent them from damaging the skin, and protect the skin from oxidative stress, thereby slowing down the aging process. However, due to the existence of the skin barrier, the absorption efficiency of these exogenous ingredients is not high, and the scope of action is limited to the surface of the skin, and it is difficult to reach the deep skin layer effectively, and it is impossible to fundamentally regulate the overall antioxidant capacity of the skin. Moreover, these exogenous ingredients are usually removed or metabolized over time, so their effects are usually temporary, and the antioxidant effect is often not lasting. There are problems such as limited skin absorption and poor persistence, which require frequent use and limited effect. There are also some anti-aging solutions that increase the antioxidant capacity of the skin and delay aging by activating the skin's own antioxidant enzyme system (such as superoxide dismutase, catalase, etc.), or supplement some endogenous antioxidant substances (such as glutathione, etc.) through oral or topical use to try to enhance the antioxidant capacity of the skin and slow down the aging process. However, the above schemes are also limited in effect: supplementing endogenous antioxidants (such as glutathione) can improve the antioxidant capacity of the skin in some cases, but its effect is often limited by bioavailability. For example, when glutathione is taken orally, due to its decomposition in the digestive system, the skin actually absorbs less effective ingredients; and when applied topically, the absorption rate is also low due to the influence of the skin barrier function. There are also anti-aging schemes that use hormone substances (such as estrogen or estrogen-like ingredients) to fight aging. Although this method can delay aging to a certain extent, it is usually accompanied by side effects, such as skin irritation or hormone imbalance, and long-term use may have a negative impact on health. In addition, many current anti-aging products or technologies are mainly focused on quickly alleviating or inhibiting the external signs of aging in the short term (such as reducing wrinkles and enhancing skin elasticity), but lack fundamental regulation of the essential process of aging, and often fail to touch the core mechanism of aging phenomena, such as the redox balance inside cells and the damage repair process. Especially at the cellular level, the current schemes are difficult to directly affect the redox state inside cells and the repair of cell damage during the aging process, which also leads to the lack of durability of the anti-aging effects of the above schemes.

[0004] Therefore, it is of great significance to solve the problems of low absorption rate and poor persistence of exogenous antioxidant ingredients, limited bioavailability of endogenous antioxidants, and side effects of hormone substances in current anti-aging products, and to develop anti-aging strategies that regulate the redox balance and damage repair inside cells from the cellular level, and provide anti-aging solutions that are highly effective, lasting, have no obvious side effects, are safe, and can regulate the essential process of aging. Summary of the invention

[0005] The present invention aims to solve at least one of the technical problems existing in the prior art. To this end, the present invention proposes an application of a metabolite of Stenotrophomonas maltophilia and its products, aiming to solve the problems of low absorption rate and poor persistence of exogenous antioxidant ingredients, limited bioavailability of endogenous antioxidant substances, and side effects of hormone substances existing in current anti-aging products.

[0006] An embodiment of the first aspect of the present invention provides an application of a metabolite of Stenotrophomonas maltophilia in anti-aging, wherein the application is for non-diagnostic or therapeutic purposes.

[0007] According to the application of the embodiment of the first aspect of the present invention, at least the following beneficial effects are achieved: the present invention first identifies a skin symbiotic bacterium, Stenotrophomonas maltophilia, and provides the application of its metabolites in anti-aging. Unlike most anti-aging technologies that use antioxidant supplementation and other means, the above-mentioned Stenotrophomonas maltophilia metabolites are unique in their anti-aging mechanism of action. They focus on regulating the balance of intracellular oxidized glutathione and glutathione, improving the intracellular redox state, and providing an anti-aging strategy for regulating the redox balance and damage repair inside cells at the cellular level. The above-mentioned anti-aging scheme can fundamentally regulate the essential process of aging, with strong effectiveness and good persistence. Secondly, the strain involved in the present invention is a skin symbiotic bacterium, and its metabolites are naturally compatible with the skin microecology, which can reduce side effects or adverse reactions, have good safety, high affinity, and are more advantageous than anti-aging schemes that rely on exogenous supplementation. Moreover, the anti-aging effect of the Stenotrophomonas maltophilia metabolites provided by the present invention has been verified by experiments with multi-dimensional and quantitative indicators, and is scientific and reliable. In addition, the above application scheme also provides another idea for developing more safe, effective and long-lasting anti-aging products. It has great application potential. It can not only be used to develop skin care products and drugs based on this metabolite to delay skin aging or treat aging-related skin diseases, but also can be used to develop skin repair drugs. It is even expected to realize personalized anti-aging solutions, which has extremely high scientific research significance and practical value.

[0008] Skin commensal bacteria play an important role in maintaining the balance of skin microecology. Their metabolites help regulate skin immunity and improve skin barrier function, which is very important for maintaining skin health. Current anti-aging programs often ignore the role of skin microbiota, basically do not have the function of regulating skin microbiota, and ignore the potential role of skin commensal bacteria in anti-aging. Therefore, exploring the application of skin commensal bacteria and their metabolites in anti-aging has important theoretical significance and practical value.

[0009] Based on this, the present invention provides an application of a metabolite of Stenotrophomonas maltophilia in skin anti-aging, wherein the application is for non-diagnostic or therapeutic purposes.

[0010] The identified Stenotrophomonas maltophilia, also known as Stenotrophomonas maltophilia, scientific name: Stenotrophomonas maltophilia, is widely present in water, soil, and animals, and is also a skin commensal bacteria. The strain can be directly purchased from Beina Biotech (BNCC, item number BNCC185982) or other commercial channels.

[0011] The identification process of the skin commensal bacteria in the present invention is as follows: Figure 1 As shown. As shown in the figure, the present invention uses metagenomic analysis technology to culture and perform metagenomic analysis on a large number of skin microbial samples, and preliminarily identifies a skin commensal bacterium with the potential to clear intracellular oxidized glutathione content and anti-aging function - Stenotrophomonas maltophilia. The specific steps are as follows: First, a multi-index test (such as skin elasticity, wrinkle score, oil content, etc.) is performed on the facial skin of multiple female volunteers, and the volunteers are divided into a younger group and an older group according to the results; then, the facial skin swab samples of the volunteers are cultured, and the obtained microbial community is subjected to metagenomic detection and non-targeted metabolomics detection, and then its microbial components and metabolite components are analyzed; finally, through a multi-faceted analysis of aging-related multi-omics biomarkers, the above-mentioned skin commensal bacteria with the potential to clear intracellular oxidized glutathione content and anti-aging function are finally obtained.

[0012] After the preliminary identification of the above-mentioned skin symbiotic bacteria, the present invention further verified the role of its metabolites in skin anti-aging. The technical process diagram is shown in FIG. Figure 2 . First, the above-mentioned Stenotrophomonas maltophilia was cultured and its metabolites were collected. By setting different action times and dose gradients, the metabolites were co-incubated with human skin fibroblasts. After the incubation was completed, the changes in the content of oxidized glutathione in skin cells were detected using GSH and GSSG detection kits. The results showed that the metabolites of Stenotrophomonas maltophilia could significantly reduce the content of oxidized glutathione in cells. Furthermore, the cell aging was detected by the β-galactosidase method. The results showed that the aging characteristics of skin cells treated with Stenotrophomonas maltophilia metabolites were significantly improved, confirming its anti-aging effect. In addition, the present invention also deeply explored the molecular mechanism behind it through bulk RNA sequencing, revealing the effects of Stenotrophomonas maltophilia metabolites on the expression of key genes, including upregulation of genes such as GCLM, PGD, SOD2 and NQO1, which play an important role in enhancing GSH synthesis and alleviating oxidative stress-induced skin aging. Through these molecular-level evidences, the present invention not only confirms the anti-aging effects of Stenotrophomonas maltophilia metabolites, but also provides in-depth molecular-level insights for understanding its mechanism of action.

[0013] In some embodiments of the present invention, the Stenotrophomonas maltophilia includes but is not limited to homologous strains, mutant strains, genetically engineered strains, and the like.

[0014] In some embodiments of the invention, the senescence comprises senescence and / or aging of skin cells or tissues.

[0015] In some embodiments of the present invention, the aging includes an increase in the content of oxidized glutathione in skin cells. The accumulation of GSSG exacerbates oxidative damage and further promotes skin aging. The intracellular GSH / GSSG ratio is an important marker of the cell's antioxidant capacity.

[0016] In some embodiments of the present invention, the anti-aging comprises reducing the intracellular oxidized glutathione content and / or improving the intracellular redox state.

[0017] In some embodiments of the present invention, the anti-aging comprises reducing the activity of β-galactosidase in cells.

[0018] In some embodiments of the present invention, the anti-aging comprises up-regulating the expression of SOD2 gene, NQO gene, TXN gene, FTH1 gene, JUN gene, APCDD1 gene, or SFRP2 gene.

[0019] In some embodiments of the present invention, the method for preparing the metabolites of Stenotrophomonas maltophilia comprises the steps of: shaking culturing the Stenotrophomonas maltophilia in a liquid culture medium for 48 to 72 hours; after the culturing, freezing and centrifuging the bacterial solution, taking the supernatant and filtering it to obtain a solution containing the metabolites of Stenotrophomonas maltophilia.

[0020] An embodiment of the second aspect of the present invention provides a use of a metabolite of Stenotrophomonas maltophilia in the preparation of an oxidized glutathione inhibitor, wherein the use is for non-diagnostic or therapeutic purposes.

[0021] An embodiment of the third aspect of the present invention provides an oxidized glutathione inhibitor, wherein the oxidized glutathione inhibitor comprises a metabolite of Stenotrophomonas maltophilia.

[0022] The Stenotrophomonas maltophilia metabolite provided by the present invention can significantly inhibit the content of intracellular oxidized glutathione, regulate the balance of oxidized glutathione and glutathione, and improve the intracellular redox state.

[0023] The embodiment of the fourth aspect of the present invention provides the use of a metabolite of Stenotrophomonas maltophilia in at least one of (a1) to (a7):

[0024] (a1) developing and / or preparing anti-aging products;

[0025] (a2) Developing and / or preparing skin antioxidant products;

[0026] (a3) developing and / or preparing skin care products;

[0027] (a4) preparing products for preventing and / or delaying skin aging;

[0028] (a5) preparing products for preventing and / or delaying skin aging;

[0029] (a6) Developing and / or preparing skin repair drugs;

[0030] (a7) Developing and / or preparing drugs for treating aging-related skin diseases.

[0031] According to the application of the fourth aspect of the present invention, there are at least the following beneficial effects: the present invention discloses the application of the metabolites of the above-mentioned strains in regulating the content of oxidized glutathione in cells and improving the redox state in cells, as well as its application in anti-aging related cell and skin care products and medical fields, such as developing skin care products and drugs based on the metabolites to delay skin aging or treat skin diseases related to aging. The application scheme of the above-mentioned Oligotrophomonas maltophilia metabolites focuses on regulating the balance of oxidized glutathione and glutathione in cells, improving the redox state in cells, and providing an anti-aging strategy for regulating the redox balance and damage repair inside cells at the cellular level. The above-mentioned application scheme can fundamentally regulate the essential process of aging, with strong effectiveness and good persistence. Moreover, the above-mentioned strains are skin symbiotic bacteria, and their metabolites are naturally compatible with the skin microecology, which can reduce side effects or adverse reactions, have good safety and high affinity. The above application scheme also provides another idea for developing more safe, effective and long-lasting anti-aging products. It has great application potential. It can not only be used to develop skin care products and drugs based on this metabolite to delay skin aging or treat aging-related skin diseases, but also can be used to develop skin repair drugs. It is even expected to realize personalized anti-aging solutions, which has extremely high scientific research significance and practical value.

[0032] The embodiment of the fifth aspect of the present invention provides at least one product of (b1) to (b7):

[0033] (b1) Skin anti-aging products;

[0034] (b2) Skin antioxidant products;

[0035] (b3) Skin care products;

[0036] (b4) products for preventing and / or delaying skin aging;

[0037] (b5) Products for preventing and / or delaying skin aging;

[0038] (b6) Skin repair drugs;

[0039] (b7) Drugs for treating skin diseases associated with aging;

[0040] The active ingredients of the product include Stenotrophomonas maltophilia metabolites.

[0041] According to the product of the fifth aspect of the present invention, there are at least the following beneficial effects: the product or medicine provided by the present invention, the active ingredient includes a metabolite of Stenotrophomonas maltophilia, and the metabolite of the above strain can regulate the intracellular oxidized glutathione content and improve the intracellular redox state. Various anti-aging related products or drugs developed based on the metabolite focus on regulating the balance of intracellular oxidized glutathione and glutathione, improving the intracellular redox state, and can regulate the internal redox balance and damage repair of cells at the cellular level, with strong effectiveness and good persistence. Moreover, the above strain is a skin commensal bacteria, and its metabolites are naturally compatible with the skin microecology, which can reduce side effects or adverse reactions, have good safety, and high affinity.

[0042] In some embodiments of the present invention, the product is an external preparation prepared by using the metabolites of Stenotrophomonas maltophilia as active ingredients and adding excipients or auxiliary ingredients acceptable in medicine or cosmetics.

[0043] In some embodiments of the present invention, the external preparation comprises a cosmetic or a skin care product.

[0044] In some embodiments of the present invention, the cosmetics or skin care products include skin cream, essence, emulsion, lotion, face cream, eye cream, freeze-dried powder, gel, foundation, pressed powder, facial cleanser, or facial mask.

[0045] In some embodiments of the present invention, the acceptable excipients include at least one of a diluent, a binder, a wetting agent, a humectant, a thickener, a solvent, an emulsifier, a cosolvent, a preservative, a pH adjuster, an osmotic pressure regulator, a surfactant, and a buffer.

[0046] In some embodiments of the present invention, the auxiliary ingredients include at least one of a whitening agent, an emollient, an anti-acne agent, an ultraviolet light absorber, and a skin conditioning agent.

[0047] In some embodiments of the present invention, the auxiliary ingredients also include at least one of collagen, vitamins, tea polyphenols, and coenzyme Q10.

[0048] An embodiment of the sixth aspect of the present invention provides a method for preparing the above-mentioned Stenotrophomonas maltophilia metabolites, comprising the steps of: shaking culturing the Stenotrophomonas maltophilia in a liquid culture medium for 48 to 72 hours; after the culturing, freezing and centrifuging the bacterial liquid, taking the supernatant and filtering it to obtain a solution containing the Stenotrophomonas maltophilia metabolites.

[0049] In some embodiments of the present invention, the liquid culture medium is brain heart infusion liquid culture medium (BHI).

[0050] Specifically, the components of the brain heart infusion liquid culture medium include: 5.0g of beef heart infusion powder, 12.5g of beef brain infusion powder, Peptone (Proteose Peptone) 10.0g, Glucose 2.0g, NaCl 5.0g, Na 2 HPO 4 2.5g, distilled water 1000.0mL. Adjust pH to 7.4, 121℃, sterilize for 15min.

[0051] In some embodiments of the present invention, the Stenotrophomonas maltophilia is cultured with shaking at 37° C. and 220 rpm for 48 to 72 hours.

[0052] In some embodiments of the present invention, the concentration of Stenotrophomonas maltophilia is 10 6 ~10 7 A bacteria.

[0053] In some embodiments of the present invention, the bacterial solution is centrifuged at 10,000 g and 4° C. for 20 minutes using a high-speed refrigerated centrifuge. After the centrifugation, the supernatant is filtered through a 0.22 μm bacterial filter to obtain a solution containing metabolites of Stenotrophomonas maltophilia.

[0054] In some embodiments of the present invention, activation of bacterial strains is also included.

[0055] Specifically, the activation of the strain includes the following steps: in a sterile clean bench, use an inoculation loop to pick up a small amount of frozen strains, inoculate them onto a brain heart infusion solid culture medium, and gently draw lines to evenly distribute the strains on the slant of the culture medium; then place the inoculated slant culture medium in a 37°C constant temperature incubator and culture upright for 24 to 48 hours until a single colony grows.

[0056] Specifically, the components of the brain heart infusion solid culture medium include: 5.0g of beef heart infusion powder, 12.5g of beef brain infusion powder, Peptone (Proteose Peptone) 10.0g, Glucose 2.0g, NaCl 5.0g, Na 2 HPO 42.5g, agar 15.0g, distilled water 1000.0mL. Adjust pH to 7.4, 121℃, sterilize for 15min.

[0057] Other features and advantages of the present invention will be described in the following description, and partly become apparent from the description, or understood by practicing the present invention. The purpose and other advantages of the present invention can be realized and obtained by the structures particularly pointed out in the description, claims and drawings. BRIEF DESCRIPTION OF THE DRAWINGS

[0058] Figure 1 A schematic diagram of the identification process of the skin commensal bacteria Stenotrophomonas maltophilia provided in an embodiment of the present invention;

[0059] Figure 2 A schematic diagram of the functional verification process of the Stenotrophomonas maltophilia metabolites provided in an embodiment of the present invention;

[0060] Figure 3 A schematic diagram of the detection result of intracellular glutathione content provided by an embodiment of the present invention;

[0061] Figure 4 A schematic diagram of the intracellular β-galactosidase detection results provided in an embodiment of the present invention;

[0062] Figure 5 Schematic diagram of bulk RNA sequencing results and KEGG enrichment analysis results provided in the embodiments of the present invention. DETAILED DESCRIPTION

[0063] The following will be combined with the embodiments to clearly and completely describe the concept of the present invention and the technical effects produced, so as to fully understand the purpose, characteristics and effects of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, other embodiments obtained by those skilled in the art without creative work are all within the scope of protection of the present invention.

[0064] In the description of the present invention, the description with reference to the terms "one embodiment", "some embodiments", "illustrative embodiments", "examples", "specific examples", or "some examples" means that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic representation of the above terms does not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described may be combined in any one or more embodiments or examples in a suitable manner.

[0065] In the description of the present invention, unless otherwise specified, the numerical range "a to b" represents an abbreviation of any real number combination between a and b, wherein a and b are real numbers. Unless otherwise specified, each reaction or operation step may be performed in sequence or not. Preferably, the reaction method of the present invention is performed in sequence.

[0066] The experimental methods in the following examples without specifying specific conditions are usually carried out under conventional conditions or under conditions recommended by the manufacturers. The materials and reagents used in the examples are commercially available unless otherwise specified.

[0067] Example 1 Cultivation of Stenotrophomonas maltophilia and Obtaining Metabolites

[0068] In this example, metagenomic analysis was performed on facial skin swab samples of multiple female volunteers using metagenomic analysis technology, and a strain of Stenotrophomonas maltophilia with the potential to clear intracellular oxidized glutathione content and anti-aging function was preliminarily identified. The identification process of the above skin commensal bacteria is as follows: Figure 1 The specific steps are as follows: First, a multi-index test (such as skin elasticity, wrinkle score, oil content, etc.) was performed on the facial skin of several Chinese female volunteers, and the volunteers were divided into a younger group (52 people in total) and an older group (51 people in total) according to the results; then, the facial skin swab samples of the volunteers were cultured, and the obtained microbiome was subjected to metagenomics and non-targeted metabolomics testing, and then its microbial composition and metabolite composition were analyzed; finally, through a comprehensive analysis of aging-related multi-omics biomarkers, the above-mentioned skin commensal bacteria with the potential to clear intracellular oxidized glutathione content and anti-aging function were finally obtained.

[0069] Afterwards, the corresponding strains were purchased from Beina Biotechnology (BNCC, catalog number BNCC185982) and functionally verified. The functional verification process is as follows: Figure 2 shown.

[0070] First, the strain is revived and cultured. The specific steps are as follows:

[0071] (1) In a sterile clean bench, use an inoculation loop to pick up a small amount of the purchased frozen strain and inoculate it onto a solid slant medium of brain heart infusion. Gently draw lines to evenly distribute the strain on the slant. Then, place the inoculated slant medium in a 37°C constant temperature incubator and culture it upright for 24 to 48 hours until a single colony grows, completing the strain activation process.

[0072] (2) Pick a single colony after activation and place it in brain heart infusion liquid culture medium. Shake and culture it at 37°C and 220 rpm for 48 to 72 hours to ensure that the bacteria are in good growth condition and the concentration reaches 10 per ml. 6~10 7 About 100 bacteria.

[0073] (3) After the culture is completed, the bacterial solution is centrifuged at 10,000 g and 4°C for 20 minutes using a high-speed refrigerated centrifuge. After the centrifugation, the lower layer is the bacterial precipitate, and the upper layer is the culture supernatant containing the strain metabolites. The supernatant is filtered with a 0.22 μm bacterial filter and stored for subsequent functional verification.

[0074] Example 2 Co-culture of Stenotrophomonas maltophilia metabolites with skin cells

[0075] In this example, human skin fibroblasts were selected as the research object (the acquisition method complies with relevant regulations), and DMEM / F12 culture medium containing 10% fetal bovine serum and 1% penicillin-streptomycin was used, and placed in a 37° C., 5% carbon dioxide incubator for routine culture.

[0076] In this example, "H 2 O 2 " to simulate the elderly group, without adding "H 2 O 2 " is used to represent the young group as the control group. When the cell density reached more than 80%, the filtered supernatant of Stenotrophomonas maltophilia obtained in Example 1 was diluted in the culture medium at a ratio of 1:100, and the cells were stimulated for 24 hours as the treatment group; at the same time, the cells stimulated with bacterial culture medium (brain heart infusion liquid culture medium, BHI) were used as the untreated group.

[0077] Example 3 Detection of intracellular glutathione content

[0078] After the cells in Example 2 were treated for 24 hours, the cells were collected and the intracellular glutathione content was detected using a GSH and GSSG detection kit (purchased from Beyotime, item number S0053). The specific operation was carried out according to the instructions of the kit.

[0079] The results are as follows Figure 3 As shown in the figure, “H 2 O 2 " represents cells using H 2 O 2 After treatment, BHI was added for stimulation; “H 2 O 2 + Stenotrophomonas maltophilia metabolites" represents the cell H 2 O 2 After treatment, the filtered supernatant of Stenotrophomonas maltophilia was added for stimulation; “Control + Stenotrophomonas maltophilia metabolites” represents cells not treated with H 2 O 2Treatment, only the filtered supernatant of Stenotrophomonas maltophilia was added for stimulation; "control" represents cells not treated with H 2 O 2 Treatment, only BHI was added for stimulation. Figure 3 It can be seen from A and B that "H 2 O 2 "The content of reduced glutathione (GSH) in the aged cells treated with simulation was lower than that in the young cells, while the content of oxidized glutathione (GSSG) was higher than that in the young cells, indicating that the aged cells suffered more oxidative stress. The metabolites of Stenotrophomonas maltophilia can increase the content of GSH in cells, remove GSSG in cells, and increase the GSH / GSSG ratio in cells ( Figure 3 These data suggest that Stenotrophomonas maltophilia has some antioxidant capacity.

[0080] Example 4 Intracellular β-galactosidase detection

[0081] After 24 hours of cell treatment in Example 2, the cells were collected and stained using a cell senescence detection kit (β-galactosidase method) (purchased from Yacoin, item number KTA3030). After staining, the cell staining was observed under an optical microscope. Cells with senescence characteristics will be stained blue (because β-galactosidase activity is enhanced in senescent cells, it can decompose the substrate X-Gal to produce a blue product). Multiple fields of view are randomly selected, and the proportion of the number of cells stained blue in each field of view to the total number of cells is counted, which is used as a measure of the degree of cell senescence.

[0082] The results are as follows Figure 4 As shown in the figure, “H 2 O 2 " represents cells using H 2 O 2 After treatment, BHI was added for stimulation; “H 2 O 2 + Stenotrophomonas maltophilia metabolites" represents the cell H 2 O 2 After treatment, the filtered supernatant of Stenotrophomonas maltophilia was added for stimulation; “Control + Stenotrophomonas maltophilia metabolites” represents cells not treated with H 2 O 2 Treatment, only the filtered supernatant of Stenotrophomonas maltophilia was added for stimulation; "control" represents cells not treated with H 2 O 2 Treatment, only BHI was added for stimulation. Figure 4 As shown in A, both in the young group and in the “H 2 O 2"In the aged group treated with simulated treatment, after being treated with the metabolites of Stenotrophomonas maltophilia, the percentage of senescence-associated β-galactosidase (SA-β-Gal)-positive cells in both groups decreased, and the statistical results were significantly different ( Figure 4 (B) Overall, the metabolites of Stenotrophomonas maltophilia can reduce the activity of senescence-related β-galactosidase in cells and have a certain anti-aging effect.

[0083] Example 5 Bulk RNA Sequencing Experiment

[0084] (1) After 24 hours of cell treatment in Example 2, the cells were collected and RNA was extracted using an RNA extraction kit (purchased from Magen / Meiji Biotechnology, catalog number R4011-02). The concentration and purity of RNA were detected by a nucleic acid quantifier (OD of RNA). 260 / OD 280 The ratio is between 1.8 and 2.2, indicating good purity and can be used for subsequent experiments). The integrity of RNA was further evaluated by agarose gel electrophoresis: 1 to 2 μg RNA sample was mixed with the loading buffer and electrophoresed in 1.5% agarose gel (electrophoresis voltage was 100 V and the electrophoresis time was about 20 to 30 minutes). The electrophoresis bands were observed. The RNA electrophoresis pattern showed clear 28S and 18S ribosomal RNA bands, and the brightness of the 28S band was about twice that of the 18S band, with no obvious diffusion phenomenon, indicating that the RNA integrity was good and met the sequencing requirements.

[0085] (2) Use the above RNA samples to construct a library according to the library construction kit requirements of the BGI RNA sequencing platform.

[0086] Specifically, the method comprises the following steps: a. using DNase I (NEB) to perform DNA digestion on the total RNA sample, adding 0.5M EDTA, denaturing at 75°C for 10 min, and cooling on ice; b. adding RNA Clean beads (Vazyme) to purify the sample; c. using mRNA Capture Beads (Vazyme) to enrich and purify the mRNA, then adding 5×HiScript II Buffer (Vazyme) and N6 primer (0.1μg / μL), and reacting at 94°C for 10 min; d. adding dNTP mix (10mM, Vazyme), RNase Inhibitor (40U / μL, Vazyme) and HiScript II (200U / μL, Vazyme) to the disrupted sample, and performing reverse transcription single-strand synthesis on a PCR instrument; e. adding 35μL reaction mix (5×second strand buffer (invitrogen), dNTP mix (10mM, Vazyme), RNaseH (5U / μL, Yeasen), DNA polI (5U / μL, Takara), Nuclease free water (invitrogen)), PCR instrument at 16℃ for 2h, and then use DNA CleanBeads (Vazyme) to purify the sample; f. End repair / addition of "A" and adapter connection: use T4 DNA Polymerase / Klenow DNA Polymerase / T4 Polynucleotide Kinase (Vazyme) to repair the ends and add dA tails, TaqDNA Polymerase (Vazyme); use AD153-primer and 2×KAPA HiFi HotStart ReadyMix (Vazyme) for PCR amplification, and then perform library purification, quantification and pooling, and then perform PE150 double-end sequencing on the MGI T7 platform.

[0087] (3) Perform quality control and preprocessing on the raw data obtained by sequencing (fastq format files), remove low-quality reads, adapter sequences, and repetitive sequences, etc., to obtain high-quality clean data, align the preprocessed clean data with the reference genome, use the alignment software STAR to determine the location of the sequencing reads on the genome, and then use the gene expression quantification software (Feature Counts) to count the number of reads corresponding to each gene, which is used as a measure of gene expression level to calculate the expression level of each gene in different experimental groups. Statistical methods (such as DESeq2, edgeR and other software packages) are used to perform differential expression analysis on the gene expression data between different experimental groups, and screen out genes with significant differential expression between different treatment groups (Fold Change>2 and FDR<0.05, indicating that the gene expression level is significantly different between the experimental group and the control group). Functional annotation and enrichment analysis were performed on the screened differentially expressed genes. The biological functions of the genes, the cellular processes they participated in, and the signaling pathways involved were annotated through databases (Gene Ontology (GO) database, Kyoto Encyclopedia of Genes and Genomes (KEGG) database) to further explore the effects of the metabolites of the skin commensal bacteria on the regulation of intracellular gene expression and the potential molecular mechanisms related to anti-aging.

[0088] The results of bulk RNA sequencing experiments are shown in Figure 5 As shown in the figure, "control + Stenotrophomonas maltophilia metabolites" represents the cells without H 2 O 2 Treatment, only the filtered supernatant of Stenotrophomonas maltophilia was added for stimulation; "control" represents cells not treated with H 2 O 2 treatment, and only BHI was added for stimulation. The results showed that in the group treated with Stenotrophomonas maltophilia metabolites, GCLM gene expression was upregulated, and this change could enhance the activity of glutamate-cysteine ​​ligase (GCL), thereby increasing the synthesis of intracellular glutathione (GSH); in addition, the increase in PGD gene expression promoted the supply of NADPH, which is a key cofactor for GSH reduction, which further provided strong support for the GSH cycle. These changes help cells resist oxidative stress and maintain the vitality of skin fibroblasts, thereby effectively preventing cell aging. These research results strongly confirm that Stenotrophomonas maltophilia can alleviate the aging process induced by oxidative stress by promoting the synthesis of GSH in skin cells.

[0089] Moreover, KEGG enrichment analysis data also showed that in the group treated with the metabolites of Stenotrophomonas maltophilia, the antioxidant enzyme genes SOD2 and NQO, as well as the antioxidant protein genes TXN and FTH1 were significantly upregulated, and several signaling pathways related to cell growth, regeneration, and tissue repair also showed enrichment. In addition, the high expression of JUN, APCDD1, and SFRP2 jointly regulated the Wnt signaling pathway, which plays a vital role in the proliferation, differentiation, and tissue repair of fibroblasts.

[0090] In summary, the present invention identified a specific skin commensal bacteria, Stenotrophomonas maltophilia, and found that the metabolites of this bacteria can significantly reduce the content of intracellular oxidized glutathione. Furthermore, the cell aging was detected by the β-galactosidase method. The results showed that the aging characteristics of skin cells treated with the bacterial metabolites were significantly improved, confirming its anti-aging effect. In addition, the present invention also deeply explored the molecular mechanism behind it through bulk RNA sequencing, revealing the effect of Stenotrophomonas maltophilia metabolites on the expression of key genes, including upregulation of genes such as GCLM, PGD, SOD2 and NQO1, which play an important role in enhancing GSH synthesis and reducing oxidative stress-induced skin aging. Through these molecular-level evidence, the present invention not only confirms the anti-aging effect of Stenotrophomonas maltophilia metabolites, but also provides in-depth molecular insights for understanding its mechanism of action.

[0091] The above is a specific description of the preferred implementation of the present application, but the present application is not limited to the above-mentioned implementation mode. Technical personnel familiar with the field can also make various equivalent deformations or substitutions without violating the spirit of the present application. These equivalent deformations or substitutions are all included in the scope defined by the claims of the present application.

Claims

1. An application of a metabolite of Stenotrophomonas maltophilia in anti-aging, wherein the application is for non-diagnostic or therapeutic purposes.

2. The use according to claim 1, characterized in that: The senescence includes senescence and / or aging of skin cells or tissues.

3. The use according to claim 1, characterized in that: Aging involves an increase in the level of oxidized glutathione within skin cells.

4. Use of a metabolite of Stenotrophomonas maltophilia in the preparation of an oxidized glutathione inhibitor, wherein the use is for non-diagnostic or therapeutic purposes.

5. An oxidized glutathione inhibitor, characterized in that The oxidative glutathione inhibitors include Stenotrophomonas maltophilia metabolites.

6. Use of a metabolite of Stenotrophomonas maltophilia in at least one of (a1) to (a7): (a1) developing and / or preparing anti-aging products; (a2) Developing and / or preparing skin antioxidant products; (a3) developing and / or preparing skin care products; (a4) preparing products for preventing and / or delaying skin aging; (a5) preparing products for preventing and / or delaying skin aging; (a6) Developing and / or preparing skin repair drugs; (a7) Developing and / or preparing drugs for treating aging-related skin diseases.

7. At least one of the following products (b1) to (b7): (b1) Skin anti-aging products; (b2) Skin antioxidant products; (b3) Skin care products; (b4) products for preventing and / or delaying skin aging; (b5) Products for preventing and / or delaying skin aging; (b6) Skin repair drugs; (b7) Drugs for treating skin diseases associated with aging; The active ingredients of the product include Stenotrophomonas maltophilia metabolites.

8. The product according to claim 7, characterized in that The product is an external preparation prepared by using the metabolites of Stenotrophomonas maltophilia as active ingredients and adding excipients or auxiliary ingredients acceptable in medicine or cosmetics.

9. The product according to claim 8, characterized in that The external preparations include cosmetics or skin care products; Preferably, the cosmetics or skin care products include skin cream, essence, lotion, toner, face cream, eye cream, freeze-dried powder, gel, foundation, pressed powder, facial cleanser, or facial mask; Preferably, the acceptable excipients include at least one of a diluent, an adhesive, a wetting agent, a humectant, a thickener, a solvent, an emulsifier, a cosolvent, a preservative, a pH regulator, an osmotic pressure regulator, a surfactant, and a buffer; Preferably, the auxiliary ingredients include at least one of a whitening agent, an emollient, an anti-acne agent, an ultraviolet light absorber, and a skin conditioning agent; Preferably, the auxiliary ingredients also include at least one of collagen, vitamins, tea polyphenols, and coenzyme Q10.

10. The method for preparing a metabolite of Stenotrophomonas maltophilia according to any one of claims 1 to 9, characterized in that: The method comprises the following steps: culturing the Stenotrophomonas maltophilia in a liquid culture medium with shaking for 48 to 72 hours; after the culturing, subjecting the bacterial liquid to refrigerated centrifugation, taking the supernatant and filtering it to obtain a solution containing metabolites of the Stenotrophomonas maltophilia.

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