Lactobacillus rhamnosus, fermentation liquor, preparation, preparation method and application

Abstract

The embodiment of the invention discloses lactobacillus rhamnosus, fermentation liquor, a preparation, a preparation method and application. The application of the lactobacillus rhamnosus can be expanded. Wherein the lactobacillus rhamnosus is preserved in the China General Microbiological Culture Collection Center, and the preservation number is CGMCC (China General Microbiological Culture Collection Center) No.31345.

Description

Technical Field

[0001] The present invention relates to the technical field of microorganisms, and specifically relates to Lactobacillus rhamnosus, fermentation broth, preparation, preparation method and uses. Background Art

[0002] Lacticaseibacillus Rhamnosus belongs to the genus Lactobacillus, and some species thereof have been publicly disclosed to be applicable to the preparation of topical skin products. For example, the application of the fermentation lysate of Lactobacillus rhamnosus LTHINK-0088 in resisting collagen loss (see the Chinese patent document with the publication number CN118620784A).

[0003] However, the types of Lactobacillus rhamnosus are still limited at present, which limits its application prospects. For example, there are few related studies on how to inhibit the expression of sebum by sebaceous gland cells with Lactobacillus rhamnosus to overcome the defect of greasy skin.

[0004] Therefore, how to obtain new Lactobacillus rhamnosus to expand the application of Lactobacillus rhamnosus is still a problem to be solved. Summary of the Invention

[0005] One object of the embodiments of the present application is to solve at least one problem in the above background and provide corresponding beneficial effects.

[0006] Another object of the embodiments of the present application is to provide a new Lactobacillus rhamnosus.

[0007] Another object of the embodiments of the present application is to provide a fermentation broth of Lactobacillus rhamnosus, which is fermented according to the said Lactobacillus rhamnosus.

[0008] Another object of the embodiments of the present application is to provide a preparation method of a fermentation broth of Lactobacillus rhamnosus, which can be used for preparing the fermentation of the said Lactobacillus rhamnosus.

[0009] Another object of the embodiments of the present application is to provide a use of Lactobacillus rhamnosus.

[0010] Another object of the embodiments of the present application is to provide a use of a fermentation broth of Lactobacillus rhamnosus.

[0011] Another object of the embodiments of the present application is to provide a preparation for skin.

[0012] The embodiments of the present application mainly achieve the above objects through the following technical solutions.

[0013] In a first aspect, an embodiment of the present application provides a Lactobacillus rhamnosus, which is deposited in the China General Microbiological Culture Collection Center (CGMCC), with the deposit number: CGMCC No. 31345, and the deposit date is July 18, 2024.

[0014] In a second aspect, an embodiment of the present application provides a Lactobacillus rhamnosus fermentation broth, which is obtained by fermenting the Lactobacillus rhamnosus described in the first aspect.

[0015] In a third aspect, an embodiment of the present application provides a method for preparing a Lactobacillus rhamnosus fermentation broth, which can be used to prepare the Lactobacillus rhamnosus fermentation broth described in the second aspect. The preparation method includes the following steps:

[0016] Obtain a Lactobacillus rhamnosus seed solution according to the Lactobacillus rhamnosus described in the first aspect;

[0017] Inoculate the Lactobacillus rhamnosus seed solution into an MRS medium to obtain an inoculum;

[0018] Ferment and culture the inoculum at a fermentation temperature of 35 - 40 °C, a shaking speed of 150 - 200 rpm, and a fermentation time of 18 - 48 hours to obtain a fermentation product;

[0019] Perform separation treatment on the fermentation product to obtain the liquid part after separation treatment, which is the Lactobacillus rhamnosus fermentation broth.

[0020] In a fourth aspect, an embodiment of the present application provides the use of the Lactobacillus rhamnosus fermentation broth described in the second aspect in the preparation of a preparation for the skin.

[0021] In some technical solutions of the fourth aspect, embodiments of the present application also provide the following further uses of the Lactobacillus rhamnosus fermentation broth.

[0022] In some technical solutions, the preparation has the use of inhibiting skin oil.

[0023] In some technical solutions, the preparation has the use of inhibiting the expression of skin cells to produce oil. It is beneficial to inhibit skin oil. Further, the skin cells are sebaceous gland cells.

[0024] In some technical solutions, the preparation has the use of inhibiting 5α - reductase.

[0025] In some technical solutions, the preparation has the use of antioxidation. It is beneficial to protect the skin.

[0026] In some technical solutions, the preparation has the use of scavenging ABTS (2,2'-Azinobis-(3-ethylbenzthiazoline-6-sulphonate)) free radicals.

[0027] In some technical solutions, scavenging ABTS free radicals is beneficial to achieving antioxidant use.

[0028] In some technical solutions, the preparation has the use of scavenging reactive oxygen species (ROS).

[0029] In some technical solutions, scavenging reactive oxygen species is beneficial for antioxidant.

[0030] In some technical solutions, the preparation has the use of repairing the skin.

[0031] In some technical solutions, the preparation has the use of repairing photo-damage. It is beneficial for repairing the skin.

[0032] In some technical solutions, the preparation has the use of preventing photo-damage. It is beneficial for repairing the skin.

[0033] In some technical solutions, the preparation has the use of repairing scratch damage. By repairing scratch damage, it is beneficial for skin repair.

[0034] In some technical solutions, the preparation has the use of promoting the migration ability of skin cells. It is beneficial for repairing scratch damage of the skin. Further, the skin cells are HaCaT cells.

[0035] In some technical solutions, the preparation has the use of anti-aging. It is beneficial for protecting the skin.

[0036] In some technical solutions, the preparation has the use of inhibiting the expression of β-galactosidase in skin cells. Further, the skin cells are HaCaT cells. β-galactosidase is a senescence marker, so inhibiting β-galactosidase is beneficial for anti-aging.

[0037] In some embodiments, the fermentation broth of Lactobacillus rhamnosus has the use of promoting the expression of type I collagen. Promoting the expression of type I collagen is beneficial for anti-aging.

[0038] In some technical solutions, the preparation has the use of inhibiting the expression of MMP-1 in skin cells. Further, the skin cells are HFF-1 cells.

[0039] In some technical solutions, the preparation has the use of inhibiting Propionibacterium acnes.

[0040] In some technical solutions, the preparation has the use of reducing the content of MDA in skin cells. By reducing the content of MDA in skin cells, the effect of anti-lipid peroxidation is achieved. Further, the skin cells are HaCaT cells.

[0041] In some technical solutions, the preparation is a cosmetic.

[0042] In some technical solutions, the preparation is a drug.

[0043] In a fifth aspect, an embodiment of the present application further provides a use of Lactobacillus rhamnosus in the preparation of a preparation for skin.

[0044] In some technical solutions, the preparation for skin includes the Lactobacillus rhamnosus fermentation broth described in the second aspect.

[0045] In some technical solutions, the preparation has one or more of the following uses:

[0046] Inhibiting skin oil;

[0047] Inhibiting the expression of skin oil in skin cells;

[0048] Inhibiting 5α-reductase;

[0049] Inhibiting Propionibacterium acnes;

[0050] Antioxidant;

[0051] Scavenging ABTS free radicals;

[0052] Scavenging reactive oxygen species;

[0053] Repairing skin photo-damage;

[0054] Preventing skin photo-damage;

[0055] Repairing scratch damage;

[0056] Promoting the migration ability of skin cells;

[0057] Anti-aging;

[0058] Inhibiting the expression of β-galactosidase in skin cells;

[0059] Promoting the expression of type I collagen;

[0060] Inhibiting the expression of MMP-1 in skin cells; and

[0061] Reducing the content of MDA in skin cells.

[0062] In another aspect, an embodiment of the present application further provides a use of the Lactobacillus rhamnosus described in the first aspect in the preparation of a Lactobacillus rhamnosus fermentation broth.

[0063] In another aspect, an embodiment of the present application provides a preparation for the skin, comprising:

[0064] The Lactobacillus rhamnosus described in the embodiment of the first aspect.

[0065] In another aspect, an embodiment of the present application provides a preparation for the skin, comprising:

[0066] The fermentation broth of Lactobacillus rhamnosus described in the embodiment of the second aspect.

[0067] The beneficial effects of the embodiments of the present invention include:

[0068] 1. The embodiment of the present application provides a new Lactobacillus rhamnosus, which enriches the varieties of Lactobacillus rhamnosus and can be used to expand the application of Lactobacillus rhamnosus, thereby solving the problems in the background technology.

[0069] 2. The new Lactobacillus rhamnosus provided by the embodiment of the present application can be used to inhibit the expression of oil by sebaceous gland cells, overcome the defect of greasy skin, and produce unexpected technical effects compared with the existing Lactobacillus rhamnosus, showing obvious progress.

[0070] 3. In some embodiments, the fermentation broth of Lactobacillus rhamnosus provided by the embodiment of the present application contains the metabolites of Lactobacillus rhamnosus and has the use of inhibiting skin oil. That is, it can be used to inhibit the expression of oil by sebaceous gland cells, overcome the defect of greasy skin, achieve unexpected technical effects, and show obvious progress.

[0071] 4. The fermentation broth of Lactobacillus rhamnosus provided by the embodiment of the present application also has various beneficial effects, such as inhibiting skin oil; inhibiting 5α-reductase; inhibiting Propionibacterium acnes; scavenging ABTS free radicals; scavenging reactive oxygen species; repairing photo-damage; preventing photo-damage; promoting the migration ability of skin cells; inhibiting the expression of β-galactosidase in skin cells; inhibiting the expression of MMP-1 in skin cells; inhibiting the expression of oil in skin cells; reducing the MDA content in skin cells, etc.

[0072] 5. In some embodiments, the preparation for the skin provided by the embodiment of the present application includes the fermentation broth of Lactobacillus rhamnosus, and can achieve the beneficial effects generated by the fermentation broth of Lactobacillus rhamnosus. BRIEF DESCRIPTION OF THE DRAWINGS

[0073] Figure 1 It is a schematic diagram of the morphology of a colony of Lactobacillus rhamnosus selected for isolation and identification in the method for obtaining Lactobacillus rhamnosus in the embodiment of the present application;

[0074] Figure 2 It is a schematic diagram of the scavenging rate of ABTS free radicals in Experimental Example 1;

[0075] Figure 3 It is a fluorescence microscopy diagram of cellular ROS in each group in Experimental Example 2 of this application;

[0076] Figure 4 It is a diagram of the relative fluorescence intensity of cellular ROS in each group in Experimental Example 2 of this application;

[0077] Figure 5 It is a diagram of cell viability of the blank group, model group and sample group in Experimental Example 3 of this application;

[0078] Figure 6 It is a diagram of cell scratch in each group in Experimental Example 4 of this application;

[0079] Figure 7 It is a microscopy diagram after staining of the blank group, model group and sample group in Experimental Example 5 of this application;

[0080] Figure 8 It is a diagram of the stained area of the blank group, model group and sample group in Experimental Example 5 of this application;

[0081] Figure 9 It is a diagram of the expression level of type I collagen in each group in Experimental Example 6 of this application;

[0082] Figure 10 It is a diagram of the expression level of MMP-1 in each group in Experimental Example 7 of this application;

[0083] Figure 11 It is a diagram of the oil production rate in each group in Experimental Example 8 of this application;

[0084] Figure 12 It is a diagram of the 5α-reductase inhibition rate of Sample Groups 1-3 in Experimental Example 9 of this application;

[0085] Figure 13 It is a diagram of the inhibition zone of Sample Groups 1 and 2 in Experimental Example 10 of this application;

[0086] Figure 14 It is a diagram of the MDA content in Experimental Example 11 of this application. Detailed implementation manners

[0087] The following further elaborates on the present invention in conjunction with the attached drawings, so that those skilled in the art can implement it with reference to the text of the specification.

[0088] The terms "first", "second", etc. in the embodiments of this application are only used for descriptive purposes, and cannot be construed as indicating or implying relative importance or implicitly specifying the quantity of the indicated technical features. Thus, features defined with "first", "second", etc. may explicitly or implicitly include at least one of such features.

[0089] Furthermore, the terms "comprising", "having", and any variations thereof are intended to cover non-exclusive inclusion. For example, a process, method, system, product, or apparatus that comprises a series of steps or units is not limited to the listed steps or units, but optionally further includes steps or units not listed, or optionally further includes other steps or units inherent to these processes, methods, products, or apparatuses.

[0090] In addition to the above, it should be emphasized that referring to "embodiments" in this document means that the specific features, structures, or characteristics described in connection with the embodiments can be included in at least one embodiment of the present application. The appearance of this phrase at various positions in the specification does not necessarily refer to the same embodiment, nor is it an independent or alternative embodiment mutually exclusive with other embodiments. Those skilled in the art will explicitly and implicitly understand that the embodiments described herein can be combined with other embodiments.

[0091] <Lactobacillus rhamnosus>

[0092] In a first aspect, an embodiment of the present application provides a new Lactobacillus rhamnosus, namely, Lactobacillus rhamnosus 31345.

[0093] The preservation information of Lactobacillus rhamnosus 31345 is as follows:

[0094] Taxonomic name: Lactobacillus rhamnosus;

[0095] Latin scientific name: Lactobacillus rhamnosus;

[0096] The preservation unit is: China General Microbiological Culture Collection Center, abbreviated as CGMCC;

[0097] The preservation number is: CGMCC No. 31345;

[0098] The preservation date is: July 18, 2024;

[0099] The preservation address: The address of CGMCC is No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing;

[0100] The biological material (strain) referred to: SLT;

[0101] Survival status: The preservation certificate shows survival.

[0102] As can be seen from the above, the embodiment of the present application provides a new Lactobacillus rhamnosus, enriching the varieties of Lactobacillus rhamnosus and being able to be used to expand the applications of Lactobacillus rhamnosus, thereby solving the problems in the background technology.

[0103] In addition, the skin helps protect various tissues and organs in the body from physical, mechanical, chemical, and pathogenic microbial invasions. The sebaceous gland is a multi-acinar holocrine tissue composed of one or several sac-like alveoli and a short duct, distributed in most areas of the whole body skin, with the highest density on the scalp and face. Under normal physiological conditions, the sebaceous gland secretes an appropriate amount of oily wax-like substance (sebum) onto the surface of the stratum corneum, thereby nourishing, lubricating the skin and hair, and inhibiting the growth and invasion of pathogenic microorganisms. However, some endogenous or exogenous factors, such as age, diet, temperature, humidity, ultraviolet rays, or endocrine factors, can cause hyperfunction of the sebaceous gland. Excessive secretion of the sebaceous gland can lead to greasy skin, rough skin, enlarged pores, and is prone to acne and seborrheic dermatitis.

[0104] The novel Lactobacillus rhamnosus provided in the embodiments of the present application can be used to inhibit the expression of oil in sebaceous gland cells, overcome the defect of greasy skin, and has an unexpected technical effect compared with the existing Lactobacillus rhamnosus, showing obvious progress.

[0105] <Fermentation broth of Lactobacillus rhamnosus>

[0106] In a second aspect, the embodiments of the present application provide a fermentation broth of Lactobacillus rhamnosus, which is obtained by fermenting the Lactobacillus rhamnosus 31345 according to the embodiments of the first aspect.

[0107] The fermentation broth of Lactobacillus rhamnosus provided in the embodiments of the present application contains the metabolites of Lactobacillus rhamnosus and has the use of inhibiting skin oil. That is, it can be used to inhibit the expression of oil in sebaceous gland cells, overcome the defect of greasy skin, achieve an unexpected technical effect, and show obvious progress.

[0108] In addition, the fermentation broth of Lactobacillus rhamnosus also has various beneficial effects, which are described as follows.

[0109] In some embodiments, the fermentation broth of Lactobacillus rhamnosus has the use of inhibiting the activity of 5α-reductase. By inhibiting the activity of 5α-reductase, the expression of oil in sebaceous gland cells is inhibited.

[0110] In some embodiments, the fermentation broth of Lactobacillus rhamnosus has the use of antioxidation.

[0111] In some embodiments, the fermentation broth of Lactobacillus rhamnosus has the use of scavenging ABTS free radicals. Further, by scavenging ABTS free radicals, the fermentation broth of Lactobacillus rhamnosus has the use of antioxidation.

[0112] In some embodiments, the fermentation broth of Lactobacillus rhamnosus has the use of scavenging reactive oxygen species (ROS).

[0113] In some embodiments, the Lactobacillus rhamnosus fermentation broth is useful for repairing photo-damage.

[0114] In some embodiments, the Lactobacillus rhamnosus fermentation broth is useful for preventing photo-damage.

[0115] In some embodiments, the Lactobacillus rhamnosus fermentation broth is useful for repairing scratches.

[0116] In some embodiments, the Lactobacillus rhamnosus fermentation broth is useful for promoting the migration ability of skin cells. Promoting the migration ability of skin cells is beneficial to repair the scratch damage of skin cells. Further, the skin cells are HaCaT cells.

[0117] In some embodiments, the Lactobacillus rhamnosus fermentation broth is useful for anti-aging.

[0118] In some embodiments, the Lactobacillus rhamnosus fermentation broth is useful for promoting the expression of type I collagen. Promoting the expression of type I collagen is beneficial to anti-aging.

[0119] In some embodiments, the Lactobacillus rhamnosus fermentation broth is useful for inhibiting the expression of β-galactosidase in skin cells. β-galactosidase is a senescence marker, so inhibiting β-galactosidase is beneficial to anti-aging. Further, the skin cells are HaCaT cells.

[0120] In some embodiments, the Lactobacillus rhamnosus fermentation broth is useful for inhibiting the expression of MMP-1 in skin cells. Further, the skin cells are HFF-1 cells.

[0121] In some embodiments, the Lactobacillus rhamnosus fermentation broth is useful for inhibiting Propionibacterium acnes.

[0122] In some embodiments, the Lactobacillus rhamnosus fermentation broth is useful for reducing the content of MDA in skin cells. By reducing the content of MDA in skin cells, the effect of anti-lipid peroxidation is achieved. Further, the skin cells are HaCaT cells.

[0123] <Preparation method of Lactobacillus rhamnosus fermentation broth>

[0124] In a third aspect, the embodiments of the present application provide a preparation method of a Lactobacillus rhamnosus fermentation broth, which can be used to prepare the Lactobacillus rhamnosus fermentation broth described in the embodiments of the second aspect; the preparation method includes steps S100 to S400 as described below.

[0125] Step S100: Obtain a Lactobacillus rhamnosus seed solution. The Lactobacillus rhamnosus seed solution is obtained according to Lactobacillus rhamnosus 31345.

[0126] Step S200: Inoculate the Lactobacillus rhamnosus seed liquid into the first culture medium to obtain an inoculum.

[0127] In some embodiments, the incubator is a shake flask. The volume ratio of the Lactobacillus rhamnosus seed liquid to the culture medium is (1 - 5):97. Optionally, it is 3:97.

[0128] In some embodiments, the first culture medium is MRS culture medium.

[0129] In some embodiments, S200 specifically includes: Under aseptic conditions, inoculate the Lactobacillus rhamnosus seed liquid into an incubator containing MRS culture medium that has been sterilized and cooled, such that the Lactobacillus rhamnosus seed liquid is inoculated onto the MRS culture medium to obtain an incubator containing the inoculum.

[0130] In some embodiments, the incubator is a shake flask.

[0131] Step S300: Ferment and culture the inoculum at a fermentation temperature of 35 - 40°C, a shaking speed of 150 - 200 rpm, and a fermentation time of 18 - 48 hours to obtain a fermentation product.

[0132] In some embodiments, the fermentation temperature is 37°C.

[0133] In some embodiments, the inoculum is placed in the inoculated incubator, and the incubator is a shake flask. Therefore, S300 can specifically be operated according to the following steps: Cultivate the shake flask containing the inoculum at 37°C at a shaking speed of 150 - 200 rpm for 18 - 48 hours to obtain a fermentation product.

[0134] The main purpose of S300 is to ferment and culture Lactobacillus rhamnosus, enabling Lactobacillus rhamnosus to grow fully and ferment, and producing metabolites that accumulate in the MRS culture medium.

[0135] Step S400: Separate and process the fermentation product to obtain the liquid part after separation, which is the Lactobacillus rhamnosus fermentation broth.

[0136] It is easy to understand that the fermentation product contains bacterial cells (solid phase) and fermentation broth (liquid phase). Therefore, the purpose of S400 is to separate and process the fermentation product to obtain the fermentation broth (liquid phase) separated from the bacterial cells (solid phase), that is, the Lactobacillus rhamnosus fermentation broth.

[0137] In some embodiments, the separation in S400 is centrifugation. It should be understood that in this embodiment, the liquid part after separation is the supernatant after centrifugation, and this supernatant belongs to the fermentation broth and contains the metabolites of Lactobacillus rhamnosus.

[0138] In some embodiments, the separation in S400 is filtration. It should be understood that in this embodiment, the liquid part after the separation treatment is the filtrate after the filtration treatment, and this filtrate belongs to the fermentation culture solution and contains the metabolites of Lactobacillus rhamnosus.

[0139] In some embodiments, S100, obtaining the Lactobacillus rhamnosus seed solution, includes S110 to S120:

[0140] S110, activating Lactobacillus rhamnosus to obtain an activated Lactobacillus rhamnosus;

[0141] Specifically, Lactobacillus rhamnosus can be activated by freeze-dried powder, cryopreservation tube or plate, etc.

[0142] S120, culturing the activated Lactobacillus rhamnosus in a second culture medium to obtain the Lactobacillus rhamnosus seed solution.

[0143] In some embodiments, the second culture medium is MRS culture medium.

[0144] In some embodiments, in S120, specifically, culturing the activated Lactobacillus rhamnosus in MRS culture medium to obtain a vigorously growing seed solution, that is, the Lactobacillus rhamnosus seed solution (i.e., the pre-culture).

[0145] <Preparation>

[0146] The embodiments of the present application provide a preparation for the skin, including:

[0147] The Lactobacillus rhamnosus fermentation broth described in the second aspect of the embodiment.

[0148] It can be understood that since the preparation includes the Lactobacillus rhamnosus fermentation broth, the beneficial effects or uses provided by the Lactobacillus rhamnosus fermentation broth can be achieved.

[0149] In some embodiments, the preparation is a cosmetic.

[0150] In some embodiments, the preparation is a drug.

[0151] In some embodiments, the preparation is a topical preparation.

[0152] In some embodiments, the preparation further includes a solvent. Optionally, the solvent includes water; optionally, the solvent is selected from water.

[0153] In some embodiments, the volume concentration of the Lactobacillus rhamnosus fermentation broth in the preparation is 0.1-1%; this embodiment has the following beneficial effects: 1. It is more conducive to inhibiting the expression of oil by sebaceous gland cells, and as the addition amount increases, the oil productivity continuously decreases; 2. It is more conducive to reducing the content of MDA in skin cells and has the ability of anti-lipid peroxidation, and the skin cells are HaCaT cells.

[0154] The main function of 5α-reductase is to convert the androgen testosterone into dihydrotestosterone with stronger receptor-binding ability, which in turn leads to a large amount of oil secretion by sebaceous gland cells. Therefore, the inhibitory activity of the Lactobacillus rhamnosus fermentation broth on 5α-reductase can be measured to represent its ability to inhibit the oil secretion of sebaceous gland cells. In some embodiments, the volume concentration (volume fraction) of the Lactobacillus rhamnosus fermentation broth in the preparation is 10-50%, which is more conducive to inhibiting the activity of 5α-reductase.

[0155] In some embodiments, the volume concentration of the Lactobacillus rhamnosus fermentation broth in the preparation is 12.5-50%; it is more conducive to scavenging ABTS free radicals.

[0156] In some embodiments, the volume concentration of the Lactobacillus rhamnosus fermentation broth in the preparation is 10%; it is more conducive to scavenging ROS in skin cells.

[0157] In some embodiments, the volume concentration of the Lactobacillus rhamnosus fermentation broth in the preparation is 2%; this embodiment has the following beneficial effects: 1. It is more conducive to repairing the photo-damage of skin cells by ultraviolet rays (the ultraviolet ray is UVA), 2. It is more conducive to preventing the photo-damage of skin cells by ultraviolet rays (the ultraviolet ray is UVA), 3. It is more conducive to inhibiting the expression of β-galactosidase in skin cells. Since β-galactosidase is a senescence marker, inhibiting β-galactosidase is beneficial to alleviating skin aging, 4. It is more conducive to promoting the expression of type I collagen in skin cells.

[0158] In some embodiments, the volume concentration of the Lactobacillus rhamnosus fermentation broth in the preparation is 4%; it is more conducive to promoting the migration ability of skin cells and has the effect of repairing skin scratches.

[0159] In some embodiments, the volume concentration of the Lactobacillus rhamnosus fermentation broth in the preparation is 10%; it is more conducive to inhibiting the expression of MMP-1 in skin cells, and the skin cells are HFF-1 cells.

[0160] In some embodiments, the volume concentration of the Lactobacillus rhamnosus fermentation broth in the preparation is 50-100%; it is more conducive to inhibiting the growth of Propionibacterium acnes and has the effect of removing acne.

[0161] <Obtaining of Lactobacillus rhamnosus>

[0162] According to the method shown in the following steps S210 to S260, Lactobacillus rhamnosus 31345 described in the first aspect embodiments can be obtained:

[0163] Step S210: Mash the Rosa Roxburghi Tratt. from Bijie City, Guizhou Province, dissolve it in an appropriate amount of water, and mix well to obtain a sample.

[0164] Step S220: Pipette 0.1 mL of the sample into 0.9 mL of sterile physiological saline to obtain a 10 -1 dilution, and then pipette 0.1 mL of the 10 -1 dilution into 0.9 mL of physiological saline to obtain a 10 -2 dilution. Perform this operation successively to obtain 10 -3 , 10 -4 , 10 -5 , 10 -6 dilutions.

[0165] Specifically, step S220 includes S221 to S226:

[0166] Step S221: Pipette 0.1 mL of the sample into 0.9 mL of sterile physiological saline to obtain a 10 -1 dilution;

[0167] Step S222: Pipette 0.1 mL of the 10 -1 dilution into 0.9 mL of sterile physiological saline to obtain a 10 -2 dilution;

[0168] Step S223: Pipette 0.1 mL of the 10 -2 dilution into 0.9 mL of sterile physiological saline to obtain a 10 -3 dilution;

[0169] Step S224: Pipette 0.1 mL of the 10 -3 dilution into 0.9 mL of sterile physiological saline to obtain a 10 -4 dilution;

[0170] Step S225: Pipette 0.1 mL of the 10 -4 dilution into 0.9 mL of sterile physiological saline to obtain a 10 -5 dilution;

[0171] Step S226: Pipette 0.1 mL of the 10 -5 dilution into 0.9 mL of sterile physiological saline to obtain a 10 -6 dilution.

[0172] Step S230: Take 0.1 mL of the dilution solution and spread it evenly on the MRS medium containing calcium carbonate (obtained by mixing calcium carbonate and MRS medium, where the mass fraction of calcium carbonate is 2%), and incubate it upside down at a temperature of 37°C.

[0173] Step S240: After 24 h, select the colonies that produce a calcium dissolution zone for streak purification culture, and preliminarily identify the strains through colony morphology and microscopic examination, and select the strains that meet the characteristics from them;

[0174] Among them, the morphology of the selected strains is as Figure 1 shown, the colonies are convex, slightly white, moist, with neat edges, the colonies are round, about 1 - 2 mm in diameter; under microscopic examination, the bacterial cells are short rod-shaped, small and numerous.

[0175] Step S250: Pick a single colony according to the strain that meets the characteristics and inoculate it into the corresponding 10 mL of sterile glycerol, culture for 24 h, take 1 mL each of the bacterial solution after 24 h of culture and sterile glycerol into a 2 - mL strain preservation tube, and store it in a -80°C refrigerator (sterile glycerol is used to protect the bacteria from freezing the cell membrane at a temperature of -80°C).

[0176] Step S260: Identify the strain according to the bacterial solution cultured for 24 h.

[0177] Step S260 specifically includes steps S261 - S266:

[0178] Step S261: Take the bacterial solution cultured for 24 h in step S250, centrifuge it at a speed of 6000 r / min for 3 min, after centrifugation, discard the supernatant to obtain the first bacterial solution.

[0179] Step S262: Add 1.5 mL of sterile water to the first bacterial solution, mix well, centrifuge it at a speed of 6000 r / min for 3 min, after centrifugation, discard the supernatant to obtain the second bacterial solution.

[0180] Step S263: Add 1.5 mL of sterile water to the second bacterial solution, mix well, centrifuge it at a speed of 6000 r / min for 3 min, discard the supernatant to obtain the third bacterial solution.

[0181] Step S264: Resuspend the third bacterial solution in 1.5 mL of sterile water, mix it, and use it as a template for bacterial identification for PCR (Polymerase chain reaction) to obtain a PCR product.

[0182] Step S265: Sequence the PCR product to obtain a sequencing result.

[0183] Among them, the PCR product is sent to Sangon Biotech (Shanghai) Co., Ltd. for sequencing.

[0184] Step S266: After splicing the sequencing results, perform a homology analysis using BLAST (Basic Local Alignment Search Tool) in the NCBI (National Center for Biotechnology Information) database, and select partial sequences with a sequence identity of over 98% for alignment;

[0185] Based on the alignment results, it is determined that the obtained strain is Lactobacillus rhamnosus, and its taxonomic name is Lactobacillus rhamnosus.

[0186] <Preparation Example of Lactobacillus rhamnosus Fermentation Broth>

[0187] Prepare the Lactobacillus rhamnosus fermentation broth according to the method described in the following steps 310 to 360, and the preparation method is as follows:

[0188] Step 310: Prepare the medium and sterilize it:

[0189] Specifically, prepare an MRS liquid medium suitable for the growth of Lactobacillus rhamnosus, dispense it into shake flasks, and perform high-pressure steam sterilization treatment, then cool and reserve for use.

[0190] Step 320: Prepare the Lactobacillus rhamnosus seed liquid:

[0191] Take Lactobacillus rhamnosus 31345 described in the first aspect of the embodiment, activate Lactobacillus rhamnosus 31345 to obtain an activated Lactobacillus rhamnosus; specifically, pick a small amount of Lactobacillus rhamnosus 31345 from -80°C, streak and activate it on an MRS medium, and the obtained activated Lactobacillus rhamnosus is when monoclonal colonies grow.

[0192] Culture the activated Lactobacillus rhamnosus in an MRS medium to obtain a vigorously growing seed liquid, that is, a pre-culture, as the Lactobacillus rhamnosus seed liquid.

[0193] Step 330: Inoculate and culture:

[0194] Under sterile conditions, inoculate about 3% (by volume fraction of the shake flask) of the Lactobacillus rhamnosus seed liquid into a shake flask containing the sterilized and cooled MRS medium (the MRS medium shake flask refers to a shake flask containing the MRS medium) to obtain an inoculated shake flask; among them, in the inoculated shake flask, the volume fraction of the MRS medium is 97%, that is, the volume ratio of the Lactobacillus rhamnosus seed liquid to the MRS medium is 3:97.

[0195] Step 340: Fermentation culture:

[0196] The inoculated shake flask was placed in a constant temperature shaker and cultured at 37 °C with a shaking speed of 200 rpm for 30 hours to obtain a fermentation product containing bacterial cells (solid phase) and fermentation broth (liquid phase).

[0197] Step 350, solid-liquid separation:

[0198] After the fermentation culture was completed, the fermentation product was centrifuged to obtain the fermentation broth separated from the bacterial cells (solid phase), and the fermentation broth contained the metabolites of Lactobacillus rhamnosus.

[0199] Step 360, collect the fermentation broth of Lactobacillus rhamnosus:

[0200] Collect the supernatant obtained by separation in step 350, and the fermentation broth of Lactobacillus rhamnosus is obtained.

[0201] <Experiment>

[0202] Prepare the experimental equipment such as cells and reagents shown in Tables 1 to 3 below for the subsequent specific experimental examples.

[0203] Table 1, Experimental Equipment

[0204]

[0205] Table 2, Experimental Equipment

[0206]

[0207] Table 3, Experimental Equipment

[0208]

[0209]

[0210] <Experimental Example 1, Free Radical Scavenging Experiment>

[0211] 1-1. Experimental Method

[0212] The method of Experimental Example 1 is as follows:

[0213] Take a 96-well plate;

[0214] Select the first well on the 96-well plate, add 50 μL of water to the first well, then add 150 μL of ABTS solution to this well, mix and react for 15 min, and then measure the absorbance value at 734 nm, which is recorded as the blank group;

[0215] Select the second well on the 96-well plate, add 50 μL of vitamin C solution to the second well, then add 150 μL of ABTS solution to this well, mix and react for 15 min, and then measure the absorbance value at 734 nm, denoted as the VC group; among them, the vitamin C solution is a mixture of vitamin C and water, and the concentration of vitamin C is 10 ppm;

[0216] Select the third well on the 96-well plate, add 50 μL of the Lactobacillus rhamnosus fermentation broth sample to the third well, then add 150 μL of ABTS solution to this well, mix and react for 15 min, and then measure the absorbance value at 734 nm, denoted as sample group 1; among them, the Lactobacillus rhamnosus fermentation broth sample is a mixture of Lactobacillus rhamnosus fermentation broth and water, and the volume concentration of Lactobacillus rhamnosus fermentation broth is 12.5%;

[0217] Select the fourth well on the 96-well plate, add 50 μL of the Lactobacillus rhamnosus fermentation broth sample to the fourth well, then add 150 μL of ABTS solution to this well, mix and react for 15 min, and then measure the absorbance value at 734 nm, denoted as sample group 2; among them, the Lactobacillus rhamnosus fermentation broth sample is a mixture of Lactobacillus rhamnosus fermentation broth and water, and the volume concentration of Lactobacillus rhamnosus fermentation broth is 25%;

[0218] Select the fifth well on the 96-well plate, add 50 μL of the Lactobacillus rhamnosus fermentation broth sample to the fifth well, then add 150 μL of ABTS solution to this well, mix and react for 15 min, and then measure the absorbance value at 734 nm, denoted as sample group 3; among them, the Lactobacillus rhamnosus fermentation broth sample is a mixture of Lactobacillus rhamnosus fermentation broth and water, and the volume concentration of Lactobacillus rhamnosus fermentation broth is 50%;

[0219] After obtaining the absorbance values of each group, calculate the scavenging rates of ABTS free radicals for the VC group and sample groups 1-3, and calculate specifically according to the following formula:

[0220]

[0221] In the formula: Asample represents the absorbance value of the sample group or the VC group; Acontrol represents the absorbance value of the blank group.

[0222] 1-2. Experimental results and analysis

[0223] The experimental results are shown in Figure 2 , Figure 2 which is a schematic diagram of the scavenging rate of ABTS free radicals in Experimental Example 1.

[0224] According to Figure 2The results of the displayed sample groups 1 to 3 show that when the concentration of the Lactobacillus rhamnosus fermentation broth is 12.5% to 50%, the Lactobacillus rhamnosus fermentation broth sample has free radical scavenging ability. This shows that the Lactobacillus rhamnosus fermentation broth provided in the embodiments of the present application has free radical scavenging ability.

[0225] <Experimental Example 2. Measurement of Cellular Reactive Oxygen Species (ROS) Level>

[0226] 2-1. Experimental Method

[0227] The experimental method is as follows:

[0228] Take a 96-well plate and inoculate HFF-1 cells into the 96-well plate at an inoculation density of 5×10 4 cells / well (the wells inoculating cells on the 96-well plate at least include the first well, the second well, and the third well), and culture them in a high-glucose DMEM medium solution containing 10% FBS and 1% double antibody for 24 h to obtain the cultured 96-well plate;

[0229] Wash the cells in the first well of the cultured 96-well plate with PBS buffer, then add 1 mL of high-glucose DMEM medium solution containing 1% double antibody, and then add 1 μL of DCFH-DA fluorescent probe; continue to culture in an incubator at 37 °C and 5% CO2 for 20 min, then discard the medium solution, and wash 3 times with high-glucose DMEM medium solution containing 1% double antibody to completely remove the probe; finally, add 1 mL of PBS buffer, observe under a fluorescence microscope, and measure the relative fluorescence intensity (relative fluorescence value) by an enzyme-labeled instrument under the conditions of an excitation wavelength of 485 nm and an emission wavelength of 525 nm, and record it as the blank group;

[0230] Perform UVA treatment on the cells in the second well of the cultured 96-well plate (the dose of UVA is 15 j / cm 2 ), wash the cells with PBS buffer, then add 1 mL of high-glucose DMEM medium solution containing 1% double antibody, and then add 1 μL of DCFH-DA fluorescent probe; continue to culture in an incubator at 37 °C and 5% CO2 for 20 min, then discard the medium solution, and wash 3 times with high-glucose DMEM medium solution containing 1% double antibody to completely remove the probe; finally, add 1 mL of PBS buffer, observe under a fluorescence microscope, and measure the relative fluorescence intensity (relative fluorescence value) by an enzyme-labeled instrument under the conditions of an excitation wavelength of 485 nm and an emission wavelength of 525 nm, and record it as the model group;

[0231] Perform UVA treatment on the cells in the third well of the cultured 96-well plate (the dose of UVA is 15 j / cm 2) Wash the cells with PBS buffer, then add 1 mL of the Lactobacillus rhamnosus fermentation broth sample, and then add 1 μL of the DCFH-DA fluorescent probe; continue to culture in an incubator at 37 °C and 5% CO2 for 20 min, then discard the culture medium solution, and wash 3 times with a high-glucose DMEM culture medium solution containing 1% double antibody to completely remove the probe; finally, add 1 mL of PBS buffer, observe under a fluorescence microscope, and measure the relative fluorescence intensity (relative fluorescence value) with a microplate reader at an excitation wavelength of 485 nm and an emission wavelength of 525 nm, denoted as the sample group; among them, the Lactobacillus rhamnosus fermentation broth sample is a mixture of Lactobacillus rhamnosus fermentation broth and a high-glucose DMEM culture medium solution containing 1% double antibody, and the volume concentration of the Lactobacillus rhamnosus fermentation broth is 10%.

[0232] Note: The DCFH-DA fluorescent probe is a reagent in the reactive oxygen species detection kit.

[0233] 2-2. Experimental results and analysis:

[0234] The experimental results are shown in Figure 3 and Figure 4 .

[0235] Figure 3 is a fluorescence micrograph of a cell ROS of the blank group, model group and sample group in Experimental Example 2.

[0236] Figure 4 is a schematic diagram of the relative fluorescence value of a cell ROS of the blank group, model group and sample group in Experimental Example 2.

[0237] ROS is an additional reactive oxygen free radical produced by mitochondria during cell metabolism, which can attack various membrane structures and organelles inside the cell, cause damage to the cell, and affect cell viability.

[0238] In the ROS detection, the ROS in the cells is presented in the form of green fluorescence. The weaker the green fluorescence, the lower the cell ROS level, which means that the test sample has a stronger ability to scavenge cell ROS or inhibit its expression.

[0239] Figure 3 The results show that the green fluorescence of the sample group is weaker than that of the model group, indicating that the Lactobacillus rhamnosus fermentation broth provided in the embodiment of the present application has the ability to scavenge ROS.

[0240] In addition, according to Figure 4 the results shown, it can also be known that the relative fluorescence value of the sample group decreased by about 44% compared with the model group, indicating that the Lactobacillus rhamnosus fermentation broth provided in the embodiment of the present application has excellent scavenging ability for ROS.

[0241] <Experimental Example 3. Cell photo-damage repair test>

[0242] 3-1. Experimental method

[0243] The experimental method is as follows:

[0244] Take a 96-well plate and inoculate HFF-1 cells into the 96-well plate at an inoculation density of 1×10 5 cells / well. Inoculate HFF-1 cells into the 96-well plate at an inoculation density of 1×10 5 cells / well (the wells inoculating cells on the 96-well plate include at least the first well, the second well, and the third well). Add 100 μL of high-glucose DMEM medium to each well and culture in an incubator at 37°C and 5% CO2 for 24 h; then aspirate the medium solution and wash away the residual medium solution with PBS buffer to obtain the washed 96-well plate;

[0245] Add 100 μL of PBS buffer to the first well of the washed 96-well plate, without UVA treatment, and then aspirate the PBS buffer therein; then add 100 μL of high-glucose DMEM medium solution containing 10% FBS and 1% double antibiotics to this well and continue to incubate in an incubator at 37°C and 5% CO2 for 24 h; then remove the medium solution in this well, add 100 μL of MTT solution to this well, place it in an incubator at 37°C and 5% CO2 for 4 h, aspirate the supernatant, and then add 100 μL of DMSO solution, and measure the absorbance value at 490 nm, which is recorded as the blank group;

[0246] Add 100 μL of PBS buffer to the second well of the washed 96-well plate, first perform UVA treatment (15 j / cm 2 ) on this well, and then aspirate the PBS buffer therein; then add 100 μL of high-glucose DMEM medium solution containing 10% FBS and 1% double antibiotics to this well and continue to incubate in an incubator at 37°C and 5% CO2 for 24 h. Then remove the medium solution, add 100 μL of MTT solution to this well, place it in an incubator at 37°C and 5% CO2 for 4 h, aspirate the supernatant and add 100 μL of DMSO solution, and measure the absorbance value at 490 nm, which is recorded as the model group;

[0247] Add 100 μL of PBS buffer to the third well of the washed 96-well plate, first perform UVA treatment (15 j / cm 2) Then, aspirate the PBS buffer solution therein; then add 100 μL of the Lactobacillus rhamnosus fermentation broth sample into the well, and continue to incubate in an incubator at 37 °C and 5% CO₂ for 24 h; afterwards, remove the culture medium solution, and then add 100 μL of the MTT solution into the well. After placing it in an incubator at 37 °C and 5% CO₂ for 4 h, aspirate the solution and add 100 μL of the DMSO solution, and measure the absorbance value at 490 nm, which is recorded as the sample group; among them, the Lactobacillus rhamnosus fermentation broth sample is a mixture of the Lactobacillus rhamnosus fermentation broth and the high-glucose DMEM culture medium solution containing 10% FBS and 1% double antibodies, and the volume concentration of the Lactobacillus rhamnosus fermentation broth is 2%;

[0248] Calculate the cell viability of the sample group and the model group, and the calculation formula is as follows:

[0249]

[0250] In the formula: As represents the absorbance value of the sample group / model group; Ac represents the absorbance value of the blank group.

[0251] 3-2. Experimental results and analysis

[0252] The experimental results are shown in Figure 5 。

[0253] Figure 5 is a schematic diagram of the cell viability of the blank group, the model group and the sample group;

[0254] Photoaging is an important factor causing exogenous aging, and among them, the damage caused by ultraviolet rays to skin cells is the greatest. As the ultraviolet ray with a longer wavelength, UVA has strong penetration to the skin and has the ability to damage human foreskin fibroblasts (HFF-1 cells).

[0255] Figure 5 The results show that the cell viability of sample group 1 is higher than that of the damaged model group, indicating that for the damage caused by UVA, the Lactobacillus rhamnosus fermentation broth has the effect of repairing photo-damage.

[0256] <Experimental Example 4. Cell scratch repair test>

[0257] 4-1. Experimental method

[0258] The experimental method is specifically as follows:

[0259] Take a 6-well plate; in the 6-well plate, place the cell scratch insert with the smooth side facing up in the center of the well, so that the other side of the cell scratch insert adheres to the bottom of the 6-well plate;

[0260] After digesting HaCaT cells with trypsin, according to 6×10 5Inoculate into the first well and the second well of the cell scratch insert, add 70 μL of high-glucose DMEM medium solution containing 10% FBS and 1% double antibiotics to each well, and culture in an incubator at 37 °C and 5% CO2 for 24 h; then take out the cell scratch insert and wash away the residual medium solution with PBS buffer;

[0261] Add 1 mL of high-glucose DMEM medium solution containing 10% FBS and 1% double antibiotics to the first well of the cell scratch insert; after culturing in an incubator at 37 °C and 5% CO2 for 24 h, take a photo under a microscope, which is recorded as the blank group.

[0262] Add 1 mL of the Lactobacillus rhamnosus fermentation broth sample to the second well of the cell scratch insert, and after culturing in an incubator at 37 °C and 5% CO2 for 24 h, take a photo under a microscope, which is recorded as the sample group; among them, the Lactobacillus rhamnosus fermentation broth sample is a mixture of Lactobacillus rhamnosus fermentation broth and high-glucose DMEM medium solution containing 10% FBS and 1% double antibiotics, and the concentration of the Lactobacillus rhamnosus fermentation broth is 4%.

[0263] 4-2. Experimental results and analysis

[0264] The experimental results are shown in Figure 6 .

[0265] Figure 6 It is a cell scratch schematic diagram of each group in Experimental Example 4.

[0266] The cell scratch repair ability depends on the ability of cells to migrate to the scratch area. The smaller the vacant area of the scratch area, the stronger the cell migration ability is proved.

[0267] Figure 6 The results show that compared with the blank group, the vacant area of the scratch area in the sample group is smaller, indicating that the Lactobacillus rhamnosus fermentation broth provided in the embodiment of the present application has a significant ability to promote cell migration.

[0268] <Experimental Example 5. β-galactosidase expression>

[0269] 5-1. Experimental method

[0270] The experimental method is as follows:

[0271] Take a 6-well plate, inoculate HaCaT cells at an inoculation density of 1.5×10 4 cells / well in the 6-well plate for culture (the wells inoculating cells on the 6-well plate at least include the first well, the second well, and the third well). When the cell aggregation degree reaches 70%, remove the high-glucose DMEM medium solution containing 10% FBS and 1% double antibiotics, and after washing the cells with PBS buffer, obtain the washed 6-well plate;

[0272] Add 2 mL of high-glucose DMEM medium solution containing 10% FBS and 1% double antibody to the first well of the 6-well plate after washing, and continue culturing for 24 h; then add 1 mL of β-galactosidase staining fixative, and let it stand at room temperature for 15 min to fix the cells; then remove the β-galactosidase staining fixative, and wash the cells 3 times with PBS buffer, 3 min each time; after the washing process is completed, remove the PBS buffer, and add 1 mL of staining working solution to this well, then incubate overnight at 37 °C; finally, observe through a standard optical microscope and record it as the blank group; among them, the blank group is not treated with UVA;

[0273] Use UVA treatment (15 j / cm 2 ) to damage the cells in the second well of the 6-well plate after washing, then add 2 mL of high-glucose DMEM medium solution containing 10% FBS and 1% double antibody, and continue culturing for 24 h; then add 1 mL of β-galactosidase staining fixative, and let it stand at room temperature for 15 min to fix the cells; then remove the β-galactosidase staining fixative, and wash the cells 3 times with PBS buffer, 3 min each time; after the washing process is completed, remove the PBS buffer, and add 1 mL of staining working solution to this well, then incubate overnight at 37 °C; finally, observe through a standard optical microscope and record it as the model group;

[0274] Use UVA treatment (15 j / cm 2 ) to treat the third well of the 6-well plate after washing, then add 2 mL of the fermentation broth sample of Lactobacillus rhamnosus, and continue culturing for 24 h; then add 1 mL of β-galactosidase staining fixative, and let it stand at room temperature for 15 min to fix the cells; then remove the β-galactosidase staining fixative, and wash the cells 3 times with PBS buffer, 3 min each time; after the washing process is completed, remove the PBS buffer, and add 1 mL of staining working solution to this well, then incubate overnight at 37 °C; finally, observe through a standard optical microscope and record it as the sample group; among them, the fermentation broth sample of Lactobacillus rhamnosus is a mixture of the fermentation broth of Lactobacillus rhamnosus and the high-glucose DMEM medium solution containing 10% FBS and 1% double antibody, and the concentration of the fermentation broth of Lactobacillus rhamnosus is 2%.

[0275] Note: Both the β-galactosidase staining fixative and the staining working solution are reagents in the cell senescence β-galactosidase staining kit.

[0276] 5-1. Experimental results and analysis

[0277] The experimental results are shown in Figures 7 - 8 .

[0278] Figure 7 A microscopic diagram after staining the blank group, model group, and sample group;

[0279] Figure 8 A diagram of the stained area of the blank group, model group, and sample group.

[0280] β-galactosidase can specifically bind to the dye to present blue. The smaller the area of the cells stained blue, the lower the expression level of β-galactosidase.

[0281] Figure 7 and Figure 8 The results showed that compared with the model group, the fermentation broth of Lactobacillus rhamnosus in the sample group had a significant ability to inhibit the expression of β-galactosidase in immortalized human keratinocytes (HaCaT cells).

[0282] In addition, when the addition amount was 2%, the fermentation broth of Lactobacillus rhamnosus could even make the ability of cells to express β-galactosidase lower than that of the blank group.

[0283] <Experimental Example 6. Type I Collagen Expression Experiment>

[0284] 6-1. Experimental Method

[0285] The experimental method is as follows:

[0286] Take a 96-well plate and inoculate HFF-1 cells at 2×10 5 cells / well into the 96-well plate (the wells inoculating cells on the 96-well plate include at least the first well, the second well, the third well, and the fourth well), and culture them in an incubator at 37°C for 24 h. Discard the culture medium in the 96-well plate to obtain the 96-well plate after inoculation;

[0287] Add 100 μL of high-glucose DMEM medium solution containing 10% FBS and 1% double antibody to the first well of the 96-well plate after inoculation, and culture it in an incubator at 37°C and 5% CO2 for 24 h. After the incubation, take the upper-layer culture medium, centrifuge it at 13,000 rpm for 5 min, and aspirate the supernatant; detect the expression level of type I collagen using a human type I collagen ELISA kit, and record it as the blank group; among them, the blank group is not irradiated with UVA;

[0288] Use UVA irradiation (15 J / cm 2 ) to damage the cells in the second well of the 96-well plate after inoculation, and then add 100 μL of high-glucose DMEM medium solution containing 10% FBS and 1% double antibody to the second well, and culture it in an incubator at 37°C and 5% CO2 for 24 h. After the incubation, take the upper-layer culture medium, centrifuge it at 13,000 rpm for 5 min, and aspirate the supernatant; detect the expression level of type I collagen using a human type I collagen ELISA kit, and record it as the model group;

[0289] Irradiate with UVA (15 J / cm 2 ) to damage the cells in the third well of a 96-well plate after inoculation. Then add 100 μL of TGF-β1 solution with a concentration of 100 ng / mL to the third well, and culture in an incubator at 37 °C and 5% CO2 for 24 h. After incubation, take the upper layer of the culture medium, centrifuge at 13,000 rpm for 5 min, and aspirate the supernatant; detect with a human type I collagen ELISA kit and record it as the TGF-β1 group;

[0290] Irradiate with UVA (15 J / cm 2 ) to damage the cells in the fourth well of a 96-well plate after inoculation. Then add 100 μL of the Lactobacillus rhamnosus fermentation broth sample to the fourth well, and culture in an incubator at 37 °C and 5% CO2 for 24 h. After incubation, take the upper layer of the culture medium, centrifuge at 13,000 rpm for 5 min, and aspirate the supernatant; detect the expression level of type I collagen with a human type I collagen ELISA kit and record it as the sample group; among them, the Lactobacillus rhamnosus fermentation broth sample is a mixture of Lactobacillus rhamnosus fermentation broth and high-glucose DMEM medium solution, and the concentration of Lactobacillus rhamnosus fermentation broth is 2%.

[0291] Note: The usage method of the human type I collagen ELISA kit can refer to the attached instruction manual.

[0292] 6-2. Experimental results and analysis

[0293] The experimental results are shown in Figure 9 .

[0294] Figure 9 It is a schematic diagram of the expression levels of type I collagen in each group in Experimental Example 6.

[0295] Type I collagen is an important extracellular matrix in the skin. Abundant type I collagen in the skin can bring significant firmness and strength to the skin.

[0296] Figure 10 As shown in the results, the expression level of type I collagen in the sample group was significantly higher than that in the blank group and the damaged model group, indicating that the Lactobacillus rhamnosus fermentation broth at an addition amount of 2% has a significant ability to promote the expression of type I collagen in human foreskin fibroblasts (HFF-1 cells). At the same time, the promoting effect of the Lactobacillus rhamnosus fermentation broth on the expression level of type I collagen in cells is better than that of the positive drug TGF-β1 with a concentration of 100 ng / mL in the TGF-β1 group.

[0297] <Experimental Example 7. MMP-1 Expression Experiment>

[0298] 7-1. Experimental method

[0299] The experimental method is as follows:

[0300] Take a 96-well plate and seed HFF-1 cells at 2×10 5 cells per well in the 96-well plate (the wells seeded with cells on the 96-well plate include at least the first well, the second well, and the third well), culture in an incubator at 37°C for 24 h, discard the medium in the 96-well plate to obtain the 96-well plate after seeding;

[0301] Add 100 μL of high-glucose DMEM medium solution containing 10% FBS and 1% double antibiotics to the first well of the 96-well plate after seeding, culture in an incubator at 37°C and 5% CO2 for 24 h, take the upper layer of the medium after incubation, centrifuge at 13000 rpm for 5 min, and aspirate the supernatant; detect the expression level of MMP-1 using the Human MMP-1 ELISA Kit, and record it as the blank group; among them, the blank group is not irradiated with UVA;

[0302] Irradiate with UVA (15 J / cm 2 ) to damage the cells in the second well of the 96-well plate after seeding, add 100 μL of high-glucose DMEM medium solution containing 10% FBS and 1% double antibiotics to the second well, culture in an incubator at 37°C and 5% CO2 for 24 h, take the upper layer of the medium after incubation, centrifuge at 13000 rpm for 5 min, and aspirate the supernatant, detect the expression level of MMP-1 using the Human MMP-1 ELISA Kit, and record it as the model group;

[0303] Irradiate with UVA (15 J / cm 2 ) to damage the cells in the third well of the 96-well plate after seeding, add 100 μL of the rhamnolipid fermentation broth sample to the third well, culture in an incubator at 37°C and 5% CO2 for 24 h, take the upper layer of the medium after incubation, centrifuge at 13000 rpm for 5 min, and aspirate the supernatant; detect the expression level of MMP-1 using the Human MMP-1 ELISA Kit, and record it as the sample group; among them, the rhamnolipid fermentation broth sample is a mixture of rhamnolipid fermentation broth and high-glucose DMEM medium solution containing 10% FBS and 1% double antibiotics, and the volume concentration of the rhamnolipid fermentation broth is 10%.

[0304] Note: The usage method of the Human MMP-1 ELISA Kit can refer to the attached instruction manual.

[0305] 7-2. Experimental results and analysis

[0306] The experimental results are shown in Figure 10 .

[0307] Figure 10It is a schematic diagram of the MMP-1 expression levels in each group in Experimental Example 7.

[0308] MMP-1 (matrix metalloproteinase-1) can degrade type I and type III collagens in the extracellular matrix of the skin, leading to skin aging.

[0309] Figure 10 The results showed that the MMP-1 expression level in the sample group was significantly lower than that in the blank group and the model group, indicating that the fermentation broth of Lactobacillus rhamnosus had a significant inhibitory effect on the MMP-1 expression level in human foreskin fibroblasts (HFF-1 cells) at a 10% addition amount.

[0310] <Experimental Example 8, Oil Control Detection Experiment>

[0311] 8-1. Experimental Method

[0312] The experimental method is as follows:

[0313] Take a 6-well plate and inoculate sebaceous gland cells at 2×10 5 cells / well into the 6-well plate (the wells inoculating cells on the 6-well plate at least include the first well, the second well, the third well, the fourth well, and the fifth well), culture overnight in an incubator at 37°C, discard the culture medium the next day, and rinse twice with PBS buffer to obtain the to-be-treated 6-well plate;

[0314] Add 2 mL of high-glucose DMEM culture medium solution containing 10% FBS and 1% double antibody to the first well of the to-be-treated 6-well plate, place it in an incubator at 37°C and 5% CO2 for 48 h; then discard the culture medium and rinse twice with PBS buffer, add 100 μL of nile red solution to this well, let it stand for 15 min in the dark environment at 37°C, digest the cells with 0.25% EDTA-trypsin digestion solution and transfer them to a black 96-well plate to obtain the sample to be measured, and detect the fluorescence intensity released by the sample with a multifunctional microplate reader under the conditions of excitation wavelength 485 nm and emission wavelength 565 nm, which is recorded as the blank group;

[0315] Add 2 mL of OL solution to the second well of the to-be-treated 6-well plate, place it in an incubator at 37°C and 5% CO2 for 48 h, then discard the liquid part and rinse twice with PBS buffer; add 100 μL of nile red solution to this well, let it stand for 15 min in the dark environment at 37°C, digest the cells with 0.25% EDTA-trypsin digestion solution and transfer them to a black 96-well plate to obtain the sample to be measured, and detect the fluorescence intensity released by the sample with a multifunctional microplate reader under the conditions of excitation wavelength 485 nm and emission wavelength 565 nm, which is recorded as the control group;

[0316] Add 2 mL of the Lactobacillus rhamnosus fermentation broth sample to the third well of the 6-well plate to be processed, incubate it in an incubator at 37°C and 5% CO2 for 48 h, then discard the culture medium and rinse it twice with PBS buffer; add 100 μL of nile red solution to this well, let it stand for 15 min in the dark at 37°C, digest the cells with 0.25% EDTA-trypsin digestion solution and transfer them to a black 96-well plate to obtain the sample to be measured, and use a multifunctional microplate reader to detect the fluorescence intensity released by the sample under the conditions of an excitation wavelength of 485 nm and an emission wavelength of 565 nm, denoted as sample group 1; among them, the Lactobacillus rhamnosus fermentation broth sample is a mixture of Lactobacillus rhamnosus fermentation broth and OL solution, and the volume concentration of Lactobacillus rhamnosus fermentation broth is 0.1%.

[0317] Add 2 mL of the Lactobacillus rhamnosus fermentation broth sample to the fourth well of the 6-well plate to be processed, incubate it in an incubator at 37°C and 5% CO2 for 48 h, then discard the culture medium and rinse it twice with PBS buffer; add 100 μL of nile red solution to this well, let it stand for 15 min in the dark at 37°C, digest the cells with 0.25% EDTA-trypsin digestion solution and transfer them to a black 96-well plate, use a multifunctional microplate reader to detect the fluorescence intensity released by the sample under the conditions of an excitation wavelength of 485 nm and an emission wavelength of 565 nm, denoted as sample group 2; among them, the Lactobacillus rhamnosus fermentation broth sample is a mixture of Lactobacillus rhamnosus fermentation broth and OL solution, and the volume concentration of Lactobacillus rhamnosus fermentation broth is 0.5%.

[0318] Add 2 mL of the Lactobacillus rhamnosus fermentation broth sample to the fifth well of the 6-well plate to be processed, incubate it in an incubator at 37°C and 5% CO2 for 48 h, then discard the culture medium and rinse it twice with PBS buffer; add 100 μL of nile red solution to this well, let it stand for 15 min in the dark at 37°C, digest the cells with 0.25% EDTA-trypsin digestion solution and transfer them to a black 96-well plate to obtain the sample to be measured, and use a multifunctional microplate reader to detect the fluorescence intensity released by the sample under the conditions of an excitation wavelength of 485 nm and an emission wavelength of 565 nm, denoted as sample group 3; among them, the Lactobacillus rhamnosus fermentation broth sample is a mixture of Lactobacillus rhamnosus fermentation broth and OL solution, and the volume concentration of Lactobacillus rhamnosus fermentation broth is 1%.

[0319] 8-2. Experimental results and analysis

[0320] The experimental results are shown in Figure 11 .

[0321] Figure 11 It is a schematic diagram of the oil production rate of each group in Experimental Example 8.

[0322] Sebaceous gland cells are mainly responsible for synthesizing neutral lipids. Excessive secretion of sebaceous glands can lead to greasy skin, rough skin, enlarged pores, and prone to acne and seborrheic dermatitis. Using oleic acid / linoleic acid (OL) to stimulate sebaceous gland cells can cause them to over-express oil. Using Nile red to stain oil and detect the fluorescence intensity can examine the inhibitory effect of the product on the oil expression ability of sebaceous gland cells. Among them, the weaker the fluorescence intensity, the lower the expression of oil, the stronger the inhibitory effect of the product on the oil expression ability of cells, and the better the oil control effect. Therefore, the corresponding oil generation rate can be determined according to the fluorescence intensity of each group.

[0323] Figure 11 The results showed that the oil production rates of sample groups 1 to 3 were all lower than those of the control group, indicating that Lactobacillus rhamnosus fermentation broth can significantly inhibit the expression of oil by sebaceous gland cells at an addition amount of 0.1 to 1%, and the oil production rate continued to decrease as the addition amount increased to 1%.

[0324] <Experimental Example 9, 5α-reductase activity detection experiment>

[0325] 9-1. Experimental methods

[0326] The main function of 5α-reductase is to convert testosterone (an androgen) into dihydrotestosterone, which has a stronger ability to bind to receptors, thereby causing sebaceous gland cells to secrete a large amount of oil. In other words, the inhibitory activity of the sample to be tested on 5α-reductase can be used to indicate its ability to inhibit the secretion of oil by sebaceous gland cells.

[0327] Experimental Example 9 mainly uses UPLC to detect the effect of the test sample on the testosterone content to infer the catalytic ability of 5α-reductase (5α-reductase concentration), determine the change in the amount of 5α-reductase, and obtain the 5α-reductase inhibition rate of the sample, which can then be used to determine the oil secretion of sebaceous gland cells.

[0328] The experimental method is as follows:

[0329] Prepare the experimental reagents of sample groups 1 to 3 according to Table 4. After preparation, mix the experimental reagents of each group, incubate the mixture at 37°C for 60 minutes, then add 1 mL of pre-cooled methanol to each group to terminate the reaction, mix well, centrifuge at 10,000 r / min for 5 minutes, take the supernatant and filter it with a 0.22 μm filter membrane to obtain the filtrate of each group for the detection of the residual testosterone concentration;

[0330] Table 4. Reagent preparation table

[0331] Reagent Name Sample Group 1 (μL) Sample Group 2 (μL) Sample Group 3 (μL) PBS Buffer (pH = 7.5) 500 500 500 Testosterone (1 mmol / L) 100 100 100 NADPH (1 mmol / L) 100 100 100 Crude Enzyme Extract 200 200 200 Lactobacillus rhamnosus Fermentation Broth Sample 50 50 50 Finasteride (2.5 g / L) - - - Ethanol or PBS / μL 50 50 50

[0332] The content of testosterone in the filtered filtrate of each group was analyzed and detected using an ultra performance liquid chromatography (UPLC) system to determine the inhibition rate of 5α-reductase. The instrument conditions for UPLC were as follows: YMC-Triart C18 (100 mm × 2.0 mm, 1.9 μm); column temperature was 30 °C; mobile phase: acetonitrile-water (70 / 30, v / v); flow rate 0.2 mL / min; detector was an ultraviolet detector at 242 nm;

[0333] Among them, the method for obtaining the crude enzyme extract was as follows: The mouse epididymis (removing adipose tissue) was extracted on ice, weighed and minced, and three times the amount of protease inhibitor was added and homogenized in a glass homogenizer; the homogenate was centrifuged at 4 °C and 10,000 r / min for 10 min to remove the white fat layer, and the supernatant was taken. Then, the supernatant was centrifuged at 4 °C and 10,000 r / min for 1 h, and the supernatant was taken again to obtain the crude enzyme extract, which was aliquoted and stored in an ultra-low temperature refrigerator at -80 °C;

[0334] The sample of the Lactobacillus rhamnosus fermentation broth in Sample Group 1 was a mixture of Lactobacillus rhamnosus fermentation broth and PBS buffer, and the concentration of the Lactobacillus rhamnosus fermentation broth was 10%;

[0335] The sample of the Lactobacillus rhamnosus fermentation broth in Sample Group 2 was a mixture of Lactobacillus rhamnosus fermentation broth and PBS buffer, and the concentration of the Lactobacillus rhamnosus fermentation broth was 25%;

[0336] The sample of the Lactobacillus rhamnosus fermentation broth in Sample Group 3 was a mixture of Lactobacillus rhamnosus fermentation broth and PBS buffer, and the concentration of the Lactobacillus rhamnosus fermentation broth was 50%.

[0337] 9-2. Experimental Results and Analysis

[0338] The experimental results showed that the Lactobacillus rhamnosus fermentation broths in Sample Groups 1-3 all had the ability to inhibit 5α-reductase at concentrations of 10% - 50%, so they had excellent sebum and oil inhibition effects.

[0339] Regarding the 5α-reductase inhibition rates of Sample Groups 1-3, refer to Figure 12 .

[0340] Figure 12 It is a schematic diagram of the 5α-reductase inhibition rates of Sample Groups 1-3 in Experimental Example 9.

[0341] <Experimental Example 10. Experiment on Inhibiting Propionibacterium acnes>

[0342] 10-1. Experimental Method

[0343] The experimental method was as follows:

[0344] Pour the RCM culture medium solution into a petri dish. After the RCM culture medium solution solidifies, pipette 100 μL of Propionibacterium acnes bacterial solution (OD 600 = 1) onto the solidified RCM culture medium, spread it evenly with a spreader, gently place 3 Oxford cups on the solidified RCM culture medium, add 200 μL of the Lactobacillus rhamnosus fermentation broth solution sample into the Oxford cups, place it in an incubator at 37 °C and 5% CO2 for 24 h, then remove the Oxford cups, measure the inhibition zone with a vernier caliper, and record it as sample group 1; among them, the Lactobacillus rhamnosus fermentation broth solution sample is a mixture of Lactobacillus rhamnosus fermentation broth and RCM liquid culture medium, and the concentration of Lactobacillus rhamnosus fermentation broth is 50%.

[0345] Pour the RCM culture medium solution into a petri dish. After the RCM culture medium solution solidifies, pipette 100 μL of Propionibacterium acnes bacterial solution (OD 600 = 1) onto the solidified RCM culture medium, spread it evenly with a spreader, gently place 3 Oxford cups on the solidified RCM culture medium, add 200 μL of the Lactobacillus rhamnosus fermentation broth solution sample into the Oxford cups, place it in an incubator at 37 °C and 5% CO2 for 24 h, then remove the Oxford cups, measure the inhibition zone with a vernier caliper, and record it as sample group 2; among them, the Lactobacillus rhamnosus fermentation broth solution sample is Lactobacillus rhamnosus fermentation broth, that is, the concentration of Lactobacillus rhamnosus fermentation broth is 100%.

[0346] 10-2. Experimental results and analysis

[0347] The experimental results are shown in Figure 13 .

[0348] Figure 13 It is a schematic diagram of an inhibition zone for sample groups 1 and 2.

[0349] Propionibacterium acnes is the pathogen causing acne. Therefore, in this efficacy test, the Oxford cup method was used to investigate the inhibitory ability of the product against Propionibacterium acnes under normal culture conditions, and the inhibitory effect of the product was characterized by the size of the inhibition zone.

[0350] The experimental results showed that when the concentration of Lactobacillus rhamnosus fermentation broth in sample group 1 was 50%, the sizes of the 3 inhibition zones formed were 1.5 cm, 1.55 cm, and 1.5 cm respectively. When the concentration of Lactobacillus rhamnosus fermentation broth in sample group 2 was 100%, the sizes of the 3 inhibition zones formed were 1.9 cm, 1.95 cm, and 1.8 cm respectively. The 3 inhibitions of sample groups 1-2 can be seen in Figure 13 the picture shown.

[0351] This indicates that the fermentation broths of Lactobacillus rhamnosus in sample groups 1 - 2 can significantly inhibit the growth of Propionibacterium acnes at concentrations of 50% and 100%, and have acne - removing effects.

[0352] <Experimental Example 11: Experiment for Detecting the MDA Content in Cells>

[0353] 11 - 1. Experimental Method

[0354] The experimental method is as follows:

[0355] Take a 6 - well plate, and inoculate HaCaT cells at 2×10 4 cells / well into the 6 - well plate (the wells inoculating cells on the 6 - well plate include at least the first well, the second well, the third well, the fourth well, and the fifth well). Add 2 mL of high - glucose DMEM medium solution containing 10% FBS and 1% double antibiotics to each well, and culture in an incubator at 37℃ and 5% CO2 for 18 h. After sucking out the complete medium solution, wash away the residual medium solution with PBS buffer to obtain the washed 6 - well plate;

[0356] To the first well of the washed 6 - well plate, add 2 mL of PBS buffer and culture for 1 h, then add high - glucose DMEM medium solution containing 10% FBS and 1% double antibiotics, and culture in an incubator at 37℃ and 5% CO2 for 24 h; then add lysis solution to lyse, centrifuge, and take the supernatant; detect the supernatant with an MDA detection kit, and record it as the blank group.

[0357] To the second well of the washed 6 - well plate, add 2 mL of H2O2 solution and culture for 1 h, then add high - glucose DMEM medium solution containing 10% FBS and 1% double antibiotics, and culture in an incubator at 37℃ and 5% CO2 for 24 h; then add lysis solution to lyse, centrifuge, and take the supernatant; detect the supernatant with an MDA detection kit, and record it as the control group.

[0358] To the third well of the washed 6 - well plate, add 2 mL of H2O2 solution and culture for 1 h, then add the sample of Lactobacillus rhamnosus fermentation broth solution, and culture in an incubator at 37℃ and 5% CO2 for 24 h; then add lysis solution to lyse, centrifuge, and take the supernatant; detect the supernatant with an MDA detection kit, and record it as sample group 1; among them, the sample of Lactobacillus rhamnosus fermentation broth solution is a mixture of Lactobacillus rhamnosus fermentation broth and high - glucose DMEM medium solution containing 10% FBS and 1% double antibiotics, and the concentration of Lactobacillus rhamnosus fermentation broth is 0.1%;

[0359] Add 2 mL of H2O2 solution to the fourth well of the washed 6-well plate, incubate for 1 h, then add the solution sample of Lactobacillus rhamnosus fermentation broth, and incubate in an incubator at 37 °C and 5% CO2 for 24 h; then add lysis buffer to lyse, centrifuge, and take the supernatant; detect the supernatant with an MDA detection kit, and record it as sample group 2; among them, the solution sample of Lactobacillus rhamnosus fermentation broth is a mixture of Lactobacillus rhamnosus fermentation broth and high-glucose DMEM medium solution containing 10% FBS and 1% double antibody, and the concentration of Lactobacillus rhamnosus fermentation broth is 0.5%.

[0360] Add 2 mL of H2O2 solution to the fifth well of the washed 6-well plate, incubate for 1 h, then add the solution sample of Lactobacillus rhamnosus fermentation broth, and incubate in an incubator at 37 °C and 5% CO2 for 24 h; then add lysis buffer to lyse, centrifuge, and take the supernatant; detect the supernatant with an MDA detection kit, and record it as sample group 3; among them, the solution sample of Lactobacillus rhamnosus fermentation broth is a mixture of Lactobacillus rhamnosus fermentation broth and high-glucose DMEM medium solution containing 10% FBS and 1% double antibody, and the concentration of Lactobacillus rhamnosus fermentation broth is 1%.

[0361] Note: The detection method of the MDA detection kit is carried out according to the instructions of the MDA detection kit.

[0362] 11-2. Experimental results and analysis

[0363] The experimental results are shown in Figure 14 .

[0364] Figure 14 It is a schematic diagram of the MDA content in Experimental Example 11.

[0365] Malondialdehyde (MDA) is the end product of lipid peroxidation, which can cause cross-linking polymerization of biological macromolecules such as proteins and nucleic acids and has cytotoxicity. Reducing the MDA content helps to maintain the viability of cells in the local area rich in sebum.

[0366] Figure 14 The results show that compared with the control group, the Lactobacillus rhamnosus fermentation broth in sample groups 1-3 can significantly reduce the intracellular MDA content at concentrations of 0.1% to 1% and has the ability of anti-lipid peroxidation.

[0367] Unless otherwise specified, the MRS medium involved in the embodiments of the present application refers to a medium composed of yeast extract and yeast extract, and is purchased from Guangdong Huankai Biotechnology Co., Ltd.

[0368] The above specific embodiments have described the present invention in detail, but these do not constitute a limitation to the present invention. The protection scope of the present invention is not limited to the above embodiments. Any equivalent modifications or changes made by those of ordinary skill in the art according to the disclosure of the present invention shall fall within the protection scope recorded in the claims.

Claims

1. Lactobacillus rhamnosus, characterized in that, It is preserved in the China General Microbiological Culture Collection Center with the preservation number: CGMCC No. 31345.

2. Preparation method of Lactobacillus rhamnosus fermentation broth, characterized in that, Including: Obtaining a Lactobacillus rhamnosus seed solution according to the Lactobacillus rhamnosus described in claim 1; Inoculating the Lactobacillus rhamnosus seed solution into a culture medium to obtain an inoculum; Fermenting and culturing the inoculum at a fermentation temperature of 35 - 40 °C, a shaking speed of 150 - 200 rpm, and a fermentation time of 18 - 48 hours to obtain a fermentation product; Separating and treating the fermentation product to obtain the liquid part after separation, namely the Lactobacillus rhamnosus fermentation broth.

3. The Lactobacillus rhamnosus fermentation broth, characterized in that The Lactobacillus rhamnosus fermentation broth is obtained by fermenting and treating the Lactobacillus rhamnosus described in claim 1.

4. Lactobacillus rhamnosus fermentation broth, characterized in that, Prepared according to the preparation method of the Lactobacillus rhamnosus fermentation broth described in claim 2.

5. A preparation for the skin, including: The Lactobacillus rhamnosus described in claim 1; Or The Lactobacillus rhamnosus fermentation broth described in claim 3 or 4.

6. Use of the Lactobacillus rhamnosus described in claim 1 in the preparation of a preparation for the skin.

7. Use according to claim 6, wherein The preparation has one or more of the following uses: Inhibiting skin oil; Inhibiting the expression of oil in skin cells; Inhibiting 5α - reductase; Inhibiting Propionibacterium acnes; Antioxidant; Scavenging ABTS free radicals; Scavenging reactive oxygen species; Repairing skin photo - damage; Repairing skin scratch damage; Promoting the migration ability of skin cells; Anti - aging; Promoting the expression of type I collagen; Inhibiting the expression of β - galactosidase in skin cells; Inhibiting the expression of MMP - 1 in skin cells; and Reducing the MDA content in skin cells.

8. Use of the Lactobacillus rhamnosus described in claim 1 in the preparation of the Lactobacillus rhamnosus fermentation broth.

9. Use of the Lactobacillus rhamnosus fermentation broth described in claim 3 or 4 in the preparation of a preparation for the skin.

10. The use according to claim 8, wherein, The preparation has one or more of the following uses: Inhibiting skin oil; Inhibiting the expression of oil in skin cells; Inhibiting 5α - reductase; Inhibiting Propionibacterium acnes; Antioxidant; Scavenging ABTS free radicals; Scavenging reactive oxygen species; Repairing skin photo - damage; Repairing skin scratch damage; Promoting the migration ability of skin cells; Anti - aging; Inhibiting the expression of β - galactosidase in skin cells; Promoting the expression of type I collagen; Inhibiting the expression of MMP - 1 in skin cells; and Reducing the MDA content in skin cells.

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