Lactobacillus gasseri bsjm25l011, extracellular protein of lactobacillus gasseri with oil-controlling efficacy, preparation method and application thereof

Abstract

The application discloses Lactobacillus gasseri BSJM25L011, Lactobacillus gasseri extracellular protein with oil-controlling efficacy and a preparation method and application thereof. Lactobacillus gasseri The Lactobacillus gasseri BSJM25L011 is classified as Lactobacillus gasseri and is preserved in the China General Microbiological Culture Collection Center on June 16, 2025, with a preservation number of CGMCC No. 34898 and a preservation address of No. 3, Xili, Beichen West Road, Chaoyang District, Beijing, with a postal code of 100101. The Lactobacillus gasseri extracellular protein with oil-controlling efficacy is obtained by fermentation culture of the Lactobacillus gasseri strain BSJM25L011. It is found that the Lactobacillus gasseri BSJM25L011 can produce a novel, mild and efficient natural biological oil-controlling active ingredient. The Lactobacillus gasseri extracellular protein has excellent oil-controlling efficacy, can significantly regulate skin sebum secretion, can be widely applied to the field of oil-controlling skin external agents, expands the application field of the Lactobacillus gasseri and has high safety in use.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology and cosmetics, specifically relating to Lactobacillus gasseri BSJM25L011, Lactobacillus gasseri extracellular protein with oil-controlling effects, its preparation method and application. Background Technology

[0002] Excessive sebum secretion is a common skin problem, especially among teenagers. Hormonal changes increase sebaceous gland activity and lead to excessive sebum production. Adults are also prone to oily skin due to stress, unhealthy lifestyle habits (such as staying up late and high-sugar, high-fat diets), and environmental factors (such as high temperature and humidity). If excessive sebum secretion is not cleaned and regulated in time, it can clog pores, providing a breeding ground for microorganisms such as Propionibacterium acnes, leading to skin diseases such as acne, pimples, and seborrheic dermatitis, seriously affecting physical and mental health.

[0003] Probiotics are live microorganisms that are beneficial to the human body. They can improve the gut microbiota by inhibiting the growth and reproduction of harmful bacteria and are commonly found in certain foods and supplements. Probiotics have broad application prospects in cosmetics, especially in regulating skin microecological balance, anti-inflammation, and anti-aging. However, because cosmetics have specific regulations on the content of microorganisms, directly adding probiotics to cosmetics is not feasible.

[0004] Lactobacillus gasseri ( Lactobacillus gasseri Lactobacillus gasseri is a facultative anaerobic probiotic found in the oral cavity and gastrointestinal mucosa. Current research on Lactobacillus gasseri focuses primarily on its probiotic properties and gut health benefits. For example, it can maintain gastrointestinal and reproductive tract stability through various pathways, such as increasing gut microbiota diversity, raising short-chain fatty acid concentrations, reducing the expression and secretion of pro-inflammatory factors, and increasing acidic mucoprotein content. It can also regulate the body's immune response through various pathways, including strengthening the immune barrier, regulating immune cell activity, anti-inflammation, enhancing mucosal immunity, anti-allergy, and anti-infection. Furthermore, it produces various bacteriocins, which significantly inhibit the growth and reproduction of various disease-causing bacteria, such as Staphylococcus aureus and Listeria monocytogenes. Currently, it is widely used in the food and medical fields. In the food sector, it is used in fermented foods, probiotic products, functional foods, food preservation, and infant formula. In the medical field, it is mainly used to regulate gut health, maintain vaginal homeostasis, treat obesity, and provide anti-inflammatory and antioxidant effects. However, there are few reports on the application of Lactobacillus gasseri in the cosmetics field, resulting in its high-value-added application potential remaining untapped. Summary of the Invention

[0005] To address the technical problem of excessive sebum secretion from sebaceous glands, this invention aims to provide *Lactobacillus gasseri* BSJM25L011, an extracellular protein of *Lactobacillus gasseri* with oil-controlling effects, its preparation method, and its applications. This invention discovers that *Lactobacillus gasseri* BSJM25L011 can produce a novel, mild, and highly effective natural biological oil-controlling active ingredient. The extracellular protein of *Lactobacillus gasseri* of this invention has excellent oil-controlling effects, can significantly regulate skin sebum secretion, and can be widely used in the field of oil-controlling topical skin agents, expanding the application scope of *Lactobacillus gasseri*, and has high safety in use.

[0006] The first aspect of this invention provides a strain of Lactobacillus gasseri BSJM25L011, whose taxonomic name is... Lactobacillus gasseri It was deposited on June 16, 2025, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 34898. The deposit address is No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, 100101, China.

[0007] The Lactobacillus gasseri strain BSJM25L011 provided by this invention can produce extracellular protein with oil-controlling effect, and the extracellular protein obtained after fermentation has excellent oil-controlling effect.

[0008] The second aspect of the present invention provides an extracellular protein of Lactobacillus gasseri with oil-controlling effects, wherein the extracellular protein of Lactobacillus gasseri is obtained by fermentation culture of Lactobacillus gasseri strain BSJM25L011 described in the first aspect of the present invention.

[0009] The third aspect of the present invention provides a method for preparing extracellular protein of Lactobacillus gasseri with oil-controlling effect, comprising inoculating Lactobacillus gasseri into a fermentation substrate for fermentation culture, separating and collecting the supernatant after fermentation culture, and filtering and sterilizing the supernatant and salting out; wherein the fermentation culture temperature is 37~45℃ and the fermentation culture time is 24~50h.

[0010] In one embodiment, the *Lactobacillus gasseri* includes strain BSJM25L011, deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 34898, and its taxonomic name is... Lactobacillus gasseri The date of deposit is June 16, 2025. The deposit address is No. 3, No. 1 Courtyard, Beichen West Road, Chaoyang District, Beijing, 100101, China.

[0011] In one embodiment, the *Lactobacillus gasseri* is inoculated in the form of a *Lactobacillus gasseri* bacterial suspension. The viable count of *Lactobacillus gasseri* in the bacterial suspension can be 10-1. 6 ~10 8 CFU / mL, preferably 107 CFU / mL.

[0012] In one embodiment, the method for preparing the Lactobacillus gasseri bacterial suspension can be conventional in the art. For example, it may specifically include activating the Lactobacillus gasseri strain BSJM25L011 in a plate, inoculating the activated strain into the culture medium using an inoculation loop, and culturing at 37°C until the viable count reaches 10^6. 6 ~10 8 CFU / mL.

[0013] In one embodiment, based on the volume of the fermentation substrate, the inoculation amount of the *Lactobacillus gasseri* culture is 1% to 7% (v / v), preferably 2% to 5% (v / v). That is, the volume ratio of the inoculation volume of the *Lactobacillus gasseri* culture to the volume of the fermentation substrate is (1~7):100, preferably (2~5):100.

[0014] In one embodiment, the Lactobacillus gasseri culture is inoculated into the fermentation substrate, mixed evenly, and then fermented.

[0015] In one embodiment, the fermentation substrate includes a culture medium, a carbon source, and water.

[0016] In one embodiment, the mass ratio of the culture medium, the carbon source, and the water is (50~150):(1~5):300, preferably (80~120):(1.5~3):300, for example 50:1:150.

[0017] In one embodiment, the culture medium may include MRS culture medium, etc.

[0018] In one embodiment, the carbon source may include glucose, fructose, galactose, mannose, etc., preferably glucose. The *Lactobacillus gasseri* strain of the present invention grows rapidly when glucose is the carbon source, while other sugar sources have lower utilization efficiency and slower metabolism.

[0019] In one embodiment, the water includes purified water and / or deionized water.

[0020] In one embodiment, the method for preparing the fermentation substrate includes: mixing the components of the fermentation substrate evenly.

[0021] In one embodiment, the fermentation substrate may further include sterilization and cooling operations before use. Further, the sterilization method for the fermentation substrate is generally high-temperature sterilization. The sterilization temperature can be a temperature conventional for this type of operation in the art, preferably 110℃~125℃, more preferably 115℃~125℃, for example 121℃; the sterilization pressure can be a pressure conventional for this type of operation in the art, preferably 0.1~0.15MPa, more preferably 0.1~0.13MPa, for example 0.12MPa; the sterilization time can be a time conventional for this type of operation in the art, preferably 20~40min, more preferably 25~35min, for example 30min.

[0022] In one embodiment, the cooling can be to room temperature.

[0023] In one embodiment, the fermentation culture is a static fermentation culture. Further, the fermentation culture can be carried out in a constant temperature incubator.

[0024] In one embodiment, the fermentation culture temperature is 40-44°C, more preferably 43°C. The inventors have found that when the fermentation temperature is 40-44°C, the extracellular protein obtained from fermentation is significantly more effective than extracellular proteins obtained from fermentation at other temperatures in controlling oil production, regulating skin sebum secretion, reducing triglyceride (TG) and free fatty acid (FFA) secretion, inhibiting sebum synthesis, inhibiting the expression of sebum secretion-related genes SREBP-1, FAS, and ACC1 in sebaceous gland cells, activating the AMPK signaling pathway, or inhibiting the PI3K / AkT / mTOR signaling pathway.

[0025] In one embodiment, the fermentation time is 36-50 hours.

[0026] In one embodiment, the separation can be performed using conventional separation methods in the art, such as centrifugation. The centrifugation speed can be a conventional speed for this type of operation in the art, preferably 4000~6000 rpm, more preferably 4500~5000 rpm, for example 4800 rpm; the centrifugation time can be a conventional time for this type of operation in the art, preferably 20~40 min, more preferably 25~35 min, for example 30 min.

[0027] In one embodiment, the filter membrane used for filtration sterilization may have a pore size of 0.15~0.45μm, preferably 0.2~0.25μm, and more preferably 0.22μm. After filtration sterilization, a sterile fermentation supernatant is obtained.

[0028] In one embodiment, the salting out may be performed using ammonium sulfate fractional salting out.

[0029] In one embodiment, ammonium sulfate fractional salting-out is performed sequentially at saturations of 20-30%, 40-60%, and 70-80%. Preferably, ammonium sulfate fractional salting-out is performed sequentially at saturations of 25%, 50%, and 75%. The ammonium sulfate saturation mentioned in this invention refers to saturation at 20-30°C, for example, saturation at 25°C.

[0030] In one specific embodiment, the ammonium sulfate fractional salting-out method includes: The ammonium sulfate saturation in the sterile fermentation supernatant is set to 20-30%, preferably 25%, for the first protein salting out. After standing, the supernatant S1 and protein precipitate P1 are collected by centrifugation. The ammonium sulfate saturation in the supernatant S1 is 40-60%, preferably 50%, and a second protein salting-out is performed. After standing, the supernatant S2 and protein precipitate P2 are collected. The ammonium sulfate saturation in the supernatant S2 is set to 70-80%, preferably 75%, for a third protein salting-out. After standing, the mixture is centrifuged to collect the protein precipitate P3.

[0031] In one embodiment, the saturation is achieved by adding ammonium sulfate solid to the supernatant.

[0032] In one embodiment, the addition of the ammonium sulfate solid and each stage of the ammonium sulfate fractional salting-out process are carried out under constant temperature and stirring conditions.

[0033] Furthermore, the constant temperature is 20~30℃, preferably 25℃; the stirring speed is 150~250rpm, preferably 180~200rpm. Specifically, the stirring can be carried out using conventional stirring methods in the art, such as on a constant temperature magnetic stirrer.

[0034] In one embodiment, the time for each stage of ammonium sulfate fractional salting-out is 0.5 to 2 hours, preferably 1 to 1.5 hours.

[0035] In one embodiment, after each stage of ammonium sulfate fractional salting out, the process further includes settling, separation, and collection of protein precipitates.

[0036] In one embodiment, the settling temperature is 2~8℃, preferably 3~4℃; the settling time is 1~4h, preferably 1.5~2h.

[0037] In one embodiment, the separation in the ammonium sulfate fractionation salting-out can be performed by centrifugation. The centrifugation speed is 10,000~15,000 rpm, preferably 11,000~13,000 rpm, for example 12,000 rpm; the centrifugation time is 10~20 min, preferably 13~16 min, for example 15 min; and the centrifugation temperature is 2~8℃, preferably 3~4℃.

[0038] In one embodiment, the method further includes a step of purifying the collected protein precipitate.

[0039] In one embodiment, the purification can be performed using dialysis. Dialysis removes salts and small molecule impurities, and the resulting contents after dialysis are the purified, high-purity Lactobacillus gasseri extracellular proteins.

[0040] In one embodiment, the dialysis method includes: first dissolving the collected protein precipitate with a buffer solution, filtering it for sterilization, transferring it into a dialysis bag, and performing dialysis to obtain the Lactobacillus gasseri extracellular protein.

[0041] In one embodiment, the buffer solution may be a Tris-HCl buffer solution, etc. The pH value of the buffer solution is 7.0 to 8.0, preferably 7.5; the amount of buffer solution used can be conventional in the art.

[0042] In one embodiment, the filter membrane used for filtration and sterilization in the dialysis method has a pore size of 0.15~0.45μm, preferably 0.22μm.

[0043] In one embodiment, the molecular weight cutoff of the dialysis bag is 300~1000Da, preferably 400~600Da, for example 500Da.

[0044] In one embodiment, the dialysis temperature is 2~8°C, preferably 4°C.

[0045] In one embodiment, the dialysis time is 2 to 5 days, with fluid changed 2 to 4 times a day.

[0046] In one embodiment, the dialysate used for dialysis is a Tris-HCl buffer solution, etc.

[0047] In one embodiment, the process of mixing the collected protein precipitate is included before dissolving it.

[0048] In one embodiment, after purification, the purified material is further subjected to freeze-drying. Further, when purification is performed using dialysis, the liquid obtained after dialysis is freeze-dried.

[0049] In one embodiment, the freeze-drying can be performed using methods conventional in the art. The freeze-dried product can be stored at -80 to -15°C, for example, -20°C.

[0050] The fourth aspect of the present invention provides an extracellular protein of Lactobacillus gasseri with oil-controlling effects, which is prepared by the method for preparing extracellular protein of Lactobacillus gasseri described in the third aspect of the present invention.

[0051] The fifth aspect of the present invention provides the application of Lactobacillus gasseri strain BSJM25L011 described in the first aspect of the present invention, or Lactobacillus gasseri extracellular protein described in the second aspect of the present invention, or Lactobacillus gasseri extracellular protein prepared by the method described in the third aspect of the present invention, in the preparation of products with oil-controlling effects.

[0052] In one embodiment, the oil control can be achieved by regulating the secretion of sebum in the skin.

[0053] In one embodiment, the oil-controlling effect has one of the following effects: Reduce sebaceous gland alveoli; It causes the sebaceous glands to shrink and become smaller; This loosens the arrangement of sebaceous glands; Inhibit sebaceous gland cells from secreting sebum; Inhibit sebum synthesis; Reduce the area of ​​sebaceous gland spots; It can lighten the color of sebaceous gland spots.

[0054] In one embodiment, the inhibition of sebum secretion by sebaceous gland cells can be achieved by intervening in the expression of genes related to sebum synthesis.

[0055] In one embodiment, the inhibition of sebum secretion by sebaceous gland cells can be achieved by inhibiting the expression of sebum secretion-related genes SREBP-1, FAS, and ACC1 in sebaceous gland cells; furthermore, by inhibiting the expression of sebum secretion-related genes SREBP-1, FAS, and ACC1 in sebaceous gland cells, the secretion of triglycerides and / or free fatty acids can be regulated, thereby reducing the level of sebum secretion.

[0056] In one embodiment, sebum synthesis is inhibited by regulating the sebum signaling pathway; further, sebum synthesis is inhibited by activating the AMPK signaling pathway or inhibiting the PI3K / AkT / mTOR signaling pathway.

[0057] In one embodiment, in the preparation of a product with oil-controlling effects, the Lactobacillus gasseri extracellular protein can be used directly as a product or as an additive.

[0058] In one embodiment, the product with oil-controlling effects can be a topical skin agent. Further, the topical skin agent can be an oil-controlling cosmetic, a topical skin care agent, etc., specifically, for example, an essence, serum, lotion, cream, or mask.

[0059] In one embodiment, the product with oil-controlling effects can be used to control oil production on the skin.

[0060] The sixth aspect of the present invention provides an oil-controlling product, comprising the Lactobacillus gasseri extracellular protein described in the second aspect of the present invention or the Lactobacillus gasseri extracellular protein described in the fourth aspect of the present invention.

[0061] In one embodiment, based on the quality of the oil-controlling product, the mass content of the Lactobacillus gasseri extracellular protein is 1% to 10%, more preferably 2.5% to 10%.

[0062] The sixth aspect of the present invention provides a topical skin agent comprising the extracellular protein of Lactobacillus gasseri as described in the second aspect of the present invention or the extracellular protein of Lactobacillus gasseri as described in the fourth aspect of the present invention.

[0063] In one embodiment, based on the mass of the topical skin agent, the mass content of the Lactobacillus gasseri extracellular protein is 1% to 10%, more preferably 2.5% to 10%.

[0064] In one embodiment, the topical skin agent further includes active ingredients commonly used in the art, which may generally include at least one of moisturizing active ingredients, whitening active ingredients, skin barrier repair active ingredients, antibacterial or microecological regulating active ingredients.

[0065] Based on common knowledge in the field, the above-mentioned preferred conditions can be combined arbitrarily to obtain various preferred embodiments of this application.

[0066] The positive and progressive effects of this application are as follows: (1) Existing technologies are mostly focused on the extraction of Lactobacillus gasseri proteins or general functional studies, but lack functional application studies. There are no public reports or applications of Lactobacillus gasseri extracellular proteins in the field of skin oil control. This invention is the first to discover and verify the unique effect of Lactobacillus gasseri extracellular proteins in regulating skin sebum secretion. It provides a new raw material for developing oil-control cosmetics or skin care products with a new mechanism. It can be used as the core raw material for oil control, breaking through the traditional thinking that existing oil-control ingredients mostly rely on chemical synthesis or plant extraction. It expands the application of Lactobacillus gasseri in the field of cosmetics and can be safely used in oil-control cosmetics.

[0067] (2) The extracellular protein of Lactobacillus gasseri in this invention can be used as a natural biological oil-controlling active ingredient. It has excellent oil-controlling effect, can significantly reduce the area of ​​sebaceous gland spots, and make the color of sebaceous gland spots lighter; reduce sebaceous acini, and make the glands shrink and loosely arranged; inhibit the secretion of sebum by sebaceous gland cells, reduce the content of triglycerides and free fatty acids in sebaceous gland cells; and inhibit sebum synthesis. Moreover, it is non-irritating to the skin, gentle, highly effective and stable.

[0068] (3) The method of the present invention obtains Lactobacillus gasseri extracellular protein with excellent performance in regulating skin sebum secretion by fermenting Lactobacillus gasseri under certain temperature and other conditions, combined with fractional salting out and fine purification process. Attached Figure Description

[0069] This application can be better understood by referring to the description given below in conjunction with the accompanying drawings. These drawings, together with the detailed description below, are included in and form part of this specification, and are used to further illustrate preferred embodiments of the application and explain the principles and advantages of the application. Wherein: Figure 1 This is a comparison of the effects of topical administration of LG-EP prepared in Example 2 at different concentrations for 28 days on the body weight of LVG golden hamsters; Figure 2 The images show the effects of different concentrations of LG-EP prepared in Example 2 on the appearance of LVG-stained sebaceous glands in golden hamsters. Figure 3 This is a graph showing the effect of LG-EP prepared in Example 2 at different concentrations on the area of ​​sebaceous gland spots in golden hamsters with LVG. Figure 4 Microscopic images showing the effect of LG-EP prepared in Example 2 at different concentrations on the microstructure of local sebaceous gland patches in golden hamsters with LVG. Figure 5 Microscopic images showing the effect of LG-EP prepared in Example 2 at different concentrations on sebum accumulation in LVG golden hamster sebaceous gland spots; Figure 6 The effect of LG-EP prepared in Example 2 at different concentrations on the content of triglycerides (TG) and free fatty acids (FFA) in the sebaceous gland patch tissue of LVG golden hamsters; Figure 7 The effect of LG-EP prepared in Example 2 at different concentrations on the transcription level of genes closely related to sebum secretion in the sebaceous gland patch tissue of golden hamsters; Figure 8The effects of LG-EP prepared in Example 2 at different concentrations on the PI3K / AkT / mTOR signaling pathway and the AMPK / mTOR signaling pathway. Detailed Implementation

[0070] The present application is further illustrated below by way of embodiments, but these embodiments are not intended to limit the scope of the present application. Experimental methods not specifically described in the following embodiments are performed according to conventional methods and conditions, or as selected in accordance with the product instructions.

[0071] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0072] In the following examples, the MRS culture medium was purchased from Shanghai Yuanye Biotechnology Co., Ltd., catalog number R42468.

[0073] In the following embodiments, the freeze-drying was performed using a CoolSafe 110-4 instrument manufactured by Gene Biotechnology International Trading (Shanghai) Co., Ltd., Beijing Branch.

[0074] In the following examples, the saturation of ammonium sulfate refers to the saturation at 25°C.

[0075] In the following examples, *Lactobacillus gasseri* strain BSJM25L011 was deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 34898, and its taxonomic name is [not specified]. Lactobacillus gasseri The date of deposit is June 16, 2025. The deposit address is No. 3, No. 1 Courtyard, Beichen West Road, Chaoyang District, Beijing, 100101, China.

[0076] In the following examples, the preparation of Lactobacillus gasseri culture was as follows: Lactobacillus gasseri strain BSJM25L011 in a plate was activated, and the activated strain was inoculated into the culture medium using an inoculation loop. The culture was then incubated at 37°C until the viable count reached 102. 7 CFU / mL.

[0077] Example 1 1. Lactobacillus gasseri strain ( Lactobacillus gasseri Separation of BSJM25L011 The *Lactobacillus gasseri* strain described in this invention is derived from fresh breast milk from healthy volunteers. Healthy lactating female volunteers who have not used antibiotics for more than two weeks were selected. Breast milk samples were collected using sterile collection tubes and processed within 2 hours of collection. The breast milk samples were added to sterile physiological saline at a ratio of 1:9 and thoroughly mixed, then... -1 ~10 -6Perform serial dilutions. Spread 100 μL of each dilution onto the surface of MRS solid medium and incubate at 37°C under anaerobic (or microaerobic) conditions for 24–48 h. Select colonies exhibiting typical lactic acid bacteria characteristics, including milky white or light yellow, round, with regular edges and a smooth, moist surface, and perform three consecutive streak purifications to obtain a single strain. Perform Gram staining, MRS plate purity checks, and microscopic observation on the purified strain to confirm the absence of contamination. Then, add the strain to MRS cryoprotectant containing 20% ​​glycerol and incubate. Store at 80℃.

[0078] 2. Lactobacillus gasseri strains ( Lactobacillus gasseri BSJM25L011 Morphological Identification Morphological identification of the isolated target strain revealed that after incubation on MRS solid medium at 37°C for 24–48 h, it formed typical Lactobacillus colonies. The colonies were milky white or pale yellow, soft in texture, smooth, slightly moist, approximately 1–3 mm in diameter, with regular edges, and no pigment deposition or hemolysis. Gram staining showed that the strain was Gram-positive, appearing as short rods with rounded ends under the microscope, distributed singly, in pairs, or in short chains. The cell size was approximately 0.5–1.2 μm × 1.5–4.0 μm, non-spore-forming, non-flagellated, and non-motile. The strain was negative for catalytic enzyme tests and exhibited typical physiological and biochemical characteristics of lactic acid bacteria.

[0079] 3. Lactobacillus gasseri strains ( Lactobacillus gasseri Preservation of strain BSJM25L011 Lactobacillus gasseri strain ( Lactobacillus gasseri BSJM25L011 was deposited on June 16, 2025, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 34898. The deposit address is No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, 100101, China.

[0080] Example 2 Weigh 100g of MRS medium and 2g of analytical grade glucose, dissolve them in 300mL of deionized water, mix thoroughly, and sterilize at 121℃ for 30min. After sterilization, cool to room temperature to obtain the fermentation medium. Inoculate 3mL of *Lactobacillus gasseri* culture into the obtained fermentation medium, shake well, and then incubate at 43℃ for 48h. After fermentation, centrifuge at 4800rpm for 30min, collect the supernatant, and obtain the *Lactobacillus gasseri* fermentation broth. Filter the obtained *Lactobacillus gasseri* fermentation broth through 0.22μm filter paper and collect the sterile fermentation supernatant.

[0081] Then, a fractional salting-out method using ammonium sulfate (25%-50%-75%) was employed. The collected sterile fermentation supernatant was placed on a magnetic stirrer and stirred at 200 rpm at 25°C. Solid ammonium sulfate was added to achieve a saturation of 25% in the supernatant. After adding the solid ammonium sulfate, stirring was continued at 25°C and 200 rpm for 1 hour to perform the first protein salting-out. After stirring, the mixture was transferred to a 4°C refrigerator and allowed to stand for 2 hours. Then, it was centrifuged at 4°C and 12000 rpm for 15 minutes, and the supernatant S1 and protein precipitate P1 were collected. Subsequently, solid ammonium sulfate was added to the collected supernatant S1 at 25°C and 200 rpm to achieve a saturation of 25% in the supernatant S1. The concentration was 50%, and the mixture was stirred at 25°C and 200 rpm for 1 hour for a second protein salting-out. After stirring, the mixture was transferred to a 4°C refrigerator and allowed to stand for 2 hours. Then, it was centrifuged at 4°C and 12000 rpm for 15 minutes, and the supernatant S2 and protein precipitate P2 were collected. Then, ammonium sulfate solid was added to the collected supernatant S2 at 25°C and 200 rpm to make the ammonium sulfate saturation in supernatant S2 75%. The mixture was stirred at 25°C and 200 rpm for 1 hour for a third protein salting-out. After stirring, the mixture was transferred to a 4°C refrigerator and allowed to stand for 2 hours. Then, it was centrifuged at 4°C and 12000 rpm for 15 minutes, and protein precipitate P3 was collected.

[0082] The collected and mixed protein precipitates P1, P2, and P3 were dissolved in Tris-HCl buffer at pH 7.5, and then filtered through a microporous membrane with a pore size of 0.22 μm to obtain a protein solution. The obtained protein solution was placed in a dialysis bag with a molecular weight cutoff of 500 Da and dialyzed with Tris-HCl buffer at pH 7.5 at 4°C. The buffer was changed 3 times a day for 3 days. The liquid obtained after dialysis was freeze-dried to finally obtain the Lactobacillus gasseri extracellular protein LG-EP with oil-controlling effect.

[0083] Example 3 Effect of external application of Lactobacillus gasseri extracellular protein (LG-EP) on the body weight of golden hamsters Experimental methods: 25 male LVG golden hamsters (6-7 weeks old), SPF grade, were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd., with the license number SCXK (Beijing). 25 LVG golden hamsters were randomly divided into 5 groups, with 5 hamsters in each group. After 7 days of adaptive feeding, LVG golden hamsters without adverse reactions and with normal diet, drinking and activity were included in the experiment. They were raised in separate cages, fed twice a day, and given free access to drinking water. The hair around the sebaceous gland spots of LVG golden hamsters was removed with depilatory cream and a depilatory razor, so as to fully expose the complete sebaceous gland spots clearly. The maximum transverse diameter and maximum longitudinal diameter of the sebaceous gland spots of LVG golden hamsters were measured with a vernier caliper, the product of which was recorded as the area of the sebaceous gland spots, and photographic records were made at the same time.

[0084] Preparation of test samples: 0.1g, 0.3g, and 0.5g of the extracellular protein LG-EP prepared in Example 2 were respectively dissolved in 1mL of water to prepare 100 mg / mL LG-EP (low-dose sample), 300 mg / mL LG-EP (medium-dose sample), and 500 mg / mL LG-EP (high-dose sample).

[0085] According to the experimental requirements, the above 5 groups of LVG golden hamsters were named respectively: blank control group (BC), positive control group (PC), high-dose sample group (High LG-EP), medium-dose sample group (Medium LG-EP) and low-dose sample group (Low LG-EP). For each group, the corresponding sample was applied respectively, twice a day, 200 μL each time, for 28 consecutive days. Among them, the blank control group (BC) was administered with deionized water; the positive control group (PC) was administered with Clindamycin and Metronidazole and Retinoic Acid Gel (purchased from Hefei Lifang Pharmaceutical Co., Ltd., 6g per tube); the low-dose sample group (Low LG-EP) was administered with 100 mg / mL LG-EP; the medium-dose sample group (Medium LG-EP) was administered with 300 mg / mL LG-EP; the high-dose sample group (High LG-EP) was administered with 500 mg / mL LG-EP. The activity, diet, drinking, fecal morphology and the condition of sebaceous gland spots (whether there were adverse reactions after administration) of LVG golden hamsters were observed every day. The body weight of LVG golden hamsters was measured every week, and the new hair growing around the sebaceous gland spots was removed in time. After 28 days of external administration, the body weight measurement results of LVG golden hamsters are shown in Figure 1 .

[0086] From Figure 1As can be seen, there was no significant difference in the initial weight of LVG golden hamsters in each group. As the experimental time increased, the weight of LVG golden hamsters in each group increased. There was no significant difference in weight between each sample group (high-dose group, medium-dose group, low-dose group) and the positive control group (PC) and the blank control group (BC), indicating that topical application of LG-EP had no effect on the weight of LVG golden hamsters.

[0087] Example 4: Changes in the appearance and area of ​​sebaceous gland spots Similar to Example 3, except that the area of ​​sebaceous gland patches in LVG golden hamsters was measured weekly, and the morphological changes of the sebaceous gland patches were photographed and recorded. During the 28-day experimental period of topical application, photographs of the appearance of sebaceous gland patches in each group of LVG golden hamsters at days 0, 14, and 28 of the experiment are shown below. Figure 2 The effects of each group of samples on the change in the area of ​​sebaceous gland patches in LVG golden hamsters are shown in Table 1 and 2. Figure 3 . p < 0.01, p < 0.001, compared with the blank control group.

[0088] Table 1. Measurement results of sebaceous gland patch area in LVG golden hamsters

[0089] Depend on Figure 2 As can be seen, in the blank control group, the color of the sebaceous gland patches did not change significantly during the experimental period, and a gray halo was present around the sebaceous gland patches. Compared with the blank control group, the color of the sebaceous gland patches in the high-dose group, medium-dose group, low-dose group, and positive control group was significantly lighter, the boundary between the sebaceous gland patches and normal skin became blurred, and the protrusion range of the spots was significantly reduced. The results indicate that topical application of LG-EP can improve the characteristics of LVG golden hamster sebaceous gland patches, making the color of the golden hamster sebaceous gland patches lighter.

[0090] From Table 1 and Figure 3 As can be seen, at the same time point, there was no significant difference in the size of sebaceous gland patches among the LVG golden hamster groups at days 0 and 7 of the experiment. At day 14 of the experiment, compared with the blank control group, the area of ​​sebaceous gland patches in the positive control group, medium-dose group, and high-dose group of LVG golden hamsters was significantly reduced. At days 21 and 28 of the experiment, compared with the blank control group, the area of ​​sebaceous gland patches in each group of LVG golden hamsters was significantly reduced, and the effect of the sample on reducing the area of ​​sebaceous gland patches became more obvious with the increase of LG-EP concentration. The results indicate that topical application of LG-EP can effectively reduce the area of ​​sebaceous gland patches in LVG golden hamsters.

[0091] Example 5 HE staining The same method as in Example 3 was used for drug administration. After 28 days of administration, the rats were deprived of water and food for 12 hours. They were then euthanized by cervical dislocation. 2cm × 2cm sebaceous gland patch tissue was excised from each group (blank control group, positive control group, high-dose sample group, medium-dose sample group, and low-dose sample group) using forceps and a scalpel, and sectioned in paraffin. The paraffin sections were dewaxed to water, stained with hematoxylin for 3-5 minutes, washed with tap water, differentiated with differentiation solution, washed with tap water, blued with blue solution, and rinsed with running water. The sections were then dehydrated in 85% and 95% graded alcohol solutions for 5 minutes each, and stained with eosin for 5 minutes. The sections were then cleared by sequentially immersing them in anhydrous ethanol I for 5 minutes, anhydrous ethanol II for 5 minutes, anhydrous ethanol III for 5 minutes, xylene I for 5 minutes, and xylene II for 5 minutes, and mounted with neutral resin. Microscopic observation was performed, and images were acquired and analyzed. The effects of each group of samples on the structure of the LVG golden hamster sebaceous gland patch tissue are shown in [the table below]. Figure 4 .

[0092] like Figure 4 As can be seen, in the blank control group, the sebaceous glands were lobulated, full, large, tightly packed, and thickened, with numerous surrounding glandular cells. Compared with the blank control group, in the positive control group, the sebaceous glands atrophied, became smaller, and were more loosely arranged. After applying high, medium, and low doses of the sample, compared with the blank control group, the sample groups showed a significant reduction in sebaceous acini, shrinkage of the glands, loosening of their arrangement, and a spindle-shaped appearance. The high-dose sample group showed the most significant changes in the microstructure of the sebaceous gland plaques.

[0093] Example 6: Oil Red O staining The same method as in Example 3 was used for drug administration. After 28 days of administration, the rats were deprived of water and food for 12 hours. LVG golden hamsters were then euthanized by cervical dislocation. Skin samples of approximately 0.5 × 0.5 cm in size and no more than 3 mm in thickness were collected from each group (i.e., blank control group, positive control group, high-dose sample group, medium-dose sample group, and low-dose sample group) using forceps and a scalpel. OCT embedding and rapid freezing were used to prepare frozen sections with a thickness of 8–10 μm. After section preparation, [the samples were then...]. Store at 80℃.

[0094] Frozen sections were thawed and fixed in fixative for 15 minutes, washed with tap water, and air-dried. Sections were then immersed in oil red stain for 8-10 minutes (covered and protected from light). After removal, sections were held for 3 seconds and then sequentially immersed in two tanks of 60% isopropanol for differentiation, 3 seconds and 5 seconds respectively. Sections were then sequentially immersed in two tanks of pure water for 10 seconds each. After removal, sections were held for 3 seconds and then counterstained with hematoxylin for 3-5 minutes, followed by rinsing in three tanks of pure water for 5 seconds, 10 seconds, and 30 seconds respectively. Differentiation was performed in differentiation solution (60% alcohol as solvent) for 2-8 seconds, followed by rinsing in two tanks of distilled water for 10 seconds each, and then in blue solution for 1 second. Sections were then gently immersed in two tanks of tap water for 5 seconds and 10 seconds respectively. The staining effect was examined under a microscope, and sections were mounted using glycerol-gelatin mounting medium. Observation was performed under a microscope, and images were acquired and analyzed. The effects of each group on sebum accumulation in the sebaceous gland spots of LVG golden hamsters are described in [reference needed]. Figure 5 .

[0095] from Figure 5 As can be seen, in the blank control group, numerous clusters of red lipid droplets were observed within the cytoplasm of sebaceous gland cells in the sebaceous gland patch tissue of golden hamsters, and these droplets merged into sheets, indicating vigorous lipid secretion. Compared with the blank control group, the accumulation of orange-red lipid droplets around the acini was significantly reduced in the positive control group. After applying high, medium, and low doses of the sample, sebum secretion in all sample groups showed a decreasing trend compared with the blank control group, with the high-dose sample group showing the most significant inhibitory effect on sebum secretion.

[0096] Example 7 Effect of Lactobacillus gasseri extracellular protein on triglyceride (TG) and free fatty acid (FFA) content in sebaceous plaques 100 mg of sebaceous gland plaque tissue samples from the blank control group, positive control group, high-dose sample group, medium-dose sample group, and low-dose sample group obtained in Example 5 were weighed separately and added to 900 μL of physiological saline. The samples were homogenized using a glass homogenizer under ice bath conditions, centrifuged at 8000g, 4℃ for 10 min, and the supernatant was collected for later use. Specific experimental procedures were performed according to the instructions for the Triglyceride (TG) Content Detection Kit (catalog number: BC0625) from Beijing Solarbio Science & Technology Co., Ltd., using an FC Multiskam microplate reader to measure the absorbance at 420 nm. The protein content in the tissue was determined using the BCA Protein Content Detection Kit from Beijing Bairui Biotechnology Co., Ltd. The triglyceride (TG) content in the sebaceous gland plaque tissue was calculated as follows: TG (mmol / g) = ((Atest - Ablank) / (Astandard - Ablank)) × 1.7 mmol / L ÷ Protein concentration of the sample (mg / mL).

[0097] The LVG golden hamster sebaceous gland patch tissues obtained in Example 5 (blank control group, positive control group, high-dose sample group, medium-dose sample group, and low-dose sample group) were rinsed with physiological saline. Excess water was absorbed with absorbent paper. 100 mg of the rinsed tissues from each group and 1 mL of the extraction solution from the Free Fatty Acid Content Detection Kit were added to a glass homogenizer, and the mixture was homogenized on ice. The homogenate was then centrifuged at 8000 rpm for 10 min at 4°C. The supernatant was collected for analysis. Specific experimental procedures were performed according to the Free Fatty Acid (FFA) Content Detection Kit instructions (catalog number: BC0595) from Beijing Solarbio Science & Technology Co., Ltd., using an FC Multiskam microplate reader to measure the absorbance at 550 nm. The protein content in the tissues was determined using the BCA protein content detection kit from Beijing Bairui Biotechnology Co., Ltd. FFA content (μmol / mg prot) = sample concentration x (μmol / mL) ÷ sample protein concentration (mg / mL). Results are shown below. Figure 6 , Figure 6 (A) shows the effect of different concentrations of LG-EP on the triglyceride content in the sebaceous gland patch tissue of LVG golden hamsters; (B) shows the effect of different concentrations of LG-EP on the free fatty acid content in the sebaceous gland patch tissue of LVG golden hamsters. p < 0.001, compared with the blank control group; ns p > 0.05 c p < 0.001, compared with the positive control group.

[0098] Table 2. Content of triglycerides (TG) and free fatty acids (FFA) in sebaceous gland plaque tissue.

[0099] From Table 2 and Figure 6 The results show that, compared with the blank control group, the extracellular protein of *Lactobacillus gasseri* in the sample group inhibited the secretion of both TG and FFA in the sebaceous gland patches of LVG golden hamsters. Furthermore, the inhibitory effect of LG-EP on the secretion of both TG and FFA increased with increasing concentration. The inhibitory effect of LG-EP in the high-dose group on the secretion of TG and FFA in the sebaceous gland patches of LVG golden hamsters was not significantly different from that in the positive control group. These results indicate that topical application of LG-EP to sebaceous gland patches can effectively reduce the secretion of TG and FFA.

[0100] Example 8: Effects of Lactobacillus gasseri extracellular proteins on the transcriptional levels of sebum secretion-related genes SREBP-1, FAS, and ACC1 in sebaceous gland plaques. The sebaceous gland plaque tissue obtained in Example 5 was homogenized in a sterile, enzyme-free glass homogenizer (1 mL Trizol was added for every 100 mg of Lactobacillus gasseri extracellular protein). After homogenization until complete lysis, the homogenate was transferred to a centrifuge tube, incubated at room temperature for 5 min, centrifuged at 12000 g and 4°C for 10 min, and the precipitate was discarded, keeping the supernatant for later use. 0.2 mL of chloroform was added to the supernatant, vortexed and mixed for 30 s, incubated at room temperature for 2 min, centrifuged at 12000 g and 4°C for 10 min, the upper aqueous phase was collected, and isopropanol was added at a 1:1 volume ratio. The mixture was inverted and mixed thoroughly, incubated at room temperature for 10 min, centrifuged at 12000 g and 4°C for 15 min, the supernatant was discarded, and the precipitate was kept. 1 mL of pre-cooled 75% ethanol was added to wash the precipitate, centrifuged at 12000 g and 4°C for 5 min, and air-dried upside down at room temperature for 10 min. 30 μL of DEPC water was added to dissolve the RNA precipitate, and the precipitate was stored at -80°C.

[0101] cDNA transfer process: Prepare the following reverse transcription system in an ice-cold, nuclease-free centrifuge tube: Table 3 Reverse Transcription System

[0102] Reverse transcription was performed using a PCR instrument, and the reaction steps are as follows: 1) 37℃, 2min; 2) 55℃, 10min; 3) 85℃, 10 sec After the reaction is complete, the obtained product should be placed on ice immediately or stored at -20°C for subsequent qPCR experiments.

[0103] Table 4 Primer Information

[0104] The qPCR reaction system is as follows: Table 5 Composition of reaction components per tube

[0105] The PCR reaction procedure was then set up as a three-step amplification method. Table 6. Parameter settings for the three-step amplification method

[0106] The calculation formula is as follows: 1. Calculate △Ct: △Ct = Ct value of the gene to be tested - Ct value of the internal reference gene.

[0107] 2. Calculate △△Ct: △△Ct = △Ct of the sample group - △Ct of the blank group.

[0108] 3. Calculate the expression level ratio: Expression level ratio = 2^(-△△Ct). See the results below. Figure 7 , Figure 7 (A) shows the effect of different concentrations of LG-EP on SREBP-1 transcriptional levels; (B) shows the effect of different concentrations of LG-EP on FAS transcriptional levels; (C) shows the effect of different concentrations of LG-EP on ACC1 transcriptional levels. NS p > 0.05 p < 0.05 p < 0.01, p < 0.001, compared with the blank control group; ns p > 0.05 a p < 0.05 b p < 0.01, c p < 0.001, compared with the positive control group.

[0109] from Figure 7 As can be seen, compared with the blank control group, the high-dose and medium-dose LG-EP groups significantly inhibited the transcription of SREBP-1 (sterol regulatory element-binding protein-1), FAS (fatty acid synthase), and ACC1 (acetyl-CoA carboxylase-1). The inhibitory effect of the high-dose LG-EP on the relative mRNA expression of these three genes was not significantly different from that of the positive control group. Moreover, the higher the concentration of the sample, the more significant the inhibitory effect on the mRNA expression of sebum synthesis-related genes. In conclusion, LG-EP can inhibit sebum secretion by suppressing the expression of SREBP-1, FAS, and ACC1, thereby alleviating acne.

[0110] Example 9: Effects of Lactobacillus gasseri extracellular proteins on the AMPK / mTOR and PI3K / AkT / mTOR signaling pathways. Same as Example 8, except for the primer information.

[0111] Table 7 Primer Information

[0112] The effects of LG-EP on the AMPK / mTOR and PI3K / AkT / mTOR pathways are as follows: Figure 8 As shown, Figure 8 (A) shows the effect of different concentrations of LG-EP on PI3K mRNA expression; (B) shows the effect of different concentrations of LG-EP on AkT mRNA expression; (C) shows the effect of different concentrations of LG-EP on mTOR mRNA expression; and (D) shows the effect of different concentrations of LG-EP on the relative mRNA expression of AMPK. NS p > 0.05 p < 0.05 p < 0.01, p < 0.001, compared with the blank control group; ns p > 0.05 a p < 0.05 b p < 0.01, compared with the positive control group.

[0113] from Figure 8 As can be seen, compared with the blank control group, LG-EP significantly upregulated the expression level of AMPK mRNA and significantly downregulated the expression levels of PI3K, AkT, and mTOR mRNA. Furthermore, the high-dose group of LG-EP showed an inhibitory effect on the expression of PI3K, AkT, and mTOR mRNA in the sebaceous gland tissue of golden hamsters, reaching the level of the positive control. In conclusion, LG-EP can exert its inhibitory effect on sebum synthesis by activating the AMPK signaling pathway or inhibiting the PI3K / AkT / mTOR signaling pathway.

[0114] Finally, it should be noted that the terms “comprising,” “including,” or any other variations thereof in this application are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such process, method, article, or apparatus.

[0115] Although this application has been disclosed above through the description of specific embodiments, it should be understood that those skilled in the art can devise various modifications, improvements, or equivalents to this application within the spirit and scope of the appended solutions. Such modifications, improvements, or equivalents should also be considered to be included within the scope of protection claimed in this application.

Claims

1. A type of Lactobacillus gasseri ( Lactobacillus gasseri strain BSJM25L011, characterized in that, The strain has the accession number CGMCC No. 34898.

2. A method for preparing extracellular protein of Lactobacillus gasseri with oil-controlling effects, characterized in that, include: Lactobacillus gasseri is inoculated into a fermentation substrate for fermentation culture. After fermentation culture is completed, the supernatant is separated and collected, and the supernatant is filtered for sterilization and salting out. The fermentation culture temperature is 40~44℃ and the fermentation culture time is 24~50h. Among them, the Lactobacillus gasseri includes the Lactobacillus gasseri strain BSJM25L011, which is deposited at the China General Microbiological Culture Collection Center with accession number CGMCC No.34898; The fermentation substrate comprises MRS medium, additional glucose and water, wherein the mass ratio of the MRS medium, additional glucose and water is (80~120):(1.5~3):300; The salting out is performed using ammonium sulfate graded salting out, with ammonium sulfate saturation of 20-30%, 40-60%, and 70-80% being performed sequentially. Each stage of the ammonium sulfate fractional salting-out process also includes a process of settling, separating, and collecting the protein precipitate. The method for preparing Lactobacillus gasseri extracellular protein further includes a step of purifying the collected protein precipitate; the purification is performed by dialysis, which includes: first dissolving the collected protein precipitate in a buffer solution, then sterilizing it, transferring it to a dialysis bag, and performing dialysis to obtain the Lactobacillus gasseri extracellular protein, wherein the molecular weight cutoff of the dialysis bag is 400~600 Da.

3. The method for preparing Lactobacillus gasseri extracellular protein according to claim 2, characterized in that, The Lactobacillus gasseri was inoculated in the form of Lactobacillus gasseri bacterial suspension; And / or, based on the volume of the fermentation substrate, the inoculum amount of Lactobacillus gasseri is 1% to 7% (v / v). And / or, the water includes purified water and / or deionized water; And / or, the fermentation substrate may further include sterilization and cooling operations before use; And / or, the fermentation culture is a static fermentation culture; And / or, the fermentation time is 36~50h.

4. The method for preparing Lactobacillus gasseri extracellular protein according to claim 3, characterized in that, The viable count of *Lactobacillus gasseri* in the *Lactobacillus gasseri* culture was 10. 6 ~10 8 CFU / mL; And / or, based on the volume of the fermentation substrate, the inoculum amount of Lactobacillus gasseri is 2% to 5% (v / v). And / or, inoculate the Lactobacillus gasseri culture into the fermentation substrate, mix thoroughly, and then carry out fermentation; And / or, the fermentation culture temperature is 43°C.

5. The method for preparing Lactobacillus gasseri extracellular protein according to claim 4, characterized in that, The viable count of *Lactobacillus gasseri* in the *Lactobacillus gasseri* culture was 10. 7 CFU / mL.

6. The method for preparing Lactobacillus gasseri extracellular protein according to any one of claims 2-5, characterized in that, The separation is performed by centrifugation; And / or, the pore size of the filter membrane used for filtration and sterilization is 0.15~0.45μm; And / or, sequentially perform fractional salting out of ammonium sulfate with saturations of 25%, 50%, and 75%; And / or, the saturation is achieved by adding ammonium sulfate solid to the supernatant; And / or, the time for each stage of salting out in the ammonium sulfate fractionation is 0.5 to 2 hours.

7. The method for preparing Lactobacillus gasseri extracellular protein according to claim 6, characterized in that, The centrifugation speed is 4000~6000 rpm; the centrifugation time is 20~40 min; And / or, the pore size of the filter membrane used for filtration and sterilization is 0.2~0.25μm; And / or, the ammonium sulfate fractional salting-out method includes: The ammonium sulfate saturation in the sterile fermentation supernatant was adjusted to 20-30% for the first protein salting out. After standing, the supernatant S1 and protein precipitate P1 were collected by centrifugation. Set the ammonium sulfate saturation in the supernatant S1 to 40-60%, perform a second protein salting-out, let stand, centrifuge, and collect the supernatant S2 and protein precipitate P2. To achieve an ammonium sulfate saturation of 70-80% in the supernatant S2, a third protein salting-out was performed. After standing, the mixture was centrifuged to collect the protein precipitate P3. And / or, the addition of the ammonium sulfate solid and each stage of the ammonium sulfate fractional salting-out process are carried out under constant temperature and stirring conditions; And / or, the time for each stage of the ammonium sulfate fractional salting-out process is 1~1.5h; And / or, the settling temperature is 2~8℃; the settling time is 1~4h.

8. The method for preparing Lactobacillus gasseri extracellular protein according to claim 7, characterized in that, The centrifugation speed is 4500~5000 rpm; the centrifugation time is 25~35 min; And / or, the ammonium sulfate fractional salting-out method includes: The ammonium sulfate saturation in the sterile fermentation supernatant was increased to 25% for the first protein salting out. After standing, the supernatant S1 and protein precipitate P1 were collected by centrifugation. To achieve a 50% ammonium sulfate saturation in the supernatant S1, a second protein salting-out was performed. After standing, the supernatant S2 and protein precipitate P2 were collected by centrifugation. To achieve an ammonium sulfate saturation of 75% in the supernatant S2, a third protein salting-out was performed. After standing, the mixture was centrifuged to collect the protein precipitate P3. And / or, the constant temperature is 20~30℃; the stirring speed is 150~250rpm; And / or, the time for each stage of the ammonium sulfate fractional salting-out process is 1~1.5h; And / or, the settling temperature is 3~4℃; the settling time is 1.5~2h; And / or, the separation in the ammonium sulfate fractionation salting-out is carried out by centrifugation, the centrifugation speed is 10000~15000 rpm, the centrifugation time is 10~20 min, and the centrifugation temperature is 2~8℃.

9. The method for preparing Lactobacillus gasseri extracellular protein according to claim 8, characterized in that, The separation in the ammonium sulfate fractionation salting-out process is carried out by centrifugation, with a centrifugation speed of 11,000-13,000 rpm, a centrifugation time of 13-16 min, and a centrifugation temperature of 3-4°C. And / or, the buffer solution is a Tris-HCl buffer solution; the pH value of the buffer solution is 7.0~8.0; And / or, the filter membrane used for sterilization in the dialysis method has a pore size of 0.15~0.45μm; And / or, the dialysis temperature is 2~8℃; And / or, the dialysis duration is 2 to 5 days, with fluid changes 2 to 4 times per day; And / or, the dialysate used in the dialysis is Tris-HCl buffer; And / or, the process further includes mixing the collected protein precipitate before dissolving it; And / or, after the purification, the purified material is further subjected to freeze drying.

10. The method for preparing Lactobacillus gasseri extracellular protein according to claim 9, characterized in that, And / or, the pH of the buffer solution is 7.5; And / or, the filter membrane used for sterilization in the dialysis method has a pore size of 0.22 μm; And / or, the dialysis temperature is 4°C.

11. The use of Lactobacillus gasseri extracellular protein prepared by the method of any one of claims 2-10 in the preparation of products with oil-controlling effects.

12. The application of the *Lactobacillus gasseri* extracellular protein prepared by the method for preparing any one of claims 2-10 according to claim 11 in the preparation of products with oil-controlling effects, characterized in that, The oil control mentioned refers to regulating the secretion of sebum in the skin; And / or, the oil-controlling effect has one of the following effects: Reduce sebaceous gland alveoli; It causes the sebaceous glands to shrink and become smaller; This loosens the arrangement of sebaceous glands; Inhibit sebaceous gland cells from secreting sebum; Inhibit sebum synthesis; Reduce the area of ​​sebaceous gland spots; It can lighten the color of sebaceous gland spots; And / or, the product with oil-controlling effect is a topical skin agent; And / or, the oil-controlling product is used for skin oil control.

13. The application of the Lactobacillus gasseri extracellular protein according to claim 12 in the preparation of products with oil-controlling effects, characterized in that, The inhibition of sebum secretion by sebaceous gland cells can be achieved by intervening in the expression of genes related to sebum synthesis; And / or, the inhibition of sebum secretion by sebaceous gland cells can be achieved by inhibiting the expression of sebum secretion-related genes SREBP-1, FAS, and ACC1 in sebaceous gland cells; And / or, by regulating the sebum signaling pathway to inhibit sebum synthesis; And / or, the topical skin agent is an oil-controlling cosmetic or a topical skin care agent.

14. A topical skin preparation comprising Lactobacillus gasseri extracellular protein prepared by the method for preparing Lactobacillus gasseri extracellular protein according to any one of claims 2-10.

15. The topical skin agent according to claim 14, characterized in that, Based on the mass of the topical skin agent, the mass content of the Lactobacillus gasseri extracellular protein is 1% to 10%. And / or, the topical skin agent further includes at least one of the following: moisturizing active ingredient, whitening active ingredient, skin barrier repair active ingredient, antibacterial or microecological regulating active ingredient.

16. The topical skin agent according to claim 15, characterized in that, Based on the mass of the topical skin agent, the mass content of the extracellular protein of Lactobacillus gasseri is 2.5% to 10%.

Images