Lactobacillus rhamnosus JMB235 as well as fermentation product and application thereof

Abstract

The invention relates to the field of daily cosmetics, health care product raw materials and biology, and discloses lactobacillus rhamnosus JMB235 with the preservation number of CCTCC (China Center for Type Culture Collection) No.M 20232530. The fermentation product of lactobacillus rhamnosus has multiple effects, and can control grease secretion, prevent alopecia, inhibit acne, promote hair growth, inhibit eczema, inhibit inflammation, maintain skin barrier, promote epidermal cell proliferation and differentiation, block sun, repair damaged skin or improve insomnia. The compound can be used for preparing skin external preparations, health-care products, beautifying oral preparations, foods or food additives. And the fermentation product has good stability, is storable and has a wide application prospect.

Description

Technical Field

[0001] The invention relates to raw materials for daily cosmetics and health products and the biological field, and in particular to a lactobacillus rhamnosus, a fermentation product thereof and an application thereof. Background Art

[0002] Lactobacillus rhamnosus is a common lactobacillus, a Gram-positive, facultative anaerobic bacterium that does not have the ability to form spores. It is acid-resistant and grows best at a pH between 5.5 and 6.2. Lactobacillus rhamnosus is commonly found in the intestinal contents, feces and reproductive tract of humans and animals, and also in milk and dairy products.

[0003] In the field of health products, Lactobacillus rhamnosus is widely used due to its significant probiotic effects. As a probiotic supplement, it can improve intestinal function, relieve digestive problems such as diarrhea and constipation, enhance intestinal barrier function, and reduce the risk of pathogen infection. Especially among infants and the elderly, Lactobacillus rhamnosus GG strain (LGG) is widely used in infant formula milk powder, probiotic drops and other products to promote the healthy development of infant intestinal flora, enhance immunity, prevent and treat antibiotic-associated diarrhea, and maintain intestinal health in the elderly and improve problems caused by decreased immunity.

[0004] In the field of cosmetics, the unique efficacy of Lactobacillus rhamnosus and its metabolites is gradually being discovered and utilized.

[0005] For example, WO2011029784A1 discloses Lactobacillus rhamnosus with a preservation number of LMGP-25211, which exhibits strong anti-inflammatory activity when administered orally or topically, but no other effects are found, and it is necessary to take or apply a microbial agent.

[0006] CN113046268A discloses a Lactobacillus rhamnosus 11-7, the lysate obtained by enzyme lysis of the bacteria after fermentation can inhibit the reproduction and growth of pathogenic bacteria and regulate the skin microecology.

[0007] CN112980892A discloses a probiotic composition fermentation product composed of plant lactobacillus, Lactobacillus casei, Bifidobacterium lactis, Bifidobacterium animalis and Lactobacillus rhamnosus, which has unique fermentation metabolites, can maintain the balance of skin flora, and has the effects of wrinkle removal, increased skin elasticity, anti-inflammatory, etc. KR1020110134151A discloses that a mixed culture fermentation product of multiple probiotics has a good effect on acne and skin inflammatory diseases, but the fermentation product of Lactobacillus rhamnosus does not improve the above skin problems.

[0008] KR1020080094459A discloses that herbal medicine (licorice, etc.) is fermented with lactobacillus, and the fermented product has antioxidant activity.

[0009] It can be seen that in the prior art, the microbial preparations, lysates or fermentation products of Lactobacillus rhamnosus have relatively single functions, or need to be fermented with other probiotics to obtain better skin care effects. Therefore, it is necessary to provide a Lactobacillus rhamnosus and its fermentation products, which can solve various skin problems without adding multiple functional ingredients to skin care products. Summary of the invention

[0010] The present invention aims to provide a Lactobacillus rhamnosus and a fermentation product thereof, and application thereof.

[0011] The technical solution is as follows:

[0012] A Lactobacillus rhamnosus JMB235 (Lactobacillus rhamnosus JMB235), with a deposit number of CCTCC No.M 20232530, is deposited in the China Center for Type Culture Collection (Address: Wuhan University, Wuhan City, Hubei Province, China, Postal Code 430072), and was deposited on December 11, 2023.

[0013] The culture method of the above-mentioned Lactobacillus rhamnosus JMB235 strain is as follows.

[0014] Seed culture medium: 0.5% to 3% glucose, 2% to 5% peptone, 0.5% to 3% skim milk powder, 0.5% to 3% yeast extract, 0.01% to 1% magnesium sulfate heptahydrate, 0.01% to 1% dipotassium hydrogen phosphate, 0.01% to 1% potassium chloride, 0.01% to 1% sodium chloride, the balance is water, the percentages are by mass. The pH of the culture medium is 5.5-6.5.

[0015] Bacteria expansion medium and fermentation medium: 0.5% to 3% glucose, 2% to 5% peptone, 0.5% to 3% skim milk powder, 0.5% to 3% yeast extract, 0.01% to 1% magnesium sulfate heptahydrate, 0.01% to 1% dipotassium hydrogen phosphate, 0.01% to 1% potassium chloride, 0.01% to 1% sodium chloride, the balance is water, the percentages are by mass. The pH of the medium is 5.5-6.5.

[0016] A single colony is inoculated into a seed culture medium, and the bacteria are activated by static culture at 26° C. to 36° C. in a shaker or fermenter for 16 to 24 hours to obtain a Lactobacillus rhamnosus seed solution.

[0017] Seed culture: The seed solution is cultured in the culture medium at 26℃~36℃ for 40~50 hours until the OD 6000.5-5.0, preferably, OD 600 It is 2.0-5.0.

[0018] Bacteria expansion culture: The seed liquid bacteria are inoculated into the fermentation medium at an inoculation rate of 0.1% to 10.0%, preferably, the inoculation rate is 1%-10%; 26°C to 36°C, culture for 40 to 50 hours, and stir at 50rpm to 100rpm during the culture process. The percentage is the mass percentage. Preferably, after 12-16 hours of culture, feed medium is added to make the final concentration of glucose 1% to 2%. The feed medium contains glucose and yeast extract, and the balance is water; the mass ratio of glucose to yeast extract is 1:0.5-3, preferably 1:1-2. Preferably, the feed medium contains 2% to 6% glucose and 1% to 2% yeast extract.

[0019] The fermentation product of Lactobacillus rhamnosus JMB235 can be used to prepare skin external preparations, health products, cosmetic oral preparations, foods or food additives.

[0020] The skin topical agent, health product, oral beauty agent, food or food additive has at least one of the following functions: controlling oil secretion, preventing hair loss, inhibiting acne, promoting hair growth, inhibiting eczema, inhibiting inflammation, maintaining skin barrier, promoting epidermal cell proliferation and differentiation, sun protection, repairing damaged skin or improving sleep.

[0021] The fermentation product and the skin external preparation, health product, cosmetic oral preparation or food additive prepared therefrom have the function of inhibiting 5α-reductase, and can control oil secretion, prevent hair loss and inhibit acne.

[0022] The fermentation product and the skin external preparation, health product, beauty oral preparation or food additive prepared therefrom also have the effects of improving sleep, preventing and alleviating insomnia.

[0023] The fermentation product and the skin topical preparation, health product, cosmetic oral preparation or food additive prepared therefrom have the functions of inhibiting the gene expression of inflammatory factors and the secretion of TNF-α protein, especially inhibiting the gene expression of inflammatory factors and the secretion of TNF-α protein caused by Staphylococcus aureus, and can be used to prevent or alleviate eczema, especially infantile eczema.

[0024] The fermentation product and the skin topical preparation, health care product, cosmetic oral preparation or food additive prepared therefrom have the function of regulating the transcription level of genes related to epidermal cell proliferation and differentiation, can be used to promote epidermal cell proliferation and differentiation, can repair damaged skin barrier and maintain skin barrier; especially skin damage caused by ultraviolet rays combined with air pollutants (such as BAP and benzopyrene).

[0025] Preferably, the expression of KRT1, KRT16, KLK5 and KLK11 can be upregulated, especially when ultraviolet rays are combined with air pollutants (such as BAP and benzopyrene), the expression of KRT1, KRT16, KLK5 and KLK11 can be increased.

[0026] The fermentation product and the skin topical agent, health product, cosmetic oral agent or food additive prepared therefrom have the function of inhibiting lipid peroxidation metabolites, can down-regulate the arachidonic acid metabolism, α-linolenic acid metabolism, and linoleic acid metabolism signal pathways, can be used to regulate the activity of sebaceous gland cells, reduce the secretion of sebum, and achieve an oil control effect.

[0027] Preferably, it can inhibit the arachidonic acid metabolism, α-linolenic acid metabolism, and linoleic acid metabolism signal pathways under ultraviolet light combined with light pollution chemicals (such as BAP benzopyrene). It is particularly suitable for sebaceous gland hyperplasia, sebaceous gland cell proliferation and differentiation, and excessive oil secretion caused by ultraviolet light combined with light pollution chemicals (such as BAP benzopyrene).

[0028] The fermentation product and the skin external preparation, health care product, cosmetic oral preparation or food additive prepared therefrom have the functions of promoting the proliferation of human immortalized keratinocytes (HaCaT), dermal fibroblasts (FB) and hair follicle dermal papilla cells (DP), resisting damage caused by ultraviolet rays and promoting the repair of ultraviolet damage, and can repair the skin and promote hair growth, as well as resist photoaging, protect against sunburn and repair after sunburn.

[0029] The method for preparing the fermentation product of the above-mentioned Lactobacillus rhamnosus JMB235 is as follows:

[0030] The Lactobacillus rhamnosus bacterial liquid after the expanded culture is inoculated into the sterilized fermentation medium, and fermented under the conditions of 26°C to 36°C and 50 to 150rpm, and the pH is controlled at 5.5-6.5, and optimally 6.0. Feeding is started 12h to 16h after inoculation, so that the final concentration of glucose is 1% to 2%, and the total culture time is 40 to 50 hours. Preferably, the Lactobacillus rhamnosus bacterial liquid after the expanded culture is added to the fermentation medium at an inoculum amount of 0.1 to 10.0%, and fermented in a shaking table or fermentation tank.

[0031] The fermentation product of Lactobacillus rhamnosus JMB235 includes the following components in dry matter by mass percentage:

[0032] Organic acids and their derivatives 46%-49%, carbohydrates and carbohydrate conjugates 43%-45%, amino acids, peptides and analogs 7%-8%, nucleosides, nucleotides and analogs 0.25%-0.5%, steroids and steroid derivatives 0.1%-0.2%, biogenic amines 0.1%-0.2%, pyrimidine and pyrimidine derivatives 0.03%-0.08%, benzene 0.03%-0.08%, lipids 0.02%-0.05%, purine and purine derivatives 0.02%-0.05%, indole and derivatives 0.001%-0.005%, nitrogen-containing heterocyclic compounds 0.001%-0.005%, pyridine and derivatives 0.001%-0.005%. Other undetected components are less than 0.06%.

[0033] The fermentation product has good stability and can be stored for a long time without adding preservatives, remaining clear and transparent. Its physical and chemical indicators such as pH, conductivity, refractive index, solid content, and color and smell all remain stable.

[0034] Compared with the prior art, the present invention has the following beneficial effects:

[0035] (1) A new strain of Lactobacillus rhamnosus was discovered. After fermentation, its fermentation product has multiple skin care functions and can be used to prepare cosmetics, beauty oral preparations, foods, health products and additives, etc., and has broad application prospects.

[0036] (2) The fermentation product of Lactobacillus rhamnosus has the following functions: controlling oil secretion, preventing hair loss, inhibiting acne, promoting hair growth, inhibiting eczema, inhibiting inflammation, maintaining skin barrier, promoting proliferation and differentiation of epidermal cells, protecting against the sun, repairing damaged skin, improving insomnia, etc. It has a wide range of functions and can be added to cosmetics or used in food and health products to achieve multiple functions without the need to compound various ingredients with different functions.

[0037] (3) The fermentation product obtained has good stability and can be stored for a long time without adding preservatives. It is clear and transparent, and its color and odor remain stable. When used in cosmetics, the use of additives such as preservatives and stabilizers can be reduced. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] Figure 1 Results of nanoparticle tracking analyzer (NTA) detection of exosomes in Lactobacillus rhamnosus JMB235 fermentation products. (A) is the particle concentration and (B) is the volume.

[0039] Figure 2 Electron microscopic image of exosomes in the fermentation product of Lactobacillus rhamnosus JMB235

[0040] Figure 3 This is the detection result of exosome protein concentration in the fermentation product of Lactobacillus rhamnosus JMB235.

[0041] Figure 4 The inhibitory effect of the fermentation product of Lactobacillus rhamnosus JMB235 on 5α-reductase activity.

[0042] Figure 5 The effect of Lactobacillus rhamnosus JMB235 fermentation products on promoting the proliferation of HaCaT cells.

[0043] Figure 6 The fermentation product of Lactobacillus rhamnosus JMB235 promotes the proliferation of FB cells.

[0044] Figure 7 The effect of Lactobacillus rhamnosus JMB235 fermentation products on promoting DP cell proliferation.

[0045] Figure 8 Effect of fermentation products of Lactobacillus rhamnosus JMB235 on repair of UV-damaged HaCaT cells

[0046] Fig. 9 This is the effect of Lactobacillus rhamnosus JMB235 fermentation products on the repair of UV damage to FB cells.

[0047] Fig.10 This is the effect of Lactobacillus rhamnosus JMB235 fermentation products on the repair of UV damage in DP cells.

[0048] Fig.11 The effect of Lactobacillus rhamnosus JMB235 fermentation product on inhibiting Sa-induced TNF-α protein secretion in HaCaT cells.

[0049] Fig.12 The effect of Lactobacillus rhamnosus JMB235 fermentation product on inhibiting Sa-induced IL-1α gene expression in HaCaT cells.

[0050] Fig.13 Volcano map of differentially expressed genes in different treatment groups of Lactobacillus rhamnosus JMB235 fermentation products

[0051] Fig.14 KEGG functional enrichment analysis annotation classification diagram of differentially expressed genes in different treatment groups of Lactobacillus rhamnosus JMB235 fermentation products.

[0052] Fig.15 The effect of Lactobacillus rhamnosus JMB235 fermentation product on improving zebrafish sleep. DETAILED DESCRIPTION

[0053] The present invention is described below by specific examples, but the protection scope of the present invention is not limited thereby. Experimental methods without specifying specific operating methods and conditions are selected according to conventional operating methods and experimental conditions, or product instructions.

[0054] Example 1 Screening of Lactobacillus rhamnosus JMB235

[0055] The strain of the present invention is isolated from the colostrum of Tibetan women in Tibet Autonomous Region of my country. The monoclonal strain is identified as Lactobacillus rhamnosus (Lactobacillus rhamnosus) by multi-phase identification and detection such as morphological characteristics, physiological and biochemical characteristics, and DNA sequence analysis (16S rDNA and pheS) by China Industrial Microbiological Culture Collection Center (CICC), and is numbered Lactobacillusrhamnosus JMB235, hereinafter referred to as JMB235.

[0056] The above-mentioned strains were preserved by the China Center for Type Culture Collection (CCTCC, address: Wuhan University, Wuhan City, Hubei Province, Postal Code: 430072) on December 11, 2023, with the preservation number: CCTCC NO.M 20232530.

[0057] Morphological characteristics of microorganisms: After anaerobic culture at 37℃ for 24 hours in MRS medium, the colonies are white, round, moist, opaque, and have neat edges. Microscopic observation shows that the bacteria are rod-shaped, 0.5-0.7μm×1.1-3.7μm, arranged singly or in pairs, and Gram-positive.

[0058] The 16s rDNA genome sequencing was completed by Sangon Biotech (Shanghai) Co., Ltd., and the nucleotide sequence is as follows (SEQ ID No. 1):

[0059]

[0060] The strain of the present invention is derived from a wild-type strain naturally found in the Himalayas, and can naturally tolerate strong ultraviolet rays, cosmic rays, low oxygen content, large temperature differences between day and night, and other extreme environments, so it has good stress tolerance and robustness, and can tolerate a wide range of temperature, acidity, and osmotic pressure. When the inorganic salt concentration of the culture medium is less than 0.1% or exceeds 2wt%, its survival and growth are not affected.

[0061] Example 2 Cultivation of Lactobacillus rhamnosus JMB235

[0062] (I) Culture medium

[0063] Seed culture medium: 0.5% to 3% glucose, 2% to 5% peptone, 0.5% to 3% skim milk powder, 0.5% to 3% yeast extract, 0.01% to 1% magnesium sulfate heptahydrate, 0.01% to 1% dipotassium hydrogen phosphate, 0.01% to 1% potassium chloride, 0.01% to 1% sodium chloride, the balance is water, the above percentages are by mass. The pH of the culture medium is 5.5-6.5.

[0064] The vessels used for the culture medium need to be sterilized with high-pressure steam at 115°C for 30 minutes before use.

[0065] Bacteria expansion medium / fermentation medium: 0.5% to 3% glucose, 2% to 5% peptone, 0.5% to 3% skim milk powder, 0.5% to 3% yeast extract, 0.01% to 1% magnesium sulfate heptahydrate, 0.01% to 1% dipotassium hydrogen phosphate, 0.01% to 1% potassium chloride, 0.01% to 1% sodium chloride, the balance is water, the above percentages are by mass. The pH of the medium is 5.5-6.5. The pH of the medium is adjusted to 5.5-6.5, and the utensils used for the culture medium need to be sterilized at 115°C high pressure steam for 30 minutes before use.

[0066] Feed medium: 2% to 6% glucose, 1% to 2% yeast extract, and the balance is water, and the percentages are by mass.

[0067] (II) Culture conditions

[0068] Seed culture: The seed solution is cultured in the culture medium at 26℃~36℃ for 40~50 hours until the OD 600 2.0-5.0;

[0069] Bacteria expansion culture: the seed liquid bacteria are inoculated into the fermentation medium at a rate of 1% to 10.0%, cultured at 26° C. to 36° C. for 40 to 50 hours, and stirred at 50 rpm to 100 rpm during the culture process.

[0070] The pH value of the whole culture process is controlled at 5.5-6.5, and the fermentation culture is carried out for 12h-16h. Then, feeding is started to make the final concentration of glucose reach 1%-2%, and the fermentation is continued to the end point.

[0071] Example 3 Preparation of Lactobacillus rhamnosus JMB235 fermentation product (LrFF-JMB235)

[0072] (I) Culture medium preparation

[0073] The seed culture medium and bacterial expansion culture medium of Example 2 were prepared according to the growth requirements of Lactobacillus rhamnosus, sterilized at 115° C. under high temperature and high pressure for 20 to 30 min, and cooled for later use.

[0074] (II) Expansion culture of bacteria

[0075] A single colony is inoculated into a seed culture medium, and the bacteria are activated by static culture at 26° C. to 36° C. in a shaker or fermentation tank for 16 to 24 hours to obtain a Lactobacillus rhamnosus seed culture solution.

[0076] The Lactobacillus rhamnosus seed solution is added to the sterilized fermentation medium at an inoculation rate of 0.1-10.0%, and the pH is stabilized at 5.5-6.5, preferably 6.0, in a shaker or fermentation tank at 26°C-36°C and 50-150rpm using ammonia water. After inoculation, the culture is carried out, and the dissolved oxygen curve and pH curve are observed. Feeding is started after about 12h-16h of fermentation, and the final concentration of glucose is 1%-2%. The total culture time is 40-50 hours.

[0077] (III) Obtaining the fermentation product LrFF-JMB235

[0078] The bacterial liquid obtained by the expanded culture of the bacteria is centrifuged (tubular centrifuge, 10000-13000rpm, preferably 12000rpm; disc centrifuge, 5000-6000rpm, preferably 5300rpm, flow rate 400L / h, slag discharge time 200-500s, preferably 300s), membrane filtration (plate and frame filter, 0.1μm or 0.1μm nylon membrane or polypropylene membrane, diatomaceous earth or perlite as filter aid, added in an amount of 1-5% w / w) or hollow fiber membrane system (0.1μm or 0.2μm), and the supernatant is collected. The collected supernatant is decolorized by activated carbon and the pH is adjusted to 3-7 to obtain LrFF-JMB235 without preservatives. It is prepared with sterile water and stored frozen at -20°C for subsequent detection and experiments.

[0079] (IV) Stability testing of LrFF-JMB235

[0080] The initial sample of LrFF-JMB235 obtained by the above preparation method is a colorless to light yellow clear liquid. Taking the batch sample with pH = 3.0-6.0 and conductivity of 200-5000 μS / cm as an example, the stability test of the prepared LrFF-JMB235 was carried out. The test conditions include: 4°C, 25°C, 40°C and light (4.5KLX, UV 1.0W / m 2 ).

[0081] The physicochemical parameters of the obtained LrFF-JMB235 are shown in Table 3.

[0082] Table 2 Physical and chemical parameters of LrFF-JMB235

[0083]

[0084] After testing, the LrFF-JMB235 obtained in this case can be placed under multiple conditions for 16 weeks and still remain clear and transparent, and its physical and chemical indicators such as pH, conductivity, refractive index, solid content, and color and odor remain stable and within the quality control index range. This shows that the fermentation product of the JMB235 strain has good stability.

[0085] (V) Detection of LrFF-JMB235 components

[0086] The target compounds were separated by chromatography on a Waters Atlantis Premier BEH ZHILIC Column (1.7 μm, 2.1 mm × 150 mm) using an ACQUITY UPLC H-Class (Waters) ultra-high performance liquid chromatograph. Phase A of the liquid chromatograph was ultrapure water: acetonitrile = 8:2, containing 10 mmol / L ammonium acetate, and phase B was acetonitrile: ultrapure water = 9:1, containing 10 mmol / L ammonium acetate; both phases AB were adjusted to pH 9 with aqueous ammonia. Sample tray temperature: 8°C, injection volume: 1 μL.

[0087] Mass spectrometry analysis was performed using a SCIEX 6500 QTRAP+ triple quadrupole mass spectrometer equipped with an IonDrive Turbo V ESI ion source in multiple reaction monitoring (MRM) mode. The ion source parameters were as follows: Curtain Gas = 35 psi, Ion Spray Voltage = +5000 V / -4500 V, Temperature = 500°C, Ion Source Gas 1 = 50 psi, Ion Source Gas 2 = 50 psi.

[0088] All mass spectrometry data acquisition and quantitative analysis of target compounds were completed using SCIEX Analyst WorkStation Software (1.7.2) and Data Driven Flow (v-1.0.1).

[0089] The results are shown in Table 3.

[0090] Table 3 LrFF-JMB235 component classification results

[0091]

[0092]

[0093] According to the results of component analysis, a total of 367 compounds were detected in LrFF-JMB235, among which amino acids, peptides and analogs were the most abundant, with a total of 97, accounting for about a quarter of the total, followed by organic acids and their derivatives, carbohydrates and carbohydrate conjugates, with 50 and 43 respectively. According to the content of ingredients, organic acids and their derivatives accounted for the highest proportion, reaching 47.497%, among which lactic acid, (S)-2-hydroxy-4-methylvaleric acid, 3-hydroxypropionic acid, α-ketoglutaric acid, glycolic acid, succinic acid, etc. were higher in content. Carbohydrates and carbohydrate conjugates followed, accounting for 44.165%, and L-arabinose, glyceric acid, galactose, glucose, galactitol, mannitol, etc. were higher in content. The amino acids, peptides and analogs with the largest number of ingredients accounted for a relatively low proportion of 7.458%, and lysine, arginine, glycine, ornithine, etc. were higher in content. Among them, compounds such as 3-hydroxypropionic acid and α-ketoglutaric acid were rarely reported in Lactobacillus rhamnosus.

[0094] In addition, the metabolites in LrFF-JMB235 were analyzed by liquid chromatography-mass spectrometry. The conditions are as follows: Chromatographic conditions:

[0095] Chromatographic column: ACQUITY UPLC HSS T3 (100 mm × 2.1 mm, 1.8 um);

[0096] Column temperature: 45°C;

[0097] Mobile phase: Mobile phase: A-water (containing 0.1% formic acid), B-acetonitrile;

[0098] Flow rate: 0.35 mL / min;

[0099] Injection volume: 5 μL.

[0100] PDA scanning range: 210-400nm;

[0101] Mass spectrometry conditions:

[0102] Ion source: HESI; sample mass spectrometry signal acquisition uses positive and negative ion scanning modes respectively.

[0103] Data acquisition mode: DDA

[0104] Scan mode: Full MS / dd-MS2 (TOP 8)

[0105] In the test results, the top 10 ingredients in terms of relative content are as follows:

[0106] Table 4 TOP 10 ingredient contents

[0107] Serial number Compound Relative content 1 Leu-Pro-Ile 11.29 2 Akodionine 9.13 3 L-Leucine 7.98 4 Hydroxyphenyl lactic acid 6.34 5 N-(1-deoxy-1-fructosyl)phenylalanine 5.83 6 Leucylphenylalanine 5.28 7 Guanosine 4.90 8 Leu Val Ala 4.44 9 3-(3-Hydroxyphenyl)propionic acid 3.96 10 N-Lactoylphenylalanine 3.26

[0108] Relative content: The ratio of the mass spectrometer response intensity of the compound in the sample to the sum of the response intensities of all compounds in the sample

[0109] Example 4 Extraction and detection of exosomes

[0110] (I) Exosome extraction

[0111] Collect the fermentation broth after the above-mentioned expanded culture and centrifuge it at 4000g for 20min in a 4℃ centrifuge. Take the supernatant and centrifuge it at 8000g for 20min in a 4℃ centrifuge. Filter the obtained supernatant with a 0.45μm pore size filter membrane and collect the filtrate. Transfer the filtrate to a HITACHI-himac ultracentrifuge tube, centrifuge it at 120000g for 2h in an ultracentrifuge, discard the supernatant, resuspend the precipitate with PBS, and centrifuge it again at 120000g for 2h in an ultracentrifuge. The precipitate obtained is the exosomes of Lactobacillus rhamnosus JMB235 cells, that is, the exosomes / exosomes LrFF-JMB235. Resuspend it with 1mL PBS and store it at -80℃ for later use.

[0112] (II) Exosome detection

[0113] Nanoparticle tracking analyzer (NTA) was used to detect Figure 1 The average particle size of EVs derived from LrFF-JMB235 is 115.3 nm. Exosomes in this particle size range can pass through blood vessel walls and other biological barriers, thus being transported over long distances in the body and improving storage stability. The original concentration of exosomes in LrFF-JMB235 is approximately 1.6×10 10 particles / mL. Figure 2 From the transmission electron microscopy results, it can be clearly observed that the exosomes in LrFF-JMB235 are spherical objects with membrane structure.

[0114] The protein content was determined using the BCA protein quantification kit, and the extracellular vesicles / exosomes in LrFF were characterized using transmission electron microscopy (TEM) and nanoparticle tracking analysis (NTA). The results are shown in Figure (3) BCA protein quantification detection

[0115] 1. Preparation of main solution

[0116] 1) Preparation of BCA working solution: dilute 5× solution A in the kit with distilled water to 1× solution A, quantitatively take 1× solution A: solution B = 50:1, and mix well to make BCA working solution.

[0117] 2) Preparation of solution E: Take distilled water and solution D in a ratio of 1:1 and mix well to make solution E.

[0118] 3) Preparation of Solution F: Weigh 3 mg of Reagent C, dissolve it in 150 μL of Solution E, and vortex for 1 min to obtain Solution F.

[0119] 4) Preparation of standard protein solution: Take 8 clean 2 mL EP tubes and add different volumes of solution D and 2 mg / mL standard bovine serum albumin (BSA) solution in sequence according to Table 1 to prepare 8 tubes of standard solutions with different concentrations from 0 to 7.

[0120] 2. Establishment of protein quantification standard curve

[0121] Select 16 wells on the ELISA plate, set up a replicate for each concentration of standard protein solution, add 5 μL of the corresponding concentration of standard protein solution to each well, then add 5 μL of solution F, and keep warm in a 37°C water bath for 30 minutes. Then add 200 μL BCA working solution to each well, mix quickly, and keep warm in a 37°C water bath for 30 minutes. After cooling to room temperature, measure the A562 value of each well on the ELISA instrument.

[0122] 3. Determination of Protein Concentration of Extracellular Vesicles from LrFF-JMB235 Cells

[0123] Select 9 wells on the ELISA plate, set up two replicates of the extracted extracellular vesicle samples, add 5 μL of the corresponding extracellular vesicle sample solution to each well, and then add 5 μL of solution F, and keep warm in a 37°C water bath for 30 minutes. Then add 200 μL BCA working solution to each well, mix quickly, and keep warm in a 37°C water bath for 30 minutes. After cooling to room temperature, measure the A562 value of each well on the ELISA instrument.

[0124] like Figure 3 As shown in the figure, a protein quality standard curve was drawn with BSA concentration as the horizontal axis and A562 value as the vertical axis. The A562=0.1142 of the extracellular vesicles was taken into account to obtain an extracellular vesicle protein concentration of 89.00 μg / mL.

[0125] Example 5 Detection of polypeptides in LrFF-JMB235

[0126] Peptidome refers to all endogenous polypeptide components in living biological organs, tissues, cells and body fluids. Peptidomics is a discipline that studies the composition, structure, function, change rules and related relationships of the polypeptide group. Peptides can reflect the synthesis, processing and degradation of proteins under different physiological and pathological conditions. At the same time, they have a regulatory effect on gene expression, material metabolism and other processes. Peptides can be used to trace their precursor proteins and analyze the metabolic small molecules regulated by them. Therefore, peptidomics has become a bridge connecting proteomics and metabolomics. The study of peptidomics first extracts and enriches the polypeptides in the sample, then detects and analyzes the polypeptides, and then conducts data mining to screen out differential polypeptides or target polypeptides, and then identifies and verifies these polypeptides. Finally, the functions of these polypeptides in the body and their effects on the occurrence and development of diseases are further studied. The extraction and enrichment of polypeptides in samples is one of the keys to the research.

[0127] (I) Enzymatic hydrolysis operation

[0128] The LrFF-JMB235 sample obtained in Example 3 was redissolved in a water bath at room temperature. Transfer 10 mL each time to a 3KD ultrafiltration centrifuge tube. Centrifuge at 4°C, 6400rpm for 40 minutes, collect the retentate, and repeat the previous step until all the samples are used up. Transfer the retentate and rinse the ultrafiltration tube 3 times with 200μL of chromatographic water, and mix it with the retentate. Centrifuge the mixed solution at 4°C, 12000rpm for 15 minutes, and take the supernatant for later use. Freeze-dry the supernatant overnight. Dissolve in SDS lysis buffer and dissolve at room temperature for 3-5 minutes. Centrifuge the solution at 12000rpm for 10 minutes at room temperature, take the supernatant, and centrifuge again to take the supernatant to fully remove insoluble impurities. Add the treated SP3 magnetic beads to the extracted protein solution, and immediately add the corresponding volume of 100% ACN, incubate at room temperature for 20 minutes, and gently shake manually every minute. After the sample is separated instantly, it is placed on the magnetic stand for two minutes, and the supernatant is removed. The corresponding volume of 70% ethanol and 70% ACN are added to wash the magnetic beads twice. After washing, it is placed on the magnetic stand for 2 minutes, and the supernatant is removed, and the solution is sucked dry as much as possible. The test tube opening is used to volatilize the remaining ACN as much as possible, and then the magnetic beads are resuspended with 30ul of 50mM ammonium bicarbonate (ABC) solution. DTT is added and incubated at 55℃ for 30min. IAA is added and reacted at room temperature in the dark for 20min. Trypsin is added for enzymatic hydrolysis, and the sample is shaken at 37℃ and 1500rpm. After the enzymatic hydrolysis is completed, formic acid is added, and the supernatant is centrifuged at 4℃, 12000rpm, and 10min. After C18 desalting, the sample is evaporated and re-dissolved on the machine.

[0129] (II) Mass spectrometry detection

[0130] 1. Chromatographic conditions:

[0131] Mobile phase A: H2O-FA (99.9%: 0.1%, v / v); Mobile phase B: ACN-FA (99.9: 0.1, v / v); The sample was loaded onto the analytical column (75μm×25cm, 1.6μm, C18, ionoptics) separation; gradient elution conditions: 0-45 min, 2%-22% B; 45-50 min, 22%-37% B; 50-55 min, 37%-80% B; 55-60 min, 80% B.

[0132] 2. Mass spectrometry conditions

[0133] The capillary voltage was 1.4 KV, the drying gas temperature was 180 °C, the drying gas flow rate was 3.0 L / min, the mass spectrometer scanning range was 100-1700 m / z, and the ion mobility range was 0.85-1.3 Vs / cm 2 , the collision energy range is 20-59eV.

[0134] 3. Data analysis

[0135] The LC-MS / MS original file was imported into MaxQuant (version 1.6.17.0) for library search. The library search sequence file was uniprot-Lactobacillus_rhamnosus-47715-2024.01.15.fasta, and the search engine was Andromeda. LFQ non-standard quantitative analysis was performed. To prevent peak shape mismatch, the library search conditions were controlled by the false positive rate (FDR, false discovery rate). The species database not only provides target sequence information, but also can eliminate null values ​​that do not meet the analysis criteria by filtering out the reverse database and the common contaminant protein database.

[0136] Table 5 Mass spectrometry search parameters

[0137]

[0138] The detection results of proteins and small peptides in LrFF-JMB235 are as follows:

[0139] Table 6 Protein and small peptide detection results

[0140]

[0141]

[0142] As shown in Table 5, 13 proteins were extracted and detected from LrFF-JMB235, and the corresponding peptides were 61. According to the accession number of the protein in the UniPort database, combined with literature retrieval, the proteins related to antitoxin effect or related enzyme cleavage effect were A0A0H0YIB4, A0A0H0YL76, A0A0H0YRV8, A0A9Q5HIE3, and Q8GC13. Among them, A0A0H0YIB4 may be related to the autoinhibition of adenylyltransferase, A0A0H0YL76 is related to serine endopeptidase activity, A0A0H0YRV8 may be an antitoxin subunit of the toxin-antitoxin system, A0A9Q5HIE3 is related to cysteine ​​exopeptidase activity, dipeptidase activity, proteolysis and other activities, Q8GC13 is related to serine endopeptidase activity, proteolysis and other activities, and may also be a cell wall-related protease.

[0143] Example 6: Inhibition of 5α-reductase by LrFF-JMB235

[0144] Androgenetic alopecia (AGA) is the most common type of hair loss. Androgenetic alopecia is also called seborrheic alopecia, which is related to the level of male hormones. After androgens are secreted in the body, they combine with 5α-reductase in the body and are reduced to dihydrotestosterone (DHT), which leads to androgenic alopecia. 5α-reductase is a membrane protease that depends on reduced coenzyme II (NADPH). It is an important male hormone metabolic enzyme in the skin and can irreversibly convert testosterone (Testosterone, T) into dihydrotestosterone (Dihydrotestosterone, DHT). DHT is the most active male hormone and plays a role in multiple organs and tissues such as the reproductive system, skin and hair. High levels of DHT are prone to cause androgen-dependent diseases, such as acne, hirsutism, androgenic alopecia, benign prostatic hyperplasia and prostate cancer. Androgen is the primary factor that promotes the development of sebaceous glands and sebum secretion. Therefore, inhibiting 5α-reductase is one of the important means to treat androgen-dependent diseases such as male pattern baldness and acne.

[0145] Experimental methods:

[0146] The fermentation product LIFF-JMB235 was added to the established 5α-reductase reaction system respectively. After the sample was incubated at 37°C for 20 minutes, the mixture was placed in an ELISA reader for detection. The absorbance was measured at 340 nm, and blank and positive control tests were performed at the same time.

[0147]

[0148] The results are as follows Figure 4LrFF-JMB235 had a significant inhibitory effect on 5α-reductase activity, indicating that the fermentation product has the effects of controlling oil secretion, preventing hair loss and removing acne. Figure 4 The LIFF-JMB235 concentration in the reaction system is the final concentration of LIFF-JMB235.

[0149] Unless otherwise specified, the concentrations in the figures are the final concentrations of LIFF-JMB235 in the reaction system or culture medium.

[0150] Example 7 Cell proliferation assay

[0151] Cell proliferation is the basis of growth, development, reproduction, and inheritance of organisms. It is the most basic physiological activity of cells and an important part of the cell life cycle. It plays an indispensable role in life activities and the development, repair, regeneration, and reproduction of organisms. Cell proliferation plays a vital role in organisms. First of all, it is the basis for the growth and development of organisms. Through cell proliferation, the tissues and organs of organisms can increase the number of cells, thereby achieving the growth and development of the body. Cell proliferation is also very important for tissue repair and regeneration. When tissues are damaged or attacked by diseases, cell proliferation can refill the damaged areas and promote the repair and recovery of tissue functions. Cell proliferation also maintains a relatively stable number of cells in the body. Through the balance between cell proliferation and cell death, the integrity and normal function of tissues and organs are maintained.

[0152] Human immortalized keratinocytes (HaCaT), dermal fibroblasts (FB), and hair follicle dermal papilla cells (DP) were inoculated into 96-well cell culture plates at a level of 5000 cells / well. After 48 hours of culture, the supernatant was removed, and the serum-free DMEM basal medium was replaced for each culture well to starve the cells. After the cells were starved for 16 hours, the sample was added, and the culture medium was the blank control group, the culture medium containing 5% serum (FBS) was the positive control, and the culture medium containing different concentrations of LrFF-JMB235 was the sample group. The cells continued to be cultured at 37°C and 5% CO2 for 24 hours, and then the cell viability was determined by CCK8. The absorbance at 450nm was measured by an ELISA instrument, and the proliferation rate was 100% for the blank control to evaluate the proliferation effect of LrFF-JMB235 on the cells relative to the blank control.

[0153]

[0154] The results are as follows Figure 5-7 LrFF-JMB235 can promote the proliferation of HaCaT, FB, and DP cells, and maintain the stability of the epidermis, dermis, and hair follicle skin.

[0155] Example 8 UV damage repair experiment

[0156] When the skin is exposed to ultraviolet rays, skin cells will be damaged, and the skin will not only become dry and rough, accelerating skin aging, but also cause skin pigmentation, forming various spots such as freckles and chloasma, as well as the occurrence of various solar skin diseases. After-sun repair is essential to protect skin health and prevent sunburn.

[0157] Human immortalized keratinocytes (HaCaT), dermal fibroblasts (FB), and hair follicle dermal papilla cells (DP) were inoculated into 96-well cell culture plates at a level of 5000 cells / well. After 48 hours of culture, the supernatant was removed, and the serum-free DMEM basal medium was replaced for each culture well to starve the cells. After 16 hours of cell starvation, UV irradiation damage was performed. After the damage was completed, the sample was added. The culture medium was the blank control group, the culture medium containing 5% serum (FBS) was the positive control, and the culture medium containing different concentrations of LrFF-JMB235 was the sample group. The cells continued to be cultured at 37°C and 5% CO2 for 24 hours, and then the cell viability was determined by CCK8. The absorbance at 450nm was measured by an ELISA instrument, and the proliferation rate was 100% for the blank control to evaluate the protection of LrFF-JMB235 on cells and the UV damage repair effect relative to the blank control.

[0158]

[0159] The results are as follows Figure 8-10 LrFF-JMB235 can promote the repair of HaCaT, FB, and DP cell damage caused by UV, and can promote the repair of UV damage to the epidermis, dermis, and hair follicles, achieve post-sun repair, and prevent sunburn.

[0160] Example 9 Inflammation inhibition experiment

[0161] Childhood eczema is affected by multiple factors, including impaired epidermal barrier, immune cell activation, and changes in related skin microbial communities. Staphylococcus aureus has been isolated from the skin of patients with eczema and atopic dermatitis. Studies have shown that the colonization rate of Staphylococcus aureus in atopic dermatitis eczema skin is significantly higher than that in healthy skin, and it is the main pathogen of eczema-like skin. Abnormal colonization of Staphylococcus aureus may also lead to skin barrier dysfunction. Staphylococcus aureus expresses a variety of factors that promote colonization and virulence and cause inflammatory responses.

[0162] Immortalized human keratinocytes (HaCaT) were inoculated into 12-well cell culture plates at a level of 0.2M cells / well. After 48 hours of culture, the supernatant was removed, and the serum-free DMEM basal medium was replaced for each culture well to starve the cells. After 16 hours of cell starvation, the cells were added with samples and bacteria. The control group and the model group were added with blank culture medium, the sample group was a culture medium containing different concentrations of LrFF-JMB235, the control group was added with PBS, and the model group and the sample group were added with Staphylococcus aureus (Sa) with OD=1.0. The cells continued to be cultured at 37°C and 5% CO2 for 24 hours, and then the supernatant was collected for ELISA experiments, and the cells were used for Real-time PCR experiments.

[0163] The results are as follows Fig.11 , 12 , showing that LrFF-JMB235 can effectively inhibit Sa-induced TNF-α protein secretion in HaCaT cells and inhibit Sa-induced IL-1α gene expression in HaCaT cells, and has a certain defensive effect on infantile eczema.

[0164] Example 10 Transcriptome Sequencing Analysis of Human Recombinant Full-skin 3D Skin Model

[0165] As air pollution worsens, the skin, as the outermost barrier, bears the brunt. Long-term air pollution can increase the incidence of sensitive skin, induce skin eczema and even dermatitis; at the same time, air pollution can significantly increase sebum secretion and change its composition, aggravate skin oxidative damage, and increase the incidence of skin sensitivity. PM2.5 has a small particle size, large area, strong activity, and is easy to carry toxic and harmful substances, which is more likely to have adverse effects on the skin. They are easy to adhere to the surface of the skin and continuously release toxic and harmful substances. These harmful substances can enter the deep layer of the skin through the hair follicles, destroy healthy skin cells, cause skin aging, and cause allergies, dermatitis, spots and other skin problems. Benzopyrene (BaP) is a representative substance of polycyclic aromatic hydrocarbons. It exists in automobile exhaust, cigarettes, kitchen fumes, indoor soot, ambient atmosphere, tunnel traffic environment and other fumes. Exposure to polycyclic aromatic hydrocarbons interferes with the normal barrier function of the skin, leading to pigmentation, acne, atopic dermatitis, premature aging and other skin problems.

[0166] Epidermal differentiation is precisely regulated, and the topmost keratinocytes fall off the skin surface every 2 to 4 weeks to maintain the normal thickness of the epidermis. This desquamation process requires kallikrein-related peptidase (KLK) to use its protease activity to degrade adhesion proteins that connect keratinocytes, including desmoglein 1 (DSG1), desmocollin 1 (DSC1), and corneal desmosin (CDSN). It is currently believed that at least 11 KLKs are localized in the skin at different expression levels, among which KLK5 and KLK11 are mainly localized in the upper granular layer and the stratum corneum. After being activated, they mainly regulate skin cell renewal and barrier thickness by promoting skin desquamation and keratinocyte proliferation. Keratin (KRT) and intermediate filament-related proteins constitute 80% to 90% of the protein content in the mammalian epidermis, and their abnormal expression directly affects the integrity of the skin barrier structure. At the same time, KRT also plays a key role in providing mechanical force to the stratum corneum, and is also involved in regulating epithelial cell growth, proliferation, migration and apoptosis pathways. Among them, keratin KRT1 is often expressed in the spinous layer of the skin, and its expression is an important marker for the early differentiation of keratinocytes. KRT16 belongs to a large class of acidic type I keratin. Studies have shown that KRT16 is an important protective barrier of the skin and is closely related to some skin diseases. As an inflammatory mediator, the linoleic acid metabolic pathway is widely involved in local inflammation. Linoleic acid is an essential fatty acid for the human body and is also a precursor of arachidonic acid. It has multiple physiological functions such as participating in fat decomposition, maintaining normal metabolism, and enhancing the body's immunity. Oxidative stress, lipid peroxidation metabolites and cytokines can induce the expression of cyclooxygenase COX-2 and the synthesis of prostaglandin E2 (PGE2), promote sebaceous gland hyperplasia and the proliferation and differentiation of sebaceous gland cells, and secrete more oil. COX-2 is the rate-limiting enzyme for the conversion of arachidonic acid (AA) to prostaglandins. AA is converted to PGH2 under the action of COX-2, and then converted to PGE2 by prostaglandin E2 synthase. PGE2 acts as a ligand to activate the upregulation of PPAR (peroxisome proliferator-activated receptor) expression, ultimately participating in the proliferation and differentiation of sebaceous gland cells and the production of sebum.

[0167] The transcriptome refers to the sum of all RNAs transcribed from specific cells, tissues or organs at a specific developmental stage or physiological state. By studying the differential expression of mRNA, we can reflect the potential targets of organisms at the gene transcription level and further explain the molecular mechanism of action from the regulatory network level.

[0168] Human fibroblasts were inoculated on the scaffolds and cultured in a cell culture incubator at 37°C and 5% CO2 for 21 days. KC were then inoculated on the scaffolds and cultured in the submerged state for 1 week before switching to gas-liquid phase culture. 0.5 mg / mL LrFF-JMB235 was added to the culture medium from the 38th day until the end of the experiment. 10 uL of 10 μM BaP was applied daily on the 40th and 41st days and UV irradiation (UVA 5 J / cm 2 , UVB 0.05J / cm 2 ); On the 42nd day, the recombinant human full skin 3D model was frozen in liquid nitrogen and sent to a third-party testing company for transcriptome sequencing. Qualified mRNA was used for subsequent library construction and sequencing. The raw data of the sequencing results were further filtered to obtain Clean Reads and compared with the reference genome. The DESeq2 software based on the negative binomial distribution was used to perform statistical analysis on the transcriptome data. |log2(Fold Change)|≥2 and P<0.05 were defined to obtain significantly differentially expressed genes and conduct subsequent analysis. The KEGG functional enrichment pathway analysis of the differentially expressed genes was performed using the online analysis tool of the third-party testing company (https: / / www.genescloud.cn / login), and P<0.05 was used as the screening criterion.

[0169] The transcriptome sequencing data were analyzed by DESeq2 software. Compared with the blank control group (BC), there were 4327 differentially expressed genes in the UV+BaP group, of which 2629 genes were up-regulated and 1698 genes were down-regulated ( Fig.13 A). Compared with the UV+BaP group, the B235+UV+BaP group had 722 differentially expressed genes, including 633 up-regulated genes and 89 down-regulated genes. The data were visualized as a volcano plot, as shown in Fig.13 . The expression levels of KRT1 and KRT16 in cells treated with UV and BaP were significantly reduced, while LrFF-JMB235 at a concentration of 0.5 mg / mL could significantly upregulate these genes, indicating that LrFF-JMB235 can promote epidermal proliferation and differentiation and repair damaged skin barrier by regulating keratin expression. The expression levels of genes involved in skin homeostasis, such as KLK5 and KLK11, were also significantly reduced after UV combined with BaP treatment, but these two genes were significantly upregulated after LrFF-JMB235 treatment, indicating that it can promote the normal expression of KLKs to regulate the skin barrier.

[0170] KEGG functional enrichment analysis found that Fig.14The differentially expressed genes in the LrFF-JMB235+UV+BaP group vs. UV+BaP group were mainly enriched in signal pathways such as arachidonic acid metabolism, α-linolenic acid metabolism, and linoleic acid metabolism, and were significantly downregulated, suggesting that LrFF-JMB235 may regulate the metabolites of lipid peroxidation through inflammatory pathways to effectively regulate the activity of sebaceous gland cells and reduce the secretion of sebum, thereby achieving oil control, acne removal, or hair loss prevention.

[0171] Example 12. Zebrafish sleep improvement effect

[0172] Pentylenetetrazol (PTZ) is an inhibitor of γ-aminobutyric acid (GABA) and its receptor complex. GABA is a naturally occurring non-protein amino acid and an important inhibitory neurotransmitter in the central nervous system of mammals. It can inhibit the excitability of brain neurons, thereby maintaining the brain's stable state. Small doses of PTZ can inhibit the synthesis and release of GABA, causing brain neurons to lose their inhibitory effect and become excited, thus causing insomnia. The changes in the amount of awakening activity (i.e., rapid movement distance) of zebrafish are used to evaluate whether the sample has the effect of improving sleep.

[0173] Zebrafish were randomly selected in a 6-well plate, with 15 in each well. The sample was given in water, and a normal control group and a model control group were set up at the same time, with a capacity of 3 mL in each well. Incubate in the dark at 28°C for 24 hours. Ten zebrafish were randomly selected from each experimental group and transferred to a 96-well plate, 200 μL / tail, 1 tail / well. Except for the normal control group, the other experimental groups were given PTZ in water to establish a zebrafish insomnia model. The behavior analyzer was used to collect data, analyze the amount of awakening activity (D) of zebrafish, and calculate and judge whether the sample has the effect of improving sleep according to the formula.

[0174]

[0175] The results are as follows Fig.15 The observations found that the amount of awakening activity in the LrFF-JMB235 group was significantly reduced compared with the model control group, revealing that the sample has the effect of improving sleep, with an improvement effect of 46.11%.

[0176] The above are preferred embodiments of the present invention, which are used to illustrate the technical concept and features of the present invention. The purpose is to enable people familiar with the technology of this project to understand the content of the present invention and implement it accordingly, and it cannot be used to limit the protection scope of the present invention. Any equivalent changes or modifications made according to the spirit of the present invention should be included in the protection scope of the present invention.

Claims

1. A Lactobacillus rhamnosus JMB235, characterized in that The deposit number is CCTCC No.M20232530.

2. Use of the Lactobacillus rhamnosus JMB235 or its fermentation product according to claim 1 in the preparation of skin topical preparations, health products, beauty oral preparations, foods or food additives that have the effects of controlling oil secretion, preventing hair loss, inhibiting acne, promoting hair growth, inhibiting eczema, inhibiting inflammation, maintaining skin barrier, promoting epidermal cell proliferation and differentiation, sun protection, repairing damaged skin and improving insomnia.

3. A method for preparing a fermentation product of Lactobacillus rhamnosus JMB235, characterized in that the steps include: The expanded cultured Lactobacillus rhamnosus JMB235 bacterial liquid is inoculated into a sterilized fermentation medium, and fermented at 26°C to 36°C and 50 to 150 rpm, with the pH controlled at 5.5 to 6.5; feeding is started 12 to 16 hours after the inoculation, so that the final concentration of glucose is 1% to 2%, and the total culture time is 40 to 50 hours; after the fermentation, the bacterial liquid is centrifuged to obtain the liquid, and the supernatant is collected by filtration.

4. A fermentation product of Lactobacillus rhamnosus JMB235, characterized in that: Prepared by the method according to claim 3.

5. The fermentation product of Lactobacillus rhamnosus JMB235 according to claim 4, characterized in that: The dry matter includes the following components in percentage by weight: Organic acids and their derivatives 46%-49%, carbohydrates and carbohydrate conjugates 43%-45%, amino acids, peptides and analogs 7%-8%, nucleosides, nucleotides and analogs 0.25%-0.5%, steroids and steroid derivatives 0.1%-0.2%, biogenic amines 0.1%-0.2%, pyrimidine and pyrimidine derivatives 0.03%-0.08%, benzene 0.03%-0.08%, lipids 0.02%-0.05%, purine and purine derivatives 0.02%-0.05%, indole and derivatives 0.001%-0.005%, nitrogen-containing heterocyclic compounds 0.001%-0.005%, pyridine and derivatives 0.001%-0.005%.

6. A skin external preparation, health product, cosmetic oral preparation, food or food additive, characterized in that: Contains the fermentation product of Lactobacillus rhamnosus JMB235 according to claim 4 or 5.

7. Use of the fermentation product of Lactobacillus rhamnosus JMB235 according to claim 4 or 5 in the preparation of 5α-reductase inhibitors.

8. The fermentation product of Lactobacillus rhamnosus JMB235 according to claim 4 or 5 is used for preparing inhibitors of inflammatory factors and TNF-α protein.

9. The fermentation product of Lactobacillus rhamnosus JMB235 according to claim 4 or 5 is used for regulating the transcription level of genes related to epidermal cell proliferation and differentiation and / or inhibiting lipid peroxidation metabolites.

10. The fermentation product of Lactobacillus rhamnosus JMB235 according to claim 4 or 5 is used for promoting the proliferation of human immortalized keratinocytes, dermal fibroblasts, and hair follicle dermal papilla cells and repairing ultraviolet damage.

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