Lactobacillus rhamnosus and its applications

Abstract

This invention relates to the field of microbiology and discloses a strain of *Lactobacillus rhamnosus* and its applications. The *Lactobacillus rhamnosus* strain provided by this invention, with accession number CGMCC No. 33153, exhibits strong reproductive capacity and high production of lactic acid and hydrogen peroxide, demonstrating strong antibacterial properties. It has potential applications in organisms; it can regulate genes related to inflammation and apoptosis, thereby reducing the production of inflammatory factors and promoting cell growth and repair; it can also upregulate the gene expression of aquaporins and collagen, thereby promoting skin repair.

Description

Technical Field

[0001] This invention relates to the field of microbiology, specifically to a strain of Lactobacillus rhamnosus and its applications. Background Technology

[0002] The skin is the largest organ in the human body, protecting various tissues and organs from physical, mechanical, chemical, and pathogenic microbial attacks. Under the combined influence of internal and external factors, various skin problems inevitably arise, including damage, bacterial infection, and collagen loss.

[0003] Microorganisms are commonly used to address the aforementioned skin problems. Among them, *Lactobacillus rhamnosus* is widely used due to its significant probiotic effects. CN112980892A discloses a microorganism derived from *Lactobacillus rhamnosus*. Lactobacillus plantarum Lactobacillus casei Lactobacillus casei Bifidobacterium lactis Bifidobacterium lactis Bifidobacterium animalis Bifidobacterium animalis Lactobacillus rhamnosus Lactobacillus rhamnosus The fermentation products of probiotic compositions can maintain the balance of skin flora and have effects such as wrinkle reduction, increased skin elasticity, and anti-inflammation. CN118620784A discloses a strain of Lactobacillus rhamnosus LTHINK0088 and its application in whitening and wrinkle-reducing cosmetics. The fermentation lysate obtained after fermentation of LTHINK0088 can scavenge free radicals on the skin surface, inhibit melanin production, inhibit metalloproteinase activity, and promote collagen synthesis, thus having antioxidant, whitening, firming, and wrinkle-reducing effects. CN113046268A discloses Lactobacillus rhamnosus 11-7, whose lysate obtained by enzymatic lysis of the fermented cells can inhibit the growth and reproduction of pathogenic bacteria and regulate the skin microecology. However, in the prior art, the microbial preparations, lysates, or fermentation products of Lactobacillus rhamnosus have relatively single functions, or need to be used in combination with other probiotics to obtain better results.

[0004] Therefore, there is an urgent need for a type of Lactobacillus rhamnosus that can simultaneously achieve anti-inflammatory effects and promote the production of skin repair substances, as well as a microbial agent that can achieve the above functions using only a single bacterial agent. Summary of the Invention

[0005] The purpose of this invention is to overcome the above-mentioned problems in the prior art and to provide a strain of Lactobacillus rhamnosus and its applications.

[0006] To achieve the above objectives, the first aspect of the present invention provides a strain of Lactobacillus rhamnosus. Lactobacillus rhamnosus The Lactobacillus rhamnosus has the accession number CGMCC No. 33153.

[0007] A second aspect of the present invention provides a microbial agent containing *Lactobacillus rhamnosus* as described above.

[0008] A third aspect of the present invention provides the use of Lactobacillus rhamnosus as described above in the preparation of a medicament for inhibiting the growth of pathogenic bacteria.

[0009] The fourth aspect of the present invention provides the use of Lactobacillus rhamnosus as described above in the preparation of medicaments for promoting cell growth or inhibiting cell apoptosis.

[0010] The fifth aspect of the present invention provides the preparation of Lactobacillus rhamnosus as described above for promoting strt Gene expression, or, repression caspase Applications of gene expression in pharmaceuticals.

[0011] The sixth aspect of the present invention provides the use of Lactobacillus rhamnosus as described above in the preparation of a medicament for reducing inflammatory responses.

[0012] The seventh aspect of the present invention provides the use of Lactobacillus rhamnosus as described above in the preparation of a medicament for inhibiting the expression of at least one of inflammatory factors, inhibiting the expression of cytotoxic molecules, and promoting the expression of immunomodulatory factors.

[0013] The eighth aspect of the present invention provides the use of Lactobacillus rhamnosus, as described above, in the preparation of a medicament for promoting protein synthesis.

[0014] The beneficial effects obtained by the present invention through the above technical solution include at least the following:

[0015] (1) The Lactobacillus rhamnosus with accession number CGMCC No.33153 provided by the present invention can regulate genes related to inflammation and apoptosis, thereby reducing the production of inflammatory factors and promoting cell growth and repair.

[0016] (2) The Lactobacillus rhamnosus with accession number CGMCC No.33153 provided by the present invention can upregulate the gene expression of aquaporin and collagen, thereby promoting skin repair;

[0017] (3) The Lactobacillus rhamnosus with accession number CGMCC No.33153 provided by the present invention has a strong reproductive capacity and can produce high levels of lactic acid and hydrogen peroxide, and can non-specifically and specifically inhibit a variety of pathogenic bacteria;

[0018] (4) The Lactobacillus rhamnosus cells with accession number CGMCC No.33153 provided by the present invention have the potential to be applied to organisms.

[0019] Biological Preservation

[0020] The strain provided by this invention is classified as Lactobacillus rhamnosus. Lactobacillus rhamnosusIt was deposited on December 23, 2024, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 33153, located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing. Attached Figure Description

[0021] Figure 1 This is a colony morphology diagram of Lactobacillus rhamnosus with accession number CGMCC No. 33153 provided by this invention;

[0022] Figure 2 This is a microscopic image of the colony of *Lactobacillus rhamnosus* with accession number CGMCC No. 33153 provided by this invention. Detailed Implementation

[0023] The endpoints and any values ​​of the ranges disclosed herein are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of the various ranges, the endpoint values ​​of the various ranges and individual point values, and individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed herein.

[0024] The inventors of this invention accidentally isolated a strain of Lactobacillus CCNH332 from fermented wine produced at a winery in Zhejiang Province. Figure 1 and Figure 2 As shown, the colony has a smooth, milky-white, round or oval surface. The cells of this strain are rod-shaped, arranged singly, in short chains, or in a palisade pattern. Morphological and 16S rDNA sequencing identification confirmed it as *Lactobacillus rhamnosus*. Lactobacillus rhamnosus Its 16S rDNA is shown in SEQ ID NO.1.

[0025] SEQ ID NO.1:

[0026] ACAACTTGTCACTTAGACGGCTCGCTCCCTAAAAGGGTTACGCCACCGGCTTCGGGTGTTACAAACTCTCATGGTGTGACGGGCGGTGTGTACAAGGCCCGGGAACGTATTCACCGCGGCGTGCTGATCCGCGATTACTAGCGATTCCGACTTCGTGTAGGCGAGTTGCAGCCTACAGTCCGAACTGAGAATGGCTTTAAGAGATTAGCTTGACCTCGCGGTCTCGCAACTCGTTGTACCATCCATTGTAGCACGTGTGTAGCCCAGGTCATAAGGGGCATGATGATTTGACGTCATCCCCACCTTCCTCCGGTTTGTCACCGGCAGTCTTACTAGAGTGCCCAACTAAATGCTGGCAACTAGTCATAAGGGTTGCGCTCGTTGCGGGACTTAACCCAACATCTCACGACACGAGCTGACGACAACCATGCACCACCTGTCATTTTGCCCCCGAAGGGGAAACCTGATCTCTCAGGTGATCAAAAGATGTCAAGACCTGGTAAGGTTCTTCGCGTTGCTTCGAATTAAACCACATGCTCCACCGCTTGTGCGGGCCCCCGTCAATTCCTTTGAGTTTCAACCTTGCGGTCGTACTCCCCAGGCGGAATGCTTAATGCGTTAGCTGCGGCACTGAAGGGCGGAAACCCTCCAACACCTAGCATTCATCGTTTACGGCATGGACTACCAGGGTATCTAATCCTGTTCGCTACCCATGCTTTCGAGCCTCAGCGTCAGTTACAGACCAGACAGCCGCCTTCGCCACTGGTGTTCTTCCATATATCTACGCATTTCACCGCTACACATGGAGTTCCACTGTCCTCTTCTGCACTCAAGTTTCCCAGTTTCCGATGCACTTCCTCGGTTAAGCCGAGGGCTTTCACATCAGACTTAAAAAACCGCCTGCGCTCGCTTTACGCCCAATAAATCCGGATAACGCTTGCCACCTACGTATTACCG

[0027] Based on the above findings, the first aspect of the present invention provides a strain of Lactobacillus rhamnosus. Lactobacillus rhamnosus The Lactobacillus rhamnosus has the accession number CGMCC No. 33153, and its 16S rDNA is shown in SEQ ID NO. 1.

[0028] "CCNH332" is the strain number assigned by the inventors during the strain screening process, and "CGMCC No.33153" is the preservation number of the strain. Both represent the same strain and can be used interchangeably in the following text.

[0029] A second aspect of the present invention provides a microbial agent containing *Lactobacillus rhamnosus* as described above.

[0030] In this invention, the microbial agent also contains excipients, which can be excipients commonly used in the preparation of microbial agents or fermentation agents in the art, such as buffers (e.g., buffer solutions, culture media, etc.), protectants (e.g., freeze-drying protectants, such as skim milk powder, trehalose, monosodium glutamate, glycerol, etc.), preferably glycerol and / or corn oil.

[0031] Preferably, based on the total weight of the bacterial agent, the viable count of the *Lactobacillus rhamnosus* is 10-1. 4 -10 12 CFU / g.

[0032] In this invention, the viable count of *Lactobacillus rhamnosus* is 10⁻¹¹ relative to 1 mL of excipient. 4 -10 12 CFU.

[0033] Preferably, the content of Lactobacillus rhamnosus in the fermentation agent provided by the present invention can be 10. 4 -10 12 CFU / g (liquid starter culture can be converted at 1g = 1mL). 10 4 -10 12 CFU / g refers to the content of Lactobacillus rhamnosus in the starter culture reaching 10. 4 -10 12 The order of magnitude of CFU / g, for example, 10 4 The order of magnitude of CFU / g represents greater than or equal to 1 × 10⁻⁶. 4 CFU / g to less than 1×10 5 The range of CFU / g, i.e., 1×10 4 CFU / g, 5×10 4 CFU / g, 9.9×10 4 CFU / g and other values ​​are all within 10. 4The concentration is on the order of CFU / g. Therefore, in the method provided by this invention, the content of *Lactobacillus rhamnosus* is greater than or equal to 1 × 10⁻⁶. 4 CFU / g to less than 1×10 12 Within the range of CFU / g. For example, the content of Lactobacillus rhamnosus in the fermentation agent provided by this invention can be 1×10⁻⁶. 4 CFU / g, 5×10 4 CFU / g, 1×10 5 CFU / g, 5×10 5 CFU / g, 1×10 6 CFU / g, 5×10 6 CFU / g, 1×10 7 CFU / g, 5×10 7 CFU / g, 1×10 8 CFU / g, 5×10 8 CFU / g, 1×10 9 CFU / g, 5×10 9 CFU / g, 1×10 10 CFU / g, 5×10 10 CFU / g, 1×10 11 CFU / g, 5×10 11 CFU / g, 1×10 12 CFU / g, 5×10 12 CFU / g, or any range or value within the range formed by any two of the above values.

[0034] A third aspect of the present invention provides the use of Lactobacillus rhamnosus, as described above, in the preparation of a medicament for inhibiting the growth of pathogenic bacteria.

[0035] According to the present invention, the pathogenic bacteria may include Escherichia coli (Escherichia coli) Escherichia coli Staphylococcus aureus ( Staphylococcus aureus ) and Salmonella ( Salmonella At least one of the following.

[0036] Preferably, the Salmonella includes Salmonella Typhimurium (Salmonella Typhimurium). Salmonella typhimurium ) and Salmonella enteritidis ( Salmonella enteritidis ).

[0037] More preferably, the Escherichia coli is numbered ATCC 25922 and / or CICC 10421.

[0038] More preferably, the Staphylococcus aureus is designated as CMCC(B)26001 and / or CMCC(B)26003.

[0039] More preferably, the Salmonella typhimurium is designated ATCC 14028.

[0040] More preferably, the Salmonella enteritidis is designated CVCC 3378.

[0041] The fourth aspect of the present invention provides the use of Lactobacillus rhamnosus as described above in the preparation of medicaments for promoting cell growth or inhibiting cell apoptosis.

[0042] Preferably, the cells are human immortalized keratinocytes.

[0043] Preferably, the cell growth promotion is achieved by a dormant information regulator (…). Sirt1 Gene control.

[0044] Preferably, the apoptosis-aspartate-specific cysteine ​​protease (CAMP) Caspase Gene control, more preferably, the apoptosis is controlled by genes located in Caspase Aspartate-specific cysteine ​​protease 3 on chromosome 4 ( Caspase-3 Gene control.

[0045] The fifth aspect of the present invention provides a method for preparing Lactobacillus rhamnosus as described above for promoting Sirt1 Gene expression, or, repression Caspase Applications of gene expression in pharmaceuticals.

[0046] Preferably, the Caspase Genes are specifically Caspase-3 Gene.

[0047] The sixth aspect of the present invention provides the use of Lactobacillus rhamnosus as described above in the preparation of a medicament for reducing inflammatory responses.

[0048] Preferably, the intensity of the inflammatory response is obtained by measuring the amount of NO generated.

[0049] The seventh aspect of the present invention provides the use of Lactobacillus rhamnosus as described above in a medicament for inhibiting the expression of at least one of inflammatory factors, inhibiting the expression of cytotoxic molecules, and promoting the expression of immunomodulatory factors.

[0050] Preferably, the inflammatory factor is a pro-inflammatory factor, more preferably interleukin-6 (IL-6).

[0051] Preferably, the immunomodulatory factor is recombinant human transforming growth factor-β1 (TGF-β1, also known as transforming growth factor-β1).

[0052] The eighth aspect of the present invention provides the use of Lactobacillus rhamnosus, as described above, in the preparation of a medicament for promoting protein synthesis.

[0053] Preferably, the protein includes aquaporins and / or collagen.

[0054] According to one embodiment of the present invention, the aquaporin is aquaporin 3 (AQP3), and the collagen is type I collagen natural protein (Col1) and / or type III collagen natural protein (Col3). The strain provided by the present invention can promote the gene expression of the above proteins, thereby promoting their synthesis.

[0055] Preferably, in the application described above, the Lactobacillus rhamnosus functions in the form of its fermentation product (fermentation supernatant).

[0056] Unless otherwise specified, all reagents and materials used in the following examples and comparative examples were commercially available products purchased from reputable chemical or biological reagent and material suppliers, and all reagents were of analytical grade.

[0057] Escherichia coli ( Escherichia coli (1) ATCC 25922, purchased from the American Center for Type Culture Collection; (2) CICC 10421, purchased from the China Industrial Microbial Culture Collection Center.

[0058] Staphylococcus aureus ( Staphylococcus aureus The items, numbered CMCC(B)26001 and CMCC(B)26003, were purchased from the China Medical Bacteriological Culture Collection Center.

[0059] Salmonella, (1) Salmonella typhimurium ( Salmonella typhimurium (2) Salmonella enteritidis (ATCC 14028), purchased from the American Center for Type Culture Collection; Salmonella enteritidis (Item number CVCC 3378), purchased from the National Veterinary Microbial Culture Collection Center.

[0060] Example 1

[0061] A strain of Lactobacillus, designated CCNH332, was isolated from a fermentation sample collected from a winery in Zhejiang Province using the gradient dilution plating method, and a pure culture of the strain was obtained by the streak plating method.

[0062] The colonies of strain CCNH332 on MRS plates are milky white, regularly round, moist, smooth, and opaque (as shown in the image). Figure 1 (As shown); then, the morphology of strain CCNH332 cells was observed under a microscope. The cells were rod-shaped, arranged singly, in short chains, or in a palisade pattern (as shown). Figure 2 As shown, it was found to be *Lactobacillus rhamnosus*. 16S rDNA sequencing confirmed that this strain is indeed *Lactobacillus rhamnosus*. Lactobacillus rhamnosus .

[0063] Example 2

[0064] Frozen glycerol tubes of strain CCNH332 and LGG were inoculated at 2 vol% into fresh MRS liquid medium and cultured overnight at 37°C for 18 h. OD 600 Normalization was performed to obtain activated bacterial culture, which was then inoculated into fresh MRS liquid medium at an inoculum of 2 vol% and cultured at 37°C and 200 rpm for 18 h to obtain expanded bacterial culture of CCNH332 and LGG.

[0065] OD values ​​of CCNH332 and LGG culture broths were measured. 600 The values ​​were 8.3 and 8.13 respectively, indicating that CCNH332 has better growth performance than LGG.

[0066] Example 3

[0067] The activated bacterial culture was inoculated into 30 mL of fresh MRS liquid medium at a 2 vol% inoculum. After static incubation at 37°C for 18 h, the supernatant was collected by centrifugation and filtered through a membrane (0.22 μm pore size). Lactic acid production was detected by HPLC. The lactic acid production of strain CCNH332 was 16.71 g / L, while that of LGG was 15.1 g / L. The lactic acid production performance of strain CCNH332 was superior to that of LGG, indicating that strain CCNH332 can metabolize and decompose more sugars during cultivation, thereby reducing the sugar content in the environment.

[0068] Example 4

[0069] H₂O₂ solutions with concentrations of 0, 0.5, 1.0, 1.5, 2.0, 5, 8, 10, 25, and 50 μg / mL were prepared, and the OD values ​​at different concentrations were measured. 505 The absorbance was measured to establish an absorbance-concentration standard curve. 30 mL of the expanded bacterial culture was centrifuged to obtain the supernatant, and its absorbance was measured. The H2O2 yield was calculated based on the absorbance-concentration standard curve. The H2O2 yields of CCNH332 and LGG were 5.91 μg / mL and 4.33 μg / mL, respectively. CCNH332 showed a stronger ability to produce H2O2 and thus non-specifically inhibit pathogenic bacteria than LGG.

[0070] Example 5

[0071] Escherichia coli (CICC 10421 and ATCC 25922), Staphylococcus aureus (CMCC(B)26001 and CMCC(B)26003), and Salmonella (ATCC 14028 and CVCC 3378) were inoculated into LB liquid medium, anaerobically cultured at 37°C for 18 h, and then transferred to the corresponding fresh medium and cultured until the viable count reached 10^6.8 CFU / mL, diluted to 10 with the appropriate culture medium. 5 CFU / mL was used as the indicator bacterial solution.

[0072] The activated bacterial solution was inoculated into 30 mL of fresh MRS liquid medium at a 2 vol% inoculum. After incubation at 37°C for 18 h, the supernatant was collected by centrifugation and then filtered through a membrane (membrane pore size of 0.22 μm) to obtain the supernatant.

[0073] Three experimental groups were set up: S1: 100 μL supernatant, 100 μL indicator bacterial suspension; S2: 50 μL supernatant, 150 μL indicator bacterial suspension; S3: 25 μL supernatant, 175 μL indicator bacterial suspension; 200 μL of MRS liquid culture medium served as the negative control group, and the bacterial suspension was replaced with ampicillin at a concentration of 16 μg / mL as the positive control group. The OD values ​​of the experimental groups and the negative control group were measured. 600 The values ​​are A and A respectively. 0， The inhibition rate was calculated according to the following formula, as shown in Table 1, where the unit of inhibition rate is %.

[0074] Inhibition rate (%) = (A0-A1) / A0×100%.

[0075] Table 1

[0076]

[0077] Example 6

[0078] The activated bacterial culture of strain CCNH332 was centrifuged at 4000 rpm for 10 min to obtain the supernatant.

[0079] H-DMEM medium: Calcium chloride 200 mg / L, sodium chloride 6400 mg / L, potassium chloride 400 mg / L, anhydrous magnesium sulfate 97.67 mg / L, anhydrous sodium dihydrogen phosphate 125 mg / L, ferric nitrate nonahydrate 0.1 mg / L, arginine 84 mg / L, cystine hydrochloride 62.57 mg / L, glutamine 584 mg / L, glycine 30 mg / L, histidine hydrochloride 42 mg / L, isoleucine 104.8 mg / L, leucine 104.78 mg / L, lysine hydrochloride 146.2 mg / L, methionine 30 mg / L. mg / L, phenylalanine 66mg / L, serine 42mg / L, threonine 95.2mg / L, tryptophan 16mg / L, L-tyrosine disodium salt dihydrate 103.79mg / L, valine 93.6mg / L, calcium pantothenate 4mg / L, choline chloride 4mg / L, folic acid 4mg / L, inositol 7mg / L, nicotinamide 4mg / L, pyridoxine hydrochloride 4mg / L, riboflavin 0.4mg / L, thiamine hydrochloride 4mg / L, glucose 4500mg / L, phenol red 15.9mg / L, sodium bicarbonate 3700mg / L.

[0080] HaCaT cells (human immortalized keratinocytes): purchased from Shangen Biotechnology, catalog number SNL-163.

[0081] HaCaT cells were seeded in multiple culture dishes containing H-DMEM medium and cultured at 37°C and 5 vol% CO2 until the cell confluence reached 80% of the culture medium surface area.

[0082] Four groups were set up, each containing 10 vol% fetal bovine serum (FBS). A negative control group containing 90 vol% H-DMEM and free of strain CCNH332 was set up, along with the supernatant from the three experimental groups (OD500 of CCNH332). 600 (1) The contents were 1 vol%, 3 vol%, 5 vol%, and 10 vol%, with the remainder being H-DMEM). The four groups were seeded into culture dishes containing HaCaT cells, with each dish containing 10 cells. 4 Live cells / mL. After culturing at 37℃ and 5 vol% CO2 for 48 h, 1 mL of the liquid was taken to measure its absorbance (OD) at 410 nm. 410 OD 410 The higher the value, the better the HaCaT cell proliferation. The relative cell activity of each group is calculated according to the following formula, where the blank well contains only FBS.

[0083] Cell viability (%) = (OD value of experimental group - OD value of blank well) / (OD value of negative control group - OD value of blank well) × 100%.

[0084] The cell viability rates in the negative control group and the groups with supernatant contents of strain CCNH332 of 1 vol%, 3 vol%, 5 vol%, and 10 vol% were 100%, 98±1%, 96±2%, 95±2%, and 94±2%, respectively. A cell viability rate greater than 90% indicates low cytotoxicity. Therefore, when the supernatant content of CCNH332 is lower than 10 vol%, it can maintain a high level of cell activity.

[0085] Example 7

[0086] Add 10 vol% FBS and 1% (wt / v) penicillin-streptomycin (by culture medium volume) to H-DMEM medium to obtain a double-antibiotic culture medium. Culture HaCaT cells in the double-antibiotic culture medium until the cell confluence reaches about 80%, then add 1 wt% sodium dodecyl sulfate (SDS) to induce cell damage to establish a cell damage model. During this period, the cell model was successfully constructed every 48 hours by methods such as cell viability test or inflammatory factor release detection.

[0087] Methods such as cell viability testing or inflammatory factor release detection:

[0088] Cell seeding and treatment: Inoculate the cell suspension in logarithmic growth phase at a rate of 10 cells per well. 4 Cells were seeded into 96-well plates and pre-cultured for 24 hours to allow them to adhere. After incubation for 96 hours with the test drug, 10 μL of cell counting reagent CCK-8 was added to each well, and incubation was continued for 14 hours. The absorbance (OD value) at 450 nm was measured using an ELISA reader. For highly turbid samples, a reference wavelength of 600-650 nm was set to correct the background.

[0089] Then, the supernatant prepared in Example 6 was added to the successfully modeled cell culture dishes. The amounts of supernatant added were 1 vol%, 3 vol%, and 5 vol%, respectively, based on the volume of the culture dish. The cells were cultured at 37°C, 5 vol% CO2, and 95% humidity for 24 hours. Additionally, a model group without SDS modeling, a positive control group with added epidermal growth factor (EGF), and a negative control group containing 10 vol% FBS + 90 vol% H-DMEM (without supernatant) were set up. After the experiment, the quiescent information regulator (KIF) in the culture medium was measured. Sirt1 The expression levels of mRNA (in fold change) are shown in Table 2.

[0090] In addition, following the same method, an SD cell injury model was constructed by replacing SDS with an equal amount of sodium deoxycholate (SD) and experiments were conducted to measure the aspartate-specific cysteine ​​protease-3 (CCP-3) in the cells. caspase-3The expression levels of mRNA (in fold change) are shown in Table 2.

[0091] Measurement Sirt1 and Caspase-3 Methods for determining mRNA expression levels:

[0092] RNA extraction: 1×10 7 Add 1 mL of RNA extraction reagent (TRIzol) to each cell, homogenize, then add chloroform for layering, precipitate RNA with isopropanol, and wash with 75 vol% ethanol for purification. Finally, dissolve in RNase-free water to obtain a sample containing RNA. RNA quality testing: Purity was assessed by measuring the A260 / A280 ratio (ideal value 1.8-2.0) using a UV spectrophotometer, and RNA integrity was confirmed by formaldehyde denaturing agarose gel electrophoresis (clear 28S and 18S rRNA bands). Reverse transcription (cDNA synthesis): Take 1 μg of sample and perform reverse transcription using the PrimeScript RT reverse transcription kit. The reaction conditions were 50℃ (15 min, reverse transcription) and 85℃ (5 s, enzyme inactivation). A control without reverse transcription was set up to exclude genomic DNA contamination.

[0093] Primer design and validation:

[0094] Sirt1 Primers (across exon junctions): forward 5'-TGGACAATTCCAGCCATCTC (SEQ ID NO.2)-3', reverse 5'-GCGTGTCTATGTTCTGGGTATAG (SEQ ID NO.3)-3';

[0095] Caspase-3 Primers: forward 5'-GAAATTGTGGAATTGATGCGTGA (SEQ ID NO.4)-3', reverse 5'-CTACAACGATCCCCTCTGAAAA (SEQ ID NO.5)-3';

[0096] Simultaneously, the glyceraldehyde-3-phosphate dehydrogenase gene (GAPDH) was used as an internal control for standardization.

[0097] Real-time quantitative PCR: Nucleic acid staining (SYBR Green) was used. The reaction system contained 2× dye-based quantitative PCR premix (SYBR qPCR Mix), primers (0.5 μM each) and cDNA template. The program was pre-denaturation at 95℃ for 30 seconds, followed by 40 cycles of 95℃ for 5 seconds and 60℃ for 30 seconds. Finally, the product specificity was analyzed by melting curve analysis.

[0098] Data analysis: The ΔΔCt method was used, and after correction with the internal reference gene (β-actin), the results were calculated. Sirt1 and Caspase-3 The multiple change (2^(-ΔΔCt)).

[0099] Table 2

[0100]

[0101] In Table 2, * indicates a highly significant difference from the model group (p < 0.01); ** indicates an extremely significant difference from the model group (p < 0.001).

[0102] As shown in Table 2, the fermentation product of Lactobacillus rhamnosus provided by this invention can upregulate repair genes. Sirt1 Expression and downregulation of apoptosis genes Caspase-3 The expression of the gene is enhanced, and its ability to upregulate or downregulate gene expression increases with increasing supernatant content.

[0103] Example 8

[0104] Mouse mononuclear macrophage leukemia cells (RAW264.7 cells): purchased from Shangen Biotechnology Co., Ltd., catalog number SNL-112.

[0105] RAW264.7 cells were incubated in an incubator at 37°C, 5 vol% CO2, and 95% relative humidity. They were cultured in DMEM complete medium (antibiotic-containing medium) containing 10 vol% FBS and 1 wt% penicillin-dextrose antibody. The medium was changed and passaged regularly to maintain the cells in the logarithmic growth phase. Cells were divided into a control group, a lipopolysaccharide (LPS) group, and an experimental group containing the supernatant prepared in Example 6. Control group: Contained only RAW264.7 cells and culture medium, without any treatment. LPS group: 10 mL of culture medium containing logarithmic growth phase RAW264.7 cells was added to a final concentration of 50 ng / mL of LPS to induce an inflammatory response. Experimental group: Based on the LPS group, 10 mL of supernatant with different volume concentrations (1 vol%, 3 vol%, 5 vol%) was added. After culturing for 24 h, the samples to be tested were obtained. The NO content (in mmol / L) and the expression levels of mRNA of pro-inflammatory factors (IL-6) and immunomodulatory factors (TGF-β1) were measured (in fold change) to test the inhibitory effect of different groups on the inflammatory response.

[0106] Method for measuring NO content: Centrifuge cultured cells to remove precipitate. Dilute sodium nitrate standard with antibiotic-resistant medium (concentration gradient 0-100 μM) to eliminate background interference; establish a standard curve for NO concentration. Add 50 μL of sample and standard to each well of a 96-well plate, followed by adding Griess reagent (prepared from equal volumes of sulfonamide and naphthylethylenediamine hydrochloride) to each well, mix well, and incubate at room temperature for 20 minutes until colorimetric stability. Measure the absorbance (OD) at 540 nm. 540 Calculate the NO concentration in the sample (unit: mmol / L), and set blank wells (no sample) and control wells (no drug or known toxic drug) to correct for background.

[0107] Methods for measuring IL-6 and TGF-β1 expression levels: Real-time quantitative PCR (qRT-PCR) was calculated based on the ΔΔCt method, with GAPDH as an internal reference, and fold change was detected.

[0108] Table 3

[0109]

[0110] In Table 3, * indicates a highly significant difference from the model group (p < 0.01); ** indicates an extremely significant difference from the model group (p < 0.001).

[0111] As shown in Table 3, the fermentation product of Lactobacillus rhamnosus provided by the present invention can reduce NO production, inhibit IL-6 expression and promote TGF-β1 expression, and its promoting or inhibiting ability increases with the increase of supernatant content.

[0112] Example 9

[0113] HaCaT cells were cultured and inoculated with supernatant according to the method in Example 7. A model group and an EGF positive control group were set up. After culturing for 48 h, the expression level of aquaporin 3 (AQP3) mRNA (in fold change) was measured to test the moisturizing ability of the fermentation product. The higher the expression level, the more aquaporin produced, and the stronger the moisturizing ability. The results of the model group, the EGF positive control group, and the experimental groups with supernatant contents of 1 vol%, 3 vol%, and 5 vol% were 0.9±0.3, 1.9±0.2, 1.3±0.3, 1.6±0.2, and 1.8±0.3, respectively (there were significant differences between the groups). The expression level of mRNA in the experimental groups was significantly increased, and the increase increased with the increase of supernatant content.

[0114] Example 10

[0115] HaCaT cells were cultured and inoculated with supernatant according to the method in Example 7. A blank control group without the bacterial strain and a control group with the bacterial strain replaced by 1 vol% EGF positive strain were set up. After culturing for 48 h, the natural collagen protein was measured. Col1 and Col3 The expression level of mRNA (in fold change) was measured to test the ability of fermentation products to promote collagen production. The higher the mRNA expression level, the stronger the ability. The blank control group, positive control group, and supernatant contents were 1 vol%, 3 vol%, and 5 vol%, respectively. Col1 The results were 1±0.3, 3.5±0.5, 1.5±0.4, 2.0±0.3 and 2.5±0.4 (significant differences were found between the groups). Col3 The results were 1±0.2, 7±1, 2.5±0.5, 2.8±0.3, and 4.5±0.8 (significant differences were observed between the groups). The results indicate that the mRNA expression level was significantly increased compared to the experimental group, and the increase increased with increasing supernatant content.

[0116] The preferred embodiments of the present invention have been described in detail above; however, the present invention is not limited thereto. Within the scope of the inventive concept, various simple modifications can be made to the technical solutions of the present invention, including combinations of various technical features in any other suitable manner. These simple modifications and combinations should also be considered as the content disclosed in the present invention and are all within the protection scope of the present invention.

Claims

1. A strain of Lactobacillus rhamnosus Lactobacillus rhamnosus Its characteristics are, The preservation number of the Lactobacillus rhamnosus is CGMCC No. 33153.

2. A microbial agent, characterized in that, The bacterial agent contains Lactobacillus rhamnosus as described in claim 1.

3. The microbial agent according to claim 2, wherein, The microbial agent contains excipients, wherein the excipients are selected from glycerin and / or corn oil; And / or, based on the total weight of the bacterial agent, the viable count of the *Lactobacillus rhamnosus* is 10-1. 4 -10 12 CFU / g.

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