Lactobacillus rhamnosus and application thereof

Abstract

The invention relates to the field of microorganisms, and discloses lactobacillus rhamnosus and application thereof. The lactobacillus rhamnosus with the preservation number of CGMCC (China General Microbiological Culture Collection Center) No.33153, provided by the invention, has relatively strong reproductive capacity, can produce lactic acid and hydrogen peroxide at high yield, and has relatively strong antibacterial performance; the polypeptide has the potential of being applied to organisms; the composition can regulate and control inflammation and apoptosis related genes, so that the generation amount of inflammatory factors is reduced, cell growth and repair are promoted, and gene expression of aquaporin and collagen can be up-regulated, so that skin repair is promoted.

Description

Technical Field

[0001] The invention relates to the field of microorganisms, and in particular to a Lactobacillus rhamnosus strain and application thereof. Background Art

[0002] Skin is the largest organ in the human body. It protects various tissues and organs in the body from physical, mechanical, chemical and pathogenic microbial invasions. Under the combined effects of internal and external factors, various skin problems are inevitable, including damage, bacterial infection and collagen loss.

[0003] Microorganisms are often used to solve the above skin problems. Among them, Lactobacillus rhamnosus is widely used due to its significant probiotic effect. CN112980892A discloses a plant lactobacillus Lactobacillus plantarum , Lactobacillus casei Lactobacillus casei , Bifidobacterium lactis Bifidobacterium lactis , Bifidobacterium animalis Bifidobacterium animalis and Lactobacillus rhamnosus Lactobacillus rhamnosus The probiotic composition fermentation product composed of the composition can maintain the balance of skin flora, and has the effects of wrinkle removal, increasing skin elasticity, and anti-inflammatory. CN118620784A discloses a strain of Lactobacillus rhamnosus LTHINK0088 and its application in whitening and wrinkle removal cosmetics. The fermentation lysate obtained by LTHINK0088 after fermentation can remove free radicals on the skin surface, inhibit melanin production, inhibit metalloproteinase activity, promote collagen synthesis, and has antioxidant and whitening, firming and wrinkle removal effects. CN113046268A discloses a Lactobacillus rhamnosus 11-7, and the lysate obtained by enzyme lysis of the fermented bacterial body can inhibit the reproduction and growth of pathogenic bacteria and regulate the skin microecology. However, in the prior art, the microbial preparations, lysates or fermentation products of Lactobacillus rhamnosus are relatively single in function, or need to be used in combination with other probiotics to obtain better results.

[0004] Therefore, there is an urgent need for a 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 agent. Summary of the invention

[0005] The purpose of the present invention is to overcome the above problems existing in the prior art and provide a Lactobacillus rhamnosus strain and its application.

[0006] In order to achieve the above-mentioned object, the present invention provides a Lactobacillus rhamnosus strain in the first aspect. Lactobacillus rhamnosus The preservation number of the Lactobacillus rhamnosus is CGMCC No.33153.

[0007] A second aspect of the present invention provides a bacterial agent, wherein the bacterial agent contains the Lactobacillus rhamnosus as described above.

[0008] The third aspect of the present invention provides the use of the aforementioned Lactobacillus rhamnosus in the preparation of a medicament for inhibiting the reproduction of pathogenic bacteria.

[0009] The fourth aspect of the present invention provides the use of the aforementioned Lactobacillus rhamnosus in the preparation of a medicament for promoting cell growth or inhibiting cell apoptosis.

[0010] The fifth aspect of the present invention provides the Lactobacillus rhamnosus described above in the preparation of a method for promoting strt Gene expression, or inhibition caspase Application of gene expression in medicine.

[0011] A sixth aspect of the present invention provides use of the aforementioned Lactobacillus rhamnosus in the preparation of a medicament for reducing inflammatory response.

[0012] A seventh aspect of the present invention provides the use of the aforementioned Lactobacillus rhamnosus in the preparation of a drug for inhibiting at least one of the expression of inflammatory factors, inhibiting the expression of cytotoxic molecules, and promoting the expression of immunomodulatory factors.

[0013] An eighth aspect of the present invention provides use of the aforementioned Lactobacillus rhamnosus in the preparation of a medicament for promoting protein synthesis.

[0014] Through the above technical solution, the beneficial effects obtained by the present invention include at least: (1) The Lactobacillus rhamnosus with the deposit 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; (2) The Lactobacillus rhamnosus with the deposit number CGMCC No. 33153 provided by the present invention can upregulate the gene expression of aquaporin and collagen, thereby promoting skin repair; (3) The Lactobacillus rhamnosus with the deposit number CGMCC No. 33153 provided by the present invention has a strong reproductive ability, can produce high amounts of lactic acid and hydrogen peroxide, and can inhibit a variety of pathogenic bacteria non-specifically and specifically; (4) The cells of Lactobacillus rhamnosus with a deposit number of CGMCC No. 33153 provided by the present invention have the potential to be applied to organisms.

[0015] Biological Deposit The strain provided by the present invention is classified and named as Lactobacillus rhamnosus Lactobacillus rhamnosus , was deposited in the General Microbiology Center of China Microorganism Culture Collection Administration (abbreviated as CGMCC) on December 23, 2024. Its deposit number is CGMCC No.33153, and the deposit address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 It is a colony morphology diagram of Lactobacillus rhamnosus with a deposit number of CGMCC No.33153 provided by the present invention; Figure 2 The present invention provides a colony microscopic examination diagram of Lactobacillus rhamnosus with a preservation number of CGMCC No. 33153. DETAILED DESCRIPTION

[0017] The endpoints and any values ​​of the ranges disclosed in this article 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 each range, the endpoint values ​​of each range and the individual point values, and the individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed in this article.

[0018] The inventor of the present invention accidentally isolated a strain of Lactobacillus CCNH332 from a wine fermented in a winery in Zhejiang Province. Figure 1 and Figure 2 As shown, the colony surface is smooth, milky white, round or oval, and the cells of the strain are rod-shaped, arranged singly, in short chains or in a fence-like manner. Morphological and 16S rDNA sequencing confirmed that it is Lactobacillus rhamnosus. Lactobacillus rhamnosus , and its 16S rDNA is shown in SEQ ID NO.1.

[0019] SEQ ID NO.1: ACAACTTGTCACTTAGACGGCTCGCTCCCTAAAAGGGTTACGCCACCGGCTTCGGGTGTTACAAACTCTCATGGTGTGACGGGCGGTGTGTACAAGGCCCGGGAACGTATTCACCGCGGCGTGCTGATCCGCGATTACTAGCGATTCCGACTTCGTGTAGGCGAGTTGCAGCCTACAGTCCGAACTGAGAATGGCTTTAAGAGATTAGCTTGACCTCGCGGTCTCGCAACTCGTTGTACCATCCATTGTAGCACGTGTGTAGCCCAGGTCATAAGGGGCATGATGATTTGACGTCATCCCCACCTTCCTCCGGTTTGTCACCGGCAGTCTTACTAGAGTGCCCAACTAAATGCTGGCAACTAGTCATAAGGGTTGCGCTCGTTGCGGGACTTAACCCAACATCTCACGACACGAGCTGACGACAACCATGCACCACCTGTCATTTTGCCCCCGAAGGGGAAACCTGATCTCTCAGGTGATCAAAAGATGTCAAGACCTGGTAAGGTTCTTCGCGTTGCTTCGAATTAAACCACATGCTCCACCGCTTGTGCGGGCCCCCGTCAATTCCTTTGAGTTTCAACCTTGCGGTCGTACTCCCCAGGCGGAATGCTTAATGCGTTAGCTGCGGCACTGAAGGGCGGAAACCCTCCAACACCTAGCATTCATCGTTTACGGCATGGACTACCAGGGTATCTAATCCTGTTCGCTACCCATGCTTTCGAGCCTCAGCGTCAGTTACAGACCAGACAGCCGCCTTCGCCACTGGTGTTCTTCCATATATCTACGCATTTCACCGCTACACATGGAGTTCCACTGTCCTCTTCTGCACTCAAGTTTCCCAGTTTCCGATGCACTTCCTCGGTTAAGCCGAGGGCTTTCACATCAGACTTAAAAAACCGCCTGCGCTCGCTTTACGCCCAATAAATCCGGATAACGCTTGCCACCTACGTATTACCG Based on the above findings, the first aspect of the present invention provides a strain of Lactobacillus rhamnosusLactobacillus rhamnosus The Lactobacillus rhamnosus has a deposit number of CGMCC No.33153, and its 16S rDNA is shown in SEQ ID NO.1.

[0020] "CCNH332" is the number given to the strain by the inventor of the present invention during the strain screening process, and "CGMCC No.33153" is the preservation number of the strain. The two represent the same strain and can be used interchangeably in the following text.

[0021] A second aspect of the present invention provides a bacterial agent, wherein the bacterial agent contains the Lactobacillus rhamnosus as described above.

[0022] In the present invention, the bacterial agent further contains auxiliary materials, which can be auxiliary materials commonly used in the preparation of bacterial agents or fermentation agents in the art, such as buffers (such as buffer solutions, culture media, etc.), protective agents (such as freeze-dried protective agents, such as skim milk powder, trehalose, sodium glutamate, glycerol, etc.), etc., preferably glycerol and / or corn oil.

[0023] Preferably, based on the total weight of the bacterial agent, the viable count of the Lactobacillus rhamnosus is 10 4 -10 12 CFU / g.

[0024] In the present invention, relative to 1 mL of auxiliary material, the viable count of Lactobacillus rhamnosus is 10 4 -10 12 CFU.

[0025] Preferably, in the fermentation agent provided by the present invention, the content of Lactobacillus rhamnosus can be 10 4 -10 12 CFU / g (liquid fermentation agent can be converted according to 1g=1mL). 10 4 -10 12 CFU / g means that the content of Lactobacillus rhamnosus in the fermentation agent reaches 10 4 -10 12 CFU / g level, for example 10 4 The CFU / g level represents a value greater than or equal to 1×10 4 CFU / g to less than 1×10 5 CFU / g range, that is, 1×10 4 CFU / g, 5×10 4 CFU / g, 9.9×10 4 CFU / g and so on are all 10 4 CFU / g. Therefore, in the method provided by the present invention, the content of Lactobacillus rhamnosus is greater than or equal to 1×10 4CFU / g to less than 1×10 12 CFU / g. For example, in the starter provided by the present invention, the content of Lactobacillus rhamnosus can be 1×10 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 may be any range or value within the range consisting of any two of the above values.

[0026] The third aspect of the present invention provides the use of the aforementioned Lactobacillus rhamnosus in the preparation of a medicament for inhibiting the reproduction of pathogenic bacteria.

[0027] According to the present invention, the pathogenic bacteria may include Escherichia coli ( Escherichia coli ), Staphylococcus aureus ( Staphylococcus aureus ) and Salmonella ( Salmonella ) at least one of the following.

[0028] Preferably, the Salmonella preferably includes Salmonella typhimurium ( Salmonella typhimurium ) and Salmonella Enteritidis ( Salmonella enteritidis ).

[0029] More preferably, the Escherichia coli is numbered ATCC 25922 and / or CICC 10421.

[0030] More preferably, the Staphylococcus aureus is numbered CMCC(B)26001 and / or CMCC(B)26003.

[0031] More preferably, the number of the Salmonella typhimurium is ATCC 14028.

[0032] More preferably, the number of the Enteritidis Salmonella is CVCC 3378.

[0033] The fourth aspect of the present invention provides the use of the aforementioned Lactobacillus rhamnosus in the preparation of a medicament for promoting cell growth or inhibiting cell apoptosis.

[0034] Preferably, the cells are human immortalized keratinocytes.

[0035] Preferably, the cell growth promotion is by a silent information regulator ( Sirt1 ) Genetic control.

[0036] Preferably, the apoptotic aspartate-specific cysteine ​​protease ( Caspase ) gene, more preferably, the cell apoptosis is controlled by Caspase Chromosome IV aspartate-specific caspase 3 ( Caspase-3 ) Genetic control.

[0037] The fifth aspect of the present invention provides a method for preparing the Lactobacillus rhamnosus for promoting Sirt1 Gene expression, or inhibition Caspase Application of gene expression in medicine.

[0038] Preferably, the Caspase The gene is Caspase-3 Gene.

[0039] A sixth aspect of the present invention provides the use of the aforementioned Lactobacillus rhamnosus in the preparation of a medicament for reducing inflammatory response.

[0040] Preferably, the intensity of the inflammatory response is obtained by measuring the amount of NO generated.

[0041] A seventh aspect of the present invention provides the use of the aforementioned Lactobacillus rhamnosus in the preparation of a drug for inhibiting at least one of the expression of inflammatory factors, inhibiting the expression of cytotoxic molecules and promoting the expression of immunomodulatory factors.

[0042] Preferably, the inflammatory factor is a pro-inflammatory factor, more preferably interleukin-6 (IL-6).

[0043] Preferably, the immunomodulatory factor is recombinant human transforming growth factor-β1 (TGF-β1, also known as transforming growth factor-β1).

[0044] An eighth aspect of the present invention provides use of the aforementioned Lactobacillus rhamnosus in the preparation of a medicament for promoting protein synthesis.

[0045] Preferably, the protein comprises aquaporin and / or collagen.

[0046] According to one embodiment of the present invention, the aquaporin is aquaporin 3 (AQP3), and the collagen is type I collagen native protein (Col1) and / or type III collagen native protein (Col3). The strain provided by the present invention can promote the gene expression of the above proteins and thus promote their synthesis.

[0047] Preferably, in the above-mentioned application, the Lactobacillus rhamnosus functions in the form of its fermentation product (fermentation supernatant).

[0048] Unless otherwise specified in the following examples and comparative examples, the reagents and materials used are all commercial products purchased from regular chemical or biological reagent and material suppliers, and the reagents are all analytically pure.

[0049] Escherichia coli ( Escherichia coli ), numbered (1) ATCC 25922, purchased from the American Type Culture Collection, (2) CICC 10421, purchased from the China Industrial Microbiological Culture Collection; Staphylococcus aureus ( Staphylococcus aureus ), numbered CMCC(B)26001 and CMCC(B)26003, both purchased from the Chinese Medical Bacteria Collection Center; Salmonella, (1) Salmonella typhimurium ( Salmonella typhimurium ), numbered ATCC 14028, purchased from the American Type Culture Collection; (2) Salmonella Enteritidis ( Salmonella enteritidis ), numbered CVCC 3378, purchased from the National Veterinary Microbiology and Bacteria (Virus) Collection Center.

[0050] Example 1 A lactobacillus strain CCNH332 was isolated from the fermentation samples collected from a winery in Zhejiang Province by gradient dilution and spreading method, and the pure culture of the strain was obtained by streak method.

[0051] The colonies of strain CCNH332 on MRS plates are regular round, milky white in color. They are moist, smooth, and opaque (e.g. Figure 1 Then the cell morphology of strain CCNH332 was observed under a microscope. The cells were rod-shaped and arranged singly, in short chains or in a fence-like manner (as shown in Figure 2 The strain was found to be Lactobacillus rhamnosus. 16S rDNA sequencing confirmed that the strain was Lactobacillus rhamnosus Lactobacillus rhamnosus .

[0052] Example 2 The frozen glycerol tubes of strain CCNH332 and LGG were inoculated into fresh MRS liquid medium at 2 vol%, and cultured at 37°C overnight for 18 h.600 Normalization was performed to obtain an activated bacterial solution, which was inoculated into fresh MRS liquid culture medium at an inoculation rate of 2 vol%, and cultured at 37°C, 200 rpm for 18 h to obtain the expanded culture solution of CCNH332 and the expanded culture solution of LGG.

[0053] Measure the OD of CCNH332 and LGG cultures 600 They are 8.3 and 8.13 respectively, indicating that the growth performance of CCNH332 is better than that of LGG.

[0054] Example 3 The activated bacterial liquid was inoculated into 30 mL of fresh MRS liquid medium at 2 vol% inoculation amount, and the supernatant was centrifuged after static culture at 37°C for 18 h, and the supernatant was processed by membrane (membrane pore size was 0.22 μm), and the lactic acid production was detected by HPLC. The lactic acid production of strain CCNH332 was 16.71 g / L, and the lactic acid production of LGG was 15.1 g / L. The lactic acid production performance of strain CCNH332 was better than that of LGG, indicating that strain CCNH332 can metabolize and decompose more sugars during the culture process and reduce the sugar content in the environment.

[0055] Example 4 Prepare H with concentrations of 0, 0.5, 1.0, 1.5, 2.0, 5, 8, 10, 25, and 50 μg / mL respectively. 2 O 2 Solution, detect OD at different concentrations 505 Take 30 mL of the culture solution, centrifuge to obtain the supernatant, measure its absorbance, and calculate H based on the absorbance-concentration standard curve. 2 O 2 Yield. H of CCNH332 and LGG 2 O 2 The yields were 5.91 μg / mL and 4.33 μg / mL, respectively. CCNH332 produced H 2 O 2 Therefore, its ability to non-specifically inhibit pathogenic bacteria is stronger than that of LGG.

[0056] Example 5 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, cultured anaerobically at 37°C and activated for 18 h, then transferred to the corresponding fresh medium and cultured until the viable count reached 10 8 CFU / mL, dilute to 10 5CFU / mL was used as the indicator bacterial solution.

[0057] The activated bacterial liquid was inoculated into 30 mL of fresh MRS liquid culture medium at an inoculum rate of 2 vol%, and after static culture at 37°C for 18 h, the supernatant was centrifuged and passed through a membrane (the pore size of the membrane was 0.22 μm) to obtain the supernatant.

[0058] Three experimental groups were set up: S1: 100 μL supernatant, 100 μL indicator bacterial solution; S2: 50 μL supernatant, 150 μL indicator bacterial solution; S3: 25 μL supernatant, 175 μL indicator bacterial solution; 200 μL of MRS liquid culture medium was used as the negative control group, and the bacterial solution was replaced with 16 μg / mL ampicillin as the positive control group. Measure the OD of the experimental group and the negative control group 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 %: Inhibition rate (%) = (A 0 -A 1 ) / A 0 ×100%.

[0059] Table 1

[0060] Example 6 The activated bacterial solution of strain CCNH332 was centrifuged at 4000 rpm for 10 min to obtain the supernatant.

[0061] H-DMEM medium: calcium chloride 200mg / L, sodium chloride 6400mg / L, potassium chloride 400mg / L, anhydrous magnesium sulfate 97.67mg / L, anhydrous sodium dihydrogen phosphate 125mg / L, ferric nitrate nonahydrate 0.1mg / L, arginine 84mg / L, cystine hydrochloride 62.57mg / L, glutamine 584mg / L, glycine 30mg / L, histidine hydrochloride 42mg / L, isoleucine 104.8mg / L, leucine 104.78mg / L, lysine hydrochloride 146.2mg / L, methionine 30 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.

[0062] HaCaT cells (human immortalized keratinocytes): purchased from Sean Biotechnology, catalog number SNL-163.

[0063] HaCaT cells were seeded in multiple culture dishes containing H-DMEM medium and incubated at 37°C and 5 vol% CO 2 The cells were cultured under the conditions of 4% paraformaldehyde until the cell confluence reached 80% of the culture medium surface area.

[0064] Four groups were set up, each containing 10 vol% fetal bovine serum (FBS), a negative control group containing 90 vol% H-DMEM without strain CCNH332, and the supernatants of the three experimental groups (OD 600 The contents of 1 vol%, 3 vol%, 5 vol% and 10 vol% were in turn, and the remainder was H-DMEM. The four groups were inoculated in culture dishes containing HaCaT cells, and the inoculation amount of each culture dish was 10 4 Then, the cells were incubated at 37°C and 5 vol% CO 2 After culturing for 48 hours under the same conditions, 1 mL of the liquid was taken and its absorbance OD at 410 nm was measured. 410 , O.D. 410 The larger the value, the better the proliferation of HaCaT cells, and the relative activity of cells in each group was calculated according to the following formula, where the blank wells only contained FBS.

[0065] Cell survival rate (%) = (OD value of experimental group - OD value of blank well) / (OD value of negative control group - OD value of blank well) × 100%.

[0066] The cell survival 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 survival 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.

[0067] Example 7 10 vol% FBS and 1% (wt / v) penicillin-streptomycin (based on the volume of the culture medium) were added to the H-DMEM culture medium to obtain a double-antibody culture medium. HaCaT cells were cultured in the double-antibody culture medium until the cell confluence reached about 80%, and then 1 wt% sodium dodecyl sulfate (SDS) was added to induce cell damage to establish a cell damage model. During this period, cell viability tests or inflammatory factor release tests were performed every 48 hours to confirm whether the cell model was successfully constructed.

[0068] Methods for cell viability testing or inflammatory factor release testing: Cell inoculation and treatment: Cell suspension in logarithmic growth phase was plated at 10 4 Each cell was seeded in a 96-well plate and pre-cultured for 24 hours to allow it to adhere to the wall. The drug to be tested was then added and incubated for 96 hours. Then, 10 μL of cell counting reagent CCK-8 was added to each well and the incubation 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 required to correct the background.

[0069] Then, the supernatant prepared in Example 6 was added to the successfully modeled cell culture dish. The amount of supernatant added was 1 vol%, 3 vol% and 5 vol% respectively based on the volume of the culture dish. The culture dish was incubated at 37°C and 5 vol% CO. 2 The cells were cultured for 24 hours under the conditions of 95% humidity. In addition, a model group without SDS modeling, a positive control group with added epidermal growth factor (EGF), and a negative control group with 10 vol% FBS + 90 vol% H-DMEM without supernatant were set up. After the experiment, the quiescent information regulator ( Sirt1 )'s mRNA expression levels (in fold change). The results are shown in Table 2.

[0070] In addition, the same method was used to replace SDS with an equal amount of sodium deoxycholate (SD) to construct an SD cell injury model and conduct experiments to measure the expression of aspartate-specific caspase-3 ( caspase-3 )'s mRNA expression levels (in fold change). The results are shown in Table 2.

[0071] Measurement Sirt1 and Caspase-3 Method of mRNA expression level: RNA extraction: 1×10 7Add 1 mL of RNA extraction reagent (TRIzol) to each cell, add chloroform to layer and isopropanol to precipitate RNA after homogenization, wash and purify with 75 vol% ethanol, and finally dissolve in RNase-free water to obtain a sample containing RNA. RNA quality detection: The purity is assessed by measuring the A260 / A280 ratio (ideal value 1.8-2.0) by UV spectrophotometer, and the RNA integrity is confirmed by formaldehyde denaturing agarose gel electrophoresis (clear 28S and 18S rRNA bands). Reverse transcription (cDNA synthesis): Take 1 μg of sample and use the reverse transcription kit PrimeScript RT for reverse transcription. The reaction conditions are 50°C (15 minutes, reverse transcription) and 85°C (5 seconds, enzyme inactivation). At the same time, a no reverse transcription control is set to exclude genomic DNA contamination.

[0072] Primer design and validation: Sirt1 Primers (across exon junctions): forward 5′-TGGACAATTCCAGCCATCTC (SEQ ID NO. 2)-3′, reverse 5′-GCGTGTCTATGTTCTGGGTATAG (SEQ ID NO. 3)-3′; Caspase-3 Primers: forward 5′-GAAATTGTGGAATTGATGCGTGA (SEQ ID NO. 4)-3′, reverse 5′-CTACAACGATCCCCTCTGAAAA (SEQ ID NO. 5)-3′; Glyceraldehyde-3-phosphate dehydrogenase gene (GAPDH) was used as an internal reference for normalization.

[0073] Real-time quantitative PCR: Nucleic acid stain (SYBR Green) was used for staining. The reaction system contained 2× dye method fluorescence quantitative premix system (SYBR qPCR Mix), primers (0.5 μM each) and cDNA template. The program was pre-denaturation at 95°C for 30 seconds, 40 cycles of 95°C for 5 seconds, and 60°C for 30 seconds. Finally, the product specificity was analyzed by melting curve.

[0074] Data analysis: The ΔΔCt method was used to calculate the Sirt1 and Caspase-3 Fold change (2^(-ΔΔCt)).

[0075] Table 2

[0076] In Table 2, * indicates a very significant difference from the model group, p < 0.01; ** indicates an extremely significant difference from the model group, p < 0.001.

[0077] As shown in Table 2, the fermentation product of Lactobacillus rhamnosus provided by the present invention can upregulate the repair gene Sirt1 downregulate apoptosis genes Caspase-3 and its ability to up-regulate or down-regulate gene expression increased with the increase of supernatant content.

[0078] Example 8 Mouse mononuclear macrophage leukemia cells (RAW264.7 cells): purchased from Sean Biotech, catalog number SNL-112.

[0079] RAW264.7 cells were placed at 37°C and 5 vol% CO 2 And incubate in an incubator with 95% relative humidity. Use DMEM complete culture medium (double antibody culture medium) containing 10vol% FBS and 1wt% double antibody for culture. Regularly change the cell medium and subculture to keep the cells in the logarithmic growth phase. The cells were divided into a control group, a lipopolysaccharide group (LPS group) and an experimental group containing the supernatant prepared in Example 6. Control group: only RAW264.7 cells and culture medium were contained without any treatment. LPS group: LPS at a final concentration of 50ng / mL was added to 10mL of culture medium containing RAW264.7 cells in the logarithmic growth phase to induce an inflammatory response. Experimental group: 10 mL of supernatant with different volume concentrations (1 vol%, 3 vol%, 5 vol%) was added to the LPS group. 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) (in fold change) were measured to test the inhibitory effects of different groups on inflammatory response.

[0080] Method for measuring NO content: centrifuge the cultured cells to remove the precipitate. Dilute the sodium nitrate standard (concentration gradient of 0-100 μM) with double-antibody culture medium to eliminate background interference; establish a standard curve of NO concentration. Add 50 μL of sample and standard to each 96-well plate, then add Gries reagent (prepared with equal volumes of sulfonamide and naphthylethylenediamine hydrochloride) to each well, mix well and incubate at room temperature for 20 minutes until the color is stable. Measure the absorbance at 540 nm (OD 540 ) Calculate the NO concentration of the sample (mmol / L), set up blank wells (no sample) and control wells (no drug or known toxic drug) to correct the background.

[0081] Method for measuring the expression levels of IL-6 and TGF-β1: Real-time quantitative PCR (qRT-PCR) was calculated based on the ΔΔCt method, with GAPDH as the internal reference, to detect the fold change.

[0082] Table 3

[0083] In Table 3, * indicates a very significant difference from the model group, p < 0.01; ** indicates an extremely significant difference from the model group, p < 0.001.

[0084] As shown in Table 3, the fermentation product of Lactobacillus rhamnosus provided by the present invention can reduce the generation of NO, inhibit the expression of IL-6 and promote the expression of TGF-β1, and its promoting or inhibiting ability increases with the increase of supernatant content.

[0085] Example 9 HaCaT cells were cultured and supernatant was inoculated according to the method of Example 7, and a model group and an EGF positive control group were set up for 48 hours of culture. After the culture, the expression level of aquaporin 3 (AQP3) mRNA was measured (in fold change) to test the moisturizing ability of the fermentation product. The higher the expression level, the more aquaporins 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 among the groups). The expression level of mRNA in the experimental group was significantly increased, and the increase increased with the increase of supernatant content.

[0086] Example 10 HaCaT cells were cultured and supernatant was inoculated according to the method of Example 7, and a blank control group without strain addition and a positive control group in which strain was replaced with 1 vol% EGF were set up for 48 h. After the end of the culture, the collagen native protein ( Col1 and Col3 The expression level of mRNA of the fermentation product was measured (in fold change) to test the ability of the fermentation product to promote collagen production. The higher the expression level of mRNA, the stronger the ability. The blank control group, positive control group and supernatant content were 1vol%, 3vol% and 5vol% 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 (there were significant differences among the groups). Col3 The results were 1±0.2, 7±1, 2.5±0.5, 2.8±0.3 and 4.5±0.8 (with significant differences between the groups). The results showed that the expression level of mRNA was significantly increased compared with the experimental group, and the increase increased with the increase of supernatant content.

[0087] The preferred embodiments of the present invention are described in detail above, but the present invention is not limited thereto. Within the technical concept of the present invention, the technical solution of the present invention can be subjected to a variety of simple modifications, including the combination of various technical features in any other suitable manner, and these simple modifications and combinations should also be regarded as the contents disclosed by the present invention and belong to the protection scope of the present invention.

Claims

1. A strain of Lactobacillus rhamnosus Lactobacillus rhamnosus , characterized in that, The preservation number of the Lactobacillus rhamnosus is CGMCC No.33153.

2. A bacterial agent, characterized in that The bacterial agent contains the Lactobacillus rhamnosus according to claim 1.

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

4. Use of the Lactobacillus rhamnosus according to claim 1 in the preparation of a medicament for inhibiting the reproduction of pathogenic bacteria.

5. Use of the Lactobacillus rhamnosus according to claim 1 in the preparation of a medicament for promoting cell growth or inhibiting cell apoptosis.

6. The Lactobacillus rhamnosus of claim 1 in the preparation of Sirt1 Gene expression, or inhibition Caspase Application of gene expression in medicine.

7. Use of the Lactobacillus rhamnosus according to claim 1 in the preparation of a medicament for reducing inflammatory response.

8. Use of the Lactobacillus rhamnosus according to claim 1 in the preparation of a medicament for inhibiting at least one of the expression of inflammatory factors, inhibiting the expression of cytotoxic molecules and promoting the expression of immunoregulatory factors.

9. Use of the Lactobacillus rhamnosus according to claim 1 in the preparation of a medicament for promoting protein synthesis.

10. The use according to claim 9, wherein: The proteins include aquaporins and / or collagen.

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