Thermus thermophilus and application thereof

Abstract

The invention relates to the technical field of functional microorganism screening and application, and particularly provides a thermophilic thermophilic bacterium and application thereof. The Thermus thermophilus is preserved in the China General Microbiological Culture Collection Center on November 18, 2024, the preservation number of the Thermus thermophilus is CGMCC NO: 32680, the strain can grow at the high temperature of 70 DEG C, the oxidation resistance is very high, the effect of effectively relieving skin injuries caused by ultraviolet rays is achieved, the effect is remarkable, and the application prospect is wide. The product is a potential functional food, cosmetic or medicine raw material, and has a wide application prospect.

Description

Technical Field

[0001] The present invention relates to the field of functional microorganism screening and application technology, and in particular to a thermophilic bacterium, in particular to a thermophilic bacterium having a protective effect on skin damage and an application thereof. Background Art

[0002] Ultraviolet (UV) radiation is ubiquitous in the environment, and UV has been classified as a recognized human carcinogen. As the largest and outermost organ of the human body, direct exposure of the skin to sunlight or UV radiation can lead to sunburn, inflammation, photoimmunosuppression, photoaging, and even skin cancer. UV radiation has a wavelength range of 100–400nm and is classified into three basic types: UVA (320-400nm), UVB (280-320nm), and UVC (100–280nm). UV radiation is the main cause of premature skin aging and can severely lead to photoaging and UV-induced skin cancer.

[0003] Thermus thermophilus is a widely spread thermophilic bacteria in the genus Thermus, which can grow and reproduce at high temperatures above 55°C. Thermus thermophilus has the characteristics of fast growth rate, high cell culture yield, efficient expression of natural components, and outstanding thermal stability of proteases, making it a model microorganism for studying the molecular basis of thermophiles.

[0004] After the Thermus thermophilus-derived SOD was expressed in Escherichia coli and purified and characterized, it was found that the enzyme was highly stable at 90°C and maintained 57% activity after heat treatment at 100°C for 1 hour; it had more than 50% activity between pH 3-10 and had a wide range of pH adaptability. In zebrafish IBD induced by 2,4,6-trinitrobenzenesulfonic acid, treatment with Thermus thermophilus-derived SOD reduced intestinal enlargement and neutrophil infiltration, thereby alleviating enterocolitis. In mice, oral administration of Thermus thermophilus-derived SOD significantly increased SOD activity in the intestine, which improved intestinal inflammation, maintained intestinal barrier function, and reduced the severity of dextran sulfate sodium-induced colitis. In addition, Thermus thermophilus-derived SOD inhibited the production of reactive oxygen species and inflammatory responses in mouse bone marrow-derived macrophages induced by lipopolysaccharide. In addition, Thermus thermophilus is also the source species of a large number of thermostable enzymes.

[0005] Therefore, developing and finding new thermophilic Thermus and expanding its application areas are of great significance for basic research in microbiology and product development in the field of life and health. Summary of the invention

[0006] The object of the present invention is to provide a novel Thermus thermophilus and its application. The present invention relates to a Thermus thermophilus, which has been deposited in the General Microbiological Center of the China Microbiological Culture Collection Administration on November 18, 2024, with a deposit number of CGMCC NO: 32680, and is named Thermus thermophilus YMD-5301, which has the ability to grow normally under high temperature conditions, has strong antioxidant capacity, and can effectively alleviate skin damage caused by ultraviolet rays, with significant effects.

[0007] In a first aspect, the present invention provides a Thermus thermophilus, comprising a 16S rRNA gene sequence having a nucleotide sequence of SEQ ID NO:1.

[0008] In a second aspect, the present invention provides a thermophilic Thermus thermophilus, the deposit number of the thermophilic Thermus thermophilus is CGMCCNO:32680.

[0009] Optionally, the Thermus thermophilus is isolated from a hot spring area.

[0010] In a third aspect, the present invention provides a derivative of the Thermus thermophilus of the present invention.

[0011] Optionally, the derivative is a culture, a lysate, an extract, an inactivated product or a combination thereof.

[0012] In a fourth aspect, the present invention provides a composition comprising an effective amount of the Thermus thermophilus or a derivative thereof of the present invention.

[0013] In a fifth aspect, the present invention provides use of the Thermus thermophilus of the present invention, its derivatives, or compositions comprising the same in the preparation of foods, health products, medicines, or cosmetics.

[0014] In a sixth aspect, the present invention provides use of the Thermus thermophilus of the present invention, its derivatives, or a composition comprising the same in the preparation of a product with antioxidant function.

[0015] In a seventh aspect, the present invention provides use of the Thermus thermophilus of the present invention, its derivatives, or compositions comprising the same in the preparation of products for preventing or treating photoaging or ultraviolet damage of the skin.

[0016] In summary, the present invention includes at least one of the following beneficial technical effects: 1. The thermophilic bacterium provided by the present invention can grow under high temperature conditions of 70°C and has good high temperature resistance; 2. The thermophilic bacteria provided by the present invention has strong antioxidant capacity, with a DPPH scavenging rate of 63.63%, a hydroxyl radical scavenging capacity of 14.86U / mL, an anti-superoxide anion capacity of 59.57U / mL, and a total antioxidant capacity of 2.49U / mL; 3. The thermophilic bacteria provided by the present invention can effectively promote the proliferation of skin cells; 4. The derivatives of Thermus thermophilus provided by the present invention can significantly reduce cell death and the content of inflammatory factors caused by ultraviolet irradiation, provide strong protection for cells, and are equivalent to the positive drug dexamethasone. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 The staining light microscope photos and colony morphology of Thermus thermophilus YMD-5301; Figure 2 The results of the toxicity test of Thermus thermophilus YMD-5301 on HaCaT cells; Figure 3 3A is a graph showing the detection results of cytokines after pretreatment with the lysate of Thermus thermophilus YMD-5301, wherein 3B is a graph showing the detection results of cytokines IL-1β, and 3C is a graph showing the detection results of cytokines TNF-α. DETAILED DESCRIPTION

[0018] The following examples are used to illustrate the present invention but are not intended to limit the scope of the present invention.

[0019] The experimental methods in the following examples without specifying specific conditions were all carried out according to conventional procedures, and the materials and reagents used were all conventional commercial products.

[0020] Material Configuration of TB medium: TB liquid medium: 8 g / L tryptone, 4 g / L yeast extract, 3 g / L sodium chloride, 367 mg / L NaHCO 3 、31.34mg / L MgSO 4 , 0.95mg / L KCl, 36.75mg / L CaCl 2 ·2H 2 O, 150.44 mg / L MgCl 2 6H 2 O, adjust pH to 7.5, sterilize at 121°C for 20 minutes; TB solid culture medium: Add 1.5% agar to the TB liquid culture medium, sterilize at 121°C for 20 minutes, and pour into a sterile culture dish to solidify.

[0021] Example 1: Screening of Thermus thermophilus YMD-5301 Hot spring water samples and hot spring sediment samples were collected from multiple hot spring spots in the Tengchong Hot Spring Geothermal Experience Area in Yunnan Province and the Eryuan Hot Spring Area in Dali. The samples were stored in sterile centrifuge tubes, placed in an incubator and transported to the laboratory.

[0022] The hot spring water samples were directly separated and screened by the concentration gradient dilution method. The original solution, three gradient dilutions (10 -1 , 10 -2 , 10 -3 ) were spread on TB solid culture medium, and cultured in a 70°C incubator for 24-48h. After a single colony grew on the plate, the single colony was streaked three times to obtain a pure strain.

[0023] Weigh 5 g of hot spring sediment sample and disperse it in 50 mL of sterile water. Shake on a shaker at 70°C and 160 rpm for more than 4 hours to fully disperse the sample. -1 , 10 -2 , 10 -3 ) were spread on TB solid culture medium, and cultured in a 70°C incubator for 24-48h. After a single colony grew on the plate, the single colony was streaked three times to obtain a pure strain.

[0024] According to the above method, a thermophilic bacteria that can stably grow genetically under 70°C culture conditions was finally screened and named YMD-5301.

[0025] Example 2: Identification of Thermus thermophilus YMD-5301 2.1 Colony morphology identification YMD-5301 was inoculated on TB solid medium and cultured at 70℃ for 24h. Figure 1 As shown, the single colony of YMD-5301 is light yellow with neat edges and moist surface. It is a Gram-negative, aerobic bacterium and appears as a short rod under a microscope.

[0026] 2.2 Molecular Biological Identification YMD-5301 genomic DNA was extracted using a bacterial genomic DNA extraction kit. YMD-5301 DNA was used as a template to amplify its 16S rRNA gene sequence by PCR, with primers 27F and 1492R (specific sequences can be found in CN117836400A), and the PCR reaction program was: 95°C pre-denaturation for 5 min, 95°C denaturation for 30 s, 55°C annealing for 30 s, 72°C extension for 60 s, 30 cycles, and 72°C extension for 10 min.

[0027] The purity of the amplified product was tested by electrophoresis and then sequenced, and the sequencing result is shown in SEQ ID NO: 1. The obtained 16SrRNA gene sequence SEQ ID NO: 1 was blasted against the NCBI database (https: / / blast.ncbi.nlm.nih.gov / Blast.cgi), and the strain with the highest homology was Thermus thermophilus HB8 (homology was 98.92%), confirming that YMD-5301 was Thermus thermophilus.

[0028] Example 3: Antioxidant activity of Thermus thermophilus YMD-5301 1. Preparation of bacterial suspension A single colony of Thermus thermophilus with good growth status was inoculated into 3 mL of TB liquid culture medium, and cultured on a shaking platform at 70°C for 12-18 hours. This culture solution was used as the inoculum, and inoculated into 50 mL of TB liquid culture medium at a 2% inoculum volume, and cultured on a shaking platform at 70°C for 12-18 hours to obtain the culture solution of the strain. After collecting the cells with 1 mL of bacterial solution, the cells were washed twice with 1 mL of PBS buffer, and then the cells were resuspended in PBS solution to adjust the cell density to 1.0×10 8 CFU / mL, as sample solution.

[0029] 2. DPPH free radical scavenging assay The experiment was conducted using the DPPH free radical scavenging ability kit (Cat. No. A153-1-1) of Nanjing Jiancheng Biological Research Institute, and the experimental process is as follows. Add 400 μL of sample solution to a test tube, and then add 600 μL of 80% methanol solution as a sample control tube, mix well, stand at room temperature 25°C in the dark for 30 minutes, centrifuge at 4000 rpm for 5 minutes, and use an ultraviolet spectrophotometer at a wavelength of 517 nm, 80% methanol to zero and measure the absorbance, which is recorded as A control. Add 400 μL of sample solution to a test tube, and then add 600 μL of DPPH test working solution as a sample determination tube, and measure the absorbance in the same way as the sample control tube, and the measured absorbance is recorded as A determination. Add 400 μL of 80% methanol solution and 600 μL of DPPH test working solution to a test tube, and measure the absorbance in the same way as the sample control tube, and the measured absorbance is recorded as A blank. Calculate the DPPH free radical scavenging rate using the following formula: DPPH free radical scavenging rate (%) = (1-(A measurement-A control) / A blank) × 100% (1) 3. Hydroxyl free radical scavenging assay The experiment was conducted using the hydroxyl radical determination kit (Cat. No. A018-1-1) of Nanjing Jiancheng Biological Research Institute, and the experimental process is as follows. The hydroxyl radical scavenging rate was determined using the Fenton system. Add 0.2 mL of substrate application solution, 0.2 mL of sample solution, and 0.4 mL of reagent three application solution to a test tube, mix quickly, react at 37°C for 1 minute (accurately timed with a stopwatch), and then immediately add 2 mL of color developer to terminate the reaction, mix well, and let stand at room temperature for 20 minutes. Use an ultraviolet spectrophotometer at a wavelength of 550 nm and double distilled water to zero. The measured absorbance is recorded as A determination. The absorbance measured by the control tube using double distilled water instead of the sample solution is recorded as A control. Add 0.2 mL of double distilled water, 0.2 mL of 0.03% H 2 O 2 The absorbance measured by using the standard application solution and 0.4 mL of reagent III application solution as the standard tube is recorded as standard A. Double distilled water is used instead of 0.03% H 2 O 2 The absorbance measured by the standard application solution as a blank tube is recorded as A blank. The hydroxyl radical scavenging rate is calculated by formula (2): Hydroxyl free radical inhibition ability (U / ml) = (A control - A determination) / (A standard - A blank) × C standard × 1 / V sample × NC standard: standard concentration, 8.824mmol / L; V sample: sampling volume, 0.2mL; N: dilution factor of sample solution before testing; (2) 4. Anti-superoxide anion assay The experiment was conducted using the Nanjing Jiancheng Biological Research Institute's Inhibition and Generation of Superoxide Anion Free Radical Assay Kit (Cat. No. A052-1-1), and the experimental process is as follows. Add 1 mL of reagent 1 application solution, 0.05 ml of sample solution, 0.1 mL of reagent 2, 0.1 mL of reagent 3, and 0.1 mL of reagent 4 application solution to the test tube and mix well. Place in a 37°C constant temperature water bath for 40 minutes, then add 2 mL of color developer, mix well, and let stand at room temperature for 10 minutes. Use an ultraviolet spectrophotometer at a wavelength of 550 nm and adjust to zero with double distilled water. The measured absorbance is recorded as A determination. Use double distilled water to replace the sample solution, and the measured absorbance is recorded as A control. Use 0.15 mg / mL Vc standard solution to replace the sample solution, and the measured absorbance is recorded as A standard. Calculate the anti-superoxide anion ability by formula (3): Anti-superoxide anion capacity (U / L) = (A control - A determination) / (A control - A standard) × C standard × 1000 × NC standard: standard concentration, 0.15 mg / mL; 1000: unit conversion, N: dilution factor of sample solution before testing; (3) 5. Determination of total antioxidant capacity The experiment was conducted using the total antioxidant capacity (T-AOC) assay kit (Cat. No. A015-1) of Nanjing Jiancheng Biological Research Institute, and the experimental process is as follows. Add 1 mL of reagent 1, 0.1 mL of sample solution, 2 mL of reagent 2 application solution, and 0.5 mL of reagent 3 application solution to a test tube, then mix thoroughly, and place in a 37°C water bath for 30 minutes. Then add 0.1 mL of reagent 4 to the test tube, mix thoroughly, and place at 25°C-37°C for 10 minutes. Use an ultraviolet spectrophotometer at 520 nm and adjust to zero with double distilled water. The measured absorbance is recorded as A determination. Take another test tube, add 1 mL of reagent 1, 2 mL of reagent 2 application solution, and 0.5 mL of reagent 3 application solution to it, then mix thoroughly, and place in a 37°C water bath for 30 minutes. Then add 0.1 mL of reagent 4 and 0.1 mL of sample solution to the test tube as a control tube, and the measured absorbance is recorded as A control. The total antioxidant capacity is calculated by formula (4): Total antioxidant capacity (U / mL) = (A assay - A control) / 0.01 / T × V total / V sample × N T: reaction time, 30 min; Vtotal: total volume of reaction system, mL; Vsample: sampling volume, mL; N: dilution factor of the sample before testing; (4) Thermus thermophilus HB8 (ATCC 27634) strain was used as the control bacteria. The test results are shown in Table 1.

[0030] Table 1 Antioxidant activity It can be seen from the data in Table 1 that the DPPH scavenging rate, superoxide anion resistance and total antioxidant capacity of the Thermus thermophilus YMD-5301 provided by the present invention are improved to varying degrees compared with the control group Thermus thermophilus HB8, indicating that the Thermus thermophilus YMD-5301 has stronger antioxidant activity.

[0031] Example 4: Effect of Thermus thermophilus YMD-5301 in promoting cell proliferation Thermus thermophilus YMD-5301 was inoculated into 100 mL TB liquid medium and cultured on a shaking platform at 70°C for 12-18 h. The culture was centrifuged at 6000 g for 10 min and the supernatant was discarded. The cells were washed twice with PBS buffer. Pure water was added to prepare the concentrations of 0.5×10 8 , 1×10 8 , 2×10 8 , 4×10 8 and 8×10 8CFU / mL of bacterial solution; heat the bacterial solution at 100°C for 20 min to inactivate the bacteria; freeze-dry the bacterial solutions of different concentrations for later use.

[0032] HaCaT cells in the logarithmic phase were collected and prepared with DMEM medium at a density of 2×10 5 The cells were inoculated with a cell suspension of 100 / mL in a 96-well culture plate. After the cells adhered to the wall, different concentrations of bacterial solution were added. 37°C, 5% CO 2 After incubation in the incubator for 24 h, the culture medium was discarded, 200 μL PBS (pH 6.8) was added for washing twice, 200 μL DMEM culture medium and 20 μL MTT solution were added to each well, and the well was placed in a CO 2 Incubate in the incubator for 3 hours. Carefully remove the culture medium in the wells and add 100 μL of dimethyl sulfoxide (DMSO) to each well. Place the 96-well culture plate on an oscillator for 10 minutes to allow the crystals to fully dissolve. Then use an ELISA reader to measure the absorbance of each well at 490 nm. Calculate the cell survival rate of each group based on the blank control group and serum control group. The results are shown in the figure. Figure 2 As shown, compared with the control group, in the treatment group with heat-killed Thermus thermophilus YMD-5301, 2×10 8 CFU / mL and 4×10 8 CFU / mL concentration, the activity of HaCaT cells was significantly improved, which shows that the heat-killed bacteria of Thermus thermophilus YMD-5301 provided by the present invention can significantly promote the proliferation of HaCaT cells.

[0033] Example 5: Application of Thermus thermophilus YMD-5301 lysate in reducing cell photodamage 1. Preparation of Lysis Buffer Thermus thermophilus YMD-5301 was inoculated into TB liquid culture medium and cultured at 70°C for 12-18h. The freshly cultured bacterial liquid was crushed using a high-pressure homogenizer at a pressure of 100MPa. The homogenization was repeated three times and then placed in a 70°C water bath for thorough inactivation to prepare a lysate.

[0034] 2. UVB induces HaCaT cells to secrete pro-inflammatory cytokines HaCaT cells were cultured in a 96-well plate for 24 hours, and the culture medium was removed; 10 μL of 12.5 μg / ml DMEM culture medium containing dexamethasone was added to the positive drug group, and the DMEM culture medium containing the above-mentioned Thermus thermophilus YMD-5301 lysate was added to the experimental group, incubated for 2 hours, and 150 μL of PBS buffer was added to each well. The blank group did not require ultraviolet irradiation, and the other groups were irradiated with ultraviolet lamps, with an ultraviolet intensity of 250 μw / cm2 and an irradiation dose of 30 mJ / cm2. After culturing with 200 μL of DMEM culture medium for 24 hours, HaCaT cells that were not pretreated with ultraviolet irradiation and Thermus thermophilus YMD-5301 lysate were used as controls. The above-mentioned cell culture supernatant after UV irradiation was taken, and the concentrations of pro-inflammatory cytokines IL-6, IL-1β and TNF-α were detected using Bio-Tech Human IL-6 ELISA Kit (Cat. No. PI330), Human IL-1β ELISA Kit (Cat. No. PI305) and Human TNF-α ELISA Kit (Cat. No. PT518), respectively.

[0035] The results are as follows Figure 3 As shown in A-3C, Figure 3 The results of A-3C show that pretreatment with the lysate of Thermus thermophilus YMD-5301 can significantly inhibit the secretion of pro-inflammatory cytokines IL-6, IL1β and TNF-α by HaCaT cells induced by UVB. After UVB irradiation, the contents of IL-6, IL1β and TNF-α in the culture supernatant of HaCaT cells increased sharply, reaching a maximum of 64.53pg / mL, 41.18pg / mL and 47.56pg / mL; but after pretreatment with the lysate of Thermus thermophilus YMD-5301, the production of IL-6, IL1β and TNF-α was greatly reduced, decreasing to 23.83pg / mL, 18.26pg / mL and 15.15pg / mL, respectively. In summary, the lysate of Thermus thermophilus YMD-5301 can effectively alleviate the inflammatory response of cells to UVB irradiation.

[0036] The embodiments described above are only preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

[0037] Sequence Listing SEQ ID NO:1 CTCGGCGCCTGCCCGTAGGCTCCCGGCGACTTCGGGTAGAGCCGCTCCCATGGCGTGAC GGGCGGTGTGTACAAGGCCCGGGAACGTATTCACCGCGGCATGGCTGATCCGCGATTAC TAGCGATTCCGGCTTCATGGGGTCGGGTTGCAGACCCCAATCCGAACTGGGCCCACCTT TTTGCGATTAGCTCCCCGTTGCCGGGTGGCATCGCTTTGAAGTGGGCATTGTAGCACGTG TGTCGCCCAGGCCGTAAGGGCCATGCTGACCAGACGTCGTCCCCTCCTTCCTCCCGCTT TCGCGGGCAGTCCCGTTAGAGTGCCCGGCCGAACCGCTGGGAACTAACGGCGGGGGTT GCGCTCGTTGCGGGACTTAACCCAACACCTCACGGCACGAGCTGACGACGGCCATGCA GCACCTGTGCTAGGGCTCCCCTCGCGGGGCACCCCAGGCTTACACCCGGGTTCCCTAGC ATCTCAAGGCCTGGTAAGGTTCTTCGCGTTGCTTCGAATTAAACCACATGCTCCACCGCT TGTGCGGGCCCCCGTCAATTCCTTTGAGTTTCAGCCTTGCGGCCGTACTCCCCAGGCGG CGCGCTTAACGCGTTAGCTTCGGCCCCCAGGAGACCCAGAGACCATGCGCGCATCGTTT AGGGCGTGGACTACCCGGGTATCTAATCCGGTTTGCTCCCCACGCTTTCGCGCCTCAGCG TCACGGGTGGACCAGGTGGCTGCCTTCGCCATCGGCGTTCCTCCCGGTATCTGCGCATTT CACCGCTACTCCGGGAATTCCACCACCCTCTCCCACAGTCTAGCCTGAGCGTATCCCACG CTCCTCCACGGTTGAGCCGTGGTCTTTCACATGGGACGCCCCTGGCCGCCTACACGCCC TTTACGCCCAGTGAATCCGGGTAACGCTCGCGCCCTCCGTATTACCGCGGCTGCTGGCAC GGAGTTGGCCGGCGCTATTACCCCGGTACCGTCAGTCCCCTCGTCGGGGGTTTCGTCCC GGGTTCAGGAGGTTTCACCCCGAAGGGCTTCTTCCTCCAAGCGGCGTCGCTCCGTCAGG CTTGCGCCCATTGCGGAAGATTCCTAACTGCTGCCTCCCGTAGGAGTGGGGCCCGTGTC TCAGTGCCCCTGTGGCCGGCCATCCTCTCAGACCGGCTACCCGTCGTCGCCTTGGTGGG CCATTACCCCACCAACTAGCTGATGGGACGCGGGCCCATCCGGAAGCGGGCAAAGCCCT TTGGACACAACCCAAGGGGCGGGTCCACATGGGGGATTAGGCCGAGTTTCCCCGGGTT GTCCCCCTCTTCCGGGTAGGTCATCCACGCGTTACTCACCCGTCCGCCGCTGACCACGG AGTAAAACCCCGCGGCCCGCACGTC

Claims

1. A Thermus thermophilus, characterized in that The thermophilic bacterium comprises a 16S rRNA gene sequence with a nucleotide sequence of SEQ ID NO:

1.

2. A thermophilic bacterium, characterized in that The deposit number of the Thermus thermophilus is CGMCC NO:32680.

3. The Thermus thermophilus according to any one of claims 1 to 2, characterized in that The Thermus thermophilus was isolated from a hot spring area.

4. A derivative of Thermus thermophilus according to any one of claims 1 to 3.

5. The derivative according to claim 4, characterized in that The derivative is a culture, a lysate, an extract, an inactivated product or a combination thereof.

6. A composition, characterized in that The method comprises an effective amount of the Thermus thermophilus according to any one of claims 1 to 3 or a derivative according to any one of claims 4 to 5.

7. Use of the Thermus thermophilus according to any one of claims 1 to 3, the derivative according to any one of claims 4 to 5, or the composition according to claim 6 in the preparation of food, health products, medicines or cosmetics.

8. Use of the Thermus thermophilus according to any one of claims 1 to 3, the derivative according to any one of claims 4 to 5, or the composition according to claim 6 in the preparation of a product with antioxidant function.

9. Use of the Thermus thermophilus according to any one of claims 1 to 3, the derivative according to any one of claims 4 to 5, or the composition according to claim 6 in the preparation of a product for preventing or treating photoaging or ultraviolet damage of the skin.

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