Application of lactobacillus mucilaginosus TG018

Abstract

The invention relates to the technical field of microorganisms, and in particular relates to application of a strain of lactobacillus mucus TG018. The invention further relates to a preparation method of the lactobacillus mucus TG018. The preservation number of the lactobacillus mucus TG018 is CCTCC (China Center for Type Culture Collection) No: M 2023514; the lactobacillus mucus TG018 can be used for producing nicotinic acid, serving as an antioxidant and / or preparing the antioxidant, or can be applied to medicines for reducing uric acid.

Description

Technical Field

[0001] The present invention relates to the field of microbial technology, and particularly to the application of a strain of Lactobacillus mucosae TG018 in fermentation. Background Art

[0002] Nicotinic acid is an important water-soluble vitamin that can participate in the human metabolic process as a coenzyme of various dehydrogenases. Nicotinic acid plays a key role in cellular energy metabolism and biosynthesis. It can not only effectively promote cellular energy metabolism, but also maintain the health of the skin and mucous membranes, and participate in the normal function regulation of the nervous system. Nicotinic acid also has antioxidant activity, can effectively scavenge hydroxyl radicals and DPPH radicals, and inhibit the generation of lipid peroxides. In addition, nicotinic acid also shows significant physiological effects in maintaining cardiovascular health, regulating uric acid and blood sugar, and preventing skin diseases, and is an important research object that has received much attention in the fields of nutrition and medicine. Uric acid, as a heterocyclic organic compound, its generation involves multiple metabolic pathways. Hyperuricemia has long been closely related to gout and may also be associated with the occurrence of coronary heart disease, hypertension, stroke, metabolic syndrome and other diseases.

[0003] At present, the microbial strains that can produce nicotinic acid mainly include Pseudomonas putida, Bacillus subtilis, engineered bacteria, etc., and most of the screening sources are from soil and water sources. Lactobacillus mucosae TG018 is disclosed in CN117778236A, which is preserved in the China Center for Type Culture Collection, with the preservation number of CCTCC No: M 2023514, and the preservation time is April 10, 2023. Experiments have proved that Lactobacillus mucosae TG018 has the function of reducing phytic acid, but the functions of this strain in producing nicotinic acid, antioxidant and reducing uric acid have not been reported. Summary of the Invention

[0004] Aiming at the deficiencies of the prior art, the purpose of the present invention is to provide the application of a strain of Lactobacillus mucosae TG018, which has the function of producing nicotinic acid, and at the same time also has the effects of antioxidant and reducing uric acid precursors.

[0005] In order to achieve the above purpose, the present invention adopts the following technical solutions: The present invention provides the application of Lactobacillus mucosae ( Limosilactobacillus fermentum ) TG018, and the preservation number of Lactobacillus mucosae ( Limosilactobacillus fermentum ) TG018 is CCTCC No: M2023514; the application is selected from at least one of the following: (I) The application of Lactobacillus mucosae TG018 in producing nicotinic acid; (II) The application of Lactobacillus mucosae TG018 as an antioxidant and / or in the preparation of an antioxidant; (III)Use of Lactobacillus mucosae TG018 in the preparation of a drug for reducing uric acid.

[0006] Furthermore, use of Lactobacillus mucosae TG018 in the production of nicotinic acid.

[0007] Furthermore, Lactobacillus mucosae TG018 has a 16s rDNA sequence as shown in SEQ ID No.1.

[0008] Furthermore, Lactobacillus mucosae TG018 can adhere to intestinal epithelial cells.

[0009] Furthermore, Lactobacillus mucosae TG018 is used as a raw material for the preparation of food, medicine or health products.

[0010] Furthermore, the method for producing nicotinic acid by Lactobacillus mucosae TG018 is to culture the Lactobacillus mucosae TG018 bacteria in a medium and collect the supernatant by centrifugation.

[0011] Furthermore, in the method for producing nicotinic acid by Lactobacillus mucosae TG018, the medium is selected from at least one of MRS medium and a special medium for nicotinic acid determination.

[0012] Furthermore, in the method for producing nicotinic acid by Lactobacillus mucosae TG018, the culture temperature used is 30 - 40 °C and the culture time is 12 - 72 h.

[0013] Compared with the prior art, the beneficial effects of the present invention are: 1. The present invention provides a new use of Lactobacillus mucosae ( Limosilactobacillus fermentum) TG018) in the production of nicotinic acid. It has been experimentally confirmed that this strain has the effect of producing nicotinic acid, and the nicotinic acid yield can reach 45.63 μg / mL.

[0014] 2. The present invention provides a new use of Lactobacillus mucosae ( Limosilactobacillus fermentum) TG018) in antioxidants and / or the preparation of antioxidants. It has been experimentally confirmed that the scavenging rate of the supernatant of this strain of bacteria on DPPH free radicals is 57.82%, the SOD enzyme activity is 86.00 U / mL, and the GSH-Px enzyme activity is 204.26 U / mL. This strain of bacteria has good antioxidant capacity.

[0015] 3. The present invention provides a new use of Lactobacillus mucosae ( Limosilactobacillus fermentum) TG018) in the preparation of a drug for reducing uric acid. It has been experimentally confirmed that the degradation rate of this strain on guanosine and adenosine is 100%, and the degradation rate on inosine is 51.03%. This strain can play a role in reducing uric acid by degrading uric acid precursor substances.

[0016] 4. The strain is a functional strain derived from the human body and is originally a component of the human gut microbiota. Using this strain as a raw material for the preparation of food, health products, and drugs has high safety in use. Description of the Drawings

[0017] Figure 1 It is the overlapping chromatogram of the nicotinic acid standard product in Example 1; Figure 2 It is the chromatogram of the liquid-phase determination of nicotinic acid in Lactobacillus mucosae TG018 and TH04007 in Example 1; Figure 3 It is the chromatogram of the liquid-phase determination of guanosine standard product in Example 3; Figure 4 It is the chromatogram of the liquid-phase determination of inosine and adenosine standard products in Example 3; Figure 5 It is the chromatogram of the liquid-phase determination of inosine and adenosine in Lactobacillus mucosae TG018 in Example 3; Figure 6 It is the chromatogram of the liquid-phase determination of guanosine in Lactobacillus mucosae TG018 in Example 3; Figure 7 It is the liquid-phase determination curve of guanosine, inosine, and adenosine standard products in Lactobacillus mucosae TG018 in Example 3. Detailed Embodiments

[0018] Next, in combination with the embodiments of the present invention, the technical solutions of the present invention will be clearly and completely described. The described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts fall within the scope of protection of the present invention. The methods are conventional methods unless otherwise specified, and the raw materials can be obtained from public commercial channels unless otherwise specified.

[0019] The present invention relates to the application of a strain of Lactobacillus mucosae ( Limosilactobacillus fermentum) TG018. Regarding the source, biological identification, and physiological characteristic information of Lactobacillus mucosae ( Limosilactobacillus fermentum) TG018 have been publicly disclosed in CN117778236A and incorporated herein by reference; Lactobacillus mucosae ( Limosilactobacillus fermentum) TG018 of the present invention is deposited in the China Center for Type Culture Collection, with the deposit number CCTCC NO: M 2023514, the deposit date being April 10, 2023, and the address being the deposit center of Wuhan University. The 16s rDNA sequence of Lactobacillus mucosae ( Limosilactobacillus fermentum) TG018 of the present invention is as follows: The components of the MRS medium used in the following examples are: casein peptone 10.0 g / L, beef extract powder 10.0 g / L, yeast extract powder 4.0 g / L, ammonium citrate 2.0 g / L, sodium acetate 5.0 g / L, magnesium sulfate 0.2 g / L, ammonium sulfate 2.0 g / L, manganese sulfate 0.05 g / L, dipotassium hydrogen phosphate 2.0 g / L, glucose 20.0 g / L, Tween-80 1.08 g / L, agar 15.0 g / L, riboflavin 1.0 g / L.

[0020] The components of the medium for nicotinic acid determination are: acid-hydrolyzed casein 10.0 g / L, glucose 40 g / L, sodium acetate 20.0 g / L, potassium dihydrogen phosphate 1 g / L, dipotassium hydrogen phosphate 1 g / L, L-tryptophan 0.1 g / L, L-cystine hydrochloride 0.4 g / L, magnesium sulfate 0.4 g / L, sodium chloride 0.02 g / L, ferrous sulfate 0.02 g / L, manganese sulfate 0.02 g / L, riboflavin 0.0004 g / L, calcium pantothenate 0.0002 g / L, biotin 0.000001 g / L.

[0021] Example 1: Determination of nicotinic acid production capacity From Limosilactobacillus fermentum TG018, Limosilactobacillus fermentum 100 μL of bacterial liquid was aspirated from the cryopreservation tubes of TG018 and TH04077 (NR_118978.1), inoculated into 10 mL of MRS liquid medium, and anaerobically cultured at 37 °C for 24 h to obtain a strain seed solution. 50 μL of the strain seed solution was inoculated into 5 mL of the medium for nicotinic acid determination (Haibo Biotech), cultured for 24 h, then the culture solution was transferred to a centrifuge tube, ultrasonically disrupted, centrifuged at 5000 rpm for 10 min, and the supernatant was collected to obtain a cell-free extract, which was filtered through a 0.22 μm organic filter membrane and reserved for use.

[0022] The method for nicotinic acid determination used is as follows: Chromatographic conditions: C 18 Chromatographic column (Waters), detection wavelength 266 nm, column temperature 25 °C, flow rate 1 mL / min, elution time 10 min, injection volume 10 μL.

[0023] Mobile phase preparation: 0.05 mol / L potassium dihydrogen phosphate solution: methanol = 90:10 (V / V).

[0024] Standard solution preparation: Accurately weigh 0.05 g of nicotinic acid standard and place it in a 50 mL volumetric flask. Dissolve it with 0.01 mol / L hydrochloric acid and dilute to the mark, then shake well to complete the preparation of the standard solution (1 mg / mL). Dilute the standard solution to concentrations of 500 μg / mL, 200 μg / mL, 100 μg / mL, 50 μg / mL, 1 μg / mL, and 0.5 μg / mL. After filtering through a 0.22 μm microporous filter membrane, take 1 mL and add it to a liquid phase injection vial for standby.

[0025] The calculation formula for nicotinic acid content is:

[0026] Note: V - Peak area of nicotinic acid in the supernatant of the tested strain; V0 - Peak area of nicotinic acid in the supernatant of the culture medium; b - Standard curve constructed based on the peak area and concentration of the nicotinic acid standard: Y = aX + b; The concentration gradients of the standard curve are 0.5 μg / mL, 1 μg / mL, 50 μg / mL, 100 μg / mL, 200 μg / mL, and 1 mg / mL ( Figure 1 ). The retention time of nicotinic acid is 3.8 min, Y = 483X + 3120, R 2 = 0.995, meeting the linear relationship of the standard curve.

[0027] Filter the cell-free extract of Lactobacillus mucosae ( Limosilactobacillus fermentum) TG018 through a 0.22 μm organic filter membrane and set it aside for standby. The detection method is the same as that for the standard. The liquid chromatography diagrams of nicotinic acid determination for Lactobacillus mucosae ( Limosilactobacillus fermentum) TG018 and TH04077 (NR_118978.1) are as Figure 2 shown. Among them, the peak area of the cell-free extract of Lactobacillus mucosae ( Limosilactobacillus fermentum) TG018 is 27415. After deducting the influence of the peak intensity of the culture medium used for nicotinic acid determination, the nicotinic acid production is 45.63 μg / mL. No characteristic peak of nicotinic acid was detected for TH04077 (NR_118978.1), which is also Lactobacillus mucosae, and there is no peak area.

[0028] Example 2: Determination of the antioxidant function of Lactobacillus mucosae TG018 Using a disposable inoculation loop, take from Lactobacillus mucosae ( Limosilactobacillus fermentum)The bacterial liquid was picked from the probiotic cryopreservation tube of TG018 and streaked on the MRS agar plate for activation, and then cultured in an anaerobic incubator at 37 °C until single colonies grew. Seed liquid activation: A disposable inoculation loop was used to pick a single colony on the MRS agar plate, inoculated into 5 mL of MRS medium, shaken evenly, and cultured in a shaker at 37 °C and 200 r / min for 24 h.

[0029] Lactobacillus mucosae fermentum ( Limosilactobacillus fermentum) Determination of the DPPH free radical scavenging ability of TG018.

[0030] Preparation of DPPH sample solution: Weigh 20.0 mg of DPPH powder precisely, place it in a 250 mL volumetric flask, dissolve it with absolute ethanol and make up to the mark, mix well, to obtain a 0.2 mmol / L DPPH solution, and store it at 4 °C (to be used within 3.5 h).

[0031] Take 1 mL of the supernatant of the strain, add 1 mL of DPPH with a concentration of 0.2 mmol / L, mix well, let it stand at room temperature for 30 min, then use absolute ethanol as a blank control, and measure the absorbance change at 517 nm.

[0032] Measure the absorbance of the bacterial liquid supernatant + DPPH solution at a wavelength of 517 nm as A1; use a mixture of 1.0 mL of DPPH solution and 1.0 mL of absolute ethanol as a negative control to measure the absorbance A2; use a mixture of 1.0 mL of absolute ethanol solution added to 1.0 mL of the measured bacterial liquid supernatant solution as a blank control to measure its absorbance A0. Calculate the scavenging rate according to the following formula:

[0033] Determination of the activities of superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) of Lactobacillus mucosae fermentum TG018 Use a kit (Elabscience) to determine the activities of SOD and GSH-Px in the supernatant of the bacterial liquid of Lactobacillus mucosae fermentum TG018.

[0034] Lactobacillus mucosae fermentum ( Limosilactobacillus fermentum) The seed liquid of strain TG018 was inoculated into MRS medium, anaerobically cultured in a shaker at 37 °C for 24 h, and then the supernatant of the strain was taken for antioxidant index detection.

[0035] The results are shown in Table 1. The scavenging rate of the supernatant of the bacterial liquid of Lactobacillus mucosae fermentum ( Limosilactobacillus fermentum) TG018 against DPPH free radicals was 57.82%, the SOD enzyme activity was 86.00 U / mL, and the GSH-Px enzyme activity was 204.26 U / mL, proving that this strain of bacteria has good antioxidant ability.

[0036] Table 1 Antioxidant capacity of the supernatant of Lactobacillus mucosae TG018 fermentation broth

[0037] Example 3: Evaluation of the ability of Lactobacillus mucosae TG018 to reduce uric acid precursors in vitro Using a disposable inoculation loop, pick the bacterial liquid from the cryopreserved tube of Lactobacillus mucosae ( Limosilactobacillus fermentum) TG018 probiotic, streak and activate it on an MRS agar plate, and culture it in an anaerobic incubator at 37 °C until single colonies grow. Culture it in an anaerobic incubator at 37 °C until single colonies grow. Seed liquid activation: Dip a disposable inoculation loop into the single colony on the MRS agar plate, inoculate it into 5 mL of MRS medium, shake well, and place it in a shaker at 37 °C and 200 r / min for 24 h to complete the preparation of the strain seed liquid.

[0038] Centrifuge the strain seed liquid at 10,000 r / min for 10 min, discard the supernatant and collect the bacterial cells, wash the bacterial cells 2 times with PBS buffer, and then resuspend them in PBS buffer. Take three 0.8 mL aliquots of the bacterial liquid and add 0.8 mL of inosine-adenosine solution and guanosine solution respectively. Using the inosine-adenosine solution and guanosine solution without bacteria as controls, after culturing in a shaker at 37 °C for 6 h, take out the reaction solution, place it in a water bath at 85 °C for 20 min to terminate the reaction and cool it to room temperature, centrifuge at 10,000 r / min for 10 min, and collect the supernatant. After passing through a 0.22 μm filter membrane, determine the contents of inosine, adenosine, and guanosine by liquid chromatography.

[0039] Set the following chromatographic conditions: The chromatographic column is Waters C 18 (4.6 mm × 250 mm); the flow rate is 1.0 mL / min, the detection wavelength is 254 nm; the elution time is 40 min; the column temperature is 40 °C; the injection volume is 10 μL; isocratic elution; the mobile phase composition: potassium dihydrogen phosphate (pH = 3.5): methanol = 100:1.

[0040] By separately measuring the guanosine control solution and the inosine-adenosine control solution by liquid chromatography, the retention time of guanosine is 13.573 min ( Figure 3 ), the retention time of inosine is 12.636 min, and the retention time of adenosine is 24.431 min ( Figure 4 ). The standard curve equations are respectively: Y 鸟苷 = 27136X + 156505, R 2 = 0.9999; Y 肌苷 = 22673X - 10640, R 2 = 0.9998; Y 腺苷=30535X-58803, R 2 =0.9998 ( Figure 5 ).

[0041] The liquid chromatogram of HPLC measured by liquid phase of the fermentation mucilaginous lactobacillus TG018 bacterial solution after adding inosine, adenosine, and guanosine solutions is as shown in Figure 5 - 6 shown. The measurement results are shown in Table 2. ( Limosilactobacillus fermentum ) The degradation rates of TG018 for guanosine and adenosine are 100%, and the degradation rate for inosine is 51.03%.

[0042] Table 2 Degradation rates of inosine, adenosine, and guanosine of mucilaginous lactobacillus TG018 in fermentation (%)

[0043] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and not to limit them; although the present invention has been described in detail with reference to the preferred embodiments, those of ordinary skill in the art should understand that: it is still possible to modify the specific implementation manners of the present invention or perform equivalent replacements for some technical features, and they should all be covered within the scope of the technical solutions claimed by the present invention.

Claims

1. Use of a strain of Lactobacillus mucosae ( Limosilactobacillus fermentum ), TG018, characterized in that The Lactobacillus mucosae fermentum ( Limosilactobacillus fermentum ) TG018 has a deposit number of CCTCC No: M 2023514; The applications include at least one of the following: (I) Application of Limosilactobacillus fermentum TG018 in nicotinic acid production; (II) Application of Limosilactobacillus fermentum TG018 as an antioxidant and / or in the preparation of an antioxidant; (III) Application of Limosilactobacillus fermentum TG018 in the preparation of a drug for reducing uric acid.

2. The application according to claim 1, characterized in that, The application of Limosilactobacillus fermentum TG018 in nicotinic acid production.

3. The application according to claim 1 or 2, characterized in that, The Limosilactobacillus fermentum TG018 has a 16s rDNA sequence shown in SEQ ID No.

1.

4. The application according to claim 1 or 2, characterized in that, The Limosilactobacillus fermentum TG018 can adhere to intestinal epithelial cells.

5. The application according to claim 1 or 2, characterized in that, Using the Limosilactobacillus fermentum TG018 as a raw material for the preparation of food, medicine or health products.

6. The application according to claim 1, wherein The method for Limosilactobacillus fermentum TG018 to produce nicotinic acid is to culture the Limosilactobacillus fermentum TG018 bacteria in a medium and collect the supernatant by centrifugation.

7. The application according to claim 6, characterized in that, In the method for Limosilactobacillus fermentum TG018 to produce nicotinic acid, the medium is selected from at least one of MRS medium and a special medium for nicotinic acid determination.

8. The application according to claim 6, characterized in that, In the method for Limosilactobacillus fermentum TG018 to produce nicotinic acid, the culture temperature used is 30 - 40 °C, and the culture time is 12 - 72 h.

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