Lactic acid bacteria with uric acid reducing effect as well as screening method and application of lactic acid bacteria

Abstract

The invention discloses lactic acid bacteria with an effect of reducing uric acid as well as a screening method and application of the lactic acid bacteria, and belongs to the field of microorganisms, the classification name of the lactic acid bacteria is Limosilicilla fermentum FOSU-YHD19, the lactic acid bacteria are preserved in Guangdong Microbial Culture Collection Center, the preservation date is March 6, 2024, and the preservation number is GDMCC NO.64389. The invention further discloses a preparation method of the lactic acid bacteria with the effect of reducing uric acid. The screened lactic acid bacteria have a remarkable effect of reducing the uric acid value, the obtained lactic acid bacteria have excellent safety and prebiotics, a new microbial strain resource is provided for treating hyperuricemia, and the lactic acid bacteria have excellent application value when being applied to preparation of products for reducing the uric acid value. The lactic acid bacteria are applied to the field of marine organism culture, purine metabolism in marine organism bodies can be promoted, and then the purine content of seafood products is effectively reduced. Besides, the lactic acid bacteria are added into the field of marine organism culture, so that pathogenic bacteria in marine organism bodies can be inhibited, the microbial environment can be adjusted, and marine products with relatively low purine can be obtained.

Description

Technical Field

[0001] The present invention relates to the field of microorganisms, and in particular to a lactic acid bacterium having a uric acid-lowering effect and a screening method and application thereof. Background Art

[0002] Hyperuricemia is a metabolic disease, mainly caused by purine metabolism disorders and uric acid formation and secretion disorders. When nucleotide metabolism is affected, purine substances in the human body will decompose in large quantities, which will lead to an increase in blood uric acid levels. In addition, exogenous purine intake should not be ignored. Excessive consumption of purine foods such as meat, seafood, alcohol and sugary drinks will also affect uric acid metabolism in the human body. Uric acid excretion disorders caused by kidney disease will also lead to an increase in blood uric acid levels. When uric acid accumulates to a certain extent in the human body, it will cause a series of diseases: the first is asymptomatic hyperuricemia, the second is gouty arthritis, and the third is uric acid nephropathy. Mild to moderate hyperuricemia usually occurs with hyperglycemia, hyperlipidemia and hypertension, and more severe hyperuricemia is mostly related to defects or dysfunction of uric acid metabolic enzymes. Many studies have shown that hyperuricemia often occurs in combination with other metabolic diseases, such as chronic kidney disease, cardiovascular disease, hypertension, type 2 diabetes, hyperlipidemia and obesity.

[0003] Clinically, the treatment strategies for hyperuricemia mainly focus on two aspects: one is to inhibit the production of uric acid, and the other is to enhance the excretion of uric acid. Xanthine oxidase is the key rate-limiting enzyme that controls the production of uric acid, and its activity is closely related to the production of uric acid. Therefore, xanthine oxidase inhibitors can directly inhibit the synthesis of uric acid by inhibiting the activity of xanthine oxidase, and can be used to treat hyperuricemia. Its representative drugs include allopurinol, febuxostat, bromobendanone and probenecid, which can reduce the blood uric acid level in the human body by accelerating the excretion of uric acid and reducing the deposition of urate. However, these drugs can cause a large number of serious adverse effects, and can cause damage to the liver, which has a greater impact on human health. Hyperuricemia can be prevented by controlling diet, which requires minimizing the intake of purine nucleosides, but the effect of dietary control treatment is limited and the compliance is poor. Therefore, it is of positive significance to study a product that can effectively reduce uric acid values. Summary of the invention

[0004] Based on this, in order to provide a lactic acid bacteria with uric acid lowering effect, the specific technical scheme is as follows:

[0005] A lactic acid bacterium with a uric acid-lowering effect. The lactic acid bacterium is classified and named Limosilactobacillusfermentum FOSU-YHD19, and is preserved by Guangdong Microbiological Culture Collection Center on March 6, 2024, with a preservation number of GDMCC NO.64389.

[0006] The present invention provides a method for screening lactic acid bacteria having a uric acid-lowering effect, the screening method comprising the following steps:

[0007] The fermented kimchi water was cultured in a culture medium for 24 to 48 hours, then diluted and spread on an MRS plate containing calcium carbonate, and colonies with calcium-dissolving circles were selected;

[0008] The colonies with calcium-dissolving circles were cultured, and Gram-positive bacteria and catalase-negative strains that did not produce bubbles were selected after microscopic examination. Then, lactic acid bacteria strains with nucleoside-lowering effects were screened, and then lactic acid bacteria strains with uric acid-lowering effects were screened. Finally, lactic acid bacteria strains with acid and bile salt resistance were screened.

[0009] Furthermore, the strain is cultured at a temperature of 35° C. to 37° C., a pH of 5.4, and facultative anaerobic culture.

[0010] The present invention also provides an application of lactic acid bacteria having a uric acid lowering effect, wherein the application is the application of the lactic acid bacteria in the preparation of a product for lowering uric acid levels.

[0011] The present invention provides an application of lactic acid bacteria with uric acid-lowering effect, and the application is the application of the lactic acid bacteria in the preparation of marine organism breeding feed.

[0012] The present invention provides an application of lactic acid bacteria with uric acid lowering effect, wherein the application is that the lactic acid bacteria is used as an additive, and the additive is in the form of freeze-dried powder and is applied to the field of marine organism breeding.

[0013] The lactic acid bacteria screened in the above scheme has a significant effect of reducing uric acid, and the lactic acid bacteria obtained have excellent safety and probiotic properties, provide a new microbial strain resource for treating hyperuricemia, and are applied to the preparation of products that reduce uric acid levels, with excellent application value. The lactic acid bacteria are applied to the field of marine organism breeding, which can promote the purine metabolism in the marine organism, and then effectively reduce the purine content of seafood products. In addition, the lactic acid bacteria are added to the field of marine organism breeding, and the pathogenic bacteria in the marine organism can also be suppressed, the microbial environment is adjusted, and then a seafood with lower purine is obtained. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 This is a schematic diagram of the results of 16S rRNA strain identification of the strains screened in Example 1;

[0015] Figure 2 Schematic diagram of some Gram-positive bacteria in the strain screening process of Example 1;

[0016] Figure 3 This is a schematic diagram of the catalase experiment during the strain screening process in Example 1. Figure 3 Figure A is a schematic diagram of no bubble generation and peroxidase negative. Figure 3 Figure B is a schematic diagram of bubble production and catalase positivity;

[0017] Figure 4 Schematic diagram of the ability of the strain in Example 1 to degrade nucleosides, wherein A1 and A2 are both inosine degradation rates; B1 and B2 are both guanosine degradation rates; C1 and C2 are both adenosine degradation rates;

[0018] Figure 5 This is a schematic diagram of the ability of the strain in Example 1 to degrade uric acid;

[0019] Figure 6 This is a schematic diagram of the acid resistance of different strains during the strain screening process in Example 1;

[0020] Figure 7 This is a schematic diagram of the bile salt tolerance of different strains during the strain screening process in Example 1;

[0021] Figure 8 The hemolytic activity experiment of strain YHD19 screened in Example 1;

[0022] Fig. 9 A schematic diagram of ornithine decarboxylase of strain YHD19 screened in Example 1;

[0023] Fig.10 A schematic diagram of the nitroreductase of strain YHD19 screened in Example 1;

[0024] Fig.11 Schematic diagram of the hydrophobicity of strain YHD19 screened in Example 1. DETAILED DESCRIPTION

[0025] In order to make the purpose, technical solution and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with its embodiments. It should be understood that the specific implementation methods described herein are only used to explain the present invention and do not limit the protection scope of the present invention.

[0026] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art of the present invention. The terms used herein in the specification of the present invention are only for the purpose of describing specific embodiments and are not intended to limit the present invention. The term "and / or" used herein includes any and all combinations of one or more related listed items.

[0027] In one embodiment of the present invention, a lactic acid bacterium with a uric acid-lowering effect is classified and named Limosilactobacillus fermentum FOSU-YHD19, which is preserved by Guangdong Provincial Microbiological Culture Collection Center on March 6, 2024, and the preservation number is GDMCCNO.64389.

[0028] In one embodiment, a method for screening lactic acid bacteria having a uric acid-lowering effect is provided, the screening method comprising the following steps:

[0029] The fermented kimchi water was cultured in a culture medium for 24 to 48 hours, then diluted and spread on an MRS plate containing calcium carbonate, and colonies with calcium-dissolving circles were selected;

[0030] The colonies with calcium-dissolving circles were cultured, and Gram-positive bacteria and catalase-negative strains that did not produce bubbles were selected after microscopic examination. Then, lactic acid bacteria strains with nucleoside-lowering effects were screened, and then lactic acid bacteria strains with uric acid-lowering effects were screened. Finally, lactic acid bacteria strains with acid and bile salt resistance were screened.

[0031] In one embodiment, the strain is cultured at a temperature of 35° C. to 37° C., a pH of 5.4, and a facultative anaerobic culture.

[0032] In one embodiment, an application of lactic acid bacteria having a uric acid lowering effect is provided, wherein the application is the application of the lactic acid bacteria in the preparation of a product for lowering uric acid value.

[0033] In one embodiment, the present invention provides an application of lactic acid bacteria having a uric acid-lowering effect, wherein the application is the application of the lactic acid bacteria in the preparation of marine organism aquaculture feed.

[0034] In one embodiment, an application of lactic acid bacteria having a uric acid-lowering effect is provided, wherein the lactic acid bacteria is used as an additive, and the additive is in the form of freeze-dried powder and is applied to the field of marine biological breeding.

[0035] In one embodiment, the freeze-dried powder uses skim milk powder as a single freeze-drying protective agent, and the skim milk powder is prepared into a 5% skim milk powder solution, centrifuged at 8000r / min for 30min, and the supernatant is removed to obtain bacterial mud, which is added at a volume ratio of 3:1 between the skim milk powder solution and the bacterial mud, pre-frozen at -20°C for 4h, and freeze-dried using a freeze dryer.

[0036] In one embodiment, the lyophilized powder is diluted to a bacterial solution concentration of 1×10 6 -10 9 CFU / mL, diluted according to 500ml / mu*m (i.e. about 0.00033ml / L to 0.0033ml / L, depending on the depth and area of ​​the pond), and then evenly sprinkled into the seafood farming waters at a water temperature of 4℃-40℃ to obtain low-purine seafood.

[0037] In one embodiment, the concentration of lactic acid bacteria added to the feed of seafood farming by freeze-dried powder is 1×10 6 -10 9 CFU / mL.

[0038] In one embodiment, lactic acid bacteria are added to seafood farming feed in an amount of 5-10%, and different intake amounts are provided according to different seafood species to obtain low-purine seafood.

[0039] The lactic acid bacteria screened in the above scheme has a significant effect of reducing uric acid levels, and the lactic acid bacteria obtained have excellent safety and probiotic properties, provide a new microbial strain resource for treating hyperuricemia, and are applied to the preparation of products that reduce uric acid levels, with excellent application value. The lactic acid bacteria are applied to the field of marine organism breeding, which can promote purine metabolism in marine organisms, and then effectively reduce the purine content of seafood products. In addition, the lactic acid bacteria are added to the field of marine organism breeding, and the pathogenic bacteria in the marine organisms can also be suppressed, the microbial environment is adjusted, and then a seafood product with lower purine is obtained.

[0040] The embodiments of the present invention will be described in detail below with reference to specific examples.

[0041] Embodiment 1:

[0042] The fermented kimchi water was cultured in a culture medium for 48 hours, then diluted and spread on an MRS plate containing calcium carbonate, and the colonies with calcium-dissolving circles were selected;

[0043] The fermented kimchi water was cultured in a culture medium for 24 to 48 hours, then diluted and spread on an MRS plate containing calcium carbonate, and colonies with calcium-dissolving circles were selected;

[0044] The colonies with calcium-dissolving circles were cultured, and Gram-positive bacteria and catalase-negative strains that did not produce bubbles were selected after microscopic examination. Then, lactic acid bacteria strains with nucleoside-lowering effects were screened, and then lactic acid bacteria strains with uric acid-lowering effects were screened. Finally, lactic acid bacteria strains with acid and bile salt resistance were screened.

[0045] The lactic acid bacteria strains screened in Example 1 were identified by 16S rRNA strain identification. Figure 1 As shown in the figure, the strain was determined to be Limosilactobacillus fermentum (fermented mucus lactobacillus), and it was named Limosilactobacillus fermentum FOSU-YHD19 (YHD19), and was deposited in Guangdong Microbiological Culture Collection Center on March 6, 2024, with the strain deposit number GDMCC NO.64389. Figure 1 As shown, Figure 1 This is a schematic diagram of the results of 16S rRNA strain identification of the strains screened in Example 1.

[0046] Specifically, Figure 2 As shown, Figure 2 This is a schematic diagram of some Gram-positive bacteria in the strain screening process of Example 1, which can illustrate that the lactic acid bacteria strains of the present application are Gram-positive strains. Figure 3 This is a schematic diagram of the catalase experiment during the strain screening process in Example 1. Figure 3 Figure A is a schematic diagram of no bubble generation and peroxidase negative. Figure 3 Figure B is a schematic diagram of bubble production and catalase positivity. In summary, Gram-positive bacteria and catalase-negative strains that do not produce bubbles after microscopic examination were selected.

[0047] Figure 4 Schematic diagram of the nucleoside degradation ability of the strain in Example 1, wherein A1 and A2 are both inosine degradation rates; B1 and B2 are both guanosine degradation rates; C1 and C2 are both adenosine degradation rates. Figure 5 This is a schematic diagram of the ability of the strain in Example 1 to degrade uric acid; Figure 6 This is a schematic diagram of the acid resistance of different strains during the strain screening process in Example 1; Figure 7 This is a schematic diagram of the bile salt tolerance of different strains during the strain screening process in Example 1; Figure 8 The hemolytic activity experiment of strain YHD19 screened in Example 1, Fig. 9 This is a schematic diagram of the ornithine decarboxylase of strain YHD19 screened in Example 1, Fig.10 Schematic diagram of the nitroreductase of strain YHD19 screened in Example 1, Figures 8 to 10It can be seen that the strain YHD19 screened in Example 1 is γ-hemolytic and has no hemolytic activity; has no ornithine decarboxylase activity; has no nitroreductase activity, and has high safety. Fig.11 Schematic diagram of the hydrophobicity of strain YHD19 screened in Example 1, Fig.11 It can be seen that YHD19 has good hydrophobicity, which is conducive to the colonization of the strain in the gastrointestinal tract.

[0048] Embodiment 2:

[0049] The fermented mucus lactobacillus FOSU-YHD19 obtained in Example 1 was streaked on an MRS solid plate and cultured under anaerobic conditions at 37°C for 24 h. After secondary activation, it was inoculated in an MRS liquid culture medium and diluted and counted. The concentration of the bacterial suspension was adjusted to 1×10 10 CFU / mL;

[0050] The bacterial suspension was placed at 4°C and centrifuged at 8000r / min for 15 minutes, and the supernatant was discarded. The bacterial mud at the bottom of the centrifuge tube was collected, washed twice with a sterile PBS buffer solution with a concentration of 0.90%, and then resuspended for 1 minute under the same conditions. Sterilized skim milk powder was added at a mass ratio of 1:4 and resuspended and mixed. After pre-freezing at -20°C for 4 hours, it was placed in a freeze dryer for 30 hours to prepare freeze-dried powder, and the strain was stored in the form of freeze-dried powder;

[0051] Under sterile conditions, the lyophilized powder was dissolved in a certain proportion of sterile water and diluted to 1×10 6 -10 9 CFU / mL. Calculate the number of viable bacteria per gram of lyophilized powder based on the colony count.

[0052] Embodiment 3:

[0053] Taking 100 mackerel as an example, the viable bacterial count of fermented mucus Lactobacillus FOSU-YHD19 with uric acid-lowering effect was adjusted to 1×10 9 CFU / mL, evenly sprinkled in the seafood farming waters at a ratio of 500ml / mu*m, added to seafood farming feed at a 10% addition, and the farming water temperature was 20°C. The changes in the purine content in farmed bluefin tuna were detected, and the purine content of bluefin tuna decreased from 400mg / 100g to 120mg / 100g.

[0054] It should be noted that the purine content of the above-mentioned mackerel is the average value of 100 mackerels.

[0055] Embodiment 4:

[0056] Taking 100 mackerel as an example, the viable bacterial count of fermented mucus Lactobacillus FOSU-YHD19 with uric acid-lowering effect was adjusted to 1×10 9CFU / mL, evenly sprinkled in the seafood farming waters at a ratio of 500ml / mu*m, added to seafood farming feed at a rate of 10%, and the farming water temperature was 20°C. The changes in the purine content in farmed mackerel were detected, and the purine content of mackerel decreased from 350mg / 100g to 110mg / 100g.

[0057] It should be noted that the purine content of the above-mentioned mackerel is the average value of 100 mackerels.

[0058] Embodiment 5:

[0059] Taking 100 sardines as an example, the viable bacterial count of fermented Lactobacillus mucosa FOSU-YHD19 with uric acid-lowering effect was adjusted to 1×10 9 CFU / mL, sprinkled evenly in the seafood farming waters at a ratio of 500ml / mu*m, added to seafood farming feed at a rate of 10%, and the farming water temperature was 20°C. The changes in the purine content in farmed mackerel were detected, and the purine content of mackerel decreased from 300mg / 100g to 100mg / 100g.

[0060] It should be noted that the purine content of the above-mentioned sardines is the average value of 100 sardines.

[0061] In summary, the lactic acid bacteria screened in the present application have a significant effect of reducing uric acid levels, and the obtained lactic acid bacteria have excellent safety and probiotic properties.

[0062] The technical features of the above-described embodiments may be arbitrarily combined. To make the description concise, not all possible combinations of the technical features in the above-described embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0063] The above-mentioned embodiments only express several implementation methods of the present invention, and the descriptions thereof are relatively specific and detailed, but they cannot be understood as limiting the scope of the invention patent. It should be pointed out that, for ordinary technicians in this field, several variations and improvements can be made without departing from the concept of the present invention, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention shall be subject to the attached claims.

Claims

1. A lactic acid bacterium having a uric acid-lowering effect, characterized in that: The lactic acid bacteria is classified and named Limosilactobacillus fermentum FOSU-YHD19, and is preserved by Guangdong Provincial Microbiological Culture Collection Center. The preservation date is March 6, 2024, and the preservation number is GDMCCNO.64389.

2. A method for screening lactic acid bacteria having uric acid-lowering effect, characterized in that: The screening method comprises the following steps: The fermented kimchi water was cultured in a culture medium for 24 to 48 hours, then diluted and spread on an MRS plate containing calcium carbonate, and colonies with calcium-dissolving circles were selected; The colonies with calcium-dissolving circles were cultured, and Gram-positive bacteria and catalase-negative strains that did not produce bubbles were selected after microscopic examination. Then, lactic acid bacteria strains with nucleoside-lowering effects were screened, and then lactic acid bacteria strains with uric acid-lowering effects were screened. Finally, lactic acid bacteria strains with acid and bile salt resistance were screened.

3. The screening method according to claim 2, characterized in that The strain culture temperature is 35°C to 37°C, the pH is 5.4, and the culture is facultative anaerobic.

4. An application of lactic acid bacteria having uric acid lowering effect, characterized in that: The application is the application of the lactic acid bacteria as claimed in claim 1 in the preparation of a product for lowering uric acid levels.

5. An application of lactic acid bacteria having uric acid lowering effect, characterized in that: The application is the application of the lactic acid bacteria as claimed in claim 1 in the preparation of marine organism breeding feed.

6. An application of lactic acid bacteria having uric acid lowering effect, characterized in that: The application is to use the lactic acid bacteria as claimed in claim 1 as an additive, wherein the additive is in the form of freeze-dried powder and is applied to the field of marine biological breeding.

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