CN116987617A - Lactobacillus rhamnosus UA260 strain and application thereof in blood uric acid regulation - Google Patents
Lactobacillus rhamnosus UA260 strain and application thereof in blood uric acid regulation Download PDFInfo
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- CN116987617A CN116987617A CN202310469163.3A CN202310469163A CN116987617A CN 116987617 A CN116987617 A CN 116987617A CN 202310469163 A CN202310469163 A CN 202310469163A CN 116987617 A CN116987617 A CN 116987617A
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- Prior art keywords
- lactobacillus rhamnosus
- uric acid
- blood uric
- strain
- food
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- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
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Classifications
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- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
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- A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
- A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
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- A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
- C12R2001/225—Lactobacillus
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
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Abstract
The application relates to the technical field of microorganisms, and discloses a lactobacillus rhamnosus UA260 strain and application thereof in regulating blood uric acid, wherein the lactobacillus rhamnosus UA260 is preserved in China center for type culture collection (China center for type culture Collection) of microorganisms, guangdong province, and the preservation number is GDMCC No. 63250. The lactobacillus rhamnosus UA260 can be used for preparing foods for reducing blood uric acid or medicines for reducing blood uric acid, and the lactobacillus rhamnosus UA260 obtained by screening has good safety, can regulate and improve intestinal microecological balance of a host, plays a beneficial role and generates exact health efficacy; in vitro and animal experiments show that the compound has the function of reducing uric acid in blood.
Description
Technical Field
The application relates to the technical field of microorganisms, in particular to a lactobacillus rhamnosus UA260 strain and application thereof in regulating blood uric acid.
Background
An increase in protein-and purine-rich food intake leads to an increased prevalence of Hyperuricemia (HUA), which is increasingly the second most common metabolic type of disease following type II diabetes. In recent years, hyperuricemia has a remarkable rising and younger trend, the total prevalence of Chinese hyperuricemia is 13.3%, the total prevalence of gout is about 1.77 million, the total prevalence of gout is about 1.1%, and the number of patients is about 1466 ten thousand, based on online research, the young hyperuricemia and gout patients of 18-35 years old account for nearly 60%, and become the fourth highest of Chinese people after hypertension, hyperglycemia and hyperlipidemia.
Hyperuricemia is mainly due to the accumulation of uric acid in the blood at elevated levels caused by body purine metabolic disorders, uric acid hypersecretion or intestinal renal excretion dysfunction, and is considered as an important risk factor for the development of gout and other diseases such as cardiovascular diseases, metabolic syndrome. Potential therapeutic mechanisms for lowering serum uric acid include: inhibition of xanthine oxidase (e.g., allopurinol and febuxostat) and use of recombinant uricase (e.g., labyrinase), uric acid excreting agents (e.g., probenecid), and injectable uricase (e.g., pegylated enzyme). Although these drugs are effective in lowering uric acid, there are numerous toxic side effects of these drugs, including hepatotoxicity and potentially fatal allopurinol hypersensitivity syndromes. Lifestyle changes, including nutritional interventions targeting the intestinal flora, may be a more desirable treatment option.
At present, researches on uric acid reduction of probiotics have been reported, and strains which are deeply researched on uric acid reduction functions mainly comprise lactobacillus bifidus, lactobacillus brevis, lactobacillus fermentum and the like, for example, lactobacillus fermentum JL-3, lactobacillus brevis DM2918 and lactobacillus bifidus PA-3 have the uric acid reduction effect, wherein the lactobacillus bifidus PA-3 has successfully proved the uric acid reduction effect through animal experiments and clinical trials of people.
Therefore, the lactobacillus with the uric acid regulating effect is developed safely and effectively, and has wide application prospect in the fields of fermented foods and medicines.
Disclosure of Invention
(one) solving the technical problems
Aiming at the defects of the prior art, the application provides a lactobacillus rhamnosus UA260 strain and application thereof in regulating blood uric acid, so as to solve the problems in the background art, and the lactobacillus rhamnosus UA260 obtained by screening has good safety, can regulate and improve intestinal microecological balance of a host, plays a beneficial role and produces definite health efficacy; in vitro and animal experiments show that the compound has the function of reducing uric acid in blood.
(II) technical scheme
In order to achieve the above purpose, the present application provides the following technical solutions: the application provides a lactobacillus rhamnosus UA260 strain, wherein the lactobacillus rhamnosus UA260 (latin name:Lacticaseibacillus rhamnosus UA 260) was deposited at the chinese collection of typical cultures of microorganisms, cantonese province, 3 months 10, 2023, taxonomic designation:Lacticaseibacillus rhamnosusthe deposit number is GDMCC No. 63250.
In addition, the application also provides application of the lactobacillus rhamnosus UA260 strain in regulating blood uric acid, and the lactobacillus rhamnosus UA260 strain is applied to preparing foods or medicines.
Preferably, the food or medicament acts to reduce uric acid.
Preferably, the food or drug is a blood uric acid lowering food or a blood uric acid lowering drug.
Further, the medicament comprises lactobacillus rhamnosus UA260 and a pharmaceutically non-toxic carrier.
Further, the dosage form of the medicine is at least one selected from tablets, granules, powder, capsules, suspending agents, emulsions and freeze-dried preparations.
Preferably, the medicament is a composite probiotic tablet.
Further, the food product comprises lactobacillus rhamnosus UA260 and a pharmaceutically acceptable additive.
Preferably, the food is a probiotic solid powder.
(III) beneficial effects
Compared with the prior art, the application provides the lactobacillus rhamnosus UA260 strain and the application thereof in the aspect of regulating blood uric acid, and the lactobacillus rhamnosus UA260 strain has the following beneficial effects:
(1) The lactobacillus rhamnosus UA260 obtained by screening is good in safety, can mediate and improve intestinal microecology balance of a host, plays a beneficial role, and has an exact health effect; in vitro and animal experiments show that the compound has the function of strongly reducing uric acid in blood;
(2) The lactobacillus rhamnosus UA260 obtained by screening can be used for preparing uric acid-reducing foods and medicines, and experiments show that the obtained foods also have the effect of reducing blood uric acid.
Drawings
FIG. 1 shows the inhibition of xanthine oxidase in vitro test of Lactobacillus rhamnosus UA260 according to example 2 of the present application;
FIG. 2 shows the inosine degradation rate of a key precursor for uric acid synthesis in an in vitro test of Lactobacillus rhamnosus UA260 according to example 3 of the present application;
FIG. 3 is a graph showing the ability of Lactobacillus rhamnosus UA260 of example 2 of the present application to reduce serum uric acid levels in animals;
FIG. 4 is a graph showing the ability of Lactobacillus rhamnosus UA260 to reduce uric acid levels in animal urine according to example 2 of the present application;
FIG. 5 is a graph showing the ability of Lactobacillus rhamnosus UA260 to reduce uric acid levels in animal feces according to example 2 of the present application;
FIG. 6 is a graph showing H & E staining results of pathological sections of colon, liver and kidney.
The accompanying drawings are included to provide a further understanding of the application and are incorporated in and constitute a part of this specification, illustrate the application and together with the embodiments of the application, serve to explain the application.
Detailed Description
The following description of the embodiments of the present application will be made clearly and fully with reference to the accompanying drawings, in which it is evident that the embodiments described are only some, but not all embodiments of the application; all other embodiments, which can be made by those skilled in the art based on the embodiments of the application without making any inventive effort, are intended to be within the scope of the application.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. In addition, any methods and materials similar or equivalent to those described herein can be used in the present application. The preferred methods and materials described herein are illustrative only and should not be construed as limiting the application.
The experimental methods in the following examples are all conventional methods unless otherwise specified; the test materials and test strains used in the examples described below, unless otherwise specified, were commercially available.
The application provides a lactobacillus rhamnosus UA260 strain and application thereof in regulating blood uric acid, wherein the lactobacillus rhamnosus UA260 (latin name:Lacticaseibacillus rhamnosus UA 260) was deposited at the chinese collection of typical cultures of microorganisms, cantonese province, 3 months 10, 2023, taxonomic designation:Lacticaseibacillus rhamnosusthe deposit number is GDMCC No. 63250.
Lactobacillus rhamnosus UA260 is used in the preparation of a food or medicament for reducing uric acid, preferably a blood uric acid-reducing food or a blood uric acid-reducing medicament.
The medicament comprises lactobacillus rhamnosus UA260 and a medical nontoxic carrier, and the dosage form of the medicament is at least one selected from tablets, granules, powder, capsules, suspending agents, emulsions and freeze-dried preparations; preferably, the medicament is a composite probiotic tablet, and the food comprises lactobacillus rhamnosus UA260 and a pharmaceutically acceptable additive; preferably, the food is a probiotic solid powder.
Further, the probiotic solid powder is prepared from xylo-oligosaccharide, inulin, fructo-oligosaccharide and lactobacillus rhamnosus UA 260; lactobacillus rhamnosus UA260 is a lyophilized powder; the freeze-dried powder is prepared by freeze-drying a bacterial suspension of lactobacillus rhamnosus UA260 in a sterile environment by adopting a freeze dryer.
Example 1:
isolation and identification of lactobacillus rhamnosus UA 260:
1. medium formulation optimization
And (3) carrying out an optimized culture medium experiment in groups to finally obtain the optimal formula of the lactobacillus culture medium:
MRS solid Medium 1L formulation: 10.0g/L of casein peptone, 10.0g/L of beef extract, 5.0g/L of yeast extract, 20.0g/L of glucose, 2.0g/L of dipotassium hydrogen phosphate, 801.0g/L of tween, 2.0g/L of tri-ammonium citrate, 5.0g/L of sodium acetate, 0.1g/L of magnesium sulfate, 0.05g/L of manganese sulfate and 17.5g of agar; the pH was 6.5 and sterilized at 121℃for 20min.
MRS broth medium formulation: 10.0g/L of casein peptone, 10.0g/L of beef extract, 5.0g/L of yeast extract, 20.0g/L of glucose, 2.0g/L of dipotassium hydrogen phosphate, 801.0g/L of tween, 2.0g/L of tri-ammonium citrate, 5.0g/L of sodium acetate, 0.1g/L of magnesium sulfate and 0.05g/L of manganese sulfate, wherein the pH value is 6.5, and sterilizing for 20min at 121 ℃.
2. Strain screening
Lactobacillus rhamnosus UA260 strain is derived from healthy female breast milk and is isolated from the milk of healthy female breast milk. Taking milk from healthy lactating mother, diluting sample with sterile physiological saline 10 times gradient to 10 times -3 The strain is coated on an MRS solid culture medium, placed in a 37 ℃ incubator for culturing for 24-48 hours, the morphological characteristics of bacterial colonies are observed, suspected bacterial colonies of lactobacillus on the MRS solid culture medium are picked, transferred into an MRS liquid culture medium for pure culture respectively, 33 strains are separated out, and each strain is numbered.
The uric acid reducing ability of the strains obtained by separation and purification is measured, the detailed experimental steps are shown in example 2 and example 3, and lactobacillus rhamnosus UA260 with stronger uric acid reducing ability is obtained by screening.
3. Identification of species
Extracting bacterial total DNA from screened lactobacillus rhamnosus UA260, and carrying out 16s rDNA amplification, wherein the primers are as follows:
sgF:5'-AGAGTTTGATCATGGCTCAG-3'；sgR:5'-TAGGGTTACCTTGTTACGACTT-3'。
and (3) performing PCR amplification and agarose gel electrophoresis by using the primers, and then cutting, recovering and sequencing.
The result of the DNA sequencing of the strain 16s obtained by separation is shown as SEQ ID NO. 1, and NCBI (National Center for Biotechnology Information) nucleic acid is used for comparison to obtain lactobacillus rhamnosus @Lacticaseibacillus rhamnosus) Coverage rate (Query Cover)>98%。
Example 2:
in vitro xanthine oxidase inhibition test of lactobacillus rhamnosus UA260
The inhibition rate of xanthine oxidase in vitro of the strain is detected according to the following steps:
transferring the strain to be tested into MRS liquid culture medium, culturing at 37deg.C for 48h, standing at 37deg.C for fermenting 48h, centrifuging 2mL culture solution at 12000×g at 4deg.C for 10min, and collecting supernatant;
preparing xanthine buffer solution: accurately weighing 0.08g of NaOH in 10mL of distilled water, 15mg of xanthine in NaOH solution, and 0.27g of KH 2 PO 4 Dissolving in 10mL, KH 2 PO 4 Adding xanthine-NaOH solution into the solution, and uniformly mixing for later use;
preparing xanthine oxidase solution: 1mg is weighed and dissolved in 1mL PBS for standby;
the medicines were added in the order of Table 1 and mixed well, reacted in a 37℃water bath for 10 minutes, the reaction was terminated, and absorbance was measured at 290 nm. The inhibition ratio (I) of the extract on xanthine oxidase is OD1-OD4, which is the absorbance measured at 290 nm of test 1-4 (OD value measured in 1 st test tube is measured by 3 rd test tube as blank control; OD value in 2 nd test tube is measured by 4 th test tube as blank control), respectively, and the calculation formula of the inhibition ratio is as follows:
。
TABLE 1 xanthine oxidase Activity inhibition assay reaction System
The experimental result is shown in fig. 1, the abscissa is the number of the separated strain, wherein UA260 is lactobacillus rhamnosus UA260, the ordinate is the in vitro inhibition rate of xanthine oxidase, and the inhibition rate of lactobacillus rhamnosus UA260 to xanthine oxidase is 100.0%, which indicates that lactobacillus rhamnosus UA260 has stronger in vitro uric acid reducing capability.
Example 3:
in vitro inosine degradation rate test of lactobacillus rhamnosus UA260
The in vitro inosine degradation rate of the strain is detected according to the following steps:
the test strain was inoculated into MRS and cultured under anaerobic conditions at 37℃for 48 hours. 2mL of the culture medium was centrifuged at 4000 Xg at 4℃for 10min, washed 2 times with 1mL of 0.85% NaCl, resuspended in 1mL of inosine buffer, incubated at 37℃for 60 min, and shaken well. Centrifugation was performed at 4000 Xg at 4℃for 10min, and the supernatant was assayed for its content using an Inosine kit (kit name: inosine (Inosine) content assay kit, inc.: shanghai Jiang Lai Biotech Co., ltd.).
Preparing inosine buffer solution: accurately weighing inosine 2mg, dissolving in 10mL 200 mmol/L KH 2 PO 4 The solution (0.27 g in 10 mL) was adjusted to pH 7.0 with NaOH (0.08 g in 10 mL).
The experimental result is shown in fig. 2, the abscissa is the number of the separated strain, wherein UA260 is lactobacillus rhamnosus UA260, the ordinate is the degradation rate of inosine, and the degradation rate of lactobacillus rhamnosus UA260 to inosine is 32.50%, which indicates that lactobacillus rhamnosus UA260 has a certain in vitro uric acid reducing capability.
Example 4:
in vivo effectiveness study (animal experiment) of lactobacillus rhamnosus UA260
(1) Preparation of the experimental strains:
lactobacillus rhamnosus UA260 activated twice is inoculated in MRS liquid culture medium, cultured at 37 ℃ for 18h, centrifuged at 6000r/min for 10min, washed by sterilized normal saline and then the thalli are collected. Then, 0.85% physiological saline was added to adjust the bacterial count to about 1.0X10 10 CFU/mL, and then the viable bacteria are sub-packaged into 5mL centrifuge tubes according to daily usage amount, and the gastric lavage dosage is 0.1 mL/each day, and the total amount of the viable bacteria is 10 per group, 4 groups are required to be filled for 60 days.
(2) Grouping and raising modes of experimental animals:
c57BL/6J strain 8 week old male mice, 40 total, free feeding for 2 weeks, average divided into 4 groups of 10:
normal group (CON group): after the stomach is irrigated with 0.85 percent of physiological saline, the stomach is irrigated with 0.85 percent of physiological saline;
hyperuricemia model group (HUA group): adenine (75 mg/kg/day) and potassium oxazinate (200 mg/kg/day) were lavaged for one hour, followed by lavage with 0.85% saline;
positive control group (ALL group): after one hour of gastric lavage of adenine (75 mg/kg/day) and potassium oxazinate (200 mg/kg/day), gastric allopurinol (5 mg/kg/day) was lavaged;
lactobacillus rhamnosus UA260 group (UA 260 group): after one hour of gastric adenine (75 mg/kg/day) and potassium oxazinate (200 mg/kg/day), lactobacillus rhamnosus UA260 strain (10) 9 CFU/per mouse/day) lavage;
model establishment and lactobacillus rhamnosus intervention for 2 months, and then collecting feces and urine.
(3) Sample collection and analysis test:
mice were anesthetized with tribromoethane and sacrificed by cervical removal after blood collection from the eyeballs. Blood was centrifuged at 4000r/m for 10min, and serum was separated, and the uric acid content was detected by using a kit (kit name: uric Acid (UA) content detection kit, co., ltd., beijing Soy Bao technology Co., ltd.) and a microplate reader (Co., molecular Devices, model: spectra MAX 190).
(4) Experimental results:
the experimental results are shown in fig. 3, 4, 5 and 6, wherein the abscissa in fig. 3 represents the group, and the ordinate represents the serum uric acid concentration; the abscissa in fig. 4 represents the group, and the ordinate represents urine uric acid concentration; the abscissa in fig. 5 represents the group, and the ordinate represents the fecal uric acid concentration; the different letters indicate significance between groups, i.e. p <0.05. After the strain suspension containing lactobacillus rhamnosus UA260 strain is used for feeding mice with high uric acid for 2 months, blood is sacrificed and blood is taken for detection, blood Uric Acid (UA) is found to be reduced by 60.3 percent compared with a model mouse, uric acid excretion is promoted mainly through a kidney way, and the lactobacillus rhamnosus UA260 strain has the effect of reducing uric acid in the body of the mice, and the effect is very similar to that of a positive control medicament. In addition, the pathological section results show that the lactobacillus rhamnosus UA260 strain inhibits the damage of colon mucous membrane, the reduction of goblet cell number and the damage of kidney glomerulus and tubular of the hyperuricemia mice, and inhibits inflammatory reaction of colon, liver and kidney, which shows that the lactobacillus rhamnosus UA260 strain has a certain protection effect on the damage of colon, liver and kidney structures caused by hyperuricemia and has potential anti-inflammatory effect.
Although embodiments of the present application have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made therein without departing from the principles and spirit of the application, the scope of which is defined in the appended claims and their equivalents.
The application and its embodiments have been described above with no limitation, but only one of the embodiments of the application is shown in the drawings, and the practical solution and application are not limited thereto. In summary, those skilled in the art, having benefit of this disclosure, will appreciate that the application can be practiced without the specific details disclosed herein.
Claims (9)
1. Lactobacillus rhamnosus UA260 strainLacticaseibacillus rhamnosus UA 260), characterized by: lactobacillus rhamnosus UA260 was deposited with the chinese collection of typical cultures of microorganisms from the cantonese province at 3 months 10 of 2023 under accession number GDMCC No. 63250.
2. Use of a lactobacillus rhamnosus UA260 strain for regulating blood uric acid, employing the lactobacillus rhamnosus UA260 of claim 1, characterized in that: the lactobacillus rhamnosus UA260 is applied to preparing foods or medicines.
3. Use of a lactobacillus rhamnosus UA260 strain according to claim 2 for the modulation of blood uric acid, characterized in that: the food or medicament acts to reduce uric acid.
4. Use of a lactobacillus rhamnosus UA260 strain according to claim 3 for the modulation of blood uric acid, characterized in that: the food is a food with the function of reducing blood uric acid, and the medicine is a medicine with the function of reducing blood uric acid.
5. The use of a lactobacillus rhamnosus UA260 strain according to claim 4 for regulating blood uric acid, wherein: the medicament comprises lactobacillus rhamnosus UA260 of claim 1 and a pharmaceutically non-toxic carrier.
6. The use of a lactobacillus rhamnosus UA260 strain according to claim 5 for regulating blood uric acid, wherein: the dosage form of the medicine is at least one selected from tablets, granules, powder, capsules, suspending agents, emulsions and freeze-dried preparations.
7. The use of a lactobacillus rhamnosus UA260 strain according to claim 6 for the modulation of blood uric acid, characterized in that: the medicine is a composite probiotic tablet.
8. The use of a lactobacillus rhamnosus UA260 strain according to claim 7 for regulating blood uric acid, characterized in that: the food product comprises lactobacillus rhamnosus UA260 of claim 1 and a food-acceptable additive.
9. The use of a lactobacillus rhamnosus UA260 strain according to claim 8 for regulating blood uric acid, wherein: the food is probiotic solid powder.
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