CN115191607A - Lactobacillus plantarum YU28 strain and application thereof - Google Patents
Lactobacillus plantarum YU28 strain and application thereof Download PDFInfo
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- CN115191607A CN115191607A CN202210505939.8A CN202210505939A CN115191607A CN 115191607 A CN115191607 A CN 115191607A CN 202210505939 A CN202210505939 A CN 202210505939A CN 115191607 A CN115191607 A CN 115191607A
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- lactobacillus plantarum
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- uric acid
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Abstract
The invention discloses a lactobacillus plantarum YU28 strain, wherein the lactobacillus plantarum YU28 has been preserved in Guangdong province microbial strain preservation center at 3, 6, 2022, the address of the preservation unit is No. 59 building of Michelia Tokyo No. 100, guangzhou city, and the preservation number is GDMCCNo.62279; the lactobacillus plantarum YU28 disclosed by the invention can be used for preparing foods or medicines for reducing blood uric acid, and the lactobacillus plantarum YU28 obtained by screening has better safety, can regulate and improve the intestinal microecological balance of a host, can play a beneficial role and can generate an exact health effect; in vitro and animal experiments show that the medicine has a strong function of reducing uric acid in blood.
Description
Technical Field
The invention relates to the technical field of microorganisms, and particularly relates to a lactobacillus plantarum YU28 strain and application thereof.
Background
With the rapid development of economy and lifestyle changes, an increase in protein-rich and purine-rich food intake leads to an increase in the prevalence of Hyperuricemia (HUA), which is gradually the second most common metabolic type disease 2 following type ii diabetes. In recent years, hyperuricemia in China is in a remarkable trend of rising and becoming younger, the total morbidity of the hyperuricemia in China is 13.3 percent, the number of patients is about 1.77 hundred million, the total morbidity of gout is 1.1 percent, the number of the patients is about 1466 ten thousand, the ratio of the young hyperuricemia and the gout patients in 18-35 years is nearly 60 percent based on-line adjustment and research, and the hyperuricemia becomes the fourth highest of the people in China after being followed by hyperglycemia, hypertension and hyperuricemia.
The HUA is mainly caused by purine metabolic disorder of the body, hyperuricemia or dysfunction of intestine and kidney excretion, so that uric acid is accumulated in blood and the level is increased. It is considered to be an important risk factor for the development of gout and other diseases such as cardiovascular diseases, metabolic syndrome. Potential therapeutic mechanisms for reducing serum urine include: inhibiting xanthine oxidase (e.g., allopurinol and febuxostat) and using recombinant uricase (e.g., labrasase), a uricosuric agent (e.g., probenecid), and injectable uricase (e.g., pegylated enzyme). Although these drugs are effective in lowering uric acid, there are a number of side effects associated with these drugs, including hepatotoxicity and possibly fatal allopurinol hypersensitivity syndrome. Lifestyle changes, including dietary adjustments to the gut flora, may be another treatment option.
At present, many studies on reducing uric acid by using probiotics have been reported, and strains deeply studied on the function of reducing uric acid are mainly concentrated on lactobacillus, including lactobacillus formaticus, lactobacillus brevis, lactobacillus fermentum and the like. The lactobacillus fermentum JL-3 discovered by the Leben sheath team of the university of Lanzhou Life science school, the lactobacillus brevis DM2918 discovered by the Leben sheath team of the university of Dalian medical science school, and the lactobacillus formaticus PA-3 discovered by the McFarff team of the research and development department of food science of Nippon Mingzhi, all have the effect of reducing uric acid, wherein the lactobacillus formaticus PA-3 has the effect of reducing uric acid successfully proved by animal experiments and human clinical experiments.
Therefore, the developed safe and effective lactobacillus with the function of regulating uric acid has wide application prospect in the fields of fermented foods and medicines.
Disclosure of Invention
The invention aims to provide a lactobacillus plantarum YU28 strain and application thereof, solves the problems, and has the advantages of low manufacturing cost, simple process, low energy consumption, high yield and good product quality.
In order to achieve the purpose, the invention is realized by the following technical scheme:
the invention provides a lactobacillus plantarum YU28 strain, wherein the lactobacillus plantarum YU28 has been preserved in the Guangdong province microbial strain preservation center at 3-6.2022, the preservation unit is No. 59-5 th of Michelia Tokyo 100, guangzhou city, and the preservation number is GDMCC No.62279.
Preferably, the lactobacillus plantarum YU28 is used in the preparation of food and pharmaceuticals.
Preferably, the food and the medicine are blood uric acid reducing food and blood uric acid reducing medicine.
Preferably, the medicament comprises the lactobacillus plantarum YU28 and a pharmaceutically acceptable non-toxic carrier.
Preferably, the dosage form of the medicine is one selected from tablets, granules, powder, capsules, suspending agents, emulsions and freeze-drying preparations.
Preferably, the medicament is a compound probiotic tablet.
Preferably, the food product comprises the lactobacillus plantarum YU28 and an additive.
Preferably, the food is probiotic solid powder, and the probiotic solid powder is prepared from xylo-oligosaccharide, inulin, fructo-oligosaccharide, lemon fruit powder and lactobacillus plantarum YU28.
Preferably, the medicament further comprises a pharmaceutic adjuvant, wherein the pharmaceutic adjuvant comprises: diluent, filling agent, binding agent, preservative, lubricant, dispersing agent, flavoring agent, humectant, antioxidant, coloring agent, stabilizer, buffering agent and pH value regulator, wherein the medicinal nontoxic carrier comprises starch, starch derivative, lactose and polyethylene glycol, and the diluent comprises starch, starch derivative, lactose, sucrose, vegetable oil, wax and fatty acid.
Preferably, the lactobacillus plantarum YU28 is freeze-dried powder, and the freeze-dried powder is prepared by freeze-drying a bacterial suspension of the lactobacillus plantarum YU28 in a freeze dryer under a sterile environment in a vacuum manner.
The invention has the beneficial effects that: (1) The lactobacillus plantarum YU28 strain obtained by screening has better safety, can regulate and improve the intestinal microecological balance of a host, plays the beneficial effect and produces exact health effect, and in vitro and animal experiments show that the lactobacillus plantarum YU28 strain has stronger function of reducing uric acid in blood.
(2) The lactobacillus plantarum YU28 obtained by screening can be used for preparing uric acid reducing food and medicines, and tests show that the obtained food also has the effect of reducing blood uric acid.
Drawings
FIG. 1 shows the inhibition of xanthine oxidase in Lactobacillus plantarum YU28 in vitro assay, with strain number on the abscissa (28 is the invention) and xanthine oxidase inhibition in vitro on the ordinate, according to specific example 2 of the present invention;
FIG. 2 shows the degradation rate of inosine, a key precursor for uric acid synthesis, in the in vitro assay of Lactobacillus plantarum YU28 according to an embodiment of the present invention, with the strain number on the abscissa (No. 28 is the present invention) and the degradation rate of inosine on the ordinate;
FIG. 3 is a graph of the ability of Lactobacillus plantarum YU28, the abscissa representing the group and the ordinate representing the serum uric acid concentration, to lower the serum uric acid level of an animal, according to an embodiment 2 of the present invention;
FIG. 4 is a graph of the ability of Lactobacillus plantarum YU28 to lower the uric acid level in animal urine according to example 2 of the present invention, with the abscissa representing the group and the ordinate representing the uric acid concentration in urine;
FIG. 5 is a graph showing the ability of Lactobacillus plantarum YU28 to reduce the urinary level of animal feces according to example 2 of the present invention, with the group on the abscissa and the concentration of uric acid in feces on the ordinate.
Detailed Description
The following examples are provided to further illustrate embodiments of the present invention. The following examples are only for illustrating the technical solutions of the present invention more clearly, and the protection scope of the present invention is not limited thereby.
Lactobacillus plantarum YU28 has been deposited at 6.3.2022 in Guangdong provincial collection of microorganisms, the location of the deposit is No. 59, no. 5, lou of Miehu 100, guangzhou city, and the deposit is GDMCC No.62279, and the Lactobacillus plantarum Latin text is named after classification: lactplantibacillus plantarum YU28.
The embodiment of the invention also provides the application of the lactobacillus plantarum YU28 in preparing food and medicines, and the food is to be understood in a broad sense, and can be in any form which can be used, namely, the food can be health-care products, drinks, fermented foods and the like besides the conventional food form.
In this example, the food and the drug are a blood uric acid lowering food and a blood uric acid lowering drug.
In this example, the drug comprises Lactobacillus plantarum YU28 and a pharmaceutically acceptable non-toxic carrier.
In this embodiment, the dosage form of the drug is one selected from tablets, granules, powders, capsules, suspensions, emulsions and lyophilized preparations.
In this embodiment, the medicament is a composite probiotic tablet.
In this embodiment, the food product includes lactobacillus plantarum YU28 and an additive.
In the embodiment, the food is probiotic solid powder, and the probiotic solid powder is prepared from xylo-oligosaccharide, chrysanthemum powder, fructo-oligosaccharide, lemon fruit powder and lactobacillus plantarum YU28.
In this embodiment, the drug further includes a pharmaceutical excipient, which includes: diluents, fillers, binders, preservatives, lubricants, dispersants, flavoring agents, humectants, antioxidants, colorants, stabilizers, buffers, pH adjusters, which may be present as required to aid in the stability of the formulation or to aid in the activity or bioavailability or to provide an acceptable taste or odor in the case of oral administration, pharmaceutically acceptable carriers which are not toxic include starch, starch derivatives, lactose, polyethylene glycols, diluents which include starch, starch derivatives, lactose, sucrose, vegetable oils, waxes, fatty acids.
In this embodiment, lactobacillus plantarum YU28 is freeze-dried powder, and the freeze-dried powder is obtained by freeze-drying a suspension of lactobacillus plantarum YU28 in a sterile environment by using a freeze dryer.
Example 1 isolation and identification of Lactobacillus plantarum YU28
1. Culture medium formula optimization
And (3) performing an optimized culture medium experiment in groups to finally obtain the optimal formula of the lactic acid bacteria culture medium:
MRS solid culture medium 1L formula: casein peptone 10.0g/L, beef extract 10.0g/L, yeast extract 5.0g/L, glucose 20.0g/L, dipotassium hydrogen phosphate 2.0g/L, tween 801.0g/L, triammonium citrate 2.0g/L, sodium acetate 5.0g/L, magnesium sulfate 0.1g/L, manganese sulfate 0.05g/L, agar 17.5g; sterilizing at 121 deg.C for 20min at pH 6.5.
MRS broth culture medium formula: casein peptone 10.0g/L, beef extract 10.0g/L, yeast extract 5.0g/L, glucose 20.0g/L, dipotassium phosphate 2.0g/L, tween 801.0g/L, triammonium citrate 2.0g/L, sodium acetate 5.0g/L, magnesium sulfate 0.1g/L, manganese sulfate 0.05g/L, pH6.5, and sterilization at 121 ℃ for 20min.
2. Strain screening
The lactobacillus plantarum YU28 strain originates from the intestinal tract of a healthy person and is isolated from the feces of a healthy adult. Taking the feces of healthy adults, diluting the sample with sterile normal saline by 10 times of gradient until the concentration is 10 -3 Spreading on MRS solid culture medium, culturing at 37 deg.C for 24-48 hr, observing colony morphology, selecting suspected colony of lactobacillus on MRS solid culture medium, respectively transferring to MRS liquid culture medium for pure culture, and separating 33 strainsThe strains were numbered for each strain.
The specific experimental steps of the determination of the uric acid reducing capability of the separated and purified strains are shown in example 2 and example 3, and the lactobacillus plantarum YU28 with strong uric acid reducing capability is obtained by screening.
3. Strain identification
Extracting bacterial total DNA from the screened lactobacillus plantarum YU28, and carrying out 16s rDNA amplification, wherein the primers are as follows:
sgF:5'-AGAGTTTGATCATGGCTCAG-3'(SEQ ID NO：1)
sgR:5'-TAGGGTTACCTTGTTACGACTT-3'(SEQ ID NO：2)
the primers are used for PCR amplification and agarose gel electrophoresis, and then gel cutting, recovery and sequencing are carried out.
The sequencing result of the 16s DNA of the strain obtained by separation is shown as SEQ ID NO. 3.
Example 2 in vitro xanthine oxidase inhibition assay of Lactobacillus plantarum YU28
The in vitro xanthine oxidase inhibition rate of the strain is detected according to the following steps:
the test strain was inoculated into MRS liquid medium, cultured at 37 ℃ for 48h, left to ferment at 37 ℃ for 48h, and 2mL of the culture was centrifuged at 12,000 Xg and 4 ℃ for 10 minutes. Taking the supernatant for later use;
preparing a xanthine buffer solution: accurately weighing 0.08g NaOH dissolved in 10ml distilled water, weighing 15mg xanthine dissolved in NaOH solution, weighing 0.27g KH2PO4 dissolved in 10ml, adding KH2PO4 solution into xanthine-NaOH solution, and mixing;
preparing a xanthine oxidase solution: weighing 1mg of the mixture and dissolving the mixture in 1mL of PBS for later use;
adding the medicines according to the sequence in the table 1, uniformly mixing, carrying out water bath reaction at 37 ℃ for 10min, terminating the reaction, and measuring the absorbance at 290 nm. The inhibition rate (I) of the extract on xanthine oxidase is that OD1-OD4 are respectively the absorbance of test 1-4 at 290nm (OD value measured in the 1 st test tube is measured by using the 3 rd test tube as blank control; OD value measured in the 2 nd test tube is measured by using the 4 th test tube as blank control). The inhibition rate calculation formula is as follows:
I＝[1-(OD1-OD3)/(OD2-OD4)]*100％
TABLE 1 xanthine oxidase Activity inhibition test reaction System
The experimental result is shown in figure 1, the inhibition rate of the lactobacillus plantarum YU28 on xanthine oxidase is 87.43%, and the lactobacillus plantarum YU28 has strong capacity of reducing uric acid in vitro.
Example 3 in vitro inosine degradation Rate test of Lactobacillus plantarum YU28
The in vitro inosine degradation rate of the strain is detected according to the following steps:
LAB strains were inoculated in MRS and grown for 48h under anaerobic conditions at 37 ℃. Taking 2mL of the culture solution, 4,000 Xg, centrifuging at 4 ℃ for 10min, washing with 1ml of 0.85% NaCl for 2 times, resuspending with 1mL of inosine buffer, incubating at 37 ℃ for 60min, and shaking up. 4,000 Xg, centrifuged at 4 ℃ for 10min, and the supernatant was subjected to content detection using an Inosine kit (kit name: inosine (Inosine) content detection kit, shanghai Jiang Lai Biotech Co., ltd.).
Preparing an inosine buffer solution: inosine (2 mg) was accurately weighed, dissolved in 10ml of 200mmol/L KH2PO4 solution (0.27 g in 10 ml), and adjusted to pH 7.0 with NaOH (0.08 g in 10 ml).
The experimental result is shown in figure 2, the degradation rate of the lactobacillus plantarum YU28 on inosine is 88.53%, and the lactobacillus plantarum YU28 has strong capacity of reducing uric acid in vitro.
EXAMPLE 4 in vivo efficacy study (animal experiments) of Lactobacillus plantarum YU28
(1) Preparation of the experimental strains:
the activated lactobacillus plantarum YU28 was inoculated in MRS liquid medium, cultured at 37 ℃ for 18h, centrifuged at 6000r/m for 10min, washed with sterilized physiological saline, and the cells were collected. Then, 0.85% physiological saline was added to adjust the number of bacteria to about 1.0X 10 10 CFU/mL, then subpackaging viable bacteria into 5mL centrifuge tubes according to daily use amount, and carrying out intragastric administration with the dosage of 0.1 mL/each daily, 10 per group and 4 groups in total, wherein the intragastric administration is required for 60 days.
(2) Grouping and feeding modes of experimental animals:
a total of 40 male mice of 8 week old C57BL/6J strain, fed freely for 2 weeks, divided on average into 4 groups of 10 mice each:
normal group (CON group): after the intragastric administration of 0.85% normal saline, intragastric administration of 0.85% normal saline;
hyperuricemia model group (HUA group): intragastric administration of adenine (75 mg/kg/day) and Potassium Oxonate (200 mg/kg/day) for one hour, and intragastric administration of 0.85% normal saline;
positive control group (ALL group): after one hour of intragastric administration of adenine (75 mg/kg/day) and Potassium Oxonate (200 mg/kg/day), allopurinol (5 mg/kg/day) was intragastric administered.
Lactobacillus plantarum YU28 group (YU 28 group): after one hour of intragastric administration of adenine (75 mg/kg/day) and potassium Oxazinate (200 mg/kg/day), lactobacillus plantarum YU28 strain (10) 9 CFU/per mouse/day);
establishing a model and intervening lactobacillus plantarum for 2 months, and then collecting excrement and urine.
(3) Sample collection and analytical testing:
mice were anesthetized with tribromoethane and sacrificed by cervical dislocation after blood was drawn from the eyeball. Blood was centrifuged at 4000 rpm for 10min, and serum was separated, and the uric acid content was detected using a kit (kit name: uric Acid (UA) content detection kit, manufactured by Beijing Solebao technologies, ltd.) and an enzyme-labeling instrument (manufactured by Molecular Devices, model: spectra MAX 190).
(4) Results of the experiment
The results of the experiments are shown in fig. 3, 4 and 5, with different letters indicating significance between groups, i.e. p <0.05. The sacrifice blood taking test after the lactobacillus plantarum strain-containing suspension is used for feeding the mouse with high uric acid for 2 months finds that the blood Uric Acid (UA) is reduced by 60.3 percent compared with the model mouse, and the uric acid intestinal tract and kidney drainage water is improved on average, which indicates that the lactobacillus plantarum YU28 has the efficacy of reducing the uric acid in the mouse body.
The above description is only for the preferred embodiment of the present invention, but the scope of the present invention is not limited thereto, and any person skilled in the art should be able to cover the technical scope of the present invention and the equivalent alternatives or modifications according to the technical solution and the inventive concept of the present invention within the technical scope of the present invention.
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Claims (10)
1. The lactobacillus plantarum YU28 strain is characterized in that the lactobacillus plantarum YU28 has been preserved in 6.3.2022 in Guangdong province, namely, the culture collection center of the strain is No. 59 Lou 5 of Michelia Tokyo 100, guangzhou city, and the preservation number is GDMCC No.62279.
2. Use of the strain of lactobacillus plantarum YU28, according to claim 1, characterized by: the lactobacillus plantarum YU28 is applied to preparation of foods and medicines.
3. The use of a strain of lactobacillus plantarum YU28 according to claim 2, characterized in that: the food and the medicine are food for reducing blood uric acid and medicine for reducing blood uric acid.
4. Use of a strain of lactobacillus plantarum YU28 according to claim 2, characterized in that: the medicine comprises the lactobacillus plantarum YU28 and a medicinal nontoxic carrier.
5. The use of a strain of lactobacillus plantarum YU28 according to claim 2, characterized in that: the dosage form of the medicine is one of tablets, granules, powder, capsules, suspending agents, emulsions and freeze-drying preparations.
6. The use of a strain of lactobacillus plantarum YU28 according to claim 2, characterized in that: the medicine is a compound probiotic tablet.
7. Use of a strain of lactobacillus plantarum YU28 according to claim 2, characterized in that: the food comprises the lactobacillus plantarum YU28 and an additive.
8. The use of a strain of lactobacillus plantarum YU28 according to claim 2, characterized in that: the food is probiotic solid powder, and the probiotic solid powder is prepared from xylo-oligosaccharide, inulin, fructo-oligosaccharide, lemon fruit powder and lactobacillus plantarum YU28.
9. The use of a strain of lactobacillus plantarum YU28 according to claim 4, characterized in that: the medicament also comprises pharmaceutic adjuvant which comprises: diluent, filler, binder, preservative, lubricant, dispersant, flavoring agent, humectant, antioxidant, colorant, stabilizer, buffer, pH regulator, wherein the nontoxic carrier for medical use comprises starch, starch derivative, lactose, polyethylene glycol, and the diluent comprises starch, starch derivative, lactose, sucrose, vegetable oil, wax, and fatty acid.
10. Use of a strain of lactobacillus plantarum YU28 according to claim 2, characterized in that: the lactobacillus plantarum YU28 is freeze-dried powder, and the freeze-dried powder is formed by freeze-drying a bacterial suspension of the lactobacillus plantarum YU28 in a freeze dryer under a sterile environment in a freeze-vacuum manner.
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| CN116286534A (en) * | 2023-03-22 | 2023-06-23 | 东北农业大学 | Lactobacillus plantarum for relieving hyperuricemia, and compound and application thereof |
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| CN108004155A (en) * | 2016-10-28 | 2018-05-08 | 深圳华大基因研究院 | Lactobacillus plantarum pc-26 bacterial strains and its application |
| CN108048368A (en) * | 2018-01-25 | 2018-05-18 | 吉林省命之元生物科技有限公司 | One UA-416 plants of lactobacillus plantarum and its application |
| US20200214328A1 (en) * | 2019-01-03 | 2020-07-09 | Glac Biotech Co., Ltd. | Food composition and pharmaceutical composition with strains of lactic acid bacteria for reducing blood uric acid concentration |
| CN110055199A (en) * | 2019-05-24 | 2019-07-26 | 吉林省命之元生物科技有限公司 | One UA149 plants of lactobacillus plantarum and its application |
| CN112795500A (en) * | 2020-12-21 | 2021-05-14 | 扬州大学 | Lactobacillus plantarum capable of relieving hyperuricemia and application thereof |
| CN113278552A (en) * | 2021-05-21 | 2021-08-20 | 深圳市华大农业应用研究院 | Pediococcus acidilactici HG-7 strain and application thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN116286534A (en) * | 2023-03-22 | 2023-06-23 | 东北农业大学 | Lactobacillus plantarum for relieving hyperuricemia, and compound and application thereof |
| CN116286534B (en) * | 2023-03-22 | 2023-11-17 | 东北农业大学 | Lactobacillus plantarum for relieving hyperuricemia, and compound and application thereof |
| WO2024193100A1 (en) * | 2023-03-22 | 2024-09-26 | 东北农业大学 | Lactiplantibacillus plantarum for alleviating hyperuricemia, complex comprising same and use thereof |
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Application publication date: 20221018 |