CN114164130A

CN114164130A - Propurine precursor-reducing probiotic strain, composition and application thereof

Info

Publication number: CN114164130A
Application number: CN202010947184.8A
Authority: CN
Inventors: 刘海峰; 李春艳; 刘艳红; 陈雅珊
Original assignee: Ningbo Beiyijia Biotechnology Co ltd
Current assignee: Ningbo Beiyijia Biotechnology Co ltd
Priority date: 2020-09-10
Filing date: 2020-09-10
Publication date: 2022-03-11
Anticipated expiration: 2040-09-10
Also published as: CN114164130B

Abstract

The invention discloses a probiotic strain for reducing purine precursors, a composition and application thereof. The capacity of screening the purine-reducing precursor of the probiotics under the condition of rich culture medium is provided for the first time, and a culture environment relative to a simulated human intestinal tract is obtained to obtain the lactobacillus reuteri KLR-4 with the preservation number of CCTCC M2020367. Meanwhile, the active ingredient of the probiotic composition provided by the invention comprises lactobacillus reuteri strain KLR-4. Lactobacillus reuteri KLR-4 capable of degrading purine precursors in an animal model is used as a new means for reducing blood uric acid and treating gout, and the ingestion of food-borne purine is obviously reduced under the condition of not reducing the quality of life (low-purine diet) so as to achieve the effect of low-purine diet; compared with the clinical chemical treatment mode, the medicine has the advantages of no toxic or side effect, higher safety and wide application prospect.

Description

Propurine precursor-reducing probiotic strain, composition and application thereof

Technical Field

The invention belongs to the field of prevention and treatment of hyperuricemia and gout diseases, and particularly relates to a probiotic strain for reducing purine precursors, a probiotic composition and application thereof.

Background

Hyperuricemia is a chronic metabolic disease with clinical manifestations of elevated blood uric acid levels above the normal range (> 420 μmol/L in men and >360 μmol/L in women). Patients with hyperuricemia may develop nephropathy, lithangiuria, arteriosclerosis, cardiovascular disorders, cerebrovascular disorders and the like in addition to gout caused by uric acid crystallization. The number of hyperuricemia patients in China is reported to reach 1.7 hundred million, wherein gout patients exceed 8000 ten thousand, and the annual growth rate is rapidly increasing by 9.7% per year. In china, gout has become the second largest metabolic disease after diabetes.

Under normal physiological conditions, the total amount of uric acid in a human body is about 1200mg, and there are two main ways for the excretion of uric acid, about 2/3 is excreted in the form of urine through the kidney, and 1/3 is excreted in the form of feces through the intestinal tract. Uric acid in a human body is mainly produced by purine nucleic acid substances in a metabolic mode, the purine nucleic acid sources mainly have two aspects, one is the ingestion of food-borne purine nucleic acid, such as animal viscera, seafood, beer and the like which are rich in purine nucleic acid, the blood uric acid can be increased, the other is uric acid generated by degrading the purine nucleic acid substances after cell apoptosis in the in vivo cell metabolism process, such as uric acid generated by the fact that a large amount of nucleic acid substances are released by tumor death in the radiotherapy and chemotherapy process of a patient with solid tumor, or intestinal inflammation (such as acute enteritis caused by rotavirus infection) can also cause the death of intestinal cells to release nucleic acid substances, and therefore the serious hyperuricemia is caused after the metabolism of the purine nucleic acid substances into uric acid. In patients with hyperuricemia after non-radiotherapy and chemotherapy, excessive uric acid production is closely related to the increase of food-borne purine nucleic acid intake. The prevention and treatment of hyperuricemia and gout by a dietary mode of restricting purine intake has achieved wide consensus in the medical field, and as a basic treatment scheme, the Chinese guideline for diagnosis and treatment of renal disease hyperuricemia is written. However, it is very difficult to strictly limit the intake of these ingredients in life because the taste-providing ingredients in animal and plant cells and food flavors which are very delicious in the diet contain purine precursor ingredients (nucleotides, nucleosides, etc.), and particularly, the purine content in seafood and animal meat is relatively high. The dietary pattern of strict restriction of purine nucleic acid intake will seriously affect the quality of life of the patient.

At present, two main strategies for clinically treating hyperuricemia and gout exist, firstly, the main strategies are to inhibit the generation of uric acid, and most of the medicines are xanthine oxidase inhibitors, such as allopurinol, febuxostat and the like; secondly, the excretion of uric acid is increased, and the drugs mostly act on uric acid transport proteins and the like, such as probenecid, benzbromarone and the like; in addition, some analgesic and anti-inflammatory drugs, such as colchicine, glucocorticoid, etc. However, these drugs have serious damage to liver and kidney, and cannot be used for many purposes or for a long time, and many patients abandon the treatment of the drugs because they cannot tolerate the side effects. And the use of uricosuric drugs can reduce the level of blood uric acid, but increase the uric acid content in urine, thereby increasing the risk of suffering uric acid calculi. Although there are several recombinant urate oxidase drugs for treating hyperuricemia, the drugs are all injection preparations, and no oral urate oxidase product exists. The urate oxidase for injection is exogenous protein, has high immunogenicity, cannot be used for a long time, has complex production process, high cost and very high selling price, and is mainly used for hyperuricemia caused by massive cell death of some cancer patients in the chemotherapy process at present.

The probiotics as an important flora member of human intestinal tract has more advantages than medicines in the aspect of treating hyperuricemia, and how to screen the probiotic strains with the function of reducing the blood uric acid becomes a hotspot of current research. Tsukamur pharmaceutical company patent (CN1812801A) discloses that a group of lactobacillus fermentum and a strain of yeast have the functions of decomposing inosine and guanosine and reducing blood uric acid, however, the bacteria can produce uric acid when degrading inosine and guanosine, and the increase of the concentration of uric acid in intestinal tract can also cause hyperuricemia by intestinal absorption, so the bacteria are not ideal probiotics for reducing blood uric acid. Yanghua bin et al, university of Dalian medical sciences, reported that Lactobacillus brevis (DM9218) was screened for its ability to reduce inosine and guanosine, however, it was not tested to investigate whether the strain has the ability to degrade nucleotides. It is well known that the majority of purine precursor species in food exist in the form of DNA (deoxyribonucleic acid) and RNA (ribonucleic acid). After entering the human digestive tract, pancreatic deoxyribonuclease (DNase) and ribonuclease (RNase) secreted by the intestinal tract are hydrolyzed into deoxynucleotides and nucleotides), which are further decomposed into nucleosides and phosphoric acid by Nucleotidase, and nucleosides are decomposed into bases and pentose sugars by Nucleotidase. Food-derived purine precursors (purine nucleotides, purine nucleosides, purine bases) are generally not used for synthesizing nucleic acids of human bodies after being absorbed by the human bodies, but are metabolized into uric acid to be discharged out of the bodies, and the absorption efficiency of the three purine precursors is different due to different solubilities. Jun OGAWA studies have shown that purine nucleotides and purine nucleosides are more readily taken up by cells than purine bases (Jun OGAWA, Noda Institute for Scientific Research GRANT, 2006). It was found that the solubility of purine nucleotides is at most 200-fold or more as high as that of purine nucleosides and 10-ten thousand-fold or more as high as that of purine bases (for example, the solubility of guanine nucleotide in water at 20 ℃ C. is 20g/100 ml; the solubility of guanosine in water is 77.6mg/100 ml; and the solubility of guanine in water is 0.17mg/100 ml). Therefore, nucleotides and deoxynucleotides are most easily absorbed into blood to cause hyperuricemia, and efficient degradation of purine nucleotides and deoxypurine nucleotides is a crucial step in order to reduce the absorption of food-borne purines. However, in the published screening studies of purine-lowering probiotics, nucleosides are mostly used as the screening substrates, and for example, japanese patent No. (CN 200480017815.5) discloses a group of purine-lowering lactic acid bacteria and yeast, and the purine substrates selected in the screening are only inosine and guanosine. Lactobacillus gasseri developed by Mingmy corporation of Japan (patent No. CN102747004B) also uses inosine and guanosine as substrates to test the decomposition capability, and the lactic acid bacteria strain is developed into a yoghourt product for sale; however, the recent clinical research results of this strain in patients with hyperuricemia showed that its effect of reducing blood uric acid is not significant (Hisashi Yamanaka, MODERN REUMATOLOGY, 2019, VOL.29, NO.1, 146-150), presumably related to its inability to efficiently degrade purine nucleotides. The article by Yanghong et al (gold square, Yanghong, microbiological announcement, 2018,45(8):1757 @ 1769) of Shanghai traffic university and the patent by Jiaxing Yinuokang (CN 108486007) both disclose a purine nucleic acid-reducing Lactobacillus casei (ZM15) which has the effect of degrading nucleosides (the rate of degrading adenosine is 2.56 mmol/(h.g), the rate of degrading guanosine is 2.57 mmol/(h.g)), and the effect of reducing blood uric acid is observed on a hyperuricemic rat model. In addition, Zhangxin, etc. also constructs urate oxidase into lactobacillus to construct engineering probiotics with uric acid reducing capacity, so as to reduce uric acid content in intestinal tract and reach blood uric acid reducing effect.

However, in analyzing the published results of probiotic studies on degradation of purine precursors, the inventors of the present application found that most of the studies were not comparable to each other in terms of their degradation ability (e.g., as a percentage of degraded nucleosides but without a definite time; or as a rate of degradation of substrate per gram of bacteria but without a definite wet or dry weight of the bacteria and with different reaction conditions in all studies). In addition, it has been found that most scientific studies and patents evaluate the degradation of purine precursors by probiotics by collecting cultured microorganisms by centrifugation, adding microbial cells to a buffer containing only purine precursor (nucleoside or nucleotide) substrate, and testing the ability of probiotics to degrade nucleotides or nucleosides. This test method has a serious drawback that living microorganisms need nutrients to maintain their own lives at all times, and in an environment where nutrients are deficient, the microorganisms live and absorb some nutrients that are not utilized in a normal state by being forced to digest, however, once nutrients are abundant, the microorganisms do not preferentially utilize these nutrients, so that probiotics selected by this reaction system containing only purine precursor substrates may not digest and decompose purine precursor (nucleoside, nucleotide, deoxynucleoside, deoxynucleotide, etc.) substrates under the conditions of abundant nutrients in the intestinal tract after entering the human body, and thus the selected probiotic strains may not have an effect in practical use.

Disclosure of Invention

In order to overcome the defects in the prior art, the invention aims to provide a probiotic strain for reducing purine precursors, a probiotic composition and application thereof. The strains and compositions are effective for degrading purine precursors.

In order to achieve the above object, the present invention provides the following technical solutions:

in a first aspect, the present invention provides a Lactobacillus reuteri strain (Lactobacillus reuteri) for reducing purine precursors, which has the ability to degrade purine precursors, including but not limited to one or more of inosine/deoxyinosine, inosinic acid/deoxyinosinic acid, guanosine/deoxyguanosine, guanylic acid/deoxyguanylic acid, adenosine/deoxyadenosine, adenylic acid/deoxyadenylic acid.

Preferably, the lactobacillus reuteri strain for reducing purine precursors has an average rate of degrading purine precursors of at least 50mg/OD h.l, or at least 100mg/OD h.l, or at least 150mg/OD h.l, or at least 200mg/OD h.l, or at least 250mg/OD h.l, or at least 300mg/OD h.l, and the purine precursor material includes one or more of inosine/deoxyinosine, inosinic acid/deoxyinosinic acid, guanosine/deoxyguanosine, guanylic acid/deoxyguanylic acid, adenosine/deoxyadenosine, adenylic acid/deoxyadenylic acid.

Preferably, the Lactobacillus reuteri strain for purine precursor (Lactobacillus reuteri) is Lactobacillus reuteri KLR-4 which has been deposited with China center for type culture Collection (CCTCC NO: M2020367) at 28.7.2020.

In a second aspect, the present invention provides a probiotic composition of precursors of norpurines, the active ingredient of which comprises lactobacillus reuteri strain KLR-4 according to the above scheme.

Preferably, the probiotic composition contains Lactobacillus reuteri KLR-4 with viable count of 1 x 10⁵～ 5*10¹²CFU/g composition.

In a third aspect, there is provided a use of the lactobacillus reuteri strain or the composition for preparing a medicament or food for preventing and treating hyperuricemia and/or gout.

Preferably, in the above application, the medicament is in a form for oral administration.

Preferably, in the above application, the dosage form is selected from the group consisting of: solutions, suspensions, emulsions, powders, lozenges, pills, syrups, troches, tablets, chewing gums, syrups, and capsules.

Preferably, in the above application, the food comprises general food, health food, or formula food for special medical use.

The invention has the advantages and beneficial effects that:

in order to more effectively screen the probiotics for degrading the purine precursor in the simulated intestinal environment, the application firstly provides the purine-reducing capability of screening the probiotics under the condition of rich culture medium. Through optimizing culture conditions and detection methods, a method for screening a culture environment simulating human intestinal tracts is obtained, probiotics with remarkable purine precursor reduction capability under two test conditions of a non-nutrient medium only containing purine precursors and a rich nutrient medium simultaneously containing purine precursors and carbon-nitrogen source nutrients are screened through multiple rounds of screening and optimization, and the capability of the lactobacillus reuteri KLR-4 is remarkably enhanced compared with that of lactobacillus casei from Japanese purine precursor lactobacillus probiotics and domestic patent-published purine precursor reduction probiotics (see example 1 specifically). The effect test of the uric acid reduction in an animal model shows that, under the same dosage, the KLR-4 strain has a more obvious uric acid reduction effect compared with a KLR-1 strain only capable of degrading purine nucleoside or deoxypurine nucleoside or a KLR-3 strain only capable of degrading purine nucleotide and deoxypurine nucleotide, and the effect of the mixed bacterium powder of the KLR-1 and the KLR-3 with the same dosage is basically achieved. Lactobacillus reuteri also has unique advantages over other reported reduced purine precursor probiotics: the intestinal tract colonization ability is strong, and the secreted Roiximab can inhibit intestinal tract harmful bacteria, enhance immunity and inhibit enteritis (Pangjie, journal of Chinese bioengineering, 2011, 31 (5): 131-. The intestinal tract cell death can be reduced by relieving enteritis, so that the generation of endogenous uric acid can be reduced, purine intake in diet can be synergistically degraded, and a better effect of reducing blood uric acid can be achieved, therefore, the lactobacillus reuteri with the function of degrading purine precursors is taken as a new means for reducing blood uric acid and treating gout, the intake of food-borne purine is obviously reduced under the condition of not reducing the quality of life (low-purine diet), and the effect of low-purine diet is achieved; compared with the clinical chemical treatment mode, the medicine has the advantages of no toxic or side effect, higher safety and wide application prospect.

Biological preservation Instructions

Lactobacillus reuteri (Lactobacillus reuteri) is preserved in China center for type culture Collection with the preservation address: china, wuhan university, zip code: 430072, preservation organization abbreviation: CCTCC, the preservation date is 28 months 7 and 2020, the biological preservation number is CCTCC M2020367, the strain name is as follows: lactobacillus reuteri KLR-4.

Drawings

FIG. 1. Standard Curve for the detected concentrations of different purine precursor substrates;

FIG. 2 is a LC-MS analysis chart of the mixed standard;

Detailed Description

The technical solution of the present invention will be further described in detail with reference to specific embodiments. It is to be understood that the following examples are only illustrative and explanatory of the present invention and should not be construed as limiting the scope of the present invention. All the technologies realized based on the above-mentioned contents of the present invention are covered in the protection scope of the present invention.

Unless otherwise indicated, the raw materials and reagents used in the following examples are all commercially available products or can be prepared by known methods. The experimental procedures used in the following examples are all conventional procedures unless otherwise specified.

Example 1: screening of probiotics

The probiotic strains to be tested (including more than 10 species of 130 lactobacillus probiotic strains such as lactobacillus rhamnosus, lactobacillus helveticus, lactobacillus casei, lactobacillus reuteri, lactobacillus plantarum, lactobacillus acidophilus, lactobacillus salivarius, lactobacillus gasseri, lactobacillus fermentum and the like, bifidobacterium animalis, bifidobacterium longum, bifidobacterium infantis and pediococcus acidilactici) screened and stored in the laboratory are activated, inoculated into an MRS culture medium for culture, and subjected to facultative anaerobic (standing) or strict anaerobic (oxygen concentration) at 37 DEG C<0.5%) for 8-20 h, centrifuging, collecting the cells, washing with phosphate buffer (100mM, pH7.0) for 3 times, and adjusting to OD₆₀₀2.7(1 OD about 2.0-3.0 x 10)⁸CFU/mL), were added to a phosphate test buffer (20mM, pH6.86) containing 0.7mg/mL of each of adenylic acid, guanylic acid, inosinic acid, adenosine, guanosine and inosine, respectively, to give a final cell concentration OD₆₀₀The reaction was incubated at 37 ℃ for 1 hour (0.6), centrifuged at 8000g for 5min, and 900. mu.l of the supernatant was collected, quenched by addition of 100. mu.l of 100mM perchloric acid solution, filtered through a 0.22 μm membrane, and then detected by High Performance Liquid Chromatography (HPLC).

Standard curve formulation procedure for purine precursor substrates: accurately weighing analytically pure (purity > 95%) adenosine, deoxyadenosine, inosine, guanosine, deoxyguanosine, adenylic acid, deoxyadenylic acid, disodium guanylate, deoxyguanylate and disodium inosinate, adding sterile water to prepare standards with different gradient concentrations, operating according to the above termination reaction flow, carrying out HPLC detection, establishing standard curves of various substrates, taking guanylate, guanosine, deoxyguanylate and deoxyguanosine as examples, and showing the standard curves in figure 1.

The specific detection method comprises the following steps: sepax Bio C18 chromatography column, mobile phase a: 20mM potassium dihydrogen phosphate buffer (pH2.5), mobile phase B: methanol at a flow rate of 1.0ml/min, a detection wavelength of 254nm, and a sample loading of 20. mu.l.

Table 1: elution gradient of HPLC

Time min	0	20	21	25	26	26
							Mobile phase A (%)	100	100	90	90	100	100
Mobile phase B (%)	0	0	10	10	0	0

Table 2: retention time of respective purine precursor substrates

And (3) carrying out purine precursor reduction test screening on the probiotic strains to be tested according to the purine precursor detection method. The results of the rate of degradation of purine precursors by each strain were determined as follows:

table 3: rates of degradation of purine precursors by different probiotic bacterial strains

The purine-reduced precursor is screened to obtain the compound with the advantages of degrading purine nucleoside and purine nucleotide (a)>100mg/OD · h · L): lactobacillus reuteri (KLR-4; KLR-5; KLR-8). Lactobacillus gasseri (PA-3) isolated and screened from the Japanese Mingzhi uric acid reducing probiotic product, ability to degrade Inosine as determined by the test method in this example and the results reported in the open literature (Narumi Yamada, et al. Lactobacillus gasseri PA-3 Uses the genes IMP, insulin and HypOxanthine and reduction thereof amplification in Rats, Microorganisms (2017), 5,10) (about 600 pmol/min/L10 pmol/L10)⁹Number of bacteria) are relatively close. However, the inosine reducing ability of the lactobacillus reuteri strain is very different from that of the lactobacillus reuteri strain KLR-4 screened by the patent.

The digestion of DNA in food was degraded into DNA, so that the selected dominant probiotic strains were further subjected to screening tests using deoxyribonucleosides (deoxyguanosine, deoxyadenosine) and deoxyribonucleotides (deoxyadenosine, deoxyguanylic acid) as substrates, and the test results are shown in table 4. The screening result shows that the advantageous probiotics for degrading the nucleoside/nucleotide have stronger capacity of degrading the deoxynucleotide/deoxynucleoside, can degrade products of DNA and RNA in food after intestinal digestion, and reduce the absorption of food-borne purine precursors.

Table 4: degradation testing of deoxynucleosides and deoxynucleotides by probiotics

Example 2: prourinalysis ability of probiotics under nutritional condition

The intestinal tract of a human body is an environment with rich nutrition, and the probiotics only contain nucleoside and nucleotide substrates and degrade purine nucleoside and nucleotide under the condition of no nutrition, so that the degrading capability of the intestinal tract under the condition of rich nutrition cannot be ensured, and therefore, the screening of a screening condition which contains nutrition and does not influence detection is very important. The inventor screens and optimizes the test system containing nutrition, which can ensure the growth of thalli and does not influence the detection, and the test system comprises the following components: 20mM phosphate; 0.2% glucose, 0.25% yeast powder and 0.2% ammonium sulfate, pH 6.86. The dominant probiotic strain selected in example 1 was rescreened in the nutrient containing test buffer described above. Adjusting candidate strain to OD₆₀₀2.7, the amount was added to the measurement reaction system (adjustment of the cell concentration OD)₆₀₀0.3), reaction 3h to terminate the reaction, OD was measured again₆₀₀Value, evaluating the proliferation status of the probiotic during the reaction. After the reaction, 900. mu.l of the supernatant was centrifuged, 100. mu.l of perchloric acid stop solution was added, and the mixture was filtered through a 0.22 μm filter and subjected to HPLC to determine the efficiency of degradation of the purine precursor. At the same time, the degradation rate was compared with that of example 1 to evaluate the difference between the probiotic bacteria in the nutrient-free reaction system and the nutrient-containing reaction system. The results are as follows:

TABLE 5 growth of probiotic strains in nutrient-containing test systems

Bacterial strains	Testing zero OD value	OD value of test endpoint
			KLR-4	0.3	1.339
KLR-5	0.3	0.632
			KLR-8	0.3	0.517

TABLE 6 degradation rate of purine precursors by probiotic strains in nutrient-containing test systems

TABLE 7 ratio of purine precursor degradation rates of probiotic strains in nutrient-containing medium to nutrient-free medium

The results in Table 5 show that the OD values of 3 species are elevated in the test buffer containing nutrients, indicating that the nutrients in the culture broth keep the species viable and allow the growth and reproduction of the probiotic. The results of the screening of tables 6 and 7 show that L.reuteri KLR-5, KLR-8 are purine-degrading under non-nutritive conditions containing only nucleotides and nucleosides, but have a significantly reduced purine-reducing capacity (at least a substrate degradation rate of > 30%) under nutritive conditions, whereas L.reuteri KLR-4 has a significant purine precursor-reducing capacity under both conditions tested, and is even more degraded in nutrient-containing media.

Example 3 purine-lowering screening of candidate Lactobacillus purine-lowering bacteria under different pH conditions

In order to screen for probiotics that can degrade purine precursors in the whole intestine, the human intestine has a pH of about 5.5 from the duodenum to the large intestine, and the candidate Lactobacillus reuteri KLR-4 screened in example 2 is tested for purine reduction at a pH (5.0-7.5) that simulates the environment of the human intestine. The test was carried out using guanosine, guanylic acid and adenosine, adenylic acid as substrates, and the test results (Table 8) showed that the activity of Lactobacillus reuteri KLR-4 for reducing guanosine, guanylic acid and adenosine, adenylic acid was relatively stable in various pH environments.

TABLE 8 degradation rates of purine precursors by Lactobacillus reuteri KLR-4 at different pH

Example 4: identification and analysis of products of probiotic bacteria degrading purine precursor substances

The identification and analysis of the products were carried out by liquid chromatography with mass spectrometry (Thermo Scientific Q active) according to the peak of the degradation product of various purine precursor substrates in examples 1-3. The HPLC conditions were the same as the liquid phase conditions of example 1 and the mass spectrometry conditions were as follows: spray Voltage 3200V; capillary Temperature: 300.00 ℃; 40.00L/min shear Gas; 15.00L/min of Aux Gas; max Spray Current 100.00 mA; probe Heater Temp: 350.00 ℃; 50.00 ℃ of S-Lens RF Level; ESI-ms is the Ion Source.

The standard substance adopts a mixed standard substance (figure 2) consisting of uric acid, hypoxanthine, xanthine, deoxyguanosine, deoxyadenosine, guanosine and adenosine, and the mass spectrum result shows that the consistency of the molecular weight and the theoretical molecular weight of the standard substance is good. The test samples are guanosine, deoxyguanosine, guanylic acid, deoxyguanylic acid, adenosine, deoxyadenosine, adenylic acid and deoxyadenosine which are used as substrates, respectively reacted for 2 hours by virtue of a KLR-4 strain, and centrifuged to remove thalli. The results of mass spectrometric identification of the degradation products are shown in table 9, and show that the final product of the purine precursor is the corresponding purine base after degradation reaction by the probiotic bacteria, and a small amount of nucleoside/deoxynucleoside substance is also found in the reaction product of the nucleotide/deoxynucleotide, and it is presumed that the purine precursor degradation reaction process by the probiotic bacteria is to degrade the nucleotide or deoxynucleotide into nucleoside or deoxynucleoside first, and then further degrade the nucleoside or deoxynucleoside into the corresponding purine base. The solubility of the final product (purine base) is greatly reduced, and the absorbable concentration of food-derived purine precursors in the gastrointestinal tract can be greatly reduced, so that the absorption of the purine precursors in food is reduced.

TABLE 9 identification and analysis of products of probiotic degradation of purine precursors

Substrate	End product
		Guanosine	Guanine and its preparing process
Deoxyguanosine	Guanine and its preparing process
		Guanylic acid	Guanine and its preparing process
Deoxyguanylic acid	Guanine and its preparing process
		Adenosine (I)	Adenine
Deoxyadenosine	Adenine
		Adenosine monophosphate	Adenine
Deoxyadenylic acid	Adenine

Example 5: testing of the ability of candidate probiotics to tolerate gastrointestinal tract

MRS liquid culture media with pH of 2.0, pH of 3.0 and pH of 4.0 are respectively prepared and used for testing the gastric acidity resistance of the candidate lactobacillus reuteri, MRS culture media containing 0.1%, 0.2% and 0.3% of bile salts are respectively prepared and used for testing the bile salt resistance of the candidate probiotic strain, and the reference is respectively MRS liquid culture media without pH adjustment or MRS liquid culture media without bile salts. Inoculating to test medium at 1.0%, standing at 37 deg.C, collecting culture solution at time points of 0, 2h, 4h, and 6h, determining viable bacteria number in the bacteria solution, and repeating the test twice. The test results are shown in table 10 and table 11, and the results show that the candidate lactobacillus reuteri KLR-4 has the advantage of good gastric acid resistance and bile salt resistance.

(1) Gastric acid tolerance test

TABLE 10 acid resistance test results of Lactobacillus reuteri at 6h

The results of 6h of culture in pH2.0, pH3.0 medium show, the lower the pH, the faster the viable count reduction speed, at pH3.0 viable count although some reduction, but the magnitude of change; the viable count in the culture medium with pH4.0 does not obviously decrease along with the prolonging of time, and the result shows that the screened strain has better tolerance to gastric acid.

(2) Bile salt tolerance test

TABLE 11 results of bile salt resistance test of Lactobacillus reuteri in 6h

The result of culturing in the medium containing 0.1%, 0.2% and 0.3% for 6h shows that the descending speed of viable count is faster along with the concentration and the rise of the bile salt, and the viable count is reduced by 3 orders of magnitude when the medium is incubated in the 0.3% bile salt for 6 h; under 0.1% of bile salt, the colony number is not reduced basically, and the result shows that the screened strain has better tolerance to 0.1% of bile salt. After the probiotic strains screened by the patent are orally taken and are destroyed by gastric acid and bile salt, the probiotic strains still have higher viable bacteria to enter the intestinal tract to play a role.

Example 6: growth characterization of candidate Lactobacillus reuteri strains

The screened candidate lactobacillus reuteri strains are biochemically identified by using lactobacillus biochemical identification strips (including esculin, cellobiose, maltose, mannitol, salicin, sorbitol, sucrose, raffinose, inulin, lactose, hippuric acid, purchased from Qingdao Haibo Biotech Co., Ltd.) according to the method of national standard GB 4789.35. The specific operation is as follows: picking single bacterial colony from the purified and cultured plate by using an inoculating needle to 2ml of sterile physiological saline, and blowing, beating and uniformly mixing to prepare bacterial suspension; taking out the biochemical identification strip, tearing off the cover film, adding 100 mul of bacterial suspension into each hole, mixing uniformly, covering the cover, putting the mixture into a bottom support, putting the bottom support into an anaerobic incubator at 37 ℃ for culturing for 24-48h, after the culture is finished, putting the bottom support on a recording card for observation, and judging the result according to the description of the specification. The results of the evaluation are shown in Table 12.

TABLE 12 characterization of growth characteristics of Lactobacillus reuteri KLR-4

Example 7: effect of oral recombinant strains on rat serum uric acid levels

(1) Establishment of hyperuricemia animal model

Selecting 84 male SD rats with the body weight of about 100g, wherein 6 male SD rats are divided into 14 groups randomly; after 3 days of adaptive feeding, the molding is started. 6 rats in the blank group normally eat water, 30ml/24h of the water is drunk, and normal saline is injected into the abdominal cavity; modeling comparison and testing of normal diet of each group of probiotics, replacing drinking water with 20% yeast powder aqueous solution every day for 30ml/24h, simultaneously injecting oteracil potassium (250mg/(kg/d)) into the abdominal cavity, continuously feeding for 5 days to construct a hyperuricemia model (modeling period), collecting blood at the tail of every 24h in the last three days, detecting serum uric acid, and detecting samples by using a uric acid detection kit of Wuhansheng source biological engineering Limited company. The detection results are shown in table 13, and the results show that a stable hyperuricemia animal model is obtained.

(2) Verification of uric acid reducing effect of probiotic strains

Separately culturing the Lactobacillus reuteri KLR-4 strain selected in example 5 and two other strains of Lactobacillus reuteri (Lactobacillus reuteri KLR-1 degrading purine nucleotides, Lactobacillus reuteri KLR-3 degrading purine nucleotides) selected in this unit in MRS medium at 37 ℃ for about 8-12 hours (in late logarithmic growth curve), centrifuging at 12000rpm to collect the cells, washing the cells 3 times with sterile physiological saline, weighing the wet weight of the cells, and adjusting the cells 5 with sterile physiological saline 10⁸CFU/ml，5*10⁹CFU/ml，5*10¹⁰CFU/ml, and mixing KLR-1 and KLR-3 bacteria at a ratio of 1:1, and adjusting total viable count to 5 × 10⁸CFU/ml， 5*10⁹CFU/ml，5*10¹⁰CFU/ml. After being mixed uniformly, the mixture is well establishedThe model rat of hyperuricemia is subjected to an intragastric administration experiment, wherein each intragastric administration of the experiment group is 1ml, and the intragastric administration is carried out for 2 times every day. The stomach is continuously perfused for 7 days for treatment (treatment period), and blood is collected from the tail every 24h in the last 3 days to detect serum uric acid. The results are shown in Table 13. The results show that serum uric acid levels can be reduced by oral administration of different doses and different lactobacillus reuteri and mixed bacteria, but there are differences. Under the condition of the same dose of live bacteria, the effect of KLR-4 is better than that of KLR-1 and KLR-3, and is equivalent to that of the mixed bacteria of the two. Indicating that degradation of both nucleosides and nucleotides is important in reducing the absorption of food-borne nucleic acids.

TABLE 13 serum uric acid concentration Change

Note: p <0.05 to the modeled control group; the ratio of p to the modeling control group is less than 0.01

Example 8: effect of oral administration of Lactobacillus reuteri strains on blood uric acid of hyperuricemia patients

Candidate strain of probiotic Roy's bacteria (KLR-1, KLR-3, KLR-4) is processed in a factory meeting probiotic production standard to produce probiotic solid beverage (mixing ratio of mixed Roy's bacteria solid beverage group KLR-1, KLR-3 is 1:1), and low dose product has viable count of about 5 x 10⁹CFU/bag, high dose product viable count about 5 x 10¹⁰CFU/bag, and storing the product at-20 deg.C or 4 deg.C to ensure the activity of the bacteria powder during storage. 90 patients with hyperuricemia (blood uric acid) were collected>420 mu mol/L) were used as volunteers, which were randomly divided into 9 groups of 10 persons each, and Lactobacillus reuteri KLR-1 (low, high dose), Lactobacillus reuteri KLR-3 (low, high dose), Lactobacillus reuteri KLR-4 (low, high dose), mixed Lactobacillus reuteri (low, high dose), 1 bag/time, 2 times/day, and the intervention time was 30 days. The blood uric acid level was measured 3 days before the intervention and 28-30 days after the intervention to evaluate the effect of the intervention.

TABLE 14 blood uric acid concentration Change

Note: *: p <0.05 to blank control; #: p <0.01 to blank control; a tangle-solidup: p <0.05 to an equivalent dose of KLR-1; ■: p <0.01 to an equivalent dose of KLR-1; o: p <0.05 to an equivalent dose of KLR-3; ●: p <0.01 to an equivalent dose of KLR-3; o: the ratio p of the mixed bacteria to the mixed bacteria with the equal dosage is less than 0.05; solid content: the ratio p of the mixed bacteria to the equivalent dose is less than 0.01.

The results of human clinical tests show that the uric acid reducing effect of the Lactobacillus reuteri KLR-4 is better than that of the Lactobacillus reuteri KLR-1 (low dose 117vs 54; high dose 186vs 98) and the Lactobacillus reuteri KLR-3 (low dose 117vs 71; high dose 186vs 120), the uric acid reducing effect is improved along with the rise of the dose of living bacteria, and the uric acid reducing effect of the KLR-4 is close to that of a mixed bacterium (the mixture of the KLR-1 and the KLR-3) with the same dose. The test results suggest that the reduction of food-borne purine precursor absorption and the degradation of nucleosides and nucleotides are very important, especially nucleotides, and the effect of different dosages of oral probiotics shows that a considerable amount of viable bacteria are lost when the probiotics pass through the gastric environment, and the high dosage is helpful for improving the number of the viable bacteria entering the intestinal tract, so that the effect is better, and therefore, the viable bacteria number of the oral probiotic product is an important factor for the effectiveness of the oral probiotic product.

Example 9: preparation of purine-degrading probiotic yogurt powder product

The yoghourt is a healthy food containing probiotics which is widely favored by consumers, and the embodiment introduces a simple preparation of a yoghourt powder product with the function of reducing blood uric acid and an operation flow of fermenting the yoghourt. The viable count of the sour milk powder product is more than or equal to 1 x 10⁵cfu/g, formulation per serving (about 250g) was as follows: 180g of whole milk powder, 35g of xylitol, 10g of fructo-oligosaccharide, 10g of resistant dextrin, 15g of fruit powder, and freeze-dried powder of lactobacillus reuteri KLR-4 (viable count 1 x 10)¹¹cfu/g)2 mg. Pouring the yoghurt powder product into a yoghurt jar, adding about 800ml of purified water or cool boiled water, stirring until the pure water or cool boiled water is completely dissolved, and continuously adding water to 1L of scale marks.Fermenting in yogurt machine at 38-40 deg.C for 8-12 hr, and solidifying yogurt. The taste is better after being refrigerated at 4 ℃.

Example 10: preparation method of chewable tablets containing purine-reducing probiotics

The embodiment provides a preparation method of a chewable tablet containing purine-reducing probiotics, and the formula of the chewable tablet is as follows: 40% of isomaltulose, 23% of citrus powder and 1 x 10 of lactobacillus reuteri KLR-4 freeze-dried powder¹²cfu/g) 20%; 12% of carboxymethyl cellulose and 5% of magnesium stearate. Crushing the materials, sieving the crushed materials with a 60-mesh sieve for later use, weighing the corresponding materials according to the formula, uniformly mixing, pouring the mixed materials into a charging barrel of a tabletting machine for tabletting, adjusting the stamping pressure to ensure that the hardness of the probiotic chewable tablets is 10-15kg and the tablets weigh about 2 g/tablet, and subpackaging the tablets into a double-layer bubble-cap plate or a high-density polyethylene bottle (a desiccant bag needs to be added into the high-density polyethylene bottle) in a clean environment. The viable count of the chewable tablet product is more than or equal to 1 × 10⁸cfu/g。

Example 11: preparation of enteric-coated pellets containing purine-reducing probiotics

The embodiment provides a preparation method of an enteric-coated pellet containing purine-reducing probiotics, which comprises the following specific formula: freeze-dried powder of Lactobacillus reuteri KLR-4 (1.5 x 10)¹²cfu/g)60 percent, is dissolved in sunflower seed oil to prepare suspension, the content of bacteria powder is 30 to 40 percent, and oil solution (core material) containing probiotics and enteric-coated gelatin skin material (containing carrageenan, sodium alginate, gelatin, pullulan, calcium chloride and the like) are canned into enteric-coated micro-capsules with 3 layers by a multi-layer micro-pill machine. Air-cooling and drying at 25 deg.C, and packaging in waterproof polyethylene aluminum foil bag. The viable count of lactobacillus of the pellet product is more than or equal to 1 x 10⁷cfu/g。

Example 12: preparation of enteric capsule containing purine-reducing probiotics

The embodiment provides a preparation method of an enteric capsule containing purine-reducing probiotics, and the specific formula is as follows: fructo-oligosaccharide 35%, Lactobacillus reuteri KLR-4 lyophilized powder (1.7 x 10)¹²cfu/g) 60% and magnesium stearate 5%. Sieving the above materials with 60 mesh sieve, weighing corresponding materials according to the formula, mixing, and filling into enteric-coated solutionAnd (4) packaging the hollow capsule shell by using a double-layer aluminum-plastic bubble cap plate. The viable count of the capsule product is more than or equal to 5 x 10¹¹cfu/g。

Example 13: preparation method of milk beverage containing purine-reducing probiotics

The embodiment provides a preparation method of a milk beverage containing purine-reducing probiotics, which comprises the following specific formula: inoculating the activated Lactobacillus reuteri KLR-4 strain to a fermentation medium (4% of glucose, 2% of fructo-oligosaccharide, 3% of whey protein, 2% of yeast powder, 0.2% of sodium citrate, 0.2% of ammonium sulfate and 0.05% of L-cysteine) which is sterilized and cooled to 37 ℃, and fermenting for 8 hours at 37 ℃ to prepare the lactobacillus stock solution. Preparing stock solution of lactobacillus with sterile water to viable count of 4 x 10⁶-8*10⁸cfu/g, adding 6% skimmed milk powder, 7% edible glucose, 2% apple gum, and citric acid and sodium citrate to adjust pH of the milk beverage to 3.5-3.8, wherein the number of viable bacteria in the milk beverage is more than or equal to 1 x 10⁶cfu/g, can be filled into sterile beverage bottles, sealed by heat sealing, and shipped and stored at 4 ℃.

The embodiments of the present invention have been described above. However, the present invention is not limited to the above embodiment. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A Lactobacillus reuteri strain (Lactobacillus reuteri) for purine precursor degradation, characterized in that it has the ability to degrade purine precursors, including but not limited to any, several or all of inosine/deoxyinosine, inosinic acid/deoxyinosinic acid, guanosine/deoxyguanosine, guanylic acid/deoxyguanylic acid, adenosine/deoxyadenosine, adenylic acid/deoxyadenylic acid.

2. Lactobacillus reuteri (Lactobacillus reuteri) for purine precursor degradation according to claim 1, wherein the mean rate of purine precursor degradation is at least 50mg/OD h L, or at least 100mg/OD h L, or at least 150mg/OD h L, or at least 200mg/OD h L, or at least 250mg/OD h L, or at least 300mg/OD h L, wherein said purine precursor material is at least one, several or all of inosine/deoxyinosine, inosinic acid/deoxyinosinic acid, guanosine/deoxyguanosine, guanylic acid/deoxyguanylic acid, adenosine/deoxyadenosine, adenylic acid/deoxyadenylic acid.

3. The Lactobacillus reuteri strain (Lactobacillus reuteri) for purine precursor reduction according to claim 1 or 2, wherein said Lactobacillus reuteri strain KLR-4 has been deposited with chinese type culture collection at 28/7/2020 with the collection number CCTCC M2020367.

4. A probiotic composition of precursors of norpurines, characterized in that its active ingredient comprises the lactobacillus reuteri strain KLR-4 according to claim 3.

5. The probiotic composition according to claim 4, characterized in that it contains the Lactobacillus reuteri strain KLR-4 with a viable count of 1 x 10⁵～5*10¹²CFU/g composition.

6. Use of a probiotic composition of a lactobacillus reuteri strain according to any of claims 1 to 3 or of a purine-lowering precursor according to claim 4 or 5 for the preparation of a medicament or food product for the prevention and treatment of hyperuricemia and/or gout.

7. The use of claim 6, wherein the medicament is in a form for oral administration.

8. The use according to claim 7, wherein said dosage form is selected from: solutions, suspensions, emulsions, powders, lozenges, pills, syrups, troches, tablets, chewing gums, syrups, and capsules.

9. The use according to claim 6, wherein the food product comprises a general food product, a health food product, or a food product formulated for special medical use.