Lactobacillus buchneri complex microbial inoculant and application thereof

Abstract

The application discloses a lactobacillus buchneri complex microbial agent and application thereof, and belongs to the technical field of microorganisms. In order to solve the problem that the direct degradation effect of lactic acid bacteria on uric acid is poor in the prior art, the application provides a complex microbial agent containing lactobacillus buchneri NEFU-1 and NEFU-2, and the complex microbial agent has the functions of degrading uric acid, relieving hyperuricemia, degrading inosine and / or guanosine, improving intestinal flora composition and reducing inflammatory factors; the complex microbial agent has important significance for preventing, improving and treating gout and hyperuricemia diseases, has high application value, and can be applied to preparation of fermented yoghourt.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of biotechnology, and particularly relates to a Lactobacillus buchneri complex microbial agent and application thereof. BACKGROUND

[0002] Hyperuricemia is a chronic metabolic disease caused by purine metabolism disorder, characterized by excess uric acid in the blood. Generally speaking, the uric acid level of men is higher than 420 μmol / L, and the uric acid level of women is higher than 350 μmol / L, which is hyperuricemia. The initial stage of hyperuricemia is a stable state, and 85%-90% of hyperuricemia patients have no clinical characteristics, which is called asymptomatic hyperuricemia. Typical damages caused by asymptomatic hyperuricemia include inflammation, oxidative stress and intestinal microecological imbalance. Persistent hyperuricemia can cause gout, i.e. the precipitation of monosodium urate crystals, thereby causing acute or chronic inflammation and bone and joint damage. A large number of clinical investigations have shown that hyperuricemia can not only develop into gout, but also often accompanied by other complications, especially high blood pressure, high blood lipids, metabolic syndrome, chronic kidney disease and other independent risk factors.

[0003] Since the metabolism of uric acid is closely related to the intestinal tract, the degradation of purine substances in the digestive tract through the intestinal tract is expected to become a new strategy to control blood uric acid level, prevent and treat hyperuricemia and gout, and control asymptomatic hyperuricemia. Lactic acid bacteria are a general term for non-pathogenic, gram-positive bacteria that can produce lactic acid by utilizing carbohydrates. In addition, high blood uric acid is usually accompanied by high inflammatory factors. High intracellular uric acid can induce white blood cell signal pathway reorganization and epigenetic modification, thereby causing persistent hypersensitivity to inflammatory signals. Soluble uric acid and urate particles can mediate potential pro-inflammatory effects through urate crystal mechanisms and soluble uric acid mechanisms, respectively, which depend on or do not depend on inflammasomes. High levels of renal inflammatory factors damage kidney function, which in turn affects uric acid excretion.

[0004] Therefore, those skilled in the art are eager to develop a new lactic acid bacteria strain with anti-inflammatory effect and high efficiency of degrading uric acid, which is of great significance for preventing and alleviating hyperuricemia and gout, an acute, chronic and recurrent inflammatory disease. SUMMARY

[0005] The present application provides a Lactobacillus buchneri complex microbial agent and application thereof to solve the problem of poor direct degradation effect of lactic acid bacteria on uric acid in the prior art.

[0006] One of the purposes of the present application is to provide a complex microbial agent, which is a complex microbial agent of Lactobacillus buchneri NEFU-1 ( ) and Lactobacillus buchneri NEFU-2 ( The sample was inoculated into MRS liquid medium and cultured at 37°C for 36 hours to prepare the sample.

[0007] In a preferred embodiment of the present invention, the Lactobacillus baumannii NEFU-1 has the accession number CCTCC NO: M20241206 and is classified as follows: It is deposited at the China Center for Type Culture Collection on June 13, 2024;

[0008] The Lactobacillus brunetti NEFU-2 described herein has the accession number CCTCC NO: M 20241207 and is classified as follows: It is deposited at the China Center for Type Culture Collection on June 13, 2024.

[0009] In a preferred embodiment of the present invention, the inoculation ratio of Lactobacillus Bruxelles NEFU-1 and Lactobacillus Bruxelles NEFU-2 is 2:1.

[0010] The second objective of this invention is to provide the application of the above-mentioned compound microbial agent in the preparation of fermented yogurt.

[0011] In a preferred embodiment of the present invention, the above-mentioned compound microbial agent is inoculated into a culture medium and cultured in an incubator at 37-45°C for 20-36 hours; fresh milk and sucrose are mixed evenly and homogenized to obtain a mixture; the mixture is heated to 70-80°C, kept warm for 15-30 minutes, cooled to 40-43°C, and the cultured compound microbial agent is added under aseptic conditions, stirred evenly, bottled, sealed, and fermented at 37-45°C for 12-18 hours; after fermentation, it is cooled at 0-4°C for post-ripening to obtain fermented yogurt.

[0012] In a preferred embodiment of the present invention, the culture medium used for inoculation of the compound microbial agent in the fermented yogurt preparation method is MRS liquid.

[0013] In a preferred embodiment of the present invention, the mass percentage of fresh milk to sucrose in the fermented yogurt preparation method is 20:1.

[0014] In a preferred embodiment of the present invention, the mass-volume ratio of the cultured compound microbial agent added in the fermented yogurt preparation method is 3%-10%.

[0015] The third objective of this invention is to provide the application of the above-mentioned compound microbial agent in the preparation of drugs.

[0016] In a preferred embodiment of the present invention, the drug has at least one of the following functions (a)-(d):

[0017] (a) Degrades uric acid and alleviates hyperuricemia;

[0018] (b) Degradation of inosine and / or guanosine;

[0019] (c) Improve the composition of gut microbiota;

[0020] (d) Reduce inflammatory factors.

[0021] Compared with the prior art, the beneficial effects of the present invention are as follows: The present invention provides a compound bacterial agent containing *Lactobacillus bruneri* NEFU-1 and *Lactobacillus bruneri* NEFU-2; the preservation number of *Lactobacillus bruneri* NEFU-1 is CCTCC NO: M20241206, and the preservation number of *Lactobacillus bruneri* NEFU-2 is CCTCC NO: M 20241207; the compound bacterial agent has a high efficiency in degrading inosine and guanosine, and directly degrading serum uric acid, compared with *Lactobacillus bruneri* NEFU-1 or NEFU-2 alone, compared with *Lactobacillus bruneri* NEFU-1 or NEFU-2 alone. The uric acid degradation rate can reach 72.41%, and the serum uric acid content in hyperuricemic mice can be reduced from 0.336 g / L to 0.0927 g / L. Therefore, the compound bacterial agent provided by the present invention has a significantly higher uric acid degradation efficiency than *Lactobacillus bruneri* strains NEFU-1 and NEFU-2 alone.

[0022] In vitro and in vivo experiments have effectively verified that the compound bacterial agent containing Lactobacillus brucellosis NEFU-1 and NEFU-2 provided by this invention also has anti-inflammatory effects, significantly reducing inflammatory factors in the kidneys of hyperuricemia model mice and improving the intestinal flora of hyperuricemia model mice. The compound bacterial agent can effectively produce purine-lowering probiotics, which can be used to degrade uric acid, alleviate hyperuricemia, degrade inosine and / or guanosine, improve intestinal flora composition, and reduce inflammatory factors. It is of great significance for the prevention, improvement and treatment of gout and hyperuricemia, and has high application value.

[0023] [Bio-Collection Information]: The accession number for *Lactobacillus brunetti* NEFU-1 is CCTCC NO: M 20241206, and its classification name is... It is deposited at the China Center for Type Culture Collection on June 13, 2024;

[0024] The preservation number for *Lactobacillus brunetti* NEFU-2 is CCTCC NO: M 20241207, and its classification name is... It is deposited at the China Center for Type Culture Collection on June 13, 2024. Attached Figure Description

[0025] Figure 1 This is a colony morphology diagram of Lactobacillus bruneri NEFU-1 in Example 1;

[0026] Figure 2 This is an electron micrograph of Lactobacillus brunetti NEFU-1 colonies from Example 1;

[0027] Figure 3 This is a colony morphology diagram of Lactobacillus bruneri NEFU-2 in Example 1;

[0028] Figure 4 This is an electron micrograph of Lactobacillus brunetti NEFU-2 colonies from Example 1;

[0029] Figure 5 This is a graph showing the results of the homology comparison analysis in Example 1;

[0030] Figure 6 The image shows the results of the indole test in Example 1; from left to right, they are the blank control, Lactobacillus bruneri NEFU-1, and Lactobacillus bruneri NEFU-2.

[0031] Figure 7 The image shows the gelatin liquefaction test results in Example 1; from left to right, they are Escherichia coli positive control, Lactobacillus Bruxelles NEFU-1, and Lactobacillus Bruxelles NEFU-2.

[0032] Figure 8 This is a graph showing the results of the hemolysis test in Example 1;

[0033] Figure 9 The bacterial cell OD in Example 2 600 Value detection statistics chart;

[0034] Figure 10 This is a statistical chart of DPPH free radical scavenging capacity in Example 2; the vertical axis represents DPPH free radical scavenging rate; the horizontal axis represents ascorbic acid and PC represents compound bacterial agent.

[0035] Figure 11 This is a statistical chart of ABTS radical scavenging capacity in Example 2; the vertical axis represents ABTS radical scavenging rate.

[0036] Figure 12 This is the automatic aggregation statistics chart in Example 2; the vertical axis, Auto-aggregation, represents the automatic aggregation rate; the horizontal axis, Time, represents time.

[0037] Figure 13 This is a statistical graph of cell surface hydrophobicity measurement in Example 2; the vertical axis represents cell surface hydrophobicity; ethyl acetate represents ethyl acetate; chloroform represents chloroform; and xylene represents xylene.

[0038] Figure 14 This is a statistical graph showing the 1L-1β content in the kidneys of different groups of mice in Example 2; the vertical axis represents the 1L-1β content in the kidneys.

[0039] Figure 15 This is a statistical graph of XOD activity in the kidneys of different groups of mice in Example 2; the vertical axis represents Kidney XOD activity.

[0040] Figure 16 This is a statistical graph showing the MDA content in the kidneys of different groups of mice in Example 2; the vertical axis represents Kidney MDA content.

[0041] Figure 17 This is a statistical graph showing the content of short-chain fatty acids in the feces of different groups of mice in Example 2; the vertical axis represents the SCFA concentration, the horizontal axis represents acetic acid, butyrate, propionate, hexanoate, valerate, and total SCFA represents the total short-chain fatty acid content. Detailed Implementation

[0042] Those skilled in the art can refer to the content of this document and appropriately improve the process parameters to achieve the desired results. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art and are considered to be included in this invention. The methods and applications of this invention have been described through preferred embodiments, and those skilled in the art can obviously make modifications or appropriate alterations and combinations to the methods and applications described herein without departing from the content and scope of this invention to implement and apply the technology of this invention.

[0043] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to specific embodiments. Unless otherwise specified, the experimental methods used in the following embodiments are conventional methods, and the materials, reagents, methods, and instruments used are all conventional materials, reagents, methods, and instruments in the art, and can be obtained commercially by those skilled in the art.

[0044] The Lactobacillus Brucella used in the following examples is Lactobacillus Brucella NEFU-1, with accession number CCTCC NO: M20241206, and classified as follows: It is deposited at the China Center for Type Culture Collection on June 13, 2024;

[0045] Lactobacillus brunetti NEFU-2, accession number CCTCC NO: M 20241207, classified and named It is deposited at the China Center for Type Culture Collection on June 13, 2024.

[0046] Example 1: Isolation, screening and identification of lactic acid bacteria

[0047] 1. Isolation and screening of lactic acid bacteria: Yanji pickled vegetables were selected as samples and diluted. The samples were then spread onto MRS solid medium plates containing uric acid (52.24 g of MRS broth and 10-20 g of agar powder were dissolved in 1 L of distilled water and sterilized at 121℃ for 20 min). The plates were allowed to stand and incubated at 37℃ for 48 h. Milky-white colonies were selected, and single colonies exhibiting typical lactobacillus characteristics were obtained. These single colonies were repeatedly streaked on MRS solid medium plates for purification until a single morphology of colony appeared. The obtained colonies were inoculated into liquid MRS medium at a 1% (v / v) inoculum. The milky-white colonies that grew on the liquid MRS medium were subjected to catalase activity testing and Gram staining to obtain strains that were catalase-negative and Gram-positive. These strains were inoculated into liquid MRS medium and incubated at 37℃ for 48 h. After two subcultures, 2 μL of the fermentation broth was collected. The volume was centrifuged at 4℃ and 6000 r / min, and the supernatant was collected. The uric acid content in the supernatant was measured. Two lactic acid bacteria strains with the highest uric acid degradation rate were selected, and their colony morphology was observed. Figures 1-4 As shown.

[0048] 2. Identification of Lactic Acid Bacteria: DNA was extracted from the obtained lactic acid bacteria strains using a bacterial genomic DNA extraction kit. PCR amplification was performed using universal primers 27F (nucleotide sequence shown in SEQ ID NO. 1) and 1492R (nucleotide sequence shown in SEQ ID NO. 2). The PCR amplification reaction system consisted of: 1 μL genomic DNA (20 ng / μL), 10× Buffer (containing 2.5 mM MgSO4), and 1 μL genomic DNA (20 ng / μL). 2+ 5 μL of Taq polymerase (5 u / μL), 1 μL of dNTP (10 mM), 1.5 μL each of primers 27F and 1492R (10 μM), and ddH2O to a final volume of 50 μL were added. The PCR amplification reaction program was as follows: pre-denaturation at 95℃ for 5 min; denaturation at 95℃ for 30 s, annealing at 58℃ for 30 s, extension at 72℃ for 1.5 min, for 35 cycles; final extension at 72℃ for 7 min. The PCR amplification products were recovered using the AxyPrep DNA gel extraction kit and sent to Shanghai Paisennong Biotechnology Co., Ltd. for sequencing. The obtained sequences were compared with the GenBank database using BLAST software for homology analysis. Figure 5As shown, the two lactic acid bacteria strains obtained from the above screening can be identified as Lactobacillus brunelli, and they are named Lactobacillus brunelli NEFU-1 and Lactobacillus brunelli NEFU-2, respectively; the 16S rRNA of Lactobacillus brunelli NEFU-1 is shown in SEQ ID NO.3, and the 16S rRNA of Lactobacillus brunelli NEFU-2 is shown in SEQ ID NO.4.

[0049] Effect Experiment:

[0050] 1. Safety Evaluation

[0051] Safety evaluations were conducted on the *Lactobacillus baumannii* strains NEFU-1 and NEFU-2 obtained from the above screening. One pathogenic bacterium, *Escherichia coli* (purchased from Yangling Ruizhi Weikang Biotechnology Co., Ltd.), was selected as an indicator strain. Indole, gelatin liquefaction, and hemolysis tests were performed on *Lactobacillus baumannii* strains NEFU-1 and NEFU-2. The specific steps are as follows:

[0052] (1) Indole test: Lactobacillus broomriae strains NEFU-1 and NEFU-2 were inoculated into peptone water medium (composition: 10 g peptone, 5 g sodium chloride, 2 g disodium hydrogen phosphate, 2 g dipotassium hydrogen phosphate, 5 g glucose, 1000 mL distilled water) at an inoculation rate of 2%, and physiological saline was used as a blank control to replace the strains. They were cultured at 35℃ for 72 h. Two drops of xylene were added and shaken thoroughly. After standing, 3-6 drops of indole reagent were added and the test results were observed.

[0053] The results are as follows Figure 6 As shown, no red rose indole appeared in the upper layer of the test tube, indicating that both Lactobacillus broomriae strains NEFU-1 and NEFU-2 were negative and did not produce indole-like substances.

[0054] (2) Gelatin liquefaction test: Prepare a semi-solid columnar gelatin culture medium (composition: 10 g peptone, 5 g sodium chloride, 3 g beef extract, 120 g gelatin, 1000 mL distilled water), and pick Lactobacillus baumannii strains NEFU-1 and NEFU-2 for stab inoculation. Culture at 35℃ for 48 h, with Escherichia coli as a positive control. When observing the results, place the gelatin in a 4℃ refrigerator for 20 min to observe the liquefaction of the gelatin.

[0055] The results are as follows Figure 7 As shown, the semi-solid columnar gelatin culture medium in the test tube turned into a solid, indicating that Lactobacillus baumannii strains NEFU-1 and NEFU-2 do not produce gelatinase.

[0056] (3) Hemolysis test: The above-mentioned Lactobacillus Brucella NEFU-1 and NEFU-2 were placed at 37℃ and allowed to stand for 18-24 h to obtain seed liquid. 1 mL of each was added to 9 mL of blank MRS medium and incubated for 10 h to obtain activated Lactobacillus Brucella NEFU-1 and NEFU-2. They were then streaked onto Columbia blood agar medium (composition: 5 g sodium chloride, 2 g glucose, 5 g peptone, 10 g trypone, 5 g yeast extract, 5 g beef extract, 15 g agar, 1000 mL distilled water, 60 mL defibrinated sheep blood). Escherichia coli was used as the control group. The mixture was cultured at 37℃ for 48 h. If a grass-green hemolysis ring appeared, it was α-hemolysis; if a colorless and transparent hemolysis ring appeared, it was β-hemolysis; if no hemolysis ring appeared, it was γ-hemolysis.

[0057] The results are as follows Figure 8 As shown, the control group of Escherichia coli showed obvious β-hemolytic rings (complete hemolysis, clear area), consistent with its known hemolytic characteristics; Lactobacillus brucellosis NEFU-1 and NEFU-2 did not show hemolytic activity, indicating that they do not have hemolysis-related pathogenic mechanisms.

[0058] Example 2: Preparation of Compound Microbial Agent

[0059] Lactobacillus Brucella NEFU-1 and NEFU-2 obtained in Example 1 were inoculated into MRS medium at a ratio of 2:1 and an inoculum volume of 1%. The medium was cultured at 37°C for 36 hours to obtain a compound bacterial agent, abbreviated as PC.

[0060] The OD values ​​of the above-mentioned compound bacterial agent, as well as Lactobacillus bruneri NEFU-1 and NEFU-2 at different growth times, were compared. 600 The value was measured, and the result is as follows: Figure 9 As shown, the *Lactobacillus bromis* strain in the compound microbial agent was in the logarithmic growth phase from 5 to 22 hours of cultivation, during which the growth rate constant R was the largest, the cell growth rate was the fastest, and lactic acid was produced in large quantities (the pH of the fermentation broth was maintained at 4). After 22 hours of cultivation, the *Lactobacillus bromis* strain in the compound microbial agent entered the stationary phase, where the cell death and growth reached a dynamic equilibrium, the growth rate constant R was almost zero, the growth rate of the strain slowed down, and it tended to stabilize. The acid production capacity of the strain was inhibited after entering the stationary phase. In addition, the OD of the fermentation broth of the *Lactobacillus bromis* strain in the compound microbial agent within 72 hours of cultivation... 600 The values ​​were all able to remain at a relatively stable level.

[0061] Effect Experiment:

[0062] 1. Determination of antioxidant capacity, self-aggregation rate, and hydrophobicity.

[0063] (1) DPPH free radical scavenging ability: 100 μL of VC (concentration of 15 µg / mL) and 100 μL of 0.4 mM DPPH solution were added to a 96-well plate as a control group; 100 μL of NEFU-1 and NEFU-2 obtained in Example 1 and the compound bacterial agent (abbreviated as PC) containing NEFU-1 and NEFU-2 prepared in this example were added to a 96-well plate with 100 μL of 0.4 mM DPPH solution as an experimental group. The reaction was carried out at 25°C in the dark for 30 min, and the DPPH free radical scavenging ability of lactic acid bacteria was determined based on the absorbance at 540 nm.

[0064] The results are as follows Figure 10 As shown, the DPPH free radical scavenging rates of *Lactobacillus bruneri* NEFU-1, NEFU-2, and the compound bacterial agent containing the above two strains were 72.93%, 68.37%, and 72.58%, respectively, which were closest to the DPPH free radical scavenging rate of the positive control vitamin C (80.46%). This indicates that the compound bacterial agent provided by the present invention has a strong DPPH free radical scavenging ability.

[0065] (2) ABTS radical scavenging ability: 7.4 mM ABTS radical solution was reacted with 2.6 mM potassium persulfate for 24 h at room temperature in the dark to generate a stable, high-concentration ABTS radical cation (ABTS•⁺) stock solution. Before the measurement, the ABTS•⁺ stock solution to be tested was diluted with PBS at a wavelength of 734 nm to make its absorbance value 0.70 ± 0.03. 20 μL of VC (concentration of 20 µg / mL) was added to a 96-well plate containing 180 μL of ABTS radical solution as a control group; 20 μL of NEFU-1 and NEFU-2 obtained in Example 1, and the bacterial solution of the compound bacterial agent containing NEFU-1 and NEFU-2 prepared in this example were added to a 96-well plate containing 180 μL of ABTS radical solution as experimental groups, and reacted at 25 °C in the dark for 10 min. The ABTS radical scavenging ability of lactic acid bacteria was determined based on the absorbance at 734 nm.

[0066] The results are as follows Figure 11 As shown, the ABTS free radical scavenging rates of *Lactobacillus bruneri* NEFU-1, NEFU-2, and the compound bacterial agent containing these two strains were 72.39%, 73.59%, and 76.50%, respectively. Among them, the ABTS free radical scavenging rate of the compound bacterial agent was closest to that of the positive control vitamin C (82.12%). This indicates that the compound bacterial agent containing *Lactobacillus bruneri* NEFU-1 and NEFU-2 provided by this invention has a strong ABTS free radical scavenging ability.

[0067] (3) Determination of autoaggregation ability: The NEFU-1 and NEFU-2 obtained in Example 1, as well as the compound bacterial agent containing NEFU-1 and NEFU-2 prepared in this example, were grown overnight in LB broth at 37°C. After centrifugation, the bacterial solutions were washed twice with sterile PBS (pH=7.2), resuspended in the supernatant, and vortexed for 30 seconds. With VC as the control group, the absorbance was measured at 600 nm using a UV-Vis spectrophotometer at different times (0, 1, 2, 3, 4 and 5 hours) to obtain the autoaggregation ability of Lactobacillus brucei NEFU-1, NEFU-2 and the compound bacterial agent.

[0068]

[0069] A0: Initial absorbance value measured at 600 nm wavelength, with a time of 0 hours (i.e., immediately after the bacterial suspension is prepared and vortexed); A t : The absorbance value of the sample taken from the upper layer of the suspension at a wavelength of 600 nm, which is t hours.

[0070] Lactic acid bacteria can form a barrier through self-aggregation, which on the one hand facilitates the intestinal colonization of probiotic strains, and on the other hand prevents pathogenic bacteria from colonizing and infecting the intestines. For example... Figure 12 As shown, the combined self-aggregation abilities of Lactobacillus Bruxelles NEFU-1, NEFU-2, and the compound bacterial agent containing the above two strains reached 46.49%, 39.45%, and 52.75% respectively after 24 h; it can be seen that the compound bacterial agent containing Lactobacillus Bruxelles NEFU-1 and NEFU-2 provided by the present invention has a strong self-aggregation ability.

[0071] (4) Cell surface hydrophobicity determination: The cell surface hydrophobicity of NEFU-1 and NEFU-2 obtained in Example 1 and the composite bacterial agent containing NEFU-1 and NEFU-2 prepared in this example was determined by the microbial adhesion hydrocarbon method (MATH).

[0072] Lactobacillus blight NEFU-1, NEFU-2, and a compound bacterial agent containing the two strains were cultured for 24 h, centrifuged, washed twice with PBS (pH=7.2), and resuspended in 5 mL PBS buffer. The absorbance of the cell suspension was measured at 600 nm using a UV-Vis spectrophotometer and recorded as A0. The cell suspension was then mixed with an equal volume of solvent for 5 min to obtain a suspension. Measurements were performed using ethyl acetate (alkaline solvent), chloroform (acidic solvent), or xylene (non-polar solvent). The suspension was incubated at room temperature for 30 min to achieve phase separation. The absorbance of the aqueous phase at 600 nm was measured and recorded as A0. tThe hydrophobicity of the cell surface of Lactobacillus bruneri NEFU-1, NEFU-2 and compound bacterial solutions was obtained.

[0073]

[0074] A0: Initial absorbance value measured at 600 nm wavelength, with a time of 0 hours (i.e., immediately after the bacterial suspension is prepared and vortexed); A t : The absorbance value of the sample taken from the upper layer of the suspension at a wavelength of 600 nm, which is t hours.

[0075] Higher cell surface hydrophobicity indicates stronger corresponding surface properties of the strain; results are as follows Figure 13 As shown, the bacterial cultures of *Lactobacillus bruneri* NEFU-1, NEFU-2, and the compound bacterial agent exhibited high adsorption rates for chloroform (PC: 60.00%, NEFU-1: 59.80%, NEFU-2: 56.27%), indicating that the cell surfaces of *Lactobacillus bruneri* NEFU-1, NEFU-2, and the compound bacterial agent are rich in electron donors (such as amino and hydroxyl groups) and possess a strong Lewis alkaline region, which is consistent with the surface characteristics of most lactic acid bacteria. The bacterial cultures of *Lactobacillus bruneri* NEFU-1, NEFU-2, and the compound bacterial agent showed moderate adsorption rates for xylene (PC: 49.99%, NEFU-1: 50.82%, NEFU-2: 36.88%) and the lowest adsorption rate for ethyl acetate (approximately 20%-25%), indicating that the electron acceptors (such as carboxyl and phosphate groups) on their surfaces are relatively weak, resulting in low affinity for alkaline solvents.

[0076] It is evident that *Lactobacillus bruneri* NEFU-1 exhibits the strongest surface hydrophobicity and electron donor properties, suggesting that it may possess excellent adhesion and colonization potential. *Lactobacillus bruneri* NEFU-2, on the other hand, has different surface properties, with relatively low overall hydrophobicity. The surface properties of the compound bacterial agent (PC) are more similar to those of NEFU-1. These differences in surface properties provide a theoretical basis for explaining the differences between the two strains in terms of autoaggregation, coagulation, and probiotic functions.

[0077] 2. Degradation of inosine and / or guanosine

[0078] Lactobacillus Bruxelles NEFU-1 and NEFU-2 obtained in Example 1, and the compound bacterial agent prepared in Example 2, were placed at 37°C and allowed to stand for 18-24 h to obtain seed culture. 1 mL of each seed culture was added to 9 mL of blank MRS medium and incubated for 10 h to obtain activated Lactobacillus Bruxelles NEFU-1 and NEFU-2, and the compound bacterial agent, respectively. 2 mL of the above-mentioned activated second-generation Lactobacillus Bruxelles NEFU-1 and NEFU-2, and the compound bacterial agent were taken and centrifuged at 4°C and 5000 r / min for 10 min to collect the bacterial cells. The cells were resuspended and washed with 1 mL of physiological saline, centrifuged at 4°C and 5000 r / min for 10 min, and repeated twice to collect the bacterial cells.

[0079] Weigh 11.3 mg of inosine and 28.3 mg of guanosine, dissolve them in potassium phosphate buffer and bring the volume up to 50 mL. If guanosine is not soluble in potassium phosphate buffer, it can be heated in an 80°C water bath to obtain inosine-neutral potassium phosphate solution and guanosine-neutral potassium phosphate solution, respectively. The concentration of inosine / guanosine in the above solutions is 2 mM.

[0080] The *Lactobacillus brunelli* NEFU-1 and NEFU-2 collected by washing, as well as the bacterial cells of the compound bacterial agent, were resuspended in 375 μL of guanosine-neutral potassium phosphate solution and 375 μL of inosine-neutral potassium phosphate solution, respectively. The reaction was carried out at 37℃ and 120 r / min with constant shaking for 1 h. After the reaction was completed, the cells were centrifuged at 4℃ and 5000 r / min for 10 min. 720 μL of the supernatant was collected and 80 μL of perchloric acid solution was added to terminate the reaction. The supernatant was filtered through a filter membrane, and the contents of inosine and guanosine in the solution were determined by high performance liquid chromatography.

[0081] The results are shown in Table 1. Lactobacillus burlii strain NEFU-1 showed a degradation rate of 68.64% for inosine and 100% for guanosine; Lactobacillus burlii strain NEFU-2 showed a degradation rate of 78.72% for inosine and 100% for guanosine; and the compound bacterial agent containing Lactobacillus burlii strain NEFU-1 and Lactobacillus burlii strain NEFU-2 showed a degradation rate of 80.23% for inosine and 100% for guanosine.

[0082] It is evident that, compared to Lactobacillus burlensis strain NEFU-1 and Lactobacillus burlensis strain NEFU-2, the compound bacterial agent provided by this invention has a better degradation ability for inosine and guanosine.

[0083] Table 1

[0084]

[0085] 3. Degrades uric acid and alleviates hyperuricemia

[0086] (1) In vitro test

[0087] The *Lactobacillus baumannii* strains NEFU-1 and NEFU-2 obtained in Example 1, and the compound bacterial agent obtained in Example 2, were placed in MRS medium and cultured statically at 37°C until the logarithmic growth phase. After centrifugation and three washings, they were suspended in PBS and inoculated at a 5% inoculum into MRS medium containing 0.336 g / L uric acid. The medium was incubated statically at 37°C for 24 h. Every 6 h, the culture medium was centrifuged and the supernatant was collected. After filtration through a 0.22 μm filter membrane, the degradation of uric acid was detected by HPLC to obtain the degradation rate of uric acid.

[0088]

[0089] : Initial concentration of uric acid in the culture medium after 0 hours of incubation (i.e., 0.336 g / L). The residual concentration of uric acid in the supernatant after centrifugation and filtration after culturing for t hours.

[0090] The results are shown in Tables 2-3. The *Lactobacillus brevicornu* strains NEFU-1 and NEFU-2, as well as the compound microbial agent, were able to degrade uric acid from 0.336 g / L to 0.1309 g / L, 0.1152 g / L, and 0.0927 g / L, respectively, within 24 h, with degradation rates of 61.02%, 65.71%, and 72.41%, respectively. It is evident that, compared to *Lactobacillus brevicornu* strains NEFU-1 and NEFU-2, the compound microbial agent provided by this invention has a highly efficient and direct uric acid degradation effect.

[0091] Table 2

[0092]

[0093] Table 3

[0094]

[0095] (2) In vivo test

[0096] Establishment and grouping of animal models:

[0097] Sixty male KM mice weighing 20-25 g (purchased from Suzhou Xishan Biotechnology Co., Ltd., license number SCXK(Liaoning)2010-0001) were used to establish the Brucella nefugax NEFU-1 group (B1), Brucella nefugax NEFU-2 group (B2), HUA hyperuricemia group (HUA), and compound bacterial agent treatment group (HB) by gavage. Specifically, starting from day 8, the mice were administered potassium oxonate + uric acid suspension at a dose of 300 mg / kg body weight by gavage daily, supplemented with ordinary mouse food and free access to water. After 14 days of continuous feeding, blood was collected from each group of mice, and the serum was collected by centrifugation at 3000 r / min for 15 min.

[0098] Determination of uric acid content:

[0099] On the last day of the 14-day continuous feeding following gavage, blood was collected from the orbital sinus of each group of mice. The blood was placed in the blood collection tube and left to stand at room temperature for 1 hour. The tube was then centrifuged at 3000 rpm and 4°C for 15 minutes. The supernatant was collected and stored as serum. The uric acid content in the mouse serum was determined using a uric acid test kit provided by Nanjing Jiancheng Bioengineering Research Institute. The principle is that uric acid produces quinone pigments under the action of uricase and peroxidase. The absorbance value was read at a wavelength of 510 nm using an ELISA reader, and the UA concentration was obtained using the following formula.

[0100] UA concentration (μmol / L) = (A measured - A blank) / (A standard - A blank) × C standard

[0101] A. Assay: Absorbance of test samples (serum from mice in the NEFU-1 group, NEFU-2 group, compound bacterial agent treatment group, and HUA hyperuricemia group, respectively); A. Blank: Absorbance of double-distilled water; A. Standard: Absorbance of uric acid standard; C. Standard: Concentration of uric acid standard (500 μmol / L).

[0102] As shown in Table 4, at 21 days (7 days of adaptive feeding + 14 days of continuous feeding after gavage), the serum uric acid levels in mice in the *Lactobacillus brucellosis* NEFU-1 group (B1), *Lactobacillus brucellosis* NEFU-2 group (B2), and the compound bacterial agent treatment group (HB) were significantly different from those in the HUA hyperuricemia group (HUA). The serum uric acid levels in the compound bacterial agent treatment group (HB) were significantly lower than those in the HUA hyperuricemia group (HUA). P <0.0001).

[0103] It is evident that *Lactobacillus bromide* strains NEFU-1 and NEFU-2, as well as the compound bacterial agent, exhibit highly efficient direct degradation of uric acid in hyperuricemic mice. Among these, the compound bacterial agent provided by this invention demonstrates significantly higher uric acid degradation efficiency compared to individual *Lactobacillus bromide* strains NEFU-1 and NEFU-2.

[0104] Table 4

[0105]

[0106] 4. Reduce inflammatory factors

[0107] The levels of 1L-1β, MDA, and XOD factors in the kidney homogenates of mice in the HUA hyperuricemia group (HUA) and the compound bacterial agent treatment group (HB) were detected using the ELISA kit from Seville Biotechnology.

[0108] Short-chain fatty acids (SCFAs) in the feces of mice in the HUA hyperuricemia group (HUA) and the compound bacterial agent treatment group (HB) were detected using an Agilent gas chromatography-mass spectrometry (GC-MS) system.

[0109] like Figure 14 As shown, compared with the HUA hyperuricemia group (HUA), the IL-1β content in the kidney homogenate of mice in the compound bacterial agent treatment group (HB) decreased from 3993.54 pg / mL to 2963.54 pg / mL (a decrease of ≈26%), indicating that the compound bacterial agent provided by the present invention can effectively inhibit the inflammatory pathway.

[0110] like Figure 15 As shown, compared with the HUA hyperuricemia group (HUA), the XOD activity in the kidney homogenate of mice in the compound bacterial agent treatment group (HB) decreased from 0.207 U / g to 0.141 U / g (a decrease of ≈32%), indicating that the compound bacterial agent provided by the present invention can effectively intervene and block the excessive activation of XOD, thereby reducing uric acid synthesis.

[0111] like Figure 16 As shown, compared with the HUA hyperuricemia group (HUA), the MDA content in the kidney homogenate of mice in the compound bacterial agent treatment group (HB) decreased from 14.75 µmol / gprot to 3.42 µmol / gprot (a decrease of ≈77%), indicating that the compound bacterial agent provided by the present invention can maintain renal tubular function by protecting the integrity of membrane structure by scavenging free radicals (consistent with in vitro free radical scavenging experiments).

[0112] like Figure 17As shown in Table 5, compared with the HUA hyperuricemia group (HUA), the levels of most short-chain fatty acids in mice treated with the compound probiotic agent (HB) were significantly increased. Butyric acid and hexanoic acid are particularly important short-chain fatty acids. Butyric acid is a major energy source for colon cells and plays a role in anti-inflammation and maintaining intestinal barrier function, while hexanoic acid has antibacterial and anti-inflammatory properties. Compared with the HUA hyperuricemia group (HUA), the compound probiotic agent treatment group (HB) increased the butyric acid content by 108.04% and the hexanoic acid content by 193.24%. This indicates that the compound probiotic agent provided by this invention has a positive effect on improving intestinal barrier function, inhibiting inflammation, and indirectly promoting uric acid excretion.

[0113] Table 5

[0114]

[0115] Example 3: Application of compound microbial agents in the preparation of fermented yogurt

[0116] The compound microbial agent obtained in Example 2 was inoculated into MRS liquid and cultured in an incubator at 37°C for 24 hours. Fresh milk and sucrose were mixed evenly at a mass ratio of 20:1 and homogenized to obtain a mixture. The mixture was heated to 75°C, kept warm for 25 min, cooled to 40°C, and 6% of the cultured compound microbial agent was added under aseptic conditions. The mixture was stirred evenly, bottled, sealed, and fermented at 37°C for 12 h. After fermentation, it was cooled at 4°C for post-fermentation to obtain fermented yogurt.

[0117] The specific embodiments of the present invention disclosed above are merely illustrative of the invention. These embodiments do not exhaustively describe all details, nor do they limit the invention to the specific embodiments described. Many modifications and variations can be made based on the content of this specification. This specification selects and specifically describes these embodiments to better explain the principles and practical applications of the invention, thereby enabling those skilled in the art to better understand and utilize the invention.

Claims

1. A compound microbial agent, characterized in that, The compound bacterial agent is Lactobacillus bromide (… Lactobacillus buchneri NEFU-1 and Lactobacillus brunelli ( Lactobacillus buchneri NEFU-2 was prepared by inoculating it into MRS liquid medium and culturing it at 37°C for 36 hours; The Lactobacillus brunetti NEFU-1 described herein has the accession number CCTCC NO: M 20241206 and is classified as follows: Lactobacillus buchneri NEFU-1 is deposited at the China Center for Type Culture Collection on June 13, 2024; the Lactobacillus brucellosis NEFU-2 has the accession number CCTCC NO: M 20241207 and is classified as follows: Lactobacillus buchneri NEFU-2 is deposited at the China Center for Type Culture Collection on June 13, 2024.

2. The compound microbial agent according to claim 1, characterized in that, The inoculation ratio of Lactobacillus Bruxelles NEFU-1 to Lactobacillus Bruxelles NEFU-2 was 2:

1.

3. The use of the compound microbial agent according to any one of claims 1 to 2 in the preparation of fermented yogurt.

4. The application according to claim 3, characterized in that, The method for preparing fermented yogurt includes the following steps: inoculating the compound microbial agent described in any one of claims 1 to 2 into a culture medium and culturing it in an incubator at 37-45°C for 20-36 hours; mixing fresh milk and sucrose evenly and homogenizing to obtain a mixture; heating the mixture to 70-80°C, keeping it warm for 15-30 minutes, cooling it to 40-43°C, adding the cultured compound microbial agent under aseptic conditions, stirring evenly, canning, sealing, and fermenting at 37-45°C for 12-18 hours; after fermentation, cooling it at 0-4°C for post-ripening to obtain fermented yogurt.

5. The application according to claim 4, characterized in that, The culture medium used for inoculating the compound microbial agent in the fermented yogurt preparation method is MRS liquid.

6. The application according to claim 4, characterized in that, In the fermented yogurt preparation method, the mass percentage of fresh milk to sucrose is 20:

1.

7. The application according to claim 4, characterized in that, In the fermented yogurt preparation method, the mass-volume ratio of the cultured compound microbial agent added is 3%-10%.

8. The use of the compound microbial agent according to any one of claims 1 to 2 in the preparation of drugs for relieving hyperuricemia.

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