Lactobacillus buchneri complex microbial inoculant and application thereof

Abstract

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

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 ( ) is prepared by inoculating in MRS liquid medium and culturing at 37 DEG C for 36 hours.

[0007] In a preferred embodiment of the present application, the Lactobacillus buchneri NEFU-1 has a preservation number of CCTCC NO: M20241206, a classification name of , and is preserved in the China Center for Type Culture Collection on June 13, 2024; The Lactobacillus buchneri NEFU-2 has a preservation number of CCTCC NO: M20241207, a classification name of , and is preserved in the China Center for Type Culture Collection on June 13, 2024.

[0008] In a preferred embodiment of the present application, the inoculation ratio of the Lactobacillus buchneri NEFU-1 and the Lactobacillus buchneri NEFU-2 is 2:1.

[0009] The second object of the present application is to provide the use of the above-mentioned complex microbial agent in the preparation of fermented yogurt.

[0010] In a preferred embodiment of the present application, the above-mentioned complex microbial agent is inoculated in a culture medium and cultured in a 37-45 DEG C incubator for 20-36 hours; fresh milk is mixed with sucrose uniformly, and a mixed solution is obtained after homogenization; the above-mentioned mixed solution is heated to 70-80 DEG C, kept for 15-30 min, cooled to 40-43 DEG C, and the above-mentioned cultured complex microbial agent is added under sterile operation conditions, stirred uniformly, canned, sealed, kept at 37-45 DEG C for 12-18 hours for fermentation, cooled at 0-4 DEG C for post-ripening after the fermentation is completed, and fermented yogurt is obtained.

[0011] In a preferred embodiment of the present application, the culture medium used for inoculation of the complex microbial agent in the preparation method of the fermented yogurt is MRS liquid.

[0012] In a preferred embodiment of the present application, the mixing mass percentage of fresh milk and sucrose in the preparation method of the fermented yogurt is 20:1.

[0013] In a preferred embodiment of the present application, the addition mass-volume ratio of the cultured complex microbial agent in the preparation method of the fermented yogurt is 3%-10%.

[0014] The third object of the present application is to provide the use of the above-mentioned complex microbial agent in the preparation of a medicine.

[0015] In a preferred embodiment of the present application, the medicine has at least one function of (a)-(d) below: (a) degrading uric acid and relieving hyperuricemia; (b) degrade inosine and / or guanosine; (c) improve the composition of intestinal flora; (d) reduce inflammatory factors.

[0016] Compared with the prior art, the beneficial effects of the present application are: the present application provides a complex microbial agent containing Lactobacillus buchneri NEFU-1 and Lactobacillus buchneri NEFU-2; the preservation number of the Lactobacillus buchneri NEFU-1 is CCTCC NO: M20241206, and the preservation number of the Lactobacillus buchneri NEFU-2 is CCTCC NO: M20241207; the complex microbial agent has the ability to degrade inosine, guanosine and directly degrade uric acid more efficiently than Lactobacillus buchneri NEFU-1 or NEFU-2 alone, and the degradation rate of uric acid can reach 72.41%, which can reduce the uric acid content in the serum of hyperuricemia mice from 0.336 g / L to 0.0927 g / L. It can be seen that the degradation efficiency of the complex microbial agent provided by the present application for uric acid is significantly higher than that of Lactobacillus strains NEFU-1 and NEFU-2 alone.

[0017] Through in vivo and in vitro tests, it is effectively verified that the complex microbial agent containing Lactobacillus buchneri NEFU-1 and NEFU-2 provided by the present application also has an anti-inflammatory effect and can significantly reduce the inflammatory factors in the kidneys of hyperuricemia model mice and improve the ability of the complex microbial agent to improve the intestinal flora of hyperuricemia model mice; the complex microbial agent can effectively produce a hypoxanthine-producing probiotic, thereby being applicable to degrade uric acid, relieve hyperuricemia, degrade inosine and / or guanosine, improve the composition of intestinal flora, reduce inflammatory factors, and has important significance for preventing, improving and treating gout and hyperuricemia diseases, and has high application value.

[0018]

Biological preservation information

[0019] Figure 1 It is a colony morphology diagram of Lactobacillus buchneri NEFU-1 in Example 1; Figure 2 It is a colony electron microscope diagram of Lactobacillus buchneri NEFU-1 in Example 1; Figure 3This is a colony morphology diagram of Lactobacillus bruneri NEFU-2 in Example 1; Figure 4 This is an electron micrograph of Lactobacillus brunetti NEFU-2 colonies from Example 1; Figure 5 This is a graph showing the results of the homology comparison analysis in Example 1; 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. 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. Figure 8 This is a graph showing the results of the hemolysis test in Example 1; Figure 9 The bacterial cell OD in Example 2 600 Value detection statistics chart; 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. Figure 11 This is a statistical chart of ABTS radical scavenging capacity in Example 2; the vertical axis represents ABTS radical scavenging rate. 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. 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. 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. 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. 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. Figure 17 Statistical chart of short-chain fatty acid content in feces of different groups of mice in Example 2; the vertical coordinate SCFAconcentration is the short-chain fatty acid content, and the horizontal coordinate Acetate is acetic acid, Butyrate is butyric acid, Propionate is propionic acid, Hexanoate is hexanoic acid, Valerate is valeric acid, and Total·SCFA is the total short-chain fatty acid content. DETAILED DESCRIPTION

[0020] Those skilled in the art can modify the process parameters according to the content herein. It is particularly pointed out that all similar substitutions and modifications are obvious to those skilled in the art, and they are considered to be included in the present application. The methods and applications of the present application have been described by preferred embodiments, and relevant personnel can obviously modify or appropriately change and combine the methods and applications described herein without departing from the content and scope of the present application, to realize and apply the present application technology.

[0021] In order to make the purpose, technical solutions and advantages of the present application clearer and more apparent, the present application will be further described in detail below in combination with specific embodiments. The experimental methods used in the following examples are conventional methods unless otherwise specified. The materials, reagents, methods and instruments used are conventional materials, reagents, methods and instruments in the art unless otherwise specified, and can be obtained by commercial channels by those skilled in the art.

[0022] The B. breve used in the following examples is: B. breve NEFU-1, with the preservation number CCTCC NO: M20241206, classified and named as , preserved in the China Center for Type Culture Collection on June 13, 2024; B. breve NEFU-2, with the preservation number CCTCC NO: M20241207, classified and named as , preserved in the China Center for Type Culture Collection on June 13, 2024.

[0023] Example 1: Isolation, screening and identification of lactic acid bacteria 1. Isolation and screening of lactic acid bacteria: select Yanji pickles as sample and dilute, spread on MRS solid medium containing uric acid (MRS broth 52.24 g, agar powder 10 g-20 g, dissolved in 1 L distilled water, sterilized at 121℃ for 20 min), stand, culture at 37℃ for 48 h, select the single colony with typical lactobacillus characteristics; the above single colony is placed on MRS solid medium plate and repeatedly streaked for purification until single morphology colony appears; the above obtained colony is inoculated into liquid MRS medium at 1% volume percentage inoculation amount, the lactobacillus with negative peroxidase activity and positive gram staining is obtained by detecting peroxidase activity and gram staining of the lactobacillus colonies grown on liquid MRS medium; the above strain is inoculated into liquid MRS medium and cultured at 37℃ for 48 h, subcultured twice, 2 mL of the above fermentation broth is centrifuged at 4℃ and 6000 r / min, the supernatant is collected, and the uric acid content in the supernatant is detected, the lactic acid bacteria with the highest uric acid degradation rate is selected, and two strains of lactic acid bacteria are obtained, and the colony morphology is observed as shown in Figures 1-4 .

[0024] 2. Identification of lactic acid bacteria: the DNA of the above obtained lactic acid bacteria strains is extracted using bacterial genomic DNA extraction kit, and universal primers 27F (nucleotide sequence is shown in SEQ ID NO. 1) and 1492R (nucleotide sequence is shown in SEQ ID NO. 2) are used for PCR amplification; the PCR amplification reaction system is: genomic DNA (20 ng / μL) 1 μL, 10×Buffer (containing 2.5 mM Mg 2+ ) 5 μL, Taq polymerase (5 u / μL) 1 μL, dNTP (10 mM) 1 μL, primers 27F and 1492R (10 μM) 1.5 μL each, ddH2O up to 50 μL; the PCR amplification reaction program is: pre-denaturation 95℃, 5 min; denaturation 95℃, 30 s, annealing 58℃, 30 s, extension 72℃, 1.5 min, 35 cycles; final extension 72℃, 7 min. The above PCR amplification product is recovered using AxyPrep DNA gel recovery kit, the PCR amplification product is sent to Shanghai Paixinnuo Biological Technology Co., Ltd. for sequencing, and the obtained sequence is subjected to homology comparison analysis with GenBank database using BLAST software, as shown in Figure 5As shown, it can be determined that the two strains of lactic acid bacteria obtained by the above screening are Lactobacillus buchneri, and they are named Lactobacillus buchneri NEFU-1 and Lactobacillus buchneri NEFU-2, respectively; the 16S rRNA of the Lactobacillus buchneri NEFU-1 is shown as SEQ ID NO. 3, and the 16S rRNA of the Lactobacillus buchneri NEFU-2 is shown as SEQ ID NO. 4.

[0025] Effect experiment: 1. Safety evaluation The safety of the Lactobacillus buchneri strains NEFU-1 and NEFU-2 obtained by the above screening was evaluated, and one pathogenic bacterium Escherichia coli (purchased from Yangling Ruizhixinweikang Biotechnology Co., Ltd.) was selected as an indicator strain. The indole test, gelatin liquefaction test and hemolysis test were performed on the Lactobacillus buchneri strains NEFU-1 and NEFU-2, and the specific steps were as follows: (1) Indole test: Lactobacillus buchneri strains NEFU-1 and NEFU-2 were inoculated into protein peptone water medium (composition: protein peptone 10 g, sodium chloride 5 g, disodium hydrogen phosphate 2 g, potassium hydrogen phosphate 2 g, glucose 5 g, distilled water 1000 mL) at an inoculation amount of 2%, and physiological saline was used as a blank control; 2 drops of dimethylbenzene were added and shaken uniformly at 35°C for 72 h, then 3-6 drops of indole reagent were added, and the test results were observed.

[0026] As shown in the results, Figure 6 no red rose indole appeared in the upper layer of the test tube, indicating that the Lactobacillus buchneri strains NEFU-1 and NEFU-2 were both negative and did not produce indole substances.

[0027] (2) Gelatin liquefaction test: semi-solid columnar gelatin medium (composition: protein peptone 10 g, sodium chloride 5 g, beef extract 3 g, gelatin 120 g, distilled water 1000 mL) was prepared, and Lactobacillus buchneri strains NEFU-1 and NEFU-2 were inoculated by puncturing, and cultured at 35°C for 48 h. Escherichia coli was used as a positive control, and the results were observed by placing the test tube in a 4°C refrigerator for 20 min, and observing the liquefaction of the gelatin.

[0028] As shown in the results, Figure 7 the semi-solid columnar gelatin medium in the test tube became solid, indicating that the Lactobacillus buchneri strains NEFU-1 and NEFU-2 did not produce gelatinase.

[0029] (3) Hemolysis test: The above L. buchneri NEFU-1 and NEFU-2 were placed at 37°C for 18-24 h to obtain seed liquid, 1 mL of which was added to 9 mL of blank MRS medium and incubated for 10 h to obtain activated L. buchneri NEFU-1 and NEFU-2, which were streaked on Columbia blood agar medium (composition: sodium chloride 5 g, glucose 2 g, peptone 5 g, tryptone 10 g, yeast extract powder 5 g, beef extract powder 5 g, agar 15 g, distilled water 1000 mL, defibrillated sheep blood 60 mL) and cultured at 37°C for 48 h, with E. coli as a control group. If a grass green hemolytic ring appeared, it was a-hemolytic; if a colorless transparent hemolytic ring appeared, it was β-hemolytic; and if no hemolytic ring appeared, it was γ-hemolytic.

[0030] The results are shown in Table 1. Figure 8 As shown in Table 1, the E. coli control group showed obvious β-hemolytic ring (complete hemolysis, transparent area), which was consistent with its known hemolytic properties; L. buchneri NEFU-1 and NEFU-2 did not show hemolytic activity, indicating that they did not have hemolytic related pathogenic mechanism.

[0031] Example 2: Preparation of composite microbial agent L. buchneri NEFU-1 and NEFU-2 obtained in Example 1 were inoculated into MRS medium at a ratio of 2:1 and a volume ratio of 1% inoculation amount, and cultured at 37°C for 36 hours to obtain a composite microbial agent, referred to as PC.

[0032] The OD 600 values of the above composite microbial agent, and L. buchneri NEFU-1 and NEFU-2 at different growth times were determined, and the results are shown in Table 2. Figure 9 As shown in Table 2, the L. buchneri strains in the composite microbial agent were in the logarithmic growth phase from 5-22 h, during which the maximum growth rate constant R and the fastest growth rate of the bacterial cells were achieved, and a large amount of lactic acid was produced (the fermentation broth PH was maintained at 4); the L. buchneri strains in the composite microbial agent entered the stationary phase after 22 h of culture, and the strain death and growth reached a dynamic balance, with the growth rate constant R being essentially zero, the growth rate of the strain slowed down, and the strain acid production capacity was inhibited after entering the stationary phase; in addition, the OD 600 values of the fermentation broth of the L. buchneri strains in the composite microbial agent could be maintained at a relatively stable level within 72 h of culture.

[0033] Effect experiment: 1. Determination of antioxidant capacity, self-aggregation rate, and hydrophobicity (1) DPPH free radical scavenging capacity: 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 composite microbial agent containing NEFU-1 and NEFU-2 prepared in the present example (referred to as: PC) were added to 100 μL of 0.4 mM DPPH solution in a 96-well plate as a test group, and reacted at 25°C for 30 min in the dark. The DPPH free radical scavenging capacity of lactic acid bacteria was determined according to the absorbance at 540 nm.

[0034] The results are shown in Table 1. Figure 10 As shown in Table 1, the DPPH free radical scavenging rates of L. buchneri NEFU-1, NEFU-2 and the composite microbial agent containing the above two strains were 72.93%, 68.37% and 72.58%, respectively, which were most similar to the DPPH free radical scavenging rate of the positive control vitamin C of 80.46%. It is shown that the composite microbial agent provided by the present application has strong DPPH free radical scavenging capacity.

[0035] (2) ABTS free radical scavenging capacity: 7.4 mM ABTS free radical solution was reacted with 2.6 mM potassium persulfate for 24 h at room temperature in the dark, and the purpose was to generate a stable, high-concentration ABTS radical cation (ABTS•⁺) stock solution. Before determination, the ABTS•⁺ stock solution to be tested was diluted with PBS at a wavelength of 734 nm so that its absorbance value was 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 free radical solution as a control group; 20 μL of NEFU-1 and NEFU-2 obtained in Example 1 and the composite microbial agent containing NEFU-1 and NEFU-2 prepared in the present example were added to 180 μL of ABTS free radical solution in a 96-well plate as a test group, and reacted at 25°C for 10 min in the dark. The ABTS free radical scavenging capacity of lactic acid bacteria was determined according to the absorbance at 734 nm.

[0036] The results are shown in Table 2. Figure 11 As shown in Table 2, the ABTS free radical scavenging rates of L. buchneri NEFU-1, NEFU-2 and the composite microbial agent containing the above two strains were 72.39%, 73.59% and 76.50%, respectively, which were most similar to the ABTS free radical scavenging rate of the positive control vitamin C of 82.12%. It is shown that the composite microbial agent containing L. buchneri NEFU-1 and NEFU-2 provided by the present application has strong ABTS free radical scavenging capacity.

[0037] (3) Automatic aggregation ability determination: NEFU-1 and NEFU-2 obtained in Example 1 and the composite microbial inoculant containing NEFU-1 and NEFU-2 prepared in the present example were respectively grown in LB broth at 37℃ overnight, centrifuged and washed twice with sterile PBS (pH = 7.2), resuspended in supernatant, vortex mixed for 30 seconds; VC was used as a control group, and the absorbance was measured at a wavelength of 600 nm at different times (0, 1, 2, 3, 4 and 5 hours) using a UV-visible spectrophotometer, and the automatic aggregation abilities of Lactobacillus bulgaricus NEFU-1, NEFU-2 and the composite microbial inoculant were obtained respectively.

[0038]

[0039] A0: The initial absorbance value measured at a wavelength of 600 nm when the time was 0 hour (i.e. immediately after the bacterial suspension was prepared and vortex mixed); A t : The absorbance value measured at a wavelength of 600 nm for the sample taken from the upper layer of the suspension at t hours.

[0040] Lactic acid bacteria can form a barrier by self-aggregation, which is beneficial to the intestinal colonization of probiotic strains and can prevent pathogenic bacteria from colonizing and infecting the intestine. Figure 12 As shown in Table 1, the combined self-aggregation abilities of Lactobacillus bulgaricus NEFU-1, NEFU-2 and the composite microbial inoculant containing the above two strains reached 46.49%, 39.45% and 52.75% respectively at 24 h; it can be seen that the composite microbial inoculant containing Lactobacillus bulgaricus NEFU-1 and NEFU-2 provided by the present application has strong automatic aggregation ability.

[0041] (4) Cell surface hydrophobicity determination: The cell surface hydrophobicity of NEFU-1 and NEFU-2 obtained in Example 1 and the composite microbial inoculant containing NEFU-1 and NEFU-2 prepared in the present example was determined by the microbial adhesion to hydrocarbons method (MATH).

[0042] The above Lactobacillus bulgaricus NEFU-1, NEFU-2 and the composite microbial inoculant containing the above two strains were respectively cultured for 24 h, centrifuged and washed twice with PBS (pH = 7.2), resuspended in 5 mL PBS buffer, and the absorbance of the cell suspension was measured at 600 nm using a UV-visible spectrophotometer, and recorded as A0; then the cell suspension was mixed with the same volume of solvent for 5 min, and the suspension was obtained, which was measured when the solvent was ethyl acetate (basic solvent), chloroform (acidic solvent) or xylene (non-polar solvent); the suspension was incubated at room temperature for 30 min to achieve two-phase separation; the absorbance of the aqueous phase at 600 nm was detected and recorded as A tThe hydrophobicity of the cell surface of Lactobacillus bruneri NEFU-1, NEFU-2 and compound bacterial solutions was obtained.

[0043]

[0044] 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.

[0045] 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.

[0046] 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.

[0047] 2. Degradation of inosine and / or guanosine The Lactobacillus buchneri strains NEFU-1 and NEFU-2 screened in Example 1 and the compound microbial agent prepared in Example 2 were respectively placed at 37 DEG C for 18-24 h to obtain seed liquid, 1 mL of each was added into 9 mL of blank MRS medium for incubation for 10 h to obtain the activated Lactobacillus buchneri strains NEFU-1 and NEFU-2 and the compound microbial agent; 2 mL of each of the activated second-generation Lactobacillus buchneri strains NEFU-1 and NEFU-2 and the compound microbial agent was centrifuged at 5000 r / min for 10 min at 4 DEG C to collect the bacterial bodies; the bacterial bodies were resuspended and washed with 1 mL of normal saline, centrifuged at 5000 r / min for 10 min at 4 DEG C, repeated twice and the bacterial bodies were collected; 11.3 mg of inosine and 28.3 mg of guanosine were weighed, respectively dissolved in potassium phosphate buffer and made up to 50 mL, if guanosine is not dissolved in potassium phosphate buffer, 80 DEG C water bath heating can be used, 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; 375 μL of guanosine-neutral potassium phosphate solution and 375 μL of inosine-neutral potassium phosphate solution were added to the washed and collected Lactobacillus buchneri strains NEFU-1 and NEFU-2 and the compound microbial agent bacterial bodies respectively to resuspend the bacterial bodies, and the reaction was carried out at 37 DEG C and 120 r / min for 1 h, after the reaction was completed, centrifugation was carried out at 5000 r / min for 10 min at 4 DEG C, 720 μL of supernatant was taken, 80 μL of perchloric acid solution was added to terminate the reaction; the supernatant was filtered using a filter membrane, and the content of inosine and guanosine in the solution was determined by high performance liquid chromatography.

[0048] As shown in Table 1, the degradation rate of Lactobacillus buchneri strain NEFU-1 on inosine was 68.64%, and the degradation rate on guanosine was 100%; the degradation rate of Lactobacillus buchneri strain NEFU-2 on inosine was 78.72%, and the degradation rate on guanosine was 100%; the degradation rate of the compound microbial agent containing Lactobacillus buchneri strain NEFU-1 and Lactobacillus buchneri strain NEFU-2 on inosine was 80.23%, and the degradation rate on guanosine was 100%.

[0049] It can be seen that, compared with Lactobacillus buchneri strain NEFU-1 and Lactobacillus buchneri strain NEFU-2, the compound microbial agent provided by the application has better degradation ability on inosine and guanosine.

[0050] Table 1

[0051] 3. Degradation of uric acid to relieve hyperuricemia (1) In vitro test The Lactobacillus buchneri strains NEFU-1 and NEFU-2 obtained in Example 1 and the complex microbial agent obtained in Example 2 were respectively placed in MRS culture medium, and incubated at 37°C until logarithmic growth phase; after centrifugation and 3 times of washing, they were suspended in PBS, and inoculated into MRS culture medium containing 0.336 g / L uric acid at a 5% inoculation amount, and incubated in a 37°C constant temperature incubator for 24 h; the incubated culture liquid was centrifuged every 6 h to collect the supernatant, which was filtered through a 0.22 μm filter membrane, and then the degradation of uric acid was detected by HPLC to obtain the degradation rate of uric acid.

[0052]

[0053] : 0 hour of culture, initial concentration of uric acid in the culture medium (i.e. 0.336 g / L); : t hours of culture, residual concentration of uric acid in the supernatant after centrifugation and filtration.

[0054] As shown in Tables 2-3, the Lactobacillus buchneri strains NEFU-1 and NEFU-2 strains and the complex microbial agent can degrade 0.336 g / L of uric acid to 0.1309 g / L, 0.1152 g / L and 0.0927 g / L respectively within 24 h, and the degradation rates are 61.02%, 65.71% and 72.41% respectively. It can be seen that, compared with the Lactobacillus buchneri strains NEFU-1 and NEFU-2 strains, the complex microbial agent provided by the present application has the effect of efficiently and directly degrading uric acid.

[0055] Table 2

[0056] Table 3

[0057] (2) In vivo test Establishment of animal model and grouping: 60 male KM mice with a body weight of 20-25 g (purchased from Suzhou Xishan Biotechnology Co., Ltd., license number SCXK (Liaoning) 2010-0001) were taken; after 7 d of adaptive feeding, the mice in the Lactobacillus buchneri NEFU-1 group (B1), the Lactobacillus buchneri NEFU-2 group (B2), the HUA hyperuricemia group (HUA) and the complex microbial agent treatment group (HB) were all modeled by gavage; specifically, from the 8th day, 300 mg / kg body weight of oxyphenisat potassium + uric acid suspension was gavaged every day, and ordinary mouse feed was supplemented, and the mice were allowed to drink water freely; after 14 d of continuous feeding, the blood of the mice in each group was taken, centrifuged at 3000 r / min for 15 min, and the serum was collected.

[0058] Determination of uric acid content: On the last day of the above-mentioned gavage for 14 d, the mice in each group were respectively taken blood from the orbit, placed in the blood collection tube at room temperature for 1 h, centrifuged at 3000 rpm, 4℃ for 15 min, and the supernatant was taken and stored as serum; the uric acid test kit provided by Nanjing Jiancheng Biological Engineering Institute was used to determine the uric acid content in the serum of mice, and the principle was that uric acid produced quinoline pigment under the action of uricase and peroxidase, and the absorbance value was read at a wavelength of 510 nm using an enzyme label instrument, and the UA concentration was obtained by the following formula: UA concentration (μmol / L) = (A measured-A blank) / (A standard-A blank) x C standard A measured: absorbance value of the test product (serum of mice in NEFU-1 group, NEFU-2 group, compound bacterial agent treatment group and HUA hyperuricemia group, respectively); A blank: absorbance value of double distilled water; A standard: absorbance value of uric acid standard; C standard: concentration of uric acid standard (500 μmol / L).

[0059] As shown in Table 4, the blood uric acid content of mice in the Lactobacillus buchneri NEFU-1 group (B1), Lactobacillus buchneri NEFU-2 group (B2) and compound bacterial agent treatment group (HB) was significantly different from that of mice in the HUA hyperuricemia group (HUA) at 21 days (7 d of adaptive feeding + 14 d of continuous feeding after gavage). Compared with the HUA hyperuricemia group (HUA), the blood uric acid content of mice in the compound bacterial agent treatment group (HB) was significantly reduced (P<0.0001). P <0.0001).

[0060] It can be seen that the Lactobacillus buchneri strains NEFU-1 and NEFU-2 and the compound bacterial agent have a high-efficiency direct degradation effect on uric acid in hyperuricemic mice. Among them, the degradation efficiency of the compound bacterial agent provided by the present application on uric acid is obviously higher than that of the Lactobacillus buchneri strains NEFU-1 and NEFU-2 alone.

[0061] Table 4

[0062] 4、Reducing inflammatory factors The IL-1β, MDA and XOD factor contents in the kidney homogenate of the above-mentioned HUA hyperuricemia group (HUA) and compound bacterial agent treatment group (HB) were detected using the Elisa kit of Seville Bio. The short-chain fatty acids (SCFA) in the feces of the above-mentioned HUA hyperuricemia group (HUA) and compound bacterial agent treatment group (HB) were detected using the Agilent gas chromatography mass spectrometer GC-MS.

[0063] As shown in Figure 14 Figure 6, compared with the HUA hyperuricemia group (HUA), the IL-1β content in the kidney homogenate of the compound probiotic treatment group (HB) mice decreased from 3993.54 pg / mL to 2963.54 pg / mL (decrease ≈ 26%), indicating that the compound probiotic provided by the application can effectively inhibit the inflammatory pathway.

[0064] As shown in Figure 15 Figure 7, compared with the HUA hyperuricemia group (HUA), the XOD activity in the kidney homogenate of the compound probiotic treatment group (HB) mice decreased from 0.207 U / g to 0.141 U / g (decrease ≈ 32%), indicating that the compound probiotic provided by the application can effectively intervene and block the overactivation of XOD, thereby reducing the synthesis of uric acid.

[0065] As shown in Figure 16 Figure 8, compared with the HUA hyperuricemia group (HUA), the MDA content in the kidney homogenate of the compound probiotic treatment group (HB) mice decreased from 14.75 µmol / gprot to 3.42 µmol / gprot (decrease ≈ 77%), indicating that the compound probiotic provided by the application can protect the membrane structure integrity to maintain the function of renal tubules by removing free radicals (consistent with the in vitro free radical scavenging test).

[0066] As shown in Figure 17 and Table 5, compared with the HUA hyperuricemia group (HUA), the level of most short-chain fatty acids in the mice of the compound probiotic treatment group (HB) was significantly improved. Among them, butyric acid and caproic acid are very important short-chain fatty acids, butyric acid is the main energy source for colon cells, has anti-inflammatory and intestinal barrier function maintenance effects, and caproic acid has antibacterial and anti-inflammatory properties. Compared with the HUA hyperuricemia group (HUA), the butyric acid content in the mice of the compound probiotic treatment group (HB) was increased by +108.04%, and the caproic acid content was increased by +193.24%. It shows that the compound probiotic provided by the application has a positive significance for improving the intestinal barrier function, inhibiting inflammation, and indirectly promoting uric acid excretion.

[0067] Table 5

[0068] Example 3: Application of compound probiotic in the preparation of fermented yogurt The complex microbial inoculant obtained in Example 2 is inoculated into MRS liquid and cultured in a 37℃ incubator for 24 hours; fresh milk is mixed with sucrose in a mass percentage of 20:1, homogenized to obtain a mixed liquid; the mixed liquid is heated to 75℃, kept for 25 min, cooled to 40℃, and then 6% of the above cultured complex microbial inoculant is added under sterile operating conditions, stirred uniformly, canned, sealed, and fermented at 37℃ for 12 hours; after fermentation, the product is cooled at 4℃ for post-ripening to obtain fermented yogurt.

[0069] The above disclosed embodiments of the present application are only used to help explain the present application. The embodiments do not describe all the details and limit the present application to the described embodiments. According to the content of the present specification, many modifications and variations can be made. The present specification selects and specifically describes these embodiments in order to better explain the principles and practical applications of the present application, so that those skilled in the art can well understand and utilize the present application.

Claims

1. A compound microbial agent, characterized in that, The compound bacterial agent is Lactobacillus bromide NEFU-1 ( Lactobacillus buchneri NEFU-1) and Lactobacillus Brucella NEFU-2 ( Lactobacillus buchneri NEFU-2 was prepared by inoculating it into MRS liquid medium and culturing it at 37°C for 36 hours.

2. The compound microbial agent according to claim 1, characterized in that, The Lactobacillus brunetti NEFU-1 described herein has the accession number CCTCC NO: M 20241206 and is classified as follows: Lactobacillus buchneri NEFU-1, deposited at the China Center for Type Culture Collection, on June 13, 2024; The Lactobacillus brunetti NEFU-2 described herein 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.

3. 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.

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

5. The application according to claim 4, 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 3 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.

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

7. The application according to claim 5, characterized in that, In the fermented yogurt preparation method, the mass percentage of fresh milk to sucrose is 20:

1.

8. The application according to claim 5, characterized in that, In the fermented yogurt preparation method, the mass-volume ratio of the cultured compound microbial agent added is 3%-10%.

9. The use of the compound microbial agent according to any one of claims 1 to 3 in the preparation of a drug.

10. The application according to claim 9, characterized in that, The drug has at least one of the following functions (a)-(d): (a) Degrades uric acid and alleviates hyperuricemia; (b) Degradation of inosine and / or guanosine; (c) Improve the composition of gut microbiota; (d) Reduce inflammatory factors.

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