Compound probiotics for degrading indole and preventing or relieving nephropathy and application thereof

Abstract

The invention relates to the technical field of microorganisms, in particular to compound probiotics capable of degrading indole and preventing or relieving nephropathy and application of the compound probiotics. The compound probiotics comprise bifidobacterium animalis subsp. Lactis YYS-J9 with the preservation number of CGMCC (China General Microbiological Culture Collection Center) NO.28185 and bifidobacterium longum subsp. Longum YYS-BSM8 with the preservation number of CGMCC NO.28611. The invention further discloses a preparation method of the compound probiotics. Compared with the use of a single YYS-J9 strain or YYS-BSM8 strain, the two strains have synergistic effects after being compounded, the viable count is remarkably increased after mixed culture, the inhibition effect on pathogenic enterobacteria and the adhesion to intestinal cells are remarkably enhanced, the degradation capacity on indole and oxalic acid is higher, the proliferation and phagocytic activity of macrophages is remarkably improved, and the application range of the YYS-J9 strain and the YYS-BSM8 strain is widened. And when being applied to food, health-care products or medicines, the exopolysaccharide can be used for effectively preventing or improving nephropathy and kidney stone, regulating intestinal health and improving intestinal immunity.

Description

Technical Field

[0001] The invention relates to the technical field of microorganisms, and in particular to a composite probiotic capable of degrading indole and preventing or alleviating kidney disease and application thereof. Background Art

[0002] Indole is a nitrogen-containing heterocyclic compound widely found in nature. It is produced during the metabolism of human intestinal microorganisms. Some bacteria in the intestine can convert substances such as tryptophan into indole, which is then absorbed into the blood circulation. Under normal circumstances, indole undergoes a series of metabolic transformations in the body, and its content is in a relatively stable state. However, when the intestinal microbial community is unbalanced, the production of indole may increase, which in turn has a potential impact on kidney health. Many studies have shown that abnormal increases in indole content may cause damage to the kidneys. High concentrations of indole and its metabolites may trigger an inflammatory response in the kidneys, and the normal filtration and reabsorption functions of the kidneys will be disturbed, leading to symptoms such as proteinuria and hematuria. In severe cases, it may even affect the overall function of the kidneys. In addition, changes in indole content may also be closely related to the oxidative stress state of the kidneys. Indole and its derivatives may induce the production of reactive oxygen species in kidney cells, breaking the original redox balance in the kidneys, causing the kidneys to produce oxidative stress reactions, resulting in free radical attacks on important structures such as the cell membrane and mitochondria of kidney cells, leading to cell apoptosis or necrosis.

[0003] Clinically, some patients with kidney disease are often accompanied by disorders of the intestinal microbial community, which further confirms the association between indole content and kidney health. For example, in patients with chronic kidney disease, intestinal flora imbalance is more common, and the metabolic pathway of indole may change, resulting in increased levels of indole and its related metabolites in the body. By regulating the intestinal flora and improving the metabolic status of indole, it is expected to become an auxiliary means of treating kidney disease. For example, the use of probiotics, prebiotics or fecal transplantation to adjust the balance of intestinal microecology may help reduce indole content, reduce inflammation and oxidative stress damage in the kidneys, and thus delay the deterioration of kidney disease.

[0004] In summary, given that excessive accumulation of high concentrations of indole in an organism will produce toxic effects and affect the normal physiological functions of the organism, and probiotics have the ability to degrade indole, how to provide a probiotic that can degrade indole is a technical problem that technical personnel in this field urgently need to solve. Summary of the invention

[0005] In order to solve the deficiencies of the prior art mentioned in the above background technology, the present invention provides a composite probiotic for degrading indole and preventing or alleviating kidney disease, including Bifidobacterium animalis subsp. lactis ( Bifidobacterium animalis subsp.lactis) YYS-J9, whose deposit number is CGMCC NO.28185, and Bifidobacterium longum subsp. longum ( Bifidobacterium longum subsp.Longum ) YYS-BSM8, whose preservation number is CGMCC No.28611, and the ratio of the number of viable bacteria of animal Bifidobacterium lactis subspecies YYS-J9 to that of Bifidobacterium longum subspecies longum YYS-BSM8 is 1-10:1-10.

[0006] The present invention also provides a composite probiotic, the components of which include the above-mentioned Bifidobacterium animalis subspecies lactis YYS-J9 and Bifidobacterium longum subspecies longum YYS-BSM8.

[0007] In one embodiment, in the composite probiotic, the ratio of the number of live bacteria of Bifidobacterium animalis subspecies lactis YYS-J9 to that of Bifidobacterium longum subspecies longum YYS-BSM8 is 1-3:1-3.

[0008] Preferably, in the composite probiotic, the ratio of the number of live bacteria of Bifidobacterium animalis subspecies lactis YYS-J9 to that of Bifidobacterium longum subspecies longum YYS-BSM8 is 1:1.

[0009] In one embodiment, the viable count of the composite probiotics is ≥ 4.5×10 9 CFU / g.

[0010] In one embodiment, the composite probiotic further comprises a lyophilization protectant, and the lyophilization protectant comprises any one or more combinations of skimmed milk powder, maltodextrin, sodium glutamate, sucrose, lactose, trehalose, sorbitol and xylitol.

[0011] In one embodiment, the composite probiotic further comprises auxiliary additives, and the auxiliary additives comprise any one or more combinations of fructooligosaccharides, galacto-oligosaccharides, xylooligosaccharides, isomaltooligosaccharides, soy oligosaccharides, inulin, spirulina, stachyose, polydextrose, α-lactalbumin or lactoferrin.

[0012] In one embodiment, the composite probiotic further comprises excipients, which include any one or more combinations of prebiotics, fillers, acidulants, solvents, solubilizers, cosolvents, emulsifiers, colorants, binders, disintegrants, lubricants, wetting agents, stabilizers, glidants, flavoring agents, preservatives, coating materials, fragrances, anti-adhesive agents, integrators, thickeners, buffers, and inclusion agents.

[0013] In one embodiment, the dosage form of the composite probiotic includes lyophilized powder, capsule, tablet or granule.

[0014] The composite probiotic provided by the present invention has at least one of the following effects: (1) Degradation of indole; (2) Producing extracellular polysaccharides; (3) Resistant to gastric acid and bile salts; (4) Inhibit intestinal pathogens; (5) Improve intestinal immunity; (6) Degradation of oxalic acid.

[0015] The present invention also provides a probiotic solid beverage, the components of which include the composite probiotic agent as described above.

[0016] The present invention also provides a use of the composite probiotic agent as described above in the preparation of food, health products or medicines for degrading indole, preventing or alleviating kidney disease, preventing or improving stones, regulating intestinal health, and improving body immunity.

[0017] Compared with the prior art, the composite probiotics for degrading indole and preventing or alleviating kidney disease provided by the present invention have the following beneficial effects: Compared with the use of a single YYS-J9 strain or a single YYS-BSM8 strain, the two bacteria have synergistic effects after compounding, and the number of live bacteria is significantly increased after mixed culture of the two. When the two bacteria are used together, the inhibitory effect on intestinal pathogens and the adhesion to intestinal cells are significantly enhanced. They are more resistant to gastric acid and bile salts, have stronger ability to degrade indole and oxalic acid, significantly improve macrophage proliferation and phagocytic activity, and increase the production of extracellular polysaccharides. When used in food, health products or medicines, they can effectively prevent or improve kidney disease and kidney stones, regulate intestinal health, and enhance intestinal immunity. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0019] Figure 1 The growth curves of YYS-J9, YYS-BSM8 and the composite bacteria of Example 1 provided by the present invention; Figure 2 The viable counts of YYS-J9, YYS-BSM8 and the composite bacteria in Example 1 provided by the present invention; Figure 3 The size of the inhibition zone of each strain of Example 2 provided by the present invention for inhibiting intestinal pathogens; Figure 4 The number of probiotics adhered to every 100 HT-29 cells of Example 3 provided by the present invention; Figure 5The glucose standard curve in the determination of the extracellular polysaccharide content in Example 5 provided by the present invention; Figure 6 The standard curve of indole concentration of Example 7 provided by the present invention; Figure 7 It is the indole degradation rate of each strain in Example 7 provided by the present invention. DETAILED DESCRIPTION

[0020] In order to make the purpose, technical solution and advantages of the embodiments of the present invention clearer, the technical solution in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0021] The classification name of the Bifidobacterium animalis subsp. lactis YYS-J9 strain involved in the following examples is Bifidobacterium animalis subsp. lactis, Latin name: Bifidobacterium animalis subsp.lactis The depository is the General Microbiology Center of China Microbiological Culture Collection Administration. The deposit date is August 17, 2023. The deposit number is CGMCCNO.28185. The deposit address is: No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing.

[0022] The classification name of the Bifidobacterium longum subspecies longum YYS-BSM8 strain involved in the following examples is Bifidobacterium longum subspecies longum, Latin name: Bifidobacterium longum subsp.Longum YYS-BSM8, the depository is the General Microbiology Center of China Microbiological Culture Collection Administration, the deposit date is October 12, 2023, the deposit number is CGMCC No.28611, and the deposit address is: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

[0023] The formula of the MRS liquid culture medium involved in the following examples is: beef extract 10.0 g, glucose 20.0 g, tryptone 10.0 g, yeast extract powder 5.0 g, Tween 80 1.0 mL, dipotassium hydrogen phosphate 2.0 g, ammonium citrate 2.0 g, anhydrous sodium acetate 5.0 g, magnesium sulfate 0.5 g, manganese sulfate monohydrate 0.25 g, deionized water 1.0 L, pH 6.5 (1.5% agar is added to make MRS solid culture medium, and 0.05% L-cysteine ​​hydrochloride is added for culturing bifidobacteria).

[0024] The preparation method of the composite probiotics involved in the following embodiments can be prepared by conventional technical methods in the art, for example: after activating the animal Bifidobacterium lactis subspecies YYS-J9 strain and the long Bifidobacterium longum subspecies YYS-BSM8 strain, they are inoculated in MRS culture medium to obtain culture solution, the culture solution is centrifuged, the bacteria are resuspended to obtain a bacterial suspension, and the two bacterial suspensions are mixed according to the ratio of viable bacteria of 1:1 to obtain composite probiotics; or further add a lyophilization protective agent for freeze drying to obtain a composite probiotic agent; a single bacterial agent can be obtained in the same way. It should be noted that the ratio of viable bacteria can be selected according to other actual ratios, for example, the ratio of viable bacteria of the animal Bifidobacterium lactis subspecies YYS-J9 strain and the long Bifidobacterium longum subspecies YYS-BSM8 strain can be any one of 1:2, 1:3, 2:1, 2:3, 3:1, and 3:2.

[0025] Example 1 Synergistic effect of YYS-J9 and YYS-BSM8 YYS-J9 and YYS-BSM8 strains were activated to the second generation of bacterial liquid using MRS liquid medium, and inoculated into MRS liquid medium supplemented with 0.05% L-cysteine ​​at a volume ratio of 1:1 at a 3% inoculation rate for mixed culture. At the same time, YYS-J9 and YYS-BSM8 were inoculated at a 3% inoculation rate for separate culture at 37°C for 24 h, and the growth curve was determined ( Figure 1 ), and the final live bacteria count was performed according to the plate pouring method. The results are shown in Figure 2 .

[0026] like Figure 1 and Figure 2 As shown, the mixed culture of YYS-J9 and YYS-BSM8 had a higher viable count than that of either culture, indicating that YYS-J9 and YYS-BSM8 had a potential synergistic symbiotic effect.

[0027] Example 2 Antagonism against intestinal pathogens The punching method was used to determine the inhibitory effect of the strain on three common intestinal pathogens (Staphylococcus aureus, Escherichia coli, and Shigella sonnei). YYS-J9, YYS-BSM8, and a mixture of the two were cultured in MRS medium until the second generation of bacterial solution was used for use. The three pathogens were cultured in LB liquid medium for 24 hours, and 100 μL of each was evenly spread on the LB solid plate medium. Then, three holes were evenly punched using a puncher with a diameter of 8 mm. 200 μL of YYS-J9, YYS-BSM8, and mixed bacterial culture solution were added to the three holes, respectively. The plate was placed at 37°C for anaerobic culture. The size of the inhibition zone (unit: mm) was measured after 36 hours. The experiment was repeated three times, and the results are shown in the table. Figure 3 .from Figure 3It can be seen that the three bacterial cultures have good inhibitory effects on the three intestinal pathogens, among which the diameter of the inhibition zone of the mixed bacterial culture solution is higher than that of the single bacterial culture solution, indicating that YYS-J9 and YYS-BSM8 also have synergistic antibacterial ability.

[0028] Example 3 Intestinal cell adhesion HT-29 cells were activated and cultured in DMEM medium containing 10% heat-inactivated fetal bovine serum and 1% double antibodies (streptomycin and penicillin). After culturing to the logarithmic phase, the cells were counted and resuspended in DMEM medium containing 10% heat-inactivated fetal bovine serum and 1% double antibodies to a concentration of 1×10 5 Cells were inoculated onto a 6-well plate (2 mL) with an acid-washed sterilized coverslip, cultured for 12 h, and washed three times with sterile PBS. The experimental strain was cultured at 37°C for 18 h, harvested by centrifugation at 8000×g, 4°C for 5 min, and washed twice with sterile PBS. The cells were resuspended in DMEM medium to 5×10 7 CFU / mL, and inoculated into 6-well plates (2 mL), incubated at 37°C for 2 h, discarded the culture medium, and washed the cells 3 times with sterile PBS. Add 2 mL of methanol to each well and fix at room temperature for 1 h. After fixation, discard the methanol and stain with crystal violet solution. Examine the coverslips in the 6-well plates under a microscope and count the number of bacteria adhered per 100 HT-29 cells ( Figure 4 ).

[0029] like Figure 4 As shown, the average number of YYS-J9 cells adhered to every 100 HT-29 cells was 54, the average number of YYS-BSM8 cells adhered to every 100 HT-29 cells was 55, and the average number of composite bacteria cells adhered to every 100 HT-29 cells was 61. Each strain had good intestinal cell adhesion; however, compared with the adhesion effect of YYS-J9 and YYS-BSM8 on intestinal cells alone, the number of composite bacteria adhering to intestinal cells was significantly increased, indicating that the adhesion effect of composite bacteria was significantly higher than that of single strains.

[0030] Example 4 Heat, acid and bile resistance of bacterial agents 1. Heat resistance YYS-J9, YYS-BSM8 and their composite were inoculated into MRS liquid culture medium at an inoculum size of 2% (w / v), cultured at 40°C and 50°C for 2 h, respectively. The plate count method was used to count the viable bacteria (LogCFU / mL). The results are shown in Table 1.

[0031] Table 1 Study on heat resistance

[0032] As shown in Table 1, there was no significant difference in the survival rate of YYS-J9, YYS-BSM8 and the composite bacteria after being cultured at 40℃ and 50℃ for 2 h.

[0033] 2. Acid and bile salt resistance YYS-J9, YYS-BSM8 and their composite were inoculated at a 2% (w / v) inoculum size at pH 4.0, pH 3.0 and a bile salt concentration of 0.2% at 37°C and cultured for 2 h. The plate count method was used to count the viable bacteria (LogCFU / mL). The results are shown in Table 2.

[0034] Table 2 Study on acid resistance and bile salt resistance

[0035] As can be seen from Table 2, under the condition of pH 3.0, the YYS-J9 and YYS-BSM8 composite bacteria still had a sufficient number of live bacteria to pass through the gastric environment after 2 h, and both had good bile salt tolerance at a bile salt concentration of 0.2%.

[0036] 3. Tolerance in artificial gastric juice and artificial intestinal juice A certain amount of pepsin was dissolved in a PBS buffer solution at pH 3.0 and its final concentration was adjusted to 3.0 g / L to obtain simulated artificial gastric juice. A certain amount of trypsin was dissolved in a PBS buffer solution at pH 8.0 and its final concentration was adjusted to 1.0 g / L to obtain simulated artificial intestinal juice. Both artificial gastric juice and artificial intestinal juice were filtered with a 0.22 μm filter membrane under sterile conditions.

[0037] YYS-J9, YYS-BSM8 and their composite were inoculated into artificial gastric fluid at a 2% (w / v) inoculation rate and cultured at 37°C for 2 h. Then, the fermentation liquid in artificial gastric fluid was inoculated into artificial intestinal fluid at a 10% inoculation rate and cultured at 37°C for 2 h. The plate count method was used to count the viable bacteria (Log CFU / mL). The results are shown in Table 3.

[0038] Table 3 Tolerance in artificial gastric juice and artificial intestinal juice

[0039] As can be seen from Table 3, after passing through artificial gastric juice and then artificial intestinal juice, YYS-J9, YYS-BSM8 and the composite bacteria were well tolerated and could successfully colonize in the intestine and exert their efficacy.

[0040] Example 5: Production of exopolysaccharides 1.1 Reagent preparation Concentrated sulfuric acid solution: Accurately measure 82 mL of concentrated sulfuric acid, add it to 18 mL of water, and set aside after cooling.

[0041] Anthrone solution: Accurately weigh 0.2 g of anthrone and add 100 mL of 82% concentrated sulfuric acid solution to dissolve it.

[0042] 1.2 EPS content determination Anthrone colorimetry was used to quantify EPS production. Prepare glucose standard sample solutions of 10, 20, 40, 60, 80, and 100 μg / mL. Pipette 1 mL of standard sample of different concentrations and add it to a 10 mL stoppered colorimetric tube with pure water as a control. Add 4 mL of anthrone solution, shake well, and cool in an ice water bath. Move to a boiling water bath and heat for 10 min, cool with ice water, and place at room temperature away from light for 10 min. Measure the absorbance at a wavelength of 620 nm and draw a standard curve (y=0.0013x+0.9429, R 2 =0.9917), the standard curve is as follows Figure 5 shown.

[0043] The experimental strain was cultured at 37°C for 18 h, then expanded and centrifuged at 4°C at 12,000×g for 10 min. The supernatant was taken, 80% (v / v) trichloroacetic acid was added to a final concentration of 4%, and the mixture was allowed to stand at 4°C for 30 min, centrifuged at 8,000×g for 10 min, and the protein precipitate was discarded. The obtained supernatant was mixed with anhydrous ethanol at a ratio of 1:3, allowed to stand at 4°C for 12 h, centrifuged at 8,000×g for 10 min, and the polysaccharide precipitate was taken. 2 mL of pure water was taken to re-dissolve the polysaccharide precipitate, and the EPS content was determined according to the standard curve method. The EPS content is expressed as mg / mL.

[0044] The results in Table 4 show that the polysaccharide production of the YYS-J9 strain was 0.75±0.13 mg / mL, the polysaccharide production of the YYS-BSM8 strain was 2.09±0.25 mg / mL, and the polysaccharide production of the composite bacteria was 2.38±0.49 mg / mL. In addition, compared with the single bacteria, the composite bacteria had a higher polysaccharide production ( P <0.05).

[0045] Table 4 EPS production of each strain

[0046] Example 6 Effects of strains on the proliferation and phagocytic ability of macrophages RAW264.7 Phagocytosis is an important defense mechanism of vertebrates against pathogen invasion, and enhanced phagocytosis is a prominent feature of macrophage activation.

[0047] (1) CCK-8 assay to determine the effect of strains on RAW264.7 cell proliferation RAW264.7 cells were inoculated into DMEM medium containing 10% heat-inactivated fetal bovine serum (BI) and 1% double antibodies (streptomycin and penicillin) for activation and passage. After culturing to the logarithmic phase, the cells were counted and resuspended in DMEM medium containing 10% heat-inactivated fetal bovine serum (BI) and 1% double antibodies (streptomycin and penicillin) to a cell concentration of 1×10 5 Inoculate 100 μL of RAW264.7 cells in a 96-well cell culture plate, and add new DMEM medium to replace the original culture medium after the cells adhere to the wall.

[0048] The experiment set up a blank group (DMEM culture medium) and an experimental group (YYS-J9, YYS-BSM8 and mixed bacteria, the concentration of the bacterial solution was 10 9 CFU / mL), with 3 replicates in each group. Culture in a 37℃, 5% CO2 incubator for 24 h. After the culture, remove the old culture medium, add 100 μL of DMEM culture medium containing 10% CCK-8 to each well, protect from light, and continue to culture in a CO2 incubator for 30 min; use an ELISA reader to detect the OD value of each well at 450nm to calculate the relative cell proliferation rate.

[0049] Relative cell proliferation rate%=OD value of experimental group / OD value of blank group.

[0050] (2) The neutral red method was used to determine the effect of the strain on the phagocytic ability of RAW264.7 cells.

[0051] RAW264.7 cells were inoculated into DMEM medium containing 10% heat-inactivated fetal bovine serum (BI) and 1% double antibodies (streptomycin and penicillin) for activation and passage. After culturing to the logarithmic phase, the cells were counted and resuspended in DMEM medium containing 10% heat-inactivated fetal bovine serum (BI) and 1% double antibodies (streptomycin and penicillin) to a cell concentration of 1×10 5 / mL. Inoculate 100μL RAW264.7 cells in a 96-well cell culture plate, and add new DMEM culture medium to replace the original culture medium after the cells adhere to the wall. The experimental grouping is the same as in Example 6 (1), with 3 replicates in each group. Culture in a 37℃, 5% CO2 incubator for 24 h; discard the supernatant culture medium in the well, then add 0.1% neutral red-PBS solution in a 37℃, 5% CO2 incubator under light-proof conditions and incubate for 1 h; after the incubation, discard the supernatant and wash three times with PBS at room temperature. Then add lysis solution (acetic acid: anhydrous ethanol = 1:1) to each well and keep it in a dark place. After the cells are completely lysed, use an enzyme reader to measure the absorbance value of each well at a wavelength of 540nm.

[0052] Cell phagocytosis index = OD value of experimental group / OD value of blank group.

[0053] Table 5 Effects of various strains on the proliferation and phagocytic ability of macrophage RAW264.7 cells

[0054] As shown in Table 5, each strain can promote the proliferation and phagocytic activity of RAW264.7 cells and effectively play an immunomodulatory role. The proliferation and phagocytic activity of the composite bacteria on RAW264.7 cells are higher than those of the single strain, indicating that YYS-J9 and YYS-BSM8 have a synergistic effect.

[0055] Example 7 Indole degradation ability of various strains Indole standard was dissolved in ethyl acetate to prepare indole mother solution of 10 mg / mL, and then diluted to 0, 100, 200, 300, 400, 500, 600, 700, 800 μg / mL, respectively. High performance liquid chromatography HPLC system (Thermo U3000) was used with C18 column (5 μm, 250×4.6 mm), injection volume 10 μL, flow rate: 0.5 mL / min, mobile phase: 80% methanol (V / V), 20% ultrapure water (V / V), column temperature: 30°C, indole peak was detected at UV wavelength of 265 nm, each sample was kept for 30 min, the relationship between indole concentration and indole peak area was obtained, and the indole concentration standard curve (y=50.149x-832554R 2 =0.9932), y is the corresponding peak area, x is the indole concentration ( Figure 6 ).

[0056] Prepare MRS medium containing 300 μg / mL indole, inoculate each strain into indole-MRS medium for anaerobically culture for 24 h, centrifuge at high speed to obtain the supernatant, extract indole with ethyl acetate, add 1 mL of ethyl acetate to 1 mL of supernatant, shake well, let stand for 30 min, and obtain the supernatant. Repeat the above steps once, filter the supernatant after extraction with a 0.22 μm filter membrane, and use a high-performance liquid chromatography HPLC system to detect the indole content, and calculate the remaining indole concentration ( Figure 7 ).

[0057] The results showed that the degradation rate of indole by YYS-J9 was about 23.6%, the degradation rate of indole by YYS-BSM8 was about 32.7%, and the degradation rate of indole by the composite bacteria was about 39.1%, which was significantly higher than the degradation rate of indole by a single strain, indicating that the two also have synergistic effects in degrading indole.

[0058] Example 8 Oxalic acid degradation ability of each strain Excessive oxalic acid levels in the human body can easily lead to the formation of calcium oxalate crystals, which in turn can cause urinary system diseases such as kidney stones. Taking specific probiotics can reduce the accumulation of oxalic acid in the body and effectively prevent the formation of kidney stones.

[0059] Each strain was cultured to the second generation bacterial liquid, and the bacterial liquid was inoculated into MRS (MRS-Ox) medium containing 5 mmol / L sodium oxalate at a 4% (v / v) inoculation rate and cultured at 37°C for 2 d. The MRS-Ox culture medium without bacteria was used as the control group, and the oxalic acid concentration in the culture medium was determined by the oxalic acid-TiCl3 reaction method.

[0060] Oxalic acid-TiCl3 reaction method: Take 10 mL of culture medium and centrifuge at 8000 r / min for 10 min, take 4 mL of supernatant, add 1 mL of 0.5 mol / L CaCl2 solution and let it settle, centrifuge at 12000 r / min for 10 min, discard the supernatant, and dry the water in the centrifuge tube. Add 2.5 mL of 0.2 mol / L HCl solution and shake to dissolve the precipitate, then add 0.25 mL of freshly prepared 5% (v / v) TiCl3 solution, shake and mix, and measure the absorbance at 405 nm.

[0061] Oxalic acid degradation rate = (OD control - OD detection solution) / OD control × 100%; The OD test solution represents the absorbance value measured by the inoculated MRS-Ox culture medium, and the OD control represents the absorbance value measured by the non-inoculated MRS-Ox culture medium control group.

[0062] Table 6 Oxalic acid degradation rate of each strain

[0063] As shown in Table 6, the degradation rate of oxalic acid by YYS-J9 was 21.76%±2.33%, the degradation rate of oxalic acid by YYS-BSM8 was 18.38%±3.95%, and the degradation rate of oxalic acid by the composite bacteria was 24.67%±4.36%, which was significantly higher than the degradation rate of oxalic acid by a single strain, indicating that the two strains had synergistic effects in degrading oxalic acid ( P <0.05).

[0064] Example 9 Application of composite bacterial agent in the preparation of solid beverage 5 parts of animal Bifidobacterium lactis subspecies YYS-J9 powder, 5 parts of Bifidobacterium longum subspecies YYS-BSM8 powder, 10 parts of fish collagen peptide, 10 parts of wheat oligopeptide, 10 parts of black mulberry powder, 10 parts of yam powder, 15 parts of maltodextrin, 15 parts of skimmed milk powder, 10 parts of oligofructose and 10 parts of oligogalactose are uniformly mixed to obtain a probiotic solid beverage.

[0065] It should be noted that: (1) Definition: The term "food" as used herein is broad and includes food and drink for humans. In certain embodiments, the food product is suitable for and designed for human consumption. The application can be used to prepare solid preparations such as powders, tablets, gels, and also dispersed in liquids to prepare liquid preparations, including but not limited to the embodiment schemes.

[0066] (2) Relevant prior art means or prior art terms involved in this application: "OD" is the abbreviation of optical density, also known as absorbance. When light passes through the object, the energy difference before and after is the energy absorbed by the object. At a specific wavelength, there is a quantitative relationship between the concentration of the same object and the absorbed energy. This quantitative relationship can be used to determine the concentration of the object. 405 " is the optical density value measured when the wavelength is set to 405nm. It is a standard indicator for tracking the density of microorganisms in liquid cultures and is usually used to indicate bacterial cell density. Among them, the "OD" value determination method is a prior art, and its principles and methods will not be repeated here.

[0067] Although the terms such as composite probiotics, MRS liquid culture medium, auxiliary additives and freeze-drying protective agents are used more frequently in this article, the possibility of using other terms is not excluded. These terms are used only to more conveniently describe and explain the essence of the present invention; interpreting them as any additional limitation is contrary to the spirit of the present invention.

[0068] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A composite probiotic for degrading indole and preventing or alleviating kidney disease, characterized in that: Including Bifidobacterium animalis subsp. lactis ( Bifidobacterium animalis subsp.lactis ) YYS-J9, whose deposit number is CGMCC NO.28185, and Bifidobacterium longum subsp. longum ( Bifidobacterium longum subsp.Longum ) YYS-BSM8, whose preservation number is CGMCC No.28611, and the ratio of the number of viable bacteria of animal Bifidobacterium lactis subspecies YYS-J9 to that of Bifidobacterium longum subspecies longum YYS-BSM8 is 1-10:1-10.

2. A composite probiotic, characterized in that: The components thereof include the composite probiotics for degrading indole and preventing or alleviating kidney disease as claimed in claim 1.

3. The composite probiotic according to claim 2, characterized in that: In the composite probiotic, the ratio of the number of live bacteria of Bifidobacterium animalis subspecies lactis YYS-J9 to that of Bifidobacterium longum subspecies longum YYS-BSM8 is 1-3:1-3.

4. The composite probiotic according to claim 2, characterized in that: The viable count of the composite probiotics is ≥4.5×10 9 CFU / g.

5. The composite probiotic according to claim 2, characterized in that: The composite probiotics further include a freeze-drying protective agent, which includes any one or more combinations of skimmed milk powder, maltodextrin, sodium glutamate, sucrose, lactose, trehalose, sorbitol and xylitol.

6. The composite probiotic according to claim 2, characterized in that: The composite probiotic also includes auxiliary additives, which include any one or more combinations of fructooligosaccharides, galacto-oligosaccharides, xylooligosaccharides, isomaltooligosaccharides, soybean oligosaccharides, inulin, spirulina, stachyose, polydextrose, α-lactalbumin or lactoferrin.

7. The composite probiotic according to claim 2, characterized in that: The composite probiotics also include excipients, which include any one or more combinations of prebiotics, fillers, acidulants, solvents, solubilizers, cosolvents, emulsifiers, colorants, adhesives, disintegrants, lubricants, wetting agents, stabilizers, glidants, flavoring agents, preservatives, coating materials, fragrances, anti-adhesive agents, integrators, thickeners, buffers and inclusion agents.

8. The composite probiotic according to claim 2, characterized in that: The dosage form of the composite probiotic includes freeze-dried powder, capsule, tablet or granule.

9. A probiotic solid beverage, characterized in that: Its components include the composite probiotic agent as claimed in claim 2.

10. Use of the composite probiotic agent according to claim 2 in the preparation of food, health products or medicines for degrading indole, preventing or alleviating kidney disease, preventing or improving stones, regulating intestinal health, and improving body immunity.

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