Medicinal and edible homologous compound fermentation product for improving hyperuricemia and application of medicinal and edible homologous compound fermentation product

Abstract

The invention provides a combined lactobacillus salivarius YS-UR90 and a preparation method of the combined lactobacillus salivarius YS-UR90. The saliva combined lactobacillus provided by the invention can secrete protease and glycosidase, and the HCT and FLS gene expression levels of the saliva combined lactobacillus are high, so that the saliva combined lactobacillus can promote phenolic acid active ingredients to generate biotransformation during fermentation, and the proportion of chlorogenic acid and the like in fermentation liquor is increased. For example, marine fish oligopeptide and casein phosphopeptides are used as a composite nitrogen source, dandelion powder is used as a functional substrate, and a composite fermentation product obtained through fermentation of saliva and lactobacillus has the effect of promoting uric acid excretion and has a certain protection effect on kidney injury. The compound fermentation product has the effect of improving hyperuricemia mainly by inhibiting XOD activity, increasing serum urea nitrogen level, improving defect expression of uric acid synthesis gene HPRT1, inhibiting expression of uric acid reabsorption URAT1 and GLUT9 genes and improving expression of excretion genes OAT1 and ABCG2.

Description

Technical Field

[0001] The present invention belongs to the field of microbial technology, and specifically relates to the application of saliva-combined lactobacillus and its fermentation products. Background Art

[0002] Hyperuricemia (HUA) is a chronic metabolic disease caused by impaired purine metabolism and characterized by excessive uric acid in the blood. The overall prevalence of hyperuricemia in my country is 13.3%, affecting 177 million people. The overall incidence of gout is 1.1%, with approximately 14.66 million new cases each year. Hyperuricemia has become the fourth most common disease in China, following diabetes, hypertension, and hyperlipidemia.

[0003] Modern medicine believes that HUA is primarily linked to a diet rich in purines, fructose, and protein, leading to purine metabolism disorders and an imbalance in uric acid, the end product of purine metabolism. Uric acid accumulates and increases in the blood, causing a range of adverse reactions and damage to organs such as the kidneys. Hyperuricemia is a serious threat to people's health and quality of life.

[0004] Currently, the main clinical treatments for hyperuricemia include dietary restriction and medication. Dietary restriction can be achieved by limiting the intake of common purine-rich foods such as meat, seafood, and mushrooms, as well as beer and fructose. However, this dietary restriction disrupts the nutritional balance between high nutritional value and low purine content. Furthermore, due to long-standing dietary habits, patient compliance is poor, making restricting the intake of purine-rich foods difficult to implement. Clinically, uric acid-lowering medications primarily include those that inhibit uric acid production and those that promote uric acid excretion. Uric acid inhibitors, such as allopurinol and febuxostat, have good clinical efficacy, but they also come with high costs, significant side effects, and low patient tolerance, including the potential for gastrointestinal bleeding, hepatotoxicity, and allergic reactions. Uric acid-promoting drugs, such as benzbromarone and probenecid, inhibit renal tubular uric acid reabsorption, thereby promoting uric acid excretion and lowering serum uric acid levels. However, these drugs may cause liver damage and increase the risk of urinary stone formation. Therefore, in clinical practice, it is urgent to use new drug alternatives that are more economical, safer and have fewer side effects to relieve and assist in the treatment of hyperuricemia.

[0005] In recent years, numerous studies have demonstrated the effectiveness of probiotics, dual-purpose Chinese medicinal and edible ingredients, bioactive peptides, and edible fungi in improving hyperuricemia. This presents new opportunities for identifying natural active ingredients with uric acid-lowering properties and developing clinical treatments for hyperuricemia. Probiotics can regulate the balance of intestinal flora, increase the number of beneficial bacteria, inhibit the growth of harmful bacteria, reduce purine absorption, promote uric acid excretion, and inhibit uric acid production. Traditional Chinese medicine (TCM) can lower uric acid through various pathways. For example, some TCM ingredients can inhibit xanthine oxidase activity, reducing uric acid production; others have diuretic properties, promoting uric acid excretion. Consumers are increasingly interested in health and wellness, preferring natural, green, and side-effect-free products. Combining probiotics with TCM for uric acid-lowering products is a relatively new area, with no widely recognized or marketed mature products. Combining probiotics with TCM for uric acid-lowering products perfectly aligns with this market trend and holds great potential for development.

[0006] Chinese patent application CN 114246933 B discloses a uric acid-lowering composition, its preparation method, and use, as well as a uric acid-lowering composition, food, its preparation method, and use. The composition comprises the following components: chicory, turmeric, and marine fish oligopeptides. Through the synergistic effect of chicory, turmeric, and marine fish oligopeptides, the composition provided by the invention, when consumed as a food by patients with hyperuricemia, can steadily lower uric acid levels and maintain normal levels without burdening the liver or kidneys or causing side effects. The uric acid-lowering composition of the invention does not require the addition of probiotics, which enhance synergy and promote biotransformation of the composition.

[0007] Chinese patent application CN 116218746 A discloses a strain of Lactobacillus plantarum that has the ability to lower uric acid. The strain was isolated from a sample of traditional yak yogurt. The resulting strain grows well in a low pH environment and high bile salt concentrations, and its survival rate exceeds 80% after 3 hours of stress in simulated artificial gastric and intestinal fluid environments, demonstrating good tolerance. This Lactobacillus plantarum NML21 can alleviate hyperuricemia by lowering uric acid levels. However, the strain selection in this patent does not screen for uric acid tolerance, and the strain is not fermented with raw materials, resulting in limited uric acid-lowering efficacy of a single strain.

[0008] Chinese patent application CN 117338883 A discloses a rapidly uric acid-lowering medicinal and edible fermentation broth, its preparation method, and oral preparation, comprising: S1. preparing a medicinal and edible composition comprising kudzu root, gorgon fruit, lily bulb, poria, coix seed, dandelion, bamboo leaf, and liquorice; S2. fermenting the medicinal and edible fermentation broth: pulverizing the medicinal and edible composition to obtain a powder, adding water, sterilizing the powder, and using it as a fermentation substrate. Lactobacillus plantarum LP45 and Lactobacillus rhamnosus LR519 are inoculated for fermentation, and solid-liquid separation is performed to produce the medicinal and edible fermentation broth. The medicinal and edible fermentation broth of the present invention exhibits a multi-target synergistic effect. Compared to the single target of small molecule drugs, it regulates multiple aspects, including uric acid reduction, uric acid excretion, urate solubility, and inhibition of pro-inflammatory factors, achieving the goal of safely and rapidly lowering uric acid. Summary of the Invention

[0009] The first object of the present invention is to provide a saliva-associated lactobacillus that can degrade uric acid and inhibit xanthine oxidase.

[0010] The second object of the present invention is to provide a composite fermentation product obtained by fermenting dandelion with saliva and lactobacillus.

[0011] The third object of the present invention is to provide a method for obtaining a composite fermentation product by using marine fish oligopeptides and casein phosphopeptides as a composite nitrogen source and dandelion powder as a functional substrate.

[0012] The fourth object of the present invention is to provide a probiotic preparation comprising the composite fermentation product.

[0013] The fifth object of the present invention is to provide an application of the composite fermentation product.

[0014] The present invention is achieved through the following technical solutions:

[0015] The present invention provides a strain of Lactobacillus salivarius YS-UR90, whose Latin name is Ligilactobacillus salivarius. It was deposited with the General Microbiology Center of the China Culture Collection Administration on April 14, 2025, with the depository code CGMCC NO. 34178. The depository address is: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, zip code 100101, and the depository number is CGMCC No. 34178. The strain was alive at the time of deposit.

[0016] A composite fermentation product is obtained by fermenting dandelion with the saliva and Lactobacillus YS-UR90.

[0017] The method for preparing the composite fermentation product comprises the following steps:

[0018] S1: fermenting the saliva combined with Lactobacillus according to claim 1 in a seed culture medium to obtain a seed liquid;

[0019] The S2 seed liquid is inoculated and fermented in a fermentation medium, and concentrated to obtain a composite raw material fermentation product.

[0020] 4. The method for preparing the composite fermentation product according to claim 3, wherein:

[0021] The concentration of the fermentation bacteria in S1 is (2-9)×10 11 CFU / mL, the bacterial inoculation ratio was 2%-4%, the fermentation pH was 6.2, the fermentation temperature was 37°C, the rotation speed was 50-100 r / min, and the fermentation time was 18 h;

[0022] The concentration of the fermentation bacteria in S2 is (2-9)×10 11 CFU / mL, the bacterial inoculation ratio is 2%-4%, the fermentation pH is 6.2, the fermentation temperature is 37°C, the rotation speed is 50-100r / min, and the fermentation time is 24-48h; the concentration includes rotary evaporation concentration.

[0023] The composite nitrogen source comprises casein phosphopeptide and marine fish oligopeptide in a mass ratio of 1:3 to 1:1;

[0024] The seed culture medium comprises, by weight percentage, 2% glucose, 0.2% yeast extract, 1.0% soybean meal, 0.03% magnesium sulfate, 0.2% dipotassium hydrogen phosphate, 0.2% complex nitrogen source, and the remainder water;

[0025] The fermentation medium comprises, by weight percentage, 2% glucose, 0.2% yeast extract, 1.0% soybean meal, 0.03% magnesium sulfate, 0.2% dipotassium hydrogen phosphate, 0.2% compound nitrogen source, and 2% dandelion powder.

[0026] A probiotic freeze-dried preparation comprises probiotic sludge obtained by fermenting saliva and Lactobacillus YS-UR90 in an MRS culture medium and a freeze-drying protective agent.

[0027] The MRS culture medium comprises the following components by weight percentage: 2% glucose, 0.2% yeast extract, 1.0% soybean meal, 0.03% magnesium sulfate, 0.2% dipotassium hydrogen phosphate, 0.2% complex nitrogen source, 2% dandelion powder, and the balance water;

[0028] The freeze-drying protective agent comprises the following components in percentage by weight: 5-15% trehalose, 2-5% skim milk powder, 1-2% octenylsuccinic acid, 2-10% maltodextrin, 2-5% glycerol and the balance water.

[0029] The composite fermentation product is used to prepare medicines or foods for reducing blood uric acid levels and / or increasing urine uric acid levels.

[0030] The composite fermentation product can be used to prepare a drug or food that inhibits XOD activity in serum and / or liver. The composite fermentation product can also be used to improve serum urea nitrogen levels to alleviate kidney damage. Compared to the prior art, the present invention has the following beneficial effects:

[0031] Saliva combined with Lactobacillus YS-UR90 and dandelion fermentation liquid can promote uric acid excretion by lowering blood uric acid levels and increasing urine uric acid levels.

[0032] Probiotics and fermentation broth can inhibit XOD activity in serum and liver in vivo.

[0033] The serum urea nitrogen levels in the probiotic and fermentation broth groups were significantly increased, which may have a protective effect on renal damage caused by hyperuricemia.

[0034] The mechanism by which probiotics and fermented liquid regulate uric acid is mainly to improve the defective expression of the uric acid synthesis gene HPRT1, inhibit the expression of uric acid reabsorption (URAT1 and GLUT9) genes and increase the expression of excretion genes (OAT1, ABCG2) to improve hyperuricemia. DETAILED DESCRIPTION

[0035] In order to obtain probiotics with therapeutic effects on hyperuricemia, the present invention selects probiotics from samples such as feces of centenarians and infants, and conducts index evaluation on the screened probiotics.

[0036] Specifically, the method for evaluating the probiotic function of the strain is shown in Example 1.

[0037] Example 1

[0038] 36 bacterial strains were obtained by screening artificial gastric juice (pH 3.0, tolerance for 3 hours) and intestinal juice (containing 0.3% bile salts, tolerance for 3 hours). The gastric juice tolerance rate >80% and intestinal juice tolerance rate >50% were used as screening criteria. The screened strains were subjected to uric acid tolerance tests, uric acid decomposition ability, and its effect on XOD enzyme.

[0039] The uric acid-lowering ability of different strains was evaluated by measuring the uric acid consumption when they were grown in MRS medium containing uric acid (250 μmol / L) using a uric acid detection kit.

[0040] Table 1 Effects of different strains on uric acid degradation ability

[0041] strain Uric acid degradation capacity (%) strain Uric acid degradation capacity (%) BS225M2 1.2 24-58T4 24.5 904M6 2.5 24-58M4 24.8 501M9 3.8 24-58M5 25.2 906M7-1 4.8 24-58M9 25.6 906M21 5.0 24-58M10 26.7 BS11M1 12.5 24-58M12 26.8 BS11M6 12.8 24-58M13 52.6 11M8 25.2 24-58T2 34.5 BS11M8 15.6 906M9 35.2 BS11M10 17.8 24-33M1 56.1 BS11M13 22.6 YS-UR90 58.5 7M4 24.1 3T8 50.4 617M8 32.1 88M5 32.5 5M8 48.9 24-56T4 42.1 5M7 50.2 24-48M3 41.5 619M22 25.4 24-43M6 12.1 619-2M21 31.2 24-42M2 12.6 BS619M13 25.4 Uric-A1 52.5

[0042] Taking the uric acid degradation ability of >40% as the screening standard, the strains that can be screened are 5M8, 5M7, 24-58M13, 24-33M1, 686M3, 3T8, and Uric-A1.

[0043] Evaluate the effects of the above strains on xanthine oxidase activity:

[0044] A xanthine oxidase (XOD) activity detection kit was used to determine the effect of probiotic metabolites on XOD activity. Xanthine oxidase catalyzes hypoxanthine in the purine metabolic pathway and converts it into uric acid, which is a key step in uric acid production. By evaluating the effect of strain metabolites on XOD activity, probiotic strains with potential uric acid-lowering effects were screened.

[0045] Sample treatment: Inoculate the bacterial culture solution into MRS broth at a volume fraction of 5% (volume fraction), incubate at 37°C for 24 hours, and then centrifuge at 4°C, 10,000 rpm for 10 minutes to collect the bacterial precipitate. Rinse 2-3 times with sterile PBS (pH 6.8) and resuspend the precipitate to adjust the bacterial concentration to 1×10 9 The cells were then pulsed for 10 minutes using ultrasonic disruption (200W) at 5s on and 5s off. The resulting liquid was centrifuged at 10,000 rpm for 10 minutes, and the supernatant was filtered to obtain the cell contents. XOD activity was determined using an XOD activity assay kit.

[0046] Table 2 Inhibition rate of XOD enzyme activity by different strains

[0047]

[0048] After the above screening, it can be seen that the inhibition rate of YS-UR90 on XOD enzyme activity is as high as 36.57%. It can be seen that this strain can achieve the effect of lowering uric acid by inhibiting the key enzymes of uric acid production. Therefore, YS-UR90 is a potential uric acid-lowering strain.

[0049] Based on the above screening results, the present invention identified strain YS-UR90.

[0050] Example 2

[0051] The strain YS-UR90 originated from the feces of centenarians.

[0052] The strain YS-UR90 was identified as Lactobacillus salivarius.

[0053] The 16S sequence of the saliva-associated lactobacillus YS-UR90 is as follows:

[0054]

[0055] Example 3

[0056] In order to find out the raw materials that can synergize with the uric acid-lowering strains, we searched for uric acid-lowering raw materials from literature and Chinese Pharmacopoeia. The specific names are galangal, chicory, burdock, licorice, black wolfberry, codonopsis, astragalus, corn silk, poria, celery seed, dandelion, kudzu root, milk thistle, artichoke, marine fish oligopeptides, red cherry powder, green coffee, cranberry, red pomegranate, turmeric, emblica fruit, bonito elastin, tuna peptide, Antarctic krill oil powder (20%), light bamboo leaf, elderberry, Alpinia oxyphylla, inulin, chitosan oligosaccharide, casein phosphopeptide, etc., a total of 31 raw materials and prebiotics.

[0057] Growth curve: Add 2% of the screening material to each medium to prepare a new medium. After activating the test strain for two generations, inoculate a loopful of the strain into 10 ml of MRS broth. Incubate at 37°C for 24 hours. Adjust the OD600 of the culture to approximately 0.8. Then, inoculate the strain into MRS broth containing the material at a 2% (v / v) addition rate. Incubate at 37°C for 16 hours. Measure OD values ​​every 2 hours. After excluding the blank control and the initial OD value, compare the difference in OD values ​​at 16 hours.

[0058] Table 3 OD difference of growth of different raw materials after 16 hours

[0059]

[0060] Compared with the basic MRS, the raw materials that can promote the growth of strain YS-UR90 include casein phosphopeptide, dandelion powder, marine fish oligopeptide, tuna peptide, light bamboo leaf, chitosan oligosaccharide, Alpinia oxyphylla, red pomegranate, red cherry, bonito elastin, inulin, and corn silk. These 12 raw materials were subsequently used to conduct XOD enzyme activity reduction tests, and the effective ones were finally selected for product compounding. Evaluate the effect of the primary screening raw materials on xanthine oxidase activity

[0061] Using xanthine as substrate, the XOD inhibitory activity of 12 edible and medicinal materials was detected by microplate reader. Reaction settings: blank group, enzyme reaction group, inhibitor group, control group, with a total volume of 200μL for each group. Blank group: xanthine + phosphate buffer; enzyme reaction group: xanthine + XOD + phosphate buffer; inhibitor group: xanthine + XOD + 200μg / mL extract; control group: xanthine + XOD + phosphate buffer, and XOD inhibitor APL was used as a positive control. Xanthine oxidase solution: 0.1U / mL; 1mg / mL allopurinol (APL) solution; 2mM xanthine solution. The experimental results are as follows:

[0062] Table 4 Inhibitory activity of different raw materials on XOD enzyme

[0063]

[0064] After raw material screening and evaluation, it was found that marine fish oligopeptides, dandelion powder, and casein phosphopeptide have high inhibitory ability against XOD enzyme.

[0065] Example 4

[0066] Prepare a fermentation medium using marine fish oligopeptides and casein phosphopeptides as a composite nitrogen source and dandelion powder as a functional substrate. Prepare the composite nitrogen source by weighing and mixing casein phosphopeptides and marine fish oligopeptides at a mass ratio of 1:3 to 1:1.

[0067] Seed culture medium: 2% glucose, 0.2% yeast extract, 1.0% soybean meal, 0.03% magnesium sulfate, 0.2% dipotassium hydrogen phosphate, 0.2% complex nitrogen source, and the rest water.

[0068] Fermentation medium: glucose 2%, yeast extract 0.2%, soybean meal 1.0%, magnesium sulfate 0.03%, dipotassium hydrogen phosphate 0.2%, 0.2% complex nitrogen source, 2% dandelion powder.

[0069] The specific fermentation process is as follows:

[0070] S1: using the saliva combined with lactobacillus described in claim 1 to ferment in a seed culture medium to increase the activity of the strain and obtain a seed liquid.

[0071] The concentration of the fermentation bacteria in S1 is (2-9)×10 11 CFU / mL, the bacterial inoculation ratio was 2%-4%, the fermentation pH was 6.2, the fermentation temperature was 37°C, the rotation speed was 50-100 r / min, and the fermentation time was 18 h;

[0072] The S2 seed liquid is inoculated and fermented in a fermentation medium, and concentrated to obtain a composite raw material fermentation liquid.

[0073] The concentration of the fermentation bacteria in S1 is (2-9)×10 11 CFU / mL, the bacterial inoculation ratio is 2%-4%, the fermentation pH is 6.2, the fermentation temperature is 37°C, the rotation speed is 50-100r / min, and the fermentation time is 24-48h; after the fermentation is completed, rotary evaporation and concentration are performed to obtain a composite raw material fermentation liquid.

[0074] Experimental group: Add 2% of the bacteria and the same proportion of compound nitrogen source and dandelion powder to the above upgraded culture medium

[0075] Control group: 2% of bacteria were added to the basic MRS medium.

[0076] Comparative group 1: 2% of bacterial strains and the same proportion of compound nitrogen source were added to the basic MRS medium.

[0077] Comparative Group 2: Add 2% of the bacteria and the same proportion of dandelion powder to the basic MRS medium

[0078] Fermentation was carried out according to the above method, and the changes in active ingredients before and after fermentation and the number of viable bacteria at the fermentation end point were compared.

[0079] Table 5 Changes in viable bacterial counts at the fermentation endpoint in different groups

[0080] Group Fermentation endpoint viable bacteria count (CFU / mL) Control group (MRS medium) 3500 Experimental group (upgraded culture medium) 8200 Comparison group 1 5800 Comparison group 2 6500

[0081] The purpose of using a prebiotic fermentation method is that a complex nitrogen source can meet the microbial needs for nitrogen and growth factors, thereby increasing the biomass of the strain. Secondly, during high-density fermentation, a complex nitrogen source can prolong the logarithmic growth phase and increase cell density. As shown in Table 5, compared with the control group, the viable bacterial count at the fermentation endpoint reached 8200 CFU / mL, a 1.34-fold increase.

[0082] Table 6 Changes in uric acid degradation rate in the experimental groups before and after fermentation

[0083] Group XOD enzyme inhibition rate (%) Before fermentation 70.25％ After fermentation 89.65％ Comparison group 1 52.58％ Comparison group 2 45.25％

[0084] As shown in Table 6, the XOD enzyme inhibition rate after fermentation was as high as 89.65%. This may be because the small peptides contained in the marine fish oligopeptides may induce the strain to synthesize functional proteins related to XOD enzyme inhibition, and the phosphoserine structure of the casein phosphopeptide (CPP) may regulate the bacterial phosphorylation signaling pathway and promote the synthesis of secondary metabolites. It can be seen that the combination of marine fish oligopeptides and casein phosphopeptides, in addition to providing nitrogen, may produce a synergistic effect between the active ingredients of the two and the substrate (dandelion powder), enhancing the production efficiency of the target product (such as the XOD enzyme inhibitor).

[0085] Example 5

[0086] In order to verify the nitrogen source utilization of the strain, the protease secretion performance of the strain was evaluated.

[0087] ①Protease activity assay

[0088] The Folin-phenol reagent colorimetric method was used. One unit (U) of enzyme activity was defined as the amount of tyrosine produced per minute per milliliter of bacterial culture at 40°C per casein hydrolysis.

[0089] ②β-glucosidase and α-galactosidase assay

[0090] YS-UR90 was inoculated into MRS liquid medium and cultured at 37°C for 24 hours. The bacterial suspension was then inoculated into liquid medium at a 2% inoculum size and cultured for 24 hours. The suspension was aspirated into a centrifuge tube and centrifuged at 8000 r / min for 20 minutes. The supernatant was used to determine enzyme activity. Enzyme activity was determined using an α-galactosidase (α-GAL) kit and a β-glucosidase (β-GC) activity assay kit.

[0091] Table 7 Determination of protease activity of strains

[0092]

[0093] Compared to control bacteria, the strain provided by the present invention produces higher protease, β-glucosidase, and α-galactosidase activities. During the lactic acid bacteria fermentation process, the strain can secrete proteases to break down complex nitrogen sources (marine fish oligopeptides and casein phosphopeptides) into small molecule peptides and amino acids. Furthermore, the secretion of β-glucosidase and α-galactosidase fully utilizes the culture medium carbon source and phenolic substances.

[0094] Example 6

[0095] Preparation method of probiotics

[0096] Probiotic fermentation culture: After streaking, single probiotic bacteria were picked and placed into liquid MRS, cultured at 37°C, activated twice, and centrifuged to obtain probiotic slurry; the culture medium comprises the following components by weight: 2% glucose, 0.2% yeast extract, 1.0% soybean meal, 0.03% magnesium sulfate, 0.2% dipotassium hydrogen phosphate, 0.2% complex nitrogen source, 2% dandelion powder, and the balance distilled water;

[0097] Preparation of probiotic freeze-dried powder: The probiotic slurry is evenly mixed with a freeze-dried protective agent, and freeze-dried to obtain the probiotic freeze-dried powder; the freeze-dried protective agent comprises the following components in percentage by weight: 5-15% trehalose, 2-5% skim milk powder, 1-2% octenylsuccinic acid, 2-10% maltodextrin, 2-5% glycerol, and the balance water.

[0098] The probiotics are Lactobacillus plantarum and Lactobacillus salivarius YS-UR90.

[0099] In order to further explore the changes in fermentation products, the active ingredients of the fermentation products were determined

[0100] Example 7

[0101] Changes in amino acid composition of fermentation broth:

[0102] Compare the changes in amino acids at fermentation hour 0 and at the end of fermentation. Analyze the above raw materials using an amino acid analyzer and calculate the amino acid ratio.

[0103] Table 8 Amino acid composition analysis of composite raw materials before and after fermentation

[0104] Test items Before fermentation (%) After fermentation (%) Aspartic acid Asp 4.25 4.38 Threonine 0.63 0.69 Serine 10.72 18.20 Glutamate 5.78 6.36 Proline Pro 2.13 3.28 Glycine Gly 18.13 25.78 Alanine Ala 4.23 4.82 Valine 4.22 4.35 MethionineMet 0.51 0.58 Isoleucine Ile 0.58 0.87 Leucine 4.25 4.95 Tyrosine 0.53 0.64 Phenylalanine Phe 0.67 0.58 Histidine 0.54 0.62 Lysine 0.72 0.89 Arginine 4.25 6.25 Cysteine ​​Cys 0.52 0.65

[0105] After fermentation, the content of arginine, glycine, and serine increased significantly, among which serine increased by 69.78%, glycine increased by 42.20%, and arginine increased by 47.06%. This is mainly because marine fish oligopeptides are rich in small molecules such as glycine and arginine. Secondly, casein phosphopeptides contain a large number of phosphorylated serine residues. After fermentation by probiotics, the serine content is greatly increased. The amino acid composition of the two is complementary. For example, marine fish oligopeptides provide basic amino acids (arginine), and casein phosphopeptides provide hydrophilic amino acids (such as serine), which meet the bacteria's needs for different amino acids, achieve the effect of synergistic enhancement of nitrogen sources, and increase the biomass after fermentation.

[0106] Analysis of active ingredients in fermentation products

[0107] 5 mL of fermentation broth was extracted with 75% ethanol and sonicated (65°C, 100 W, 60 min). After cooling, the broth was adjusted to the required weight loss with 75% ethanol, and the volume was fixed to a 50 mL volumetric flask. The broth was then filtered through a 0.22 μm microporous membrane. The contents of chlorogenic acid, protocatechuic acid, caffeic acid, quercetin, chicoric acid, and luteolin were determined using high-performance liquid chromatography.

[0108] Table 9 Changes in active ingredients of dandelion fermentation products

[0109]

[0110] After fermentation with saliva and Lactobacillus YS-UR90, the chlorogenic acid content of dandelion increased by 53.04%, caffeic acid by 71.16%, quercetin by 47.11%, and luteolin by 50.90%. This is primarily due to the secretion of enzymes such as proteases, β-glucosidases, and α-galactosidases by saliva and Lactobacillus YS-UR90, which release bound phenolic acids (chlorogenic acid and chicoric acid) from the dandelion. Chlorogenic acid is likely resynthesized through the binding of hydroxycinnamoyl-CoA with quinic acid. Secondly, bound phenolic acids are converted to free phenolic acids (caffeic acid and ferulic acid). Chlorogenic acid is hydrolyzed into caffeic acid and quinic acid by microbial esterases, and caffeic acid is further converted into ferulic acid or m-coumaric acid. Finally, under the enzymatic action of lactic acid bacteria, kaempferol is converted to quercetin, which is then degraded into apigenin and luteolin.

[0111] In order to further explore the role of saliva combined with Lactobacillus YS-UR90, the levels of HCT and F3H genes in the fermentation product completed in Example 4 were evaluated. The HCT gene mainly catalyzes the bidirectional reaction of chlorogenic acid synthesis and decomposition, and may be involved in the metabolic regulation of chlorogenic acid by microorganisms, promoting the combination of hydroxycinnamoyl Coenzyme A and quinic acid to form chlorogenic acid and hydrolyzing chlorogenic acid to form caffeoyl Coenzyme A and quinic acid. F3H is the rate-limiting enzyme in the synthesis of flavonoids, catalyzing the conversion of kaempferol to quercetin under the action of lactic acid bacteria, and further producing luteolin. The expression levels of mRNA of two key genes for polyphenol biotransformation were detected using RT-qPCR technology, and the experimental results are as follows.

[0112]

[0113] Table 10 mRNA expression levels of two key genes for polyphenol biotransformation in different groups

[0114]

[0115] It can be seen that the biotransformation of phenolic acid active ingredients occurred during the fermentation process. The possible reason is that saliva combined with Lactobacillus YS-UR90 promoted the increase of chlorogenic acid, caffeic acid and luteolin in dandelion by increasing the expression of HCT and FLS genes. In order to further verify the uric acid-lowering effect of probiotics and dandelion fermentation products,

[0116] Example 8

[0117] Hyperuricemia model establishment: 100 mg / kg / day of potassium oxonate (PO) and 500 mg / kg / day of hypoxanthine (HX) were used in combination to induce hyperuricemia in mice. The animal model was established and divided into the following groups:

[0118] Blank group: mice without any treatment

[0119] Model group: mice treated with potassium oxonate (PO) and hypoxanthine (HX)

[0120] Fermentation liquid group: 200mg / kg / day of dandelion fermentation liquid

[0121] Probiotic group: Administered according to a viable bacterial count of 500 million cfu / mL

[0122] Positive control group: 10 mg / kg / day allopurinol (ALLOP) was used as the positive drug.

[0123] The experimental period was 7 days, urine was collected every day, and 1 hour after the last administration on the seventh day, all mice were anesthetized, whole blood was collected through orbital bleeding, and then they were killed.

[0124] Index determination:

[0125] ① Blood urea nitrogen

[0126] ② Urine / serum uric acid level: test according to the corresponding kit

[0127] ③Serum / liver xanthine oxidase (XOD) activity

[0128] ④RT-qPCR technology was used to detect the expression levels of five uric acid transporter mRNAs in the kidney

[0129]

[0130] Table 10 Effects of different groups on blood urea nitrogen, blood uric acid and urine uric acid levels

[0131] Group Blood urea nitrogen (mmol / L) Serum uric acid (μmol / L) Urine uric acid (μmol / L) Blank group 4.52±0.13 72.35±2.12 112.35±5.89 Model Group 5.38±0.25 235.45±5.89 50.28±2.52 Fermentation broth group 4.58±0.18 98.47±5.28 105.28±1.28 Probiotic group 4.25±0.13 105.25±8.26 125.25±5.87 Positive control group 7.02±0.25 92.56±2.35 82.15±2.58

[0132] The final metabolite of purine metabolism is uric acid, a key indicator of hyperuricemia. The results for serum uric acid are as follows: compared with the blank group, the model group had a significant increase in serum uric acid levels to 235.45 μmol / L (P < 0.01), indicating that the hyperuricemia model was successfully established. Based on this model, allopurinol (ALLOP) reduced serum uric acid levels to 92.56 μmol / L (P < 0.01). The fermentation broth group showed no significant difference in serum uric acid reduction compared to the positive drug group, reducing it to 98 μmol / L. The probiotic group also had a significant uric acid-lowering effect. Regarding urine uric acid, compared with the blank group's UUA level (112.35), the model group had a significant decrease in urine uric acid levels to 50.28 μmol / L (P < 0.01). Treatment with the positive drug group, probiotics, and dandelion fermentation all increased urine uric acid levels, suggesting that probiotics and fermentation products may promote uric acid excretion.

[0133] Urea nitrogen (BUN) is a key biomarker of renal function. Compared with the blank control group, BUN levels were significantly elevated in the model group. Allopurinol-treated groups elevated BUN levels to 7.02, indicating that allopurinol treatment significantly impairs renal function after combined PO and HX modeling. However, serum BUN levels in the fermentation product and probiotic groups gradually approached those in the control group. This suggests a protective effect against renal damage caused by hyperuricemia.

[0134] Table 11 Effects of different groups on serum and liver XOD activity in hyperuricemia mice

[0135] Group Serum XOD activity (U / L) Liver XOD activity (U / g protein) Blank group 7.58±0.21 5.82±0.35 Model Group 10.25±0.35 12.48±0.95 Fermentation broth group 7.25±0.40 6.05±0.21 Probiotic group 7.68±0.31 6.24±0.24 Positive control group 5.25±0.54 10.28±0.46

[0136] Compared with the blank group, the serum and liver XOD activity of the model group was significantly increased, while the serum and liver XOD activities in the probiotic group and fermentation liquid group were significantly decreased, which indicated that the probiotics and fermentation liquid had the effect of inhibiting XOD activity in vivo.

[0137] Table 12 Effects of different groups on the expression of HPRT1, ABCG2, OAT1, URAT1 and GLUT9 mRNA in kidney

[0138]

[0139] In the human body, the metabolism of uric acid (UA) mainly occurs in the kidneys, involving processes such as reabsorption and secretion, which rely on the coordinated excretion of multiple transport proteins to complete metabolism.

[0140] Uric acid synthesis gene: HPRT1 (hypoxanthine phosphoribosyltransferase 1) participates in the salvage pathway for purine recycling. HPRT1 deficiency → purine salvage is blocked → XOD substrate accumulation → increased uric acid production.

[0141] Renal urate reabsorption genes: URAT1 (membrane transporter gene) and GLUT9 (glucose transporter 9), transporters involved in renal tubular urate reabsorption, enable urate to be reabsorbed from the renal tubular cell membrane back into the blood; high expression of URAT1 / GLUT9 → enhanced uric acid reabsorption → increased blood uric acid → inflammation / oxidative stress → upregulated XOD activity.

[0142] Renal uric acid excretion genes: OAT1 (organic anion transporter) and ABCG2 act as transporters for uric acid excretion, absorbing uric acid from the blood into renal tubular cells. High OAT1 / ABCG2 expression leads to increased uric acid excretion, which in turn leads to decreased serum uric acid, which in turn negatively inhibits XOD. Therefore, abnormal expression and functional changes of these five genes are closely linked to the development of hyperuricemia.

[0143] Compared with the blank group, the expression levels of HPRT1, ABCG2, and OAT1 mRNA in the kidneys of the model group mice were significantly decreased, while those in the probiotic and fermentation broth groups were significantly increased, approaching those in the normal group. This suggests that the probiotic and fermentation broth groups can improve the expression of uric acid synthesis genes and significantly enhance the expression of uric acid excretion genes. Furthermore, compared with the blank group, the expression levels of GLUT9 and URAT1 mRNA in the kidneys of the model group mice were significantly increased, while those in the probiotic and fermentation broth groups were significantly decreased, indicating reduced uric acid reabsorption. In summary, the fermentation broth group was superior to the probiotic group in improving uric acid levels, and both groups can inhibit the overexpression of uric acid synthesis genes, inhibit uric acid reabsorption, and enhance the expression of excretion genes, thereby exerting their effects on hyperuricemia.

Claims

1. A salivary Lactobacillus (Ligilactobacillus salivarius) YS-UR90, characterized in that: Its accession number is CGMCC No.34178.

2. A composite fermentation product, characterized in that: The method is obtained by fermenting dandelion with the saliva combined with Lactobacillus YS-UR90 as claimed in claim 1.

3. The method for preparing the composite fermentation product according to claim 2, wherein: The steps include: S1: fermenting the saliva combined with Lactobacillus according to claim 1 in a seed culture medium to obtain a seed liquid; The S2 seed liquid is inoculated and fermented in a fermentation medium, and concentrated to obtain a composite raw material fermentation product.

4. The method for preparing the composite fermentation product according to claim 3, wherein: The concentration of the fermentation bacteria in S1 is (2-9)×10 11 CFU / mL, the bacterial inoculation ratio was 2%-4%, the fermentation pH was 6.2, the fermentation temperature was 37°C, the rotation speed was 50-100 r / min, and the fermentation time was 18 h; The concentration of the fermentation bacteria in S2 is (2-9)×10 11 CFU / mL, the bacterial inoculation ratio is 2%-4%, the fermentation pH is 6.2, the fermentation temperature is 37°C, the rotation speed is 50-100r / min, and the fermentation time is 24-48h; the concentration includes rotary evaporation concentration.

5. The method for preparing the composite fermentation product according to claim 3, wherein: The composite nitrogen source comprises casein phosphopeptide and marine fish oligopeptide in a mass ratio of 1:3 to 1:1; The seed culture medium comprises, by weight percentage, 2% glucose, 0.2% yeast extract, 1.0% soybean meal, 0.03% magnesium sulfate, 0.2% dipotassium hydrogen phosphate, 0.2% complex nitrogen source, and the remainder water; The fermentation medium comprises, by weight percentage, 2% glucose, 0.2% yeast extract, 1.0% soybean meal, 0.03% magnesium sulfate, 0.2% dipotassium hydrogen phosphate, 0.2% compound nitrogen source, and 2% dandelion powder.

6. A freeze-dried probiotic preparation, characterized in that: The invention comprises probiotic sludge obtained by fermenting the saliva combined with Lactobacillus YS-UR90 in MRS culture medium according to claim 1 and a freeze-drying protective agent.

7. The freeze-dried probiotic preparation according to claim 6, wherein: The MRS culture medium comprises the following components by weight percentage: 2% glucose, 0.2% yeast extract, 1.0% soybean meal, 0.03% magnesium sulfate, 0.2% dipotassium hydrogen phosphate, 0.2% complex nitrogen source, 2% dandelion powder, and the balance water; The freeze-drying protective agent comprises the following components in percentage by weight: 5-15% trehalose, 2-5% skim milk powder, 1-2% octenylsuccinic acid, 2-10% maltodextrin, 2-5% glycerol and the balance water.

8. The use of the composite fermentation product according to claim 2, characterized in that: The invention can be used for preparing medicines or foods for reducing blood uric acid levels and / or increasing urine uric acid levels.

9. The use of the composite fermentation product according to claim 2, characterized in that: The invention can be used for preparing medicine or food for inhibiting XOD activity in serum and / or liver.

10. The use of the composite fermentation product according to claim 2, characterized in that: Drugs or foods that are used to relieve kidney damage by increasing serum urea nitrogen levels.

Images