Lactobacillus gasseri capable of converting rutin and significantly improving its relief of high uric acid and its postbiotic

Abstract

The present application discloses a lactobacillus gasseri capable of transforming rutin and significantly improving its high uric acid relief, and a postbiotic thereof, and belongs to the technical field of microorganisms.The present application provides a lactobacillus gasseri CCFM1346 capable of transforming rutin to produce endogenous metabolites such as quercetin and isoquercitrin (but not limited to quercetin and isoquercitrin), and capable of promoting rutin to relieve hyperuricemia alone or by fermenting and metabolizing rutin.The strain and the postbiotic thereof can be used for preparing food, health products or drugs, and have wide application value.

Description

Technical Field

[0001] This invention relates to a strain of Lactobacillus gasseri capable of converting rutin and significantly enhancing its ability to alleviate hyperuricemia, and its post-biotics, belonging to the field of microbial technology. Background Technology

[0002] Rutin is a flavonoid compound widely found in the roots, stems, leaves, flowers, fruits, and seeds of edible and medicinal plants. It is present in many commonly used traditional Chinese medicines, such as Sophora japonica buds, Rutaecarpa, Leonurus japonicus, Hippophae rhamnoides, Ginkgo biloba, Lycium barbarum, Leonurus japonicus, Bupleurum chinense, Prunella vulgaris, Aloe vera, eucalyptus leaves, and tobacco leaves. As a safe and low-toxicity natural active ingredient, rutin possesses antioxidant, antihypertensive, antidiabetic, anti-inflammatory, antibacterial, anti-allergic, and anticancer effects. However, it has poor water solubility and exists in the glycosylated form of quercetin and rhamnose-glucose disaccharide. Its glycosides prevent it from passing through the intestinal epithelium, making it difficult to absorb into the bloodstream and resulting in poor bioavailability.

[0003] Studies have shown that rutin's stability and bioavailability are significantly improved after microbial metabolism, and most metabolites retain the original physiological activity of the parent compound, with some even exceeding that of the parent compound itself. In recent years, rutin and its processed products have been very popular in domestic and international markets, and its nutritional quality and deep processing utilization have become a research hotspot for domestic scholars. It is understood that there are already various health products on the US market using rutin as the main ingredient, such as rutin-vitamin combinations (e.g., rutin + VC, rutin + VE, rutin + VK, etc.) and combinations of rutin with plant-based health ingredients, such as rutin-ginkgo leaf extract tablets, rutin-sea buckthorn fruit extract tablets, rutin-hawthorn fruit extract tablets, and rutin-echinacea extract tablets, etc. The biotransformation products of rutin, such as quercetin and isoquercetin, possess superior physiological activity and medicinal value. Therefore, screening edible probiotics capable of converting rutin into active metabolites is beneficial for the development of post-fermentation bioactive products derived from both food and medicine.

[0004] The main cause of hyperuricemia is disordered purine metabolism: excessive uric acid synthesis in the liver and reduced excretion in the kidneys (2 / 3) and intestines (1 / 3). Currently, drugs for treating hyperuricemia are mainly divided into those that inhibit uric acid production (such as febuxostat and allopurinol) and those that promote uric acid excretion (such as probenecid and benzbromarone); however, these drugs address limited pathways and have side effects on the liver and kidneys. Studies have shown that rutin can inhibit the activity of xanthine oxidase (XOD) in vitro. XOD is a key enzyme responsible for the production of uric acid from the target group of uric acid metabolism. Another study showed that rutin can reduce serum uric acid levels, and its effect in improving hyperuricemia is partly attributed to the inhibition of XOD and xanthine dehydrogenase (XDH) activity in mouse liver. Furthermore, rutin can inhibit ROS production and NLRP3 inflammasome activation, such as the expression of NLRP3, ASC, Caspase-1, and IL-1β, and reduce the levels of pro-inflammatory factors IL-1β, IL-18, and TNF-α in serum and kidneys.

[0005] Lactobacillus gasseri ( Lactobacillus gasseri Lactobacillus gasseri is a type of lactic acid bacteria, Gram-positive, capable of growing in both aerobic and anaerobic environments. It is a facultative heterotrophic acidic strain with an optimal growth temperature of 30°C. It is a rare probiotic strain that can improve the digestive system and prevent recurrent indigestion; its function is to regulate the digestive system. Due to its probiotic effects, Lactobacillus gasseri is often added to dairy products and probiotic supplements as a probiotic ingredient. Currently, there are no strains capable of effectively converting rutin and alleviating high uric acid levels. Therefore, screening for strains capable of biotransforming rutin and alleviating high uric acid levels is of great significance for developing new functional probiotics. Summary of the Invention

[0006] This invention provides a strain of Lactobacillus gasseri ( Lactobacillus gasseri The Lactobacillus gasseri, described in CCFM1346, is deposited at the Guangdong Provincial Center for Microbial Culture Collection, with accession number GDMCC No: 63916 and accession date of October 25, 2023.

[0007] The Lactobacillus gasseri ( Lactobacillus gasseri The CCFM1346 strain was derived from the feces of healthy individuals. Sequencing analysis of the strain revealed its 16S rDNA sequence as shown in SEQ ID NO.1.

[0008] After being cultured on MRS solid plates for 24-48 hours, the Lactobacillus gasseri CCFM1346 was grayish-white, round, glossy, with slightly undulating and rough edges, and the colony diameter was 0.5-1 mm.

[0009] This invention provides a method for the biotransformation of rutin, wherein Lactobacillus gasseri CCFM1346 is fermented in a culture medium containing rutin.

[0010] In one embodiment, the fermentation medium comprises: 5 g / L rutin, 10 g / L tryptone, 5 g / L yeast extract, 2 g / L dipotassium hydrogen phosphate, 2 g / L diamine citrate, and pH = 6.8.

[0011] In one embodiment, the fermentation medium contains rutin, wherein the rutin content is ≥5 mg / mL.

[0012] In one embodiment, the reaction conditions are 30-37°C, 150-200 rpm, and 24-48 h.

[0013] In one embodiment, the Lactobacillus gasseri CCFM1346 is carried out under aerobic conditions.

[0014] In one embodiment, the Lactobacillus gasseri CCFM1346 is cultured with shaking at 150-250 rpm.

[0015] In one embodiment, the reaction is carried out in a biochemical incubator at 30-37°C.

[0016] In one embodiment, the reaction takes 24-48 hours.

[0017] The present invention also provides a metabiotic prepared using the Lactobacillus gasseri CCFM1346.

[0018] In one embodiment, the metabiotic is the supernatant obtained by centrifuging the culture of Lactobacillus gasseri CCFM1346 in a rutin-containing medium.

[0019] In one embodiment, the metabiotic is obtained by centrifuging the culture of Lactobacillus gasseri CCFM1346 in a rutin-containing medium, heat-treating it at 60-70°C for 20-30 min, centrifuging it again, and collecting the supernatant.

[0020] The present invention also provides compositions containing the Lactobacillus gasseri CCFM1346 or containing the postbiotic.

[0021] In one embodiment, the metabiotic is the supernatant collected after centrifuging the fermentation broth of Lactobacillus gasseri CCFM1346.

[0022] In one embodiment, the composition is a lyophilized powder prepared using a cell culture of *Lactobacillus gasseri* CCFM1346, containing ≥1.0 × 10⁻⁶ ppm. 6 Active Lactobacillus gasseri CCFM1346, cfu / g.

[0023] In one embodiment, the lyophilized powder is prepared by inoculating Lactobacillus gasseri CCFM1346 at an inoculum of 2%–4% into MRS medium, culturing at 35–37°C for 20–24 h, collecting the bacterial cells by centrifugation, washing 2–4 times with phosphate buffer (pH 7.0–7.2), and resuspending with a cryoprotectant to achieve a concentration of 1 × 10⁻⁶. 10 cfu / mL; freeze-drying to obtain the lyophilized powder.

[0024] In one embodiment, the protective agent contains 100 g / L skim milk powder, 30 mL / L glycerin, 100 g / L maltodextrin, 150 g / L trehalose, and 10 g / L... Monosodium glutamate (MSG)

[0025] The present invention also provides a method for converting rutin, which involves culturing the Lactobacillus gasseri CCFM1346 in a rutin culture medium.

[0026] This invention also provides the use of Lactobacillus gasseri CCFM1346 alone or in combination with rutin in the preparation of pharmaceuticals.

[0027] This invention provides the application of Lactobacillus gasseri CCFM1346 or Lactobacillus gasseri CCFM1346 postbiotic in the preparation of drugs to relieve hyperuricemia.

[0028] In one embodiment, the relief of high uric acid symptoms includes, but is not limited to, the following aspects: (1) Improve the condition of increased kidney index caused by kidney inflammation; (2) It alleviates the hydropic degeneration and cell swelling of renal tubular epithelial cells; (3) Reduces individual serum uric acid levels; (4) Inhibits the activity of hepatic xanthine oxidase; (5) Enhances the activity of hepatic uricase; (6) Enhances the expression of renal transport proteins; In one embodiment, the enhancement of renal transporter expression includes regulating the expression of renal transporters URAT1, GLUT9, and ABCG2.

[0029] In one embodiment, the composition includes, but is not limited to, microbial preparations, fermented foods, health products, or pharmaceuticals.

[0030] In one embodiment, the microbial preparation contains *Lactobacillus gasseri* CCFM1346, or fermentation broth containing *Lactobacillus gasseri* CCFM1346, or lyophilized powder containing *Lactobacillus gasseri* CCFM1346; or inactivated cells containing *Lactobacillus gasseri* CCFM1346, or lysate containing *Lactobacillus gasseri* CCFM1346, or extract containing *Lactobacillus gasseri* CCFM1346.

[0031] In one embodiment, the content of Lactobacillus gasseri CCFM1346 in the microbial agent is not less than 1×10⁻⁶. 9 CFU / mL or 1×10 10 CFU / g.

[0032] In one embodiment, the fermented food includes, but is not limited to, using Lactobacillus gasseri CCFM1346 as a fermenting microorganism to ferment food raw materials.

[0033] In one embodiment, the food ingredients include milk and dairy beverages, as well as fruit and vegetable products.

[0034] In one embodiment, the fruit and vegetable products include fruit juice beverages and fruit and vegetable purees made from Sophora japonica buds, Rutaecarpa, buckwheat, etc.

[0035] In one embodiment, the health food is a dairy product, soy product, or fruit and vegetable product produced using a fermentation agent containing the aforementioned Lactobacillus gasseri CCFM1346.

[0036] In one embodiment, the drug or pharmaceutical composition further includes a pharmaceutically acceptable excipient; the pharmaceutically acceptable excipient refers to any diluent, adjuvant, and / or carrier that can be used in the pharmaceutical field.

[0037] In one embodiment, the food ingredient is an ingredient containing the substrate rutin, including but not limited to: sophora japonica buds, rue, buckwheat, asparagus, apples, cherries, lemons, grapes, dates, apricots, tomatoes, etc.

[0038] Beneficial effects: This invention provides a strain of Lactobacillus gasseri CCFM1346 capable of converting rutin, which has the following advantages: (1) It has the ability to convert rutin. After fermentation in BHI medium containing rutin (50 μg / mL) for 48 h, the rutin content decreased by 81.6% compared with that before fermentation. The non-target metabolomics results showed that the differential metabolites of Lactobacillus gasseri CCFM1346 before and after fermentation conversion included quercetin, isoquercetin, kaempferol, isorhamnetin, p-coumaric acid and p-hydroxybenzaldehyde. These methylated forms (isoquercetin), hydroxylated forms (kaempferol and quercetin) and key phenolic acids (p-coumaric acid and p-hydroxybenzaldehyde) can effectively enhance the anti-inflammatory effect of rutin in vivo and play a role in reducing uric acid.

[0039] (2) The strain itself and the fermented biogener prepared by it can significantly improve the symptoms of watery degeneration, cell swelling, loose cytoplasm and light staining of renal tubular epithelial cells in the renal tissue of individuals with hyperuricemia, and reduce the kidney index; can effectively reduce the serum uric acid level of individuals and alleviate renal function damage; can significantly inhibit the activity of hepatic xanthine oxidase to reduce uric acid production, significantly increase the activity of uric acid oxidase and promote uric acid excretion; can improve the expression of renal uric acid transport proteins URAT1, GLUT9 and ABCG2.

[0040] This invention also provides the application of this strain to promote the relief of hyperuricemia by fermenting and metabolizing rutin, and develops the potential of rutin as a post-biotic or synbiotic preparation in the treatment of hyperuricemia, which has broad application value in the pharmaceutical field.

[0041] Preservation of biological materials Lactobacillus gasseri ( Lactobacillus gasseri (CCFM1346, categorized as follows) Lactobacillus gasseri It was deposited on October 25, 2023, at the Guangdong Provincial Center for Microbial Culture Collection, with accession number GDMCC No: 63916, located at 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou. Attached Figure Description

[0042] Figure 1 It is Lactobacillus gasseri ( Lactobacillus gasseri Colony morphology diagram of CCFM1346; Figure 2 The images show the liquid chromatograms of rutin fermented by CCFM1346 at 0h and 48h. Figure 3 The changes in the content of each component after rutin fermentation with CCFM1346 for 48 hours; Figure 4 This is the total ion chromatogram of rutin fermented on CCFM1346 at 0h and 48h. Figure 5 This is a graph showing the changes in body weight of ICR mice in different treatment groups; Figure 6These are kidney index graphs of ICR mice in different treatment groups; Figure 7 Morphology of kidney tissue (HE staining) from ICR mice in different treatment groups. Figure 8 This is a graph showing the serum uric acid content in ICR mice from different treatment groups; Figure 9 The levels of xanthine oxidase and uricase oxidase in the livers of ICR mice in different treatment groups; Figure 10 The expression levels of renal transport proteins in ICR mice under different treatment groups. Detailed Implementation

[0043] The present invention will be further described below with reference to specific embodiments and accompanying drawings. The following examples used SPF-grade 4-week-old male ICR mice purchased from Vital River Laboratory Animal Co., Ltd.; the following examples used potassium oxonate, xanthine, allopurinol, and sodium carboxymethyl cellulose purchased from Shanghai Sigma-Aldrich; the following examples used ELISA kits purchased from Nanjing Senbeijia Biotechnology Co., Ltd.; the following examples used brain-heart infusion broth (BHI) purchased from Qingdao High-Tech Industrial Park Haibo Biotechnology Co., Ltd.; rutin standard (HPLC ≥ 98%) purchased from Shanghai Chuangsai Technology Co., Ltd.; dimethyl sulfoxide purchased from Aladdin Chemical Reagent Co., Ltd.; acetic acid and chromatographic grade methanol purchased from Sigma-Aldrich (Shanghai) Trading Co., Ltd.; and Sophora japonica extract (rutin accounting for 75% of the total mass) purchased from Shaanxi Evergreen Biotechnology Co., Ltd.

[0044] The culture media involved in the following examples are as follows: MRS liquid culture medium (g / L): peptone 10 g / L, yeast extract 5 g / L, beef extract 10 g / L, glucose 20 g / L, anhydrous sodium acetate 2 g / L, diammonium citrate 2 g / L, K2HPO4·3H2O 2.6 g / L, MgSO4·7H2O 0.58 g / L, MnSO4·7H2O 0.25 g / L, Tween-80 1 g / L, distilled water 1000 g / L.

[0045] MRS solid culture medium (g / L): peptone 10 g / L, yeast extract 5 g / L, beef extract 10 g / L, glucose 20 g / L, anhydrous sodium acetate 2 g / L, diammonium citrate 2 g / L, K2HPO4·3H2O 2.6 g / L, MgSO4·7H2O 0.58 g / L, MnSO4·7H2O 0.25 g / L, Tween-80 1 g / L, agar 20 g / L, distilled water 1000 g / L.

[0046] Fermentation substrate containing rutin (g / L): rutin 5 g / L, tryptone 10 g / L, yeast powder 5 g / L, dipotassium hydrogen phosphate 2 g / L, diamine citrate 2 g / L, pH=6.8.

[0047] BHI liquid medium: tryptone 10 g / L, ox heart extract 17.5 g / L, sodium chloride 5.0 g / L, glucose 2.0 g / L, disodium hydrogen phosphate 2.5 g / L.

[0048] The detection methods involved in the following embodiments are as follows: The contents of rutin, quercetin, and isoquercetin were determined by HPLC, and the presence of the products was further confirmed by Q Exactive liquid chromatography-mass spectrometry. HPLC detection: A Waters e2695 liquid chromatograph with an X Bridge RC18 column (250 × 4.6 mm, 5 µm) was used. The mobile phase consisted of 0.1% phosphoric acid aqueous solution (A) and 100% acetonitrile solution (B). Spectral scanning was performed to determine the maximum absorption wavelength, 360 nm. Gradient elution was performed at a flow rate of 1.00 mL / min, column temperature 30 °C, and injection volume 10 μL. The gradient elution program was as follows: 0 min, 10% B; 0–5 min, 10%–24% B; 5–10 min, 24%–24% B; 10–12 min, 24%–50% B; 12–17 min, 50%–50% B; 17–18 min, 50%–10% B; 18–20 min, 10% B. Rutin eluted at 8.6 min; quercetin eluted at 15.4 min. Isoquercitrin elution time: 9.1 min.

[0049] LC-MS detection: The presence of the product was further confirmed using a Q Exactive LC-MS system with a C18 column and mobile phases of 0.1% formic acid in water (phase A) and acetonitrile (phase B). Qualitative and quantitative analyses were performed using a UPLC-Q Exactive quadrupole-electrostatic field orbital trap high-resolution mass spectrometer (Thermo Fisher Scientific, USA) with parallel-reaction-monitoring (PRM). The chromatographic column was an ACQUITY UPLC® HSS T3 1.8 µm (2.1 × 100 mm); the column temperature was 35 °C; the mobile phase was: A - 0.1% formic acid water, B - acetonitrile; the flow rate was 0.35 min / L; the injection volume was 2 μL; gradient elution was used: 0 min, 5% B; 0–3 min, 5%–20% B; 3–5 min, 20%–60% B; 5–6.5 min, 60% B; 6.5–10 min, 60%–100% B; 10–12 min, 100% B; 12–12.5 min, 100%–5%, 12.5–15 min, 5% B. The ion source was a HESI (heated ESI) source, with a spray voltage of 3.5 kV (+) and 3.2 kV (-), and a sheath gas flow rate of 35 µL·min. -1 Ion transfer tube temperature: 320°C; Assist gas flow rate: 15 µL·min -1 ; Auxiliary gas temperature: 320°C. Scan mode: PRM (100-500 m / z); Resolution: 35000; Acquisition polarity: positive; AGC target: 5e 5; Maximum IT: 100 ms.

[0050] Example 1: Screening, identification, observation and preservation of Lactobacillus gasseri CCFM1346.

[0051] 1. Screening Strains derived from healthy human feces and listed in the edible fungi catalog were obtained from the microbial bank of the Food Biotechnology Center of Jiangnan University. These strains were then subjected to in vitro rutin fermentation. After extraction, freeze-drying, and reconstitution of the fermentation broth, the results were analyzed by HPLC and LCMS / MS to screen out edible strains that can convert rutin into quercetin. Finally, one target strain was obtained.

[0052] 2. Identification The whole genome DNA of strain CCFM1346 was extracted for 16S rDNA amplification. The amplified DNA fragments were collected and sequenced (performed by Suzhou Genewiz Biotechnology Co., Ltd.). The sequence was aligned with the NCBI sequence, and the results showed that the strain was *Lactobacillus gasseri*, and it was named *Lactobacillus gasseri*. Lactobacillus gasseri ) CCFM1346.

[0053] 3. Observation Dip in Lactobacillus gasseri ( Lactobacillus gasseri The CCFM1346 bacterial culture was streaked on MRS solid medium and incubated aerobicly at 37°C for 48 h. Colonies were observed to be round, white, and smooth (see details). Figure 1 ).

[0054] 4. Save Pick Lactobacillus gasseri ( Lactobacillus gasseri A single colony of CCFM1346 was inoculated into MRS liquid medium and cultured aerobicly at 37°C for 24 h to obtain a bacterial suspension. 1 mL of the bacterial suspension was placed in a sterile centrifuge tube, centrifuged at 8000 r / min for 3 min, and the upper culture medium was discarded. The bacterial sludge was resuspended in 30% glycerol solution and stored at -80°C.

[0055] Example 2: Transformation of rutin by Lactobacillus gasseri CCFM1346 Lactobacillus gasseri CCFM1346 from Example 1 was streaked on MRS solid medium and incubated upside down at 37°C for 48 h. A single colony was picked and inoculated into 5 mL of MRS liquid medium and incubated at 37°C for 48 h to prepare a seed culture.

[0056] Preparation of BHI liquid medium: 10 g / L tryptone, 17.5 g / L ox heart extract, 5.0 g / L sodium chloride, 2.0 g / L glucose, and 2.5 g / L disodium hydrogen phosphate. Rutin powder was weighed and prepared into a concentrated dimethyl sulfoxide (DMSO) solution at a concentration of 100 mg / mL. This solution was placed in a clean bench and irradiated with UV light for 30 min. The solution was then poured into sterilized BHI medium, thoroughly mixed, and dispensed to prepare the fermentation medium for subsequent experiments. The activated strain was inoculated into BHI liquid medium at a 4% (v / v) inoculum to achieve a final rutin concentration of 50 μg / mL. The medium was incubated at 37℃ for 48 h. Fermentation broth at 0 h, 24 h, and 48 h was collected into 1 mL to 2 mL EP tubes, centrifuged at 8000 rpm for 5 min to remove bacterial cells, and the supernatant was transferred to 5 mL EP tubes.

[0057] Add the same volume of ethyl acetate to each EP tube containing the fermentation broth for extraction. After sonicating for 30 min to mix thoroughly, centrifuge at 8000 rpm for 5 min. Take 1 ml of the supernatant and aliquot it into 2 ml EP tubes. Use a freeze dryer at 45℃ and 2000 rpm to freeze concentrate the fermentation broth. Add 200 µL of methanol to redissolve and shake to mix.

[0058] The reference solution and the sample solution were injected and determined according to chromatographic conditions. The rutin content in the fermentation broth was calculated using the external standard method. The results are shown in Table 1. Figures 2-3 As shown.

[0059] Table 1. Content of each component after fermentation of Lactobacillus gasseri CCFM1346 in 50 μg / mL rutin.

[0060] The results showed that after 48 hours of fermentation, the rutin content in the fermentation medium decreased by 81.6% compared to before fermentation. This indicates that after fermentation with *Lactobacillus gasseri* CCFM1346 of this invention, rutin was significantly converted into isoquercetin, quercetin, and other active small molecules.

[0061] Example 3: Analysis of rutin metabolites before and after fermentation with Lactobacillus gasseri CCFM1346 1. Sample pretreatment Following the method in Example 2, activated Lactobacillus gasseri CCFM1346 was inoculated into BHI liquid medium at an inoculation rate of 4% (v / v). Rutin was added to bring the final concentration of rutin in the medium to 50 μg / mL. After incubation at 37°C for 48 h, the fermentation broth at 0 h and 48 h was taken into 1 mL to 2 mL EP tubes, centrifuged at 8000 rpm for 5 min to remove the bacterial cells, and the supernatant was transferred into 5 mL EP tubes.

[0062] (1) Transfer 100 μL of supernatant into a 1.5 mL centrifuge tube; (2) Add 400 μL of methanol:acetonitrile = (1:1, v / v) (pre-cooled at -20℃ in advance) to precipitate the protein; (3) Vortex 30 s; (4) Sonication in an ice bath for 10 min; (5) Place the sample in a -20℃ refrigerator for 1 h to increase the protein precipitation rate (secondary precipitation removes protein). (6) Centrifuge at 15000 rpm for 15 min at 4℃. (7) Take the supernatant and concentrate it under vacuum; (8) Redissolve by adding 200 μL of acetonitrile:water (1:1) and vortexing for 30 s; (9) Centrifuge at 15,000 rpm for 15 min at 4℃, take the supernatant and filter it through a 0.22 μm filter membrane, transfer an appropriate volume into a vial for instrument testing; (10) QC preparation: Transfer an equal volume from the sample to be tested into a new sample vial and mix them evenly to obtain QC (Quality Control). (11) Blank preparation: 1 mL acetonitrile: water (1:1, v / v) into the sample vial and mix thoroughly; 2. LC-MS / MS detection The rutin fermentation broth was further analyzed using a high-resolution liquid chromatography-mass spectrometry (LC-MS / MS) system (Q Exactive LC-MS / MS). Column: Hypersil GOLD C18 (1.9 µm × 2.1 mm × 100 mm), sheath gas flow rate: 35 arb, auxiliary gas flow rate: 15 arb, ion transport capillary temperature: 320 °C, autosampler temperature and column temperature: 4 °C and 30 °C, respectively. Mobile phase: 0.1% formic acid in water (phase A), acetonitrile (phase B). Elution conditions: gradient elution, elution gradient curves: 0–3 min, 5% B; 3–9 min, 5–30% B; 9–15 min, 30–100% B; 15–16 min, 100% B; 16–16.5 min, 100–5% B; 16.5–20 min, 5% B; flow rate: 0.3 mL / min, injection volume: 2 μL.

[0063] 3. Screening of active ingredients in rutin samples The full phenolic profile of rutin, including free and bound phenols, was investigated using non-targeted metabolomics methods after 0 h of fermentation and 48 h of fermentation with Lactobacillus gasseri CCFM1346 (before and after fermentation). ESI mode was used to acquire the rutin extract in the sensitivity mode of an LC-MS system. The total ion chromatogram is shown below. Figure 4 As shown.

[0064] Literature review and online traditional Chinese medicine databases were used to collect information such as compound names, molecular formulas, molecular weights, and retention times. The database was imported into Compound Discoverer 3.3 software for data analysis. By studying fragment ions, lost groups, and mass spectrometry fragmentation patterns of molecular ions in high-collision-energy channels, the possible structures of the compounds were inferred. Differential metabolite screening was conducted based on P-value < 0.05 and Log2 Fold Change > 2. After 48 hours of fermentation with Lactobacillus gasseri CCFM1346, the main metabolites of rutin were organic aromatic compounds and amino acids, specifically including benzoyl derivatives, benzoic acid derivatives, benzaldehyde derivatives, cinnamic acid and its derivatives, coumarin and its derivatives, phenylpropionic acid, glycerol and its derivatives, and hydrocarbon derivatives.

[0065] Table 2. Phenolic substances detected in rutin fermentation broth and changes in substances before and after fermentation.

[0066] "+" indicates that the amount of substance increased after fermentation compared to before fermentation, and "-" indicates that the amount of substance decreased after fermentation compared to before fermentation. Example 4: Effects of Lactobacillus gasseri CCFM1346 and its postbiotics on disease symptoms in hyperuricemic mice Preparation of rutin extract solution: Weigh 252 mg of Sophora japonica extract powder (purchased from Shaanxi Evergreen Biotechnology Co., Ltd., rutin content 75%), dissolve it in 22.4 ml of 0.9% (w / v) physiological saline to obtain a rutin extract solution with a rutin concentration of 75 mg / mL.

[0067] Preparation of the post-fermentation rutin suspension of CCFM1346: Activated Lactobacillus gasseri CCFM1346 bacterial suspension in MRS medium was inoculated into a culture medium containing Sophora japonica extract at an inoculation rate of 2% (v / v), resulting in a final concentration of rutin of 5 mg / ml. The culture was incubated at 37℃ with shaking at 200 rpm for 48 h, yielding a bacterial concentration of 5 × 10⁻⁶. 9 The fermentation broth was heated at 65°C for 30 min, centrifuged, and the supernatant was collected to obtain the post-fermentation biogenic suspension.

[0068] Preparation of CCFM1346 bacterial suspension: The *Lactobacillus gasseri* CCFM1346 bacterial suspension activated on MRS medium was centrifuged at 3000 rpm for 15 min. The supernatant was discarded, and the bacterial sludge was stored in 30% glycerol. Before gavage, the supernatant glycerol was removed by centrifugation, and 0.9% (w / v) physiological saline was added to adjust the bacterial suspension concentration to 5 × 10⁻⁶. 9 cfu / mL.

[0069] Fifty-six healthy male ICR mice aged 4 weeks were randomly divided into 7 groups of 8 mice each after one week of acclimatization. The 7 groups were: blank control group, model group, *Gynostemma pentaphyllum* group, rutin dietary group (SJ), rutin dietary post-biotic group (CCFM1346-D), *Lactobacillus gasseri* CCFM1346 group (CCFM1346-L), and rutin-containing dietary compound CCFM1346 synergistic preparation group (SJ+CCFM1346).

[0070] All modeling drugs were administered using 0.9% (w / v) physiological saline as a solvent, with a gavage dose of 0.2 mL per animal per day, at the same time each day. The model group, the *Gynostemma pentaphyllum* group, and the drug administration group were administered potassium oxonate (280 mg / kg) by gavage at 8:00 AM daily, while the control group received 0.5% CMC-Na for two consecutive weeks. At 10:00 AM on the same day, the model group and the control group received 0.9% (w / v) physiological saline treatment, the *Gynostemma pentaphyllum* group received 10 mg / kg allopurinol treatment; the SJ group was administered 75 mg / mL rutin extract solution by gavage, the CCFM1346-L group was administered CCFM1346 bacterial suspension by gavage, the CCFM1346-D group was administered CCFM1346 fermented probiotic suspension by gavage; the SJ+CCFM1346 group was administered rutin extract containing 75 mg / mL and 5×10... 9 A mixture of cfu / mL CCFM1346 bacterial suspension.

[0071] Mice were sacrificed on day 22, and serum and tissue samples were collected for relevant indicator measurements. The grouping and treatment of experimental animals are shown in Table 3.

[0072] Table 3 Animal grouping and treatment methods

[0073] During the modeling period (weeks 1-3) via gavage, the weight of mice was measured daily and the percentage change in weight was calculated. After sacrifice, the weight of the kidneys was measured, the kidney index was calculated for each group, and the kidneys were stained with hematoxylin and eosin (HE) for sectioning. Results are as follows: Figures 5-7As shown, compared with the control group, the model group mice had significantly increased body weight and kidney index, suggesting the possibility of renal hyperplasia and hypertrophy. The decrease in body weight and increase in kidney index were significantly alleviated in the SJ group, CCFM1346-L live bacteria group, and SJ+CCFM1346 combined preparation group. Compared with the model group, the CCFM1346-L live bacteria group significantly reduced the kidney index in hyperuricemic mice. Furthermore, HE sections of the kidneys in the model group showed obvious hydropic degeneration of renal tubular epithelial cells, cell swelling, and loose, pale cytoplasm, compared to the blank group; while the SJ group, CCFM1346-L group, CCFM1346-D group, and SJ+CCFM1346 group alleviated kidney damage associated with improving renal interstitial and tubular dilatation inflammatory cell infiltration. These experimental results demonstrate that SJ, CCFM1346-L, CCFM134-D, and SJ+CCFM1346 are effective in alleviating disease symptoms in mice with high uric acid, and that the live bacteria group of CCFM1346-L and the synergistic preparation of SJ+CCFM1346 are effective.

[0074] Example 5: Lactobacillus gasseri CCFM1346 and its postbiotics reduced serum uric acid levels in hyperuricemic mice. The grouping, modeling, and treatment methods for ICR mice are the same as in Example 4.

[0075] Mice were sacrificed on day 22, and their serum was collected. Mice were anesthetized with 1%-1.5% isoflurane by inhalation, and blood was collected from the ocular venous plexus. The blood was allowed to coagulate naturally at room temperature for 30-60 minutes, then centrifuged at 2000 g for 20 minutes. The supernatant was collected, and the serum uric acid content was determined according to the enzyme-linked immunosorbent assay (ELISA) kit for mice.

[0076] The effect of Lactobacillus gasseri CCFM1346 on serum uric acid levels in mice is as follows: Figure 8 Compared with hyperuricemia model mice, the SJ, CCFM1346-L, CCFM1346-D, and SJ+CCFM1346 groups reduced serum uric acid concentrations in hyperuricemia mice by 60.34%, 72.40%, 67.58%, and 74.54%, respectively, approaching the levels of the control group. The SJ+CCFM1346 synergistic formulation showed uric acid-lowering effects comparable to the clinical drug allopurinol, and can prevent and reduce the occurrence of hyperuricemia and gout. Furthermore, the uric acid-lowering effect of SJ was increased by 18.25% after fermentation with CCFM1346.

[0077] Example 6: Lactobacillus gasseri CCFM1346 and its postbiotics inhibit xanthine oxidase activity and enhance uricase oxidase activity in the livers of hyperuricemic mice. The grouping, modeling, and treatment methods for ICR mice were the same as in Example 4. The detection of xanthine oxidase and uricase oxidase were performed according to the methods described in the kit (Beijing Solarbio).

[0078] like Figure 9 As shown, compared with hyperuricemic mice, the SJ, CCFM1346-D, CCFM1346-L, and SJ+CCFM1346 groups reduced hepatic xanthine oxidase activity in hyperuricemic mice by 23.28%, 61.23%, 51.83%, and 56.10%, respectively. It can be seen that rutin extract (SJ) increased hepatic xanthine oxidase inhibitory activity by 49.45% after fermentation with strain CCFM1346, and its effect was similar to that of allopurinol. By inhibiting xanthine oxidase activity, uric acid synthesis in the body is reduced, which is beneficial for the prevention and treatment of hyperuricemia and gout.

[0079] like Figure 9 As shown, compared with hyperuricemic mice, the SJ, CCFM1346-D, CCFM1346-L, and SJ+CCFM1346 groups increased the activity of hepatic uricase in hyperuricemic mice by 25.77%, 51.35%, 28.71%, and 55.00%, respectively. Among them, the SJ+CCFM1346 and CCFM1346-D groups showed a greater ability to increase hepatic uricase activity than allopurinol, indicating that rutin enhances its effect through in vivo and in vitro conversion, thereby promoting further uric acid breakdown in mice, which is beneficial for the prevention and treatment of hyperuricemia and gout. Furthermore, the uricase-promoting effect of rutin extract (SJ) after fermentation with strain CCFM1346 was better than that before fermentation, with an activity increase of 34.46%.

[0080] Example 7: Lactobacillus gasseri CCFM1346 and its postbiotics regulate the expression of renal transport proteins URAT1, GLUT9, and ABCG2 in hyperuricemic mice. The grouping, modeling, and treatment methods for ICR mice are the same as in Example 4.

[0081] Mice were sacrificed on day 22, and kidney tissue was collected. The kidney tissue was homogenized with PBS at a weight-to-volume ratio of 1:5, RNA was extracted, and reverse transcribed into cDNA using an RT-PCR reverse transcription kit. -△△Ct The expression levels of URAT1 mRNA, GLUT9 mRNA, and ABCG2 mRNA were calculated using the formula, with GAPDH as the internal reference. The primers are shown in Table 4.

[0082] Table 4 Primers

[0083] like Figure 10As shown, the expression of renal transport proteins URAT1, GLUT9, and ABCG2 mRNA was significantly increased in the model group mice. The SJ, CCFM1346-L, CCFM1346-D, and SJ+CCFM1346 groups significantly reduced the renal ABCG2 mRNA level in hyperuricemic mice. This indicates that rutin extract (SJ) showed even lower levels of renal transport protein ABCG2 after fermentation with strain CCFM1346, similar to the therapeutic effect of allopurinol. The CCFM1346-L, CCFM1346-D, and SJ+CCFM1346 groups significantly reduced the renal URAT1 mRNA level in hyperuricemic mice, with better effects than allopurinol. Furthermore, the live strain CCFM1346 and the synergistic preparation of rutin and CCFM1346 showed greater inhibitory effects on renal transport protein URAT1 than rutin diet and rutin diet fermented with strain CCFM1346. The SJ, CCFM1346-L, CCFM1346-D, and SJ+CCFM1346 groups significantly reduced the mRNA level of GLUT9 in the kidneys of hyperuricemic mice. This indicates that rutin extract (SJ) further reduced the mRNA level of GLUT9 in the kidneys after fermentation with strain CCFM1346, and the effect was similar to that of allopurinol. Renal transport proteins URAT1, GLUT9, and ABCG2 play important roles in intestinal uric acid excretion. Lactobacillus gasseri CCFM1346 can promote uric acid excretion by reducing the expression of URAT1, GLUT9, and ABCG2 in the kidneys.

[0084] Example 8: Preparation of a synergistic formulation of Lactobacillus gasseri CCFM1346 and a rutin-containing dietary composition MRS medium preparation: 10 g / L peptone, 10 g / L beef extract, 20 g / L glucose, 2 g / L sodium acetate, 5 g / L yeast extract, 2 g / L diammonium citrate, 2.6 g / L K₂PO₄·3H₂O, 0.1 g / L MgSO₄·7H₂O, 0.05 g / L MnSO₄, 1 mL / L Tween 80, 0.5 g / L cysteine ​​phosphate, pH 6.8.

[0085] Preparation of freeze-drying protectant: A freeze-drying protectant containing 100 g / L skim milk powder, 30 mL / L glycerol, 100 g / L maltodextrin, 150 g / L trehalose, and 10 g / L lyophilization protectant was prepared by mixing water with the protectant raw materials. A protectant for monosodium glutamate.

[0086] Lactobacillus gasseri CCFM1346 was inoculated into the MRS medium at an inoculum size of 2%–4% and cultured aerobically at 37°C for 24 h. The cells were collected by centrifugation, washed 2–4 times with phosphate-buffered saline (pH 7.0–7.2), and resuspended in the protective agent to a concentration of 10. 10 The concentration of cfu / mL was then increased; the suspension was cultured at 37°C under aerobic conditions for 1 h, followed by freeze-drying to obtain the *Lactobacillus gasseri* CCFM1346 bacterial agent. The prepared bacterial agent was mixed with rutin extract to ensure that the viable count of *Lactobacillus gasseri* CCFM1346 in the composition was not less than 1.0 × 10⁻⁶. 6 cfu / mL or 1.0×10 6 cfu / g, rutin content not less than 10 mg / mL.

[0087] In addition to the above preparation methods, functional microbial preparations, fermented foods, drugs and / or drug compositions containing Lactobacillus gasseri CCFM1347 and rutin can also be prepared using Lactobacillus gasseri CCFM1347.

[0088] Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Anyone skilled in the art can make various modifications and alterations without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be determined by the claims.

Claims

1. A strain of Lactobacillus gasseri ( Lactobacillus gasseri CCFM1346 was deposited at the Guangdong Provincial Center for Microbial Culture Collection on October 25, 2023, with accession number GDMCC No: 63916.

2. The postbiotic prepared using Lactobacillus gasseri CCFM1346 as described in claim 1.

3. The epigenetic agent according to claim 2, characterized in that, The supernatant is obtained by centrifuging the culture of Lactobacillus gasseri CCFM1346 in a rutin-containing medium and collecting it.

4. A composition comprising Lactobacillus gasseri CCFM1346 of claim 1 and / or its postbiotic; said composition comprising a microbial preparation or a pharmaceutical.

5. A method for converting rutin, characterized in that, The Lactobacillus gasseri CCFM1346 of claim 1 was cultured in a medium containing rutin.

6. The use of Lactobacillus gasseri CCFM1346, alone or in combination with rutin, in the preparation of a drug according to claim 1, characterized in that... The medication is for relieving and / or treating hyperuricemia.

7. The use of Lactobacillus gasseri CCFM1346 as described in claim 1 or the metabiotic as described in claim 2 in the preparation of a drug for relieving symptoms of hyperuricemia.

8. A drug for relieving hyperuricemia, characterized in that, The product contains Lactobacillus gasseri CCFM1346 as described in claim 1 or the product of fermentation of Lactobacillus gasseri CCFM1346 in a rutin-containing medium.

9. The application of Lactobacillus gasseri CCFM1346 as described in claim 1 in the preparation of fermented products, characterized in that, The fermented product uses food containing rutin as raw material.

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