An animal bifidobacterium with both lipid-lowering and anti-aging effects and its application in enhancing the efficacy of licorice.

Abstract

This invention discloses a strain of Bifidobacterium animalis with both lipid-lowering and anti-aging effects, and its application in enhancing the efficacy of licorice through transformation, belonging to the fields of microbial technology and pharmaceutical technology. This strain can ferment licorice extract, increasing the content of active ingredients such as glycyrrhetinic acid, glycyrrhetinic acid 3-O-glucuronide, and 5-O-methyl genistein, thereby enhancing its efficacy. Specifically, it inhibits weight gain and reduces four blood lipid levels under a high-fat diet; reduces the levels of aging-related secretory phenotypes IL-1β and IL-6; reduces the content of hepatic β-galactosidase; reduces damage caused by hepatic lipid accumulation; and promotes the expression of lipid metabolism genes MGL, HSL, ATGL, and PPAR-α, while inhibiting the expression of lipid synthesis genes PPAR-γ and Fasn. Therefore, the combined application of Bifidobacterium animalis subsp. lactis CCFM1363 and licorice extract has broad potential for the development of lipid-lowering and anti-aging products.

Description

TECHNICAL FIELD

[0001] The present application relates to a strain of animal Bifidobacterium lactis subsp. CCFM1363 and its prepared anti-aging and lipid-lowering licorice postbiotic and the application of transformed licorice synergism, belonging to the field of microbial technology and medical technology. BACKGROUND

[0002] With the continuous intensification of population aging, anti-aging research has become a core issue in the field of biomedical and microbial technology. Aging is a complex biological process influenced by multiple factors, which not only involves oxidative stress, chronic inflammation, metabolic dysfunction and immune function decline, but also directly leads to gradual decline of body function and significantly increases the risk of chronic diseases such as diabetes, cardiovascular disease and neurodegenerative disease. It is worth noting that unhealthy diet and lifestyle play an important role in this process, which triggers metabolic disorders, promotes the accumulation of lipids, and induces a series of diseases such as hyperlipidemia, cardiovascular disease, fatty liver and diabetes. These disease states in turn exacerbate chronic inflammation, oxidative stress and DNA damage in the body, forming a vicious cycle that further accelerates the aging process.

[0003] In recent years, medicinal and edible materials and their microbial metabolites have attracted widespread attention due to their safety and effectiveness. Licorice is a commonly used medicinal and edible material with a wide range of applications, and related studies have found that licorice also exhibits good effects in terms of antioxidant, anti-inflammatory, anti-tumor, anti-viral and other aspects. In addition, the application potential of licorice extract in anti-aging has attracted widespread attention. Triterpenoid saponins in licorice are considered to be the main active substances, mainly including glycyrrhizic acid (GL), glycyrrhizinic acid (GAMG) and glycyrrhizinic acid (GA), among which glycyrrhizic acid has the highest content.

[0004] Studies have shown that glycyrrhizic acid is not as effective as active small molecules such as glycyrrhizinic acid and glycyrrhizinic acid in terms of absorption and physiological activity. Direct use of licorice extract is difficult to achieve direct effects, and the efficacy of licorice extract is poor when consumed. Therefore, how to promote the efficacy of licorice extract is the key to developing licorice extract as a lipid-lowering and anti-aging related product. SUMMARY

[0005] The technical problem to be solved by the present application is to overcome the defects and deficiencies of the above-mentioned prior art, and to provide the application of animal Bifidobacterium lactis subsp. CCFM1363 in enhancing the efficacy of licorice extract in anti-aging and lipid-lowering.

[0006] Another object of the present application is to provide a fermented licorice extract and composition that can improve the lipid-lowering and anti-aging efficacy.

[0007] Another object of the present application is to provide a method for increasing the content of glycyrrhetic acid in licorice extract.

[0008] The above object of the present application is achieved by the following technical solutions.

[0009] The present application provides a strain of Bifidobacterium animalis subsp. lactis CCFM1363, which has been deposited with the Guangdong Microbial Culture Collection Center on December 6, 2023, and has the accession number GDMCC No: 64111.

[0010] In an embodiment of the present application, the Bifidobacterium animalis subsp. lactis CCFM1363 of the present application is cultured on MRS medium for 48 hours, and the colonies are round, convex or lens-shaped, white, opaque, with a smooth to mucoid soft surface.

[0011] In an embodiment of the present application, the use of the Bifidobacterium animalis subsp. lactis CCFM1363 to ferment licorice extract can significantly increase the content of glycyrrhetic acid and promote the licorice extract to exert lipid-lowering and anti-aging effects. Specifically, it can: (1) control the increase of body weight in obese organisms; (2) reduce the level of aging-related secretory phenotype in organisms; (3) reduce the content of beta-galactosidase in organisms; (4) inhibit liver fat accumulation and lesions in organisms; (5) reduce blood lipid levels in organisms; (6) effectively increase the expression of lipid metabolism-related genes MGL, HSL, ATGL, PPAR-α, and reduce the expression of lipid synthesis-related genes PPAR-γ, Fasn.

[0012] The present application also provides a postbiotic prepared from the Bifidobacterium animalis subsp. lactis CCFM1363, which is obtained by fermenting licorice extract with the Bifidobacterium animalis subsp. lactis CCFM1363.

[0013] In an embodiment of the present application, the preparation method of the postbiotic comprises the following steps:

[0014] (1) activating the Bifidobacterium animalis subsp. lactis CCFM1363 to obtain a bacterial solution;

[0015] (2) inoculating the bacterial solution of (1) into a fermentation base containing licorice extract, with an inoculum of 5x10 7 CFU / mL, and fermenting in an anaerobic environment for 48 hours;

[0016] (3) after fermentation, the viable count is 1x10 9 CFU / mL, heat treating the fermentation broth, and then homogenizing under high pressure to obtain the postbiotic.

[0017] In one embodiment of the present invention, step (1) involves streaking Bifidobacterium lactis subsp. CCFM1363 on MRS solid medium, inverting the plate for anaerobic culture (preferably inverted culture at 37°C for 48h), and then picking a single colony and inoculating it into MRS liquid medium (preferably 5mL) for culture (preferably incubated at 37°C for 48h) to obtain bacterial solution.

[0018] In one embodiment of the present invention, the concentration of licorice extract in the fermentation substrate containing licorice extract in step (2) is 5-15 mg / mL (preferably 10 mg / mL).

[0019] In one embodiment of the present invention, the inoculation amount of bacterial solution in step (2) is 1-8% (v / v).

[0020] In one embodiment of the present invention, the fermentation in step (2) is a constant temperature fermentation at 30-40℃ for 24-72 hours (preferably a constant temperature fermentation at 37℃ for 48 hours).

[0021] In one embodiment of the present invention, the heat treatment conditions in step (3) are 60-70°C for 25-35 min (preferably 65°C for 30 min).

[0022] In one embodiment of the present invention, the high-pressure homogenization conditions in step (3) are 300-1500 bar, and the bacterial strain is circulated 4-8 times (preferably 1200 bar, 6 times).

[0023] The present invention also provides a synbiotic preparation made from the animal Bifidobacterium lactis subspecies CCFM1363.

[0024] In one embodiment of the present invention, the preparation method of the synbiotic preparation includes the following steps:

[0025] (1) Activate Bifidobacterium lactis subsp. CCFM1363 to obtain bacterial solution;

[0026] (2) In MRS medium, inoculate the bacterial suspension from (1) with a viable count of 5 × 10⁶. 7 CFU / mL, cultured in an anaerobic environment until the logarithmic growth phase of the strain to obtain Bifidobacterium animalis subsp. lactis bacterial culture;

[0027] (3) Collect bacterial cells from liquid culture medium by centrifugation, mix with freeze-drying protectant (12% skim milk powder), and freeze-dry to obtain freeze-dried bacterial powder;

[0028] (4) Mix the freeze-dried bacterial strain powder and licorice extract according to the dosage to obtain the product.

[0029] In one embodiment of the present invention, step (1) involves streaking Bifidobacterium lactis subsp. CCFM1363 on MRS solid medium, inverting the plate for anaerobic culture (preferably inverted culture at 37°C for 48h), and then picking a single colony and inoculating it into MRS liquid medium (preferably 5mL) for culture (preferably incubated at 37°C for 48h) to obtain bacterial solution.

[0030] In one embodiment of the present invention, the concentration of licorice extract in the fermentation substrate containing licorice extract in step (2) is 5-15 mg / mL (preferably 10 mg / mL).

[0031] In one embodiment of the present invention, the inoculation amount of bacterial solution in step (2) is 1-8% (v / v).

[0032] In one embodiment of the present invention, the fermentation in step (2) is a constant temperature fermentation at 30-40℃ for 24-72 hours (preferably a constant temperature fermentation at 37℃ for 48 hours).

[0033] In one embodiment of the present invention, the heat treatment conditions in step (3) are 60-70°C for 25-35 min (preferably 65°C for 30 min).

[0034] In one embodiment of the present invention, the high-pressure homogenization conditions in step (3) are 300-1500 Bar, and the bacterial strain is circulated 4-8 times (preferably 1200 Bar, 6 times).

[0035] The present invention provides a product containing the aforementioned Bifidobacterium lactis subsp. CCFM1363 and / or its postbiotic and / or its synergistic preparation.

[0036] In one embodiment of the present invention, the product is food, medicine, or health product.

[0037] In one embodiment of the present invention, the product is a lyophilized powder of the post-biotic or synbiotic preparation, or a reconstitute containing the lyophilized powder of the post-biotic or synbiotic preparation.

[0038] This invention provides a method for degrading glycyrrhizic acid by adding Bifidobacterium animalis subsp. lactis CCFM1363 to a culture medium containing glycyrrhizic acid for fermentation.

[0039] This invention provides a method for preparing glycyrrhetinic acid, wherein the animal Bifidobacterium lactis subsp. CCFM1363 is added to a culture medium containing glycyrrhetinic acid for fermentation.

[0040] This invention provides a method for enhancing the medicinal efficacy of licorice, wherein the method is any one of the following:

[0041] (1) The animal Bifidobacterium lactis subsp. CCFM1363 was added to a culture medium containing licorice products for fermentation;

[0042] (2) Mix the animal Bifidobacterium lactis subsp. CCFM1363 with licorice products.

[0043] In one embodiment of the present invention, the licorice product comprises licorice or licorice extract.

[0044] The present invention also provides the use of the aforementioned Bifidobacterium lactis subsp. CCFM1363 and / or its postbiotics and / or its synergistic preparations in the preparation of lipid-lowering and / or anti-aging pharmaceuticals.

[0045] In one embodiment of the present invention, the anti-aging drug is used to alleviate age-related diseases.

[0046] In one embodiment of the present invention, the aging-related diseases include oxidative stress-induced injury, chronic inflammation, liver lipid accumulation, hyperlipidemia, or metabolic syndrome.

[0047] In one embodiment of the present invention, the pharmaceutical product includes at least one of the following functions:

[0048] (1) Reduce weight gain under a high-fat diet;

[0049] (2) Reduce the levels of aging-related secretory phenotypes IL-1β and IL-6 in the body;

[0050] (3) Reduce the body's β-galactosidase content;

[0051] (4) Inhibits liver fat accumulation and damage;

[0052] (5) Lowering blood lipid levels;

[0053] (6) Effectively enhances the expression of lipid metabolism-related genes MGL, HSL, ATGL, and PPAR-α, and reduces the expression of lipid synthesis-related genes PPAR-γ and Fasn.

[0054] The present invention also provides an anti-aging medicine containing Bifidobacterium animalis subsp. lactis CCFM1363 and licorice extract.

[0055] The present invention also provides a medicine with anti-aging effects, which is a product obtained by fermenting licorice extract with Bifidobacterium lactis subsp. CCFM1363.

[0056] In one embodiment of the present invention, the amount of Bifidobacterium lactis subsp. CCFM1363 added to the product is at least 1 × 10⁻⁶. 9 CFU / mL.

[0057] In one embodiment of the present invention, the drug contains Bifidobacterium lactis subsp. CCFM1363, a drug carrier, and / or pharmaceutical excipients.

[0058] Beneficial effects:

[0059] The animal Bifidobacterium lactis subsp. CCFM1363 of the present invention has the ability to convert glycyrrhizic acid. After fermentation in a fermentation substrate rich in licorice extract, the degradation rate of glycyrrhizic acid reaches 37.42%, and active substances such as glycyrrhetinic acid, glycyrrhetinic acid 3-O-glucuronide, and apiosyl glycyrrhizin are generated.

[0060] The Bifidobacterium lactis subsp. CCFM1363 described in this invention can enhance the efficacy of licorice extract, including alleviating the degree of weight gain and liver damage caused by a high-fat diet, improving the lipid metabolism capacity of licorice extract, and reducing the body's anti-aging ability.

[0061] Products with enhanced anti-aging effects can be produced using Bifidobacterium animalis subsp. lactis CCFM1363 and licorice extract. The production process, using glycyrrhizic acid extract as a raw material, enhances the efficacy of licorice extract. This process is safe, efficient, low-cost, and mild, making it suitable for large-scale industrial production. Therefore, the application of Bifidobacterium animalis subsp. lactis CCFM1363 to products containing licorice extract has significant application prospects and a solid foundation.

[0062] Preservation of biological materials

[0063] Bifidobacterium animalis subsp. lactis (CCFM1363), taxonomically named Bifidobacterium animalis subsp. lactis, was deposited on December 6, 2023, at the Guangdong Provincial Center for Microbial Culture Collection (GDMCC No.: 64111), located at Building 59, No. 100 Xianlie Middle Road, Guangzhou. Attached Figure Description

[0064] Figure 1 HPLC chromatograms of licorice extract fermented by Bifidobacterium animalis subsp. lactis CCFM1363 at 0h and 48h, and HPLC chromatograms of glycyrrhizic acid and glycyrrhetinic acid standards;

[0065] Figure 2 Weight gain in mice of different groups;

[0066] Figure 3 Levels of IL-1β and IL-6, age-related secretory phenotypes, in mice from different groups;

[0067] Figure 4 : β-galactosidase content in mice of different groups;

[0068] Figure 5 H&E stained liver tissue sections from different groups of mice;

[0069] Figure 6 Oil Red O stained tissue sections of mouse liver from different groups;

[0070] Figure 7 Comparison of four lipid parameters (TG, TC, LDL-c, and HDL-c) in mice from different groups;

[0071] Figure 8 Comparison of ALT and AST levels in mice from different groups;

[0072] Figure 9 Expression of lipid metabolism-related genes in mice from different groups.

[0073] "*" indicates a significant difference from the Model group (P<0.05), and "**" indicates an extremely significant difference from the Model group (P<0.01). Detailed Implementation

[0074] The Bifidobacterium animalis subsp. lactis used in this invention, CCFM1363, was deposited on December 6, 2023, at the Guangdong Provincial Center for Microbial Culture Collection, with accession number GDMCC No: 64111, located at 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou.

[0075] The C57BL / 6J mice used in the following examples were purchased from Beijing Vital River Company.

[0076] The culture media involved in the following examples are as follows:

[0077] MRS liquid culture medium: yeast extract 5.0 g / L, beef extract 10.0 g / L, peptone 10.0 g / L, glucose 20.0 g / L, anhydrous sodium acetate 2.0 g / L, diammonium citrate 2.0 g / L, dipotassium hydrogen phosphate 2.6 g / L, manganese sulfate monohydrate 0.25 g / L, magnesium sulfate heptahydrate 0.5 g / L, L-cysteine ​​0.5 g / L, and Tween-80 1 mL / L, pH 6.2–6.4.

[0078] MRS solid medium: yeast extract 5.0 g / L, beef extract 10.0 g / L, peptone 10.0 g / L, glucose 20.0 g / L, anhydrous sodium acetate 2.0 g / L, diammonium citrate 2.0 g / L, dipotassium hydrogen phosphate 2.6 g / L, manganese sulfate monohydrate 0.25 g / L, magnesium sulfate heptahydrate 0.5 g / L, Tween-80 1 mL / L, L-cysteine ​​0.5 g / L, and agar 20.0 g / L, pH 6.2–6.4.

[0079] Fermentation substrate containing licorice extract (g / L): licorice extract 10g / L, peptone 10g / L, yeast extract 5g / L, beef extract 10g / L, glucose 10g / L, anhydrous sodium acetate 2g / L, diamine hydrogen citrate 2g / L, K2HPO4·3H2O 2.6g / L, MgSO4·7H2O 0.58g / L, MnSO4·7H2O 0.25g / L, Tween-80 1g / L, distilled water 1000g / L, L-cysteine ​​0.5g / L.

[0080] The extraction method for licorice extract is as follows: water extraction, with a first extraction temperature of 90-95℃, a material-to-water ratio of 1:10, and an extraction time of 45 min; and a second extraction temperature of 90-95℃, a material-to-water ratio of 1:5, and an extraction time of 30 min. After drying, the final yield of licorice extract is 12%, meaning that 1g of licorice extract is equivalent to 8.33g of licorice raw material.

[0081] The preparation method of the Bifidobacterium lactis subsp. animalis-licorice extract synbiotic formulation involved in the following examples is as follows:

[0082] (1) Activate Bifidobacterium lactis subsp. CCFM1363 to obtain bacterial solution;

[0083] (2) In MRS medium, inoculate the bacterial suspension from (1) with a viable count of 5 × 10⁶. 7 CFU / mL, cultured in an anaerobic environment until the logarithmic growth phase of the strain to obtain Bifidobacterium animalis subsp. lactis bacterial culture;

[0084] (3) Collect bacterial cells from liquid culture medium by centrifugation, mix with freeze-drying protectant (12% skim milk powder), and freeze-dry to obtain freeze-dried bacterial powder;

[0085] (4) Adjust the dosage according to the mouse's body weight (in kg), and mix to a final concentration of 5 × 10⁻⁶. 9 The product is obtained by adding CFU (Bifidobacterium animalis subsp. lactis) CCFM1363 to 78 mg / kg of licorice extract.

[0086] The following examples illustrate the preparation method of Bifidobacterium lactis subsp. animalis-licorice fermentation biogenic agent:

[0087] (1) Streaking of Bifidobacterium animalis subsp. lactis CCFM1363 on MRS solid medium, incubating at 37°C upside down for 48h, then picking a single colony and inoculating it into 5ml MRS liquid medium and incubating at 37°C for 48h to obtain bacterial solution.

[0088] (2) In a fermentation substrate containing licorice extract, the bacterial solution from (1) was inoculated and fermented at a constant temperature of 37°C for 48 hours until the viable cell count reached 1×10⁻⁶. 9 CFU / mL;

[0089] (3) After fermentation, the fermentation broth was heat-treated at 65°C for 30 minutes, followed by high-pressure homogenization at 1200 Bar for 6 times to obtain the final product. The obtained post-biotic solution was freeze-dried and resuspended in physiological saline before gavage according to the required dose (78 mg / kg licorice extract, calculated based on the concentration of licorice extract before fermentation).

[0090] Example 1: Isolation, screening, identification and preservation of Bifidobacterium animalis subsp. lactis CCFM1363

[0091] The specific steps are as follows:

[0092] 1. Screening

[0093] The samples were derived from feces of healthy individuals. After pretreatment, the samples were stored in 20% glycerol at -80°C. After thawing, the samples were mixed and 0.5 mL was added to 4.5 mL of physiological saline. The samples were then serially diluted with physiological saline. The appropriate serial dilutions were spread on MRS solid medium and incubated at 37°C for 48 h. Typical colonies of Bifidobacterium animalis subsp. lactis were picked and streaked onto MRS solid medium for purification. Single colonies were then transferred to MRS liquid medium for enrichment and preserved in 30% glycerol to obtain the strain, which was named CCFM1363. The typical colonies of Bifidobacterium animalis subsp. lactis were convex, milky white, and smooth.

[0094] 2. Identification

[0095] The genome of strain CCFM1363 was extracted, and the 16S rDNA of strain CCFM1363 was amplified and sequenced (performed by Suzhou Genewise Biotechnology Co., Ltd., and the nucleotide sequence of the amplified 16S rDNA of CCFM1363 is shown in SEQ ID NO.1). The sequence was compared with the nucleic acid sequence in NCBI, and the results showed that the strain was Bifidobacterium lactis subsp. animalis, and was named Bifidobacterium lactis subsp. animalis CCFM1363.

[0096] 3. Preservation of microbial strains

[0097] Bifidobacterium animalis subsp. lactis CCFM1363 was inoculated into 5 mL LMRS liquid medium and cultured anaerobically at 37 °C for 24 h. 1 mL of bacterial culture was taken into 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.

[0098] 4. Preservation of bacterial strains

[0099] The Bifidobacterium lactis subspecies CCFM1363 was deposited at the Guangdong Provincial Microbial Culture Collection Center (GDMCC) on December 6, 2023, with accession number GDMCC No: 64111, located at 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou.

[0100] Example 2: Transformation of licorice extract by Bifidobacterium animalis subsp. lactis CCFM1363

[0101] 1. Fermentation method:

[0102] (1) The animal Bifidobacterium lactis subsp. CCFM1363 in Example 1 was streaked on MRS solid medium and the plate was 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 seed culture.

[0103] (2) Add 4% (v / v) of the seed culture of Bifidobacterium lactis subsp. CCFM1363 (live bacteria inoculation concentration of 5×10⁻⁶) to the fermentation substrate containing licorice extract. 7 The concentration of licorice extract in the culture medium was adjusted to 10 mg / mL (CFU / mL). Fermentation was carried out at 37℃ for 48 h. Fermentation broth at 0 h and 48 h was transferred to 2 mL EP tubes, centrifuged at 8000 rpm for 5 min to remove the bacterial cells, and the supernatant was transferred to 5 mL EP tubes.

[0104] The fermentation broth was diluted 3 times with methanol, shaken well, and 1 mL of the sample was passed through a 0.22 μm organic phase filter membrane to obtain the test solution.

[0105] Inject the reference solution and sample solution into the HPLC system and perform detection under the following conditions: Waters e2695 HPLC system; X Bridge RC18 column (250 × 4.6 mm, 5 μm); mobile phase: water (A); 0.1% acetonitrile phosphate (B); detector: UV detector (UV) 237 nm; column temperature: 35℃; injection volume: 10 μL; elution conditions: flow rate 1.0 mL / min, gradient elution program: 0 min, 38% B; 0–3 min, 38–50% B; 3–10 min, 50–52% B; 10–20 min, 52–85% B; 20–30 min, 85–90% B; 30–40 min, 90–38% B. Glycyrrhizic acid peak time: 6.8 min; glycyrrhetinic acid peak time: 22.6 min.

[0106] The contents of glycyrrhizic acid and glycyrrhetinic acid in the fermentation broth were calculated using the external standard method. The results are shown in Table 1. Figure 1 As shown.

[0107] Table 1. Content of each component after fermentation of Bifidobacterium animalis subsp. lactis CCFM1363 in licorice extract-containing medium.

[0108] Component content (μg / mL) Fermentation 0h Fermentation 48h Glycyrrhizin 239.45±5.61 149.87±4.51 Glycyrrhetic acid 0 7.15±1.51

[0109] Note: The glycyrrhetinic acid content at 0h of fermentation was far below the detection limit of 100ppm.

[0110] The results showed that after 48 hours of fermentation, the glycyrrhizic acid content in the fermentation medium decreased by 37.41% compared to before fermentation. This indicates that after fermenting licorice extract using *Bifidobacterium animalis* subsp. *lactamase* of the present invention, the licorice extract was significantly converted into glycyrrhetinic acid and other active small molecules.

[0111] Example 3: Analysis of metabolites of licorice extract before and after fermentation with Bifidobacterium lactis subsp. CCFM1363

[0112] 1. Metabolite extraction

[0113] Transfer 100 μL of liquid sample to an EP tube, add 400 μL of extraction buffer (methanol:acetonitrile = 1:1 (v / v)), the extraction buffer contains an isotope-labeled internal standard; vortex mix for 30 s, sonicate for 10 min (ice-water bath); let stand at -40℃ for 1 h; centrifuge the sample at 4℃, 12000 rpm (centrifugal force 13800 (×g), radius 8.6 cm) for 15 min, and collect the supernatant; centrifuge the supernatant at 4℃, 12000 rpm (centrifugal force 13800 (×g), radius 8.6 cm) for 15 min; collect the supernatant in a sample vial for instrumental analysis; mix equal amounts of supernatant from all samples to form a QC sample for instrumental analysis.

[0114] 2. On-machine testing

[0115] For nonpolar metabolites, this project used a Vanquish (Thermo Fisher Scientific) ultra-high performance liquid chromatograph (UHPLC) with a Phenomenex Kinetex C18 (2.1 mm × 50 mm, 2.6 μm) column for chromatographic separation of target compounds. Phase A of the HPLC was aqueous containing 0.01% acetic acid, and phase B was isopropanol:acetonitrile (1:1, v / v). Column temperature: 25 °C, sample tray temperature: 4 °C, injection volume: 2 μL. An Orbitrap Exploris 120 mass spectrometer was used for primary and secondary mass spectrometry data acquisition under the control software (Xcalibur, version 4.4, Thermo). Detailed parameters are as follows: Sheathgas flow rate: 50 Arb, Aux gas flow rate: 15 Arb, Capillary temperature: 320℃, SweepGas: 1 Arb, Vaporizer Temp: 350℃, Full ms resolution: 60000, MS / MS resolution: 15000, Collision energy: SNCE 20 / 30 / 40, SprayVoltage: 3.8kV (positive) or -3.4kV (negative).

[0116] 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 CompoundDiscoverer 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 fermentation with Lactobacillus curlis CCFM1362 for 48 hours, the main metabolites of licorice extract were terpenes, shikimic acid and phenylpropionic acid, fatty acids, amino acids, and short peptides.

[0117] Table 2. Changes in terpenoids, shikimic acid, and phenylpropionic acid detected in the fermentation broth of licorice extract before and after fermentation.

[0118] Molecular weight Molecular formula Name log2 fold change Change after fermentation 821.3975 C 42 H 62 O 16 ]]> Glycyrrhizin -0.956 - 629.3663 C 36 H 54 O 10 ]]> Glycyrrhetic acid 3-O-glucuronide 8.563 + 471.3452 C 30 H 46 O4]]> Glycyrrhetic acid 5.878 + 551.1751 C 26 H 30 O 13 ]]> Isoliquiritigenin 3.826 + 353.1008 C 20 H 16 O6]]> Hemicumamarin B 4.436 + 287.0908 C 16 H 14 O5]]> Liquiritigenin 3.627 + 255.067 C 15 H 12 O4]]> Glycyrrhizin 3.026 + 285.075 C 16 H 12 O5]]> 5-O-methylgenistein 5.572 + 195.0668 C 10 H 12 O4]]> Hydroxytyrosol acetate 4.436 + 405.1717 C 25 H 26 O5]]> 6',8'-diisopentenylgenistein 2.161 + 301.0697 C 16 H 12 O6]]> Formononetin 3.627 + 455.354 C 30 H 48 O3]]> Ursolic acid 3.314 +

[0119] Example 4: Effects of a synbiotic preparation made from Bifidobacterium animalis subsp. lactis CCFM1363 and a licorice fermented biogener on weight control in mice on a high-fat diet.

[0120] Forty-eight healthy male C57BL / 6J mice aged 6 weeks were randomly divided into 6 groups of 8 mice each. The 6 groups were: blank control group, model group, licorice extract group (78 mg / kg licorice extract), and Bifidobacterium lactis subsp. animalis group (5 × 10⁻⁶ mg / kg licorice extract). 9 CFU Bifidobacterium animalis subsp. lactis CCFM1363), Bifidobacterium animalis subsp. lactis-licorice extract synergistic preparation (5×10) 9 CFU Bifidobacterium animalis subsp. lactis CCFM1363, 78 mg / kg licorice extract, and Bifidobacterium animalis subsp. lactis-licorice fermentation post-biotic group (78 mg / kg licorice extract, calculated based on the concentration of licorice extract before fermentation).

[0121] The experiment lasted 13 weeks: After one week of acclimatization, starting from the second week, the control group was fed a low-fat, low-sugar diet, while the other groups were fed a high-fat diet. The modeling period was 8 weeks. From the 10th week, each experimental group was administered 0.2 mL / mouse / day of a mixture of lyophilized bacterial strain powder and licorice extract or fermented lyophilized powder (dissolved in physiological saline at the appropriate dose). The control group and the model group were administered an equal volume of physiological saline by gavage until the end of the experiment. All groups had free access to water and food.

[0122] After intervention, the mice's mental state was observed daily at regular intervals, and their weight was measured weekly. The weight of mice in each group after intervention is shown below. Figure 2 As shown.

[0123] Depend on Figure 2 It was found that the weight gain rate of mice administered Bifidobacterium lactis subsp. CCFM1363 and licorice extract by gavage decreased by 14.632% and 14.152% respectively compared with the model group, which was less effective than the combined preparation and the licorice fermentation group (22.455% and 40.072% respectively). After the drug intervention, both the combined preparation made from Bifidobacterium lactis subsp. CCFM1363 fermented with licorice extract and the licorice fermentation group had the effect of alleviating weight gain in mice, and the licorice fermentation group showed the best weight control effect.

[0124] Table 3 Grouping of experimental animals

[0125]

[0126] Example 5: Effects of a synbiotic preparation made from Bifidobacterium animalis subsp. lactis CCFM1363 and a glycogenase-derived probiotic on the aging-related secretion phenotype in mice.

[0127] The methods for grouping, modeling, and treating mice are the same as in Example 4.

[0128] Weigh a portion of frozen liver tissue, add PBS (pH 7.4, 4℃) at a ratio of 1:9 (m / V), homogenize, centrifuge at 12000×g for 30 min at 4℃, collect the supernatant, and determine the levels of IL-1β and IL-6 according to the instructions of the corresponding ELISA kit.

[0129] Depend on Figure 3 The results showed that the IL-1β levels in mice treated with oral administration of either *Bifidobacterium lactis* subsp. CCFM1363 or licorice extract decreased by 22.176% and 19.901% respectively compared to the model group, which was less effective than the 31.220% and 57.129% decreases achieved with equal doses of the combined preparation and licorice fermented emulsion. Furthermore, in terms of IL-6 levels, the IL-6 levels in mice treated with oral administration of either *Bifidobacterium lactis* subsp. CCFM1363 or licorice extract decreased by 10.644% and 5.129% respectively compared to the model group, which was also less effective than the 40.382% and 25.249% decreases achieved with equal doses of the combined preparation and licorice fermented emulsion. After drug intervention, both the combined preparation obtained from fermenting licorice extract with *Bifidobacterium lactis* subsp. CCFM1363 and the licorice fermented emulsion reduced the levels of age-related secretory phenotypes in mice, indicating that this drug has strong potential in the anti-aging field.

[0130] Example 6: Effects of a synbiotic preparation made from Bifidobacterium animalis subsp. lactis CCFM1363 and a glycoside fermented emodin on the β-galactosidase (β-GAL) content in mice.

[0131] The methods for grouping, modeling, and treating mice are the same as in Example 4.

[0132] Weigh a portion of frozen liver tissue, add PBS (pH 7.4, 4℃) at a ratio of 1:9 (m / V), homogenize, centrifuge at 12000×g for 30 min at 4℃, collect the supernatant, and determine the level of aging-related β-galactosidase according to the instructions of the corresponding ELISA kit.

[0133] In senescent cells, both the number and size of lysosomes increase. β-galactosidase, as an enzyme directly related to lysosome quality levels, is an important biomarker characterizing aging levels. Figure 4 It was found that, compared with the model group, the β-galactosidase content in mice treated with oral administration of Bifidobacterium lactis subsp. CCFM1363 and licorice extract decreased by 7.782% and 17.056%, respectively. The effect was not as good as that of the same dose of the combined preparation and the licorice fermented emulsifier group (24.644% and 29.431%, respectively). After the drug intervention, both the combined preparation made from Bifidobacterium lactis subsp. CCFM1363 fermented with licorice extract and the licorice fermented emulsifier group had the effect of alleviating the increase of aging markers in mice, and the licorice fermented emulsifier group showed the best anti-aging control effect.

[0134] Example 7: Effects of a synbiotic preparation made from Bifidobacterium animalis subsp. lactis CCFM1363 and a licorice fermented biogener on hepatic fat accumulation and lesions in mice fed a high-fat diet.

[0135] The methods for grouping, modeling, and treating mice are the same as in Example 4.

[0136] After the experiment, blood was collected from mice and they were euthanized. Liver tissue was fixed in 4% paraformaldehyde solution for 24 hours and then stained with hematoxylin and eosin (H&E) to assess the degree of hepatic steatosis and inflammation. Frozen sections of fresh liver tissue embedded in OCT were cut and stained with Oil Red O to observe the accumulation of lipids in the liver. The stained tissue sections were observed under an optical microscope. The staining results are shown below. Figure 5 , Figure 6 .

[0137] Depend on Figure 5 , Figure 6 It was found that the liver tissues of mice in the blank group and the group treated with licorice fermentation prebiotic prepared from Bifidobacterium lactis CCFM1363 showed uniform staining, less fat accumulation, and tightly packed, uniform hepatocytes with regular shapes. The liver tissues of mice in the other groups showed more fat accumulation, obvious pathological changes, numerous fat vacuoles around cells, loose connections between hepatocytes, leakage of cellular contents, cell swelling, and disruption of cell integrity. The experimental results indicate that the mixture of Bifidobacterium lactis CCFM1363 and licorice extract, or the licorice fermentation prebiotic prepared in this invention, effectively inhibited liver fat accumulation and lesions in mice on a high-fat diet. In particular, the prebiotic significantly reduced the degree of liver damage and fatty lesions.

[0138] Example 8: Effects of a synbiotic preparation made from Bifidobacterium animalis subsp. lactis CCFM1363 and a glycoside fermented emodin on blood lipids in mice on a high-fat diet.

[0139] The methods for grouping, modeling, and treating mice are the same as in Example 4.

[0140] After the experiment, blood was collected from the mice and they were euthanized. Whole blood was allowed to stand for 1 hour, then centrifuged at 3000g for 15 minutes to obtain serum. A fully automated biochemical analyzer was used to detect lipid-related indicators, including TG (triglycerides), TC (total cholesterol), HDL-C (high-density lipoprotein cholesterol), and LDL-C (low-density lipoprotein cholesterol). The results are as follows: Figure 7 .

[0141] Depend on Figure 7It was found that, compared with the control group, the TC and TG levels in the model group were significantly increased, indicating that lipid accumulation in mice led to elevated blood lipids. Drug intervention results showed that, compared with the model group, mice administered Bifidobacterium lactis subsp. CCFM1363 and licorice extract by gavage alone had TC levels decreased by 6.301% and 9.032%, respectively, and TG levels decreased by 20.152% and 22.222%, respectively. Both the combined preparation and the licorice fermentation-derived prebiotic preparation reduced these levels to varying degrees. Specifically, the licorice fermentation-derived prebiotic preparation, derived from Bifidobacterium lactis subsp. CCFM1363 and licorice extract, reduced TC and TG levels by 21.075% and 29.885%, respectively, while the combined preparation reduced TC and TG levels by 8.081% and 29.502%, respectively.

[0142] Meanwhile, compared with the control group, the levels of LDL-c and HDL-c in the model group were significantly higher than those in the blank group. The results of drug intervention showed that, compared with the model group, the LDL-c and HDL-c of mice that were administered Bifidobacterium lactis subsp. CCFM1363 by gavage decreased by 2.848% and 1.607%, respectively. Compared with the model group, the LDL-c and HDL-c of mice that were administered licorice extract by gavage decreased by 15.913% and 0.952%, respectively. Both of these results were not as good as the combined preparation and the licorice fermentation-derived probiotic. Among them, the combined preparation of Bifidobacterium animalis subsp. lactis CCFM1363 and licorice extract reduced LDL-c and HDL-c levels by 21.441% and 1.964%, respectively, while the licorice fermented biogenic acid reduced LDL-c and HDL-c levels by 22.111% and 18.869%, respectively. This indicates that the combined preparation of Bifidobacterium animalis subsp. lactis CCFM1363 and licorice extract and the licorice fermented biogenic acid have great potential in reducing blood lipid levels. According to the four blood lipid indicators in mice, it is further shown that the combined preparation of Bifidobacterium animalis subsp. lactis CCFM1363 and licorice extract and the licorice fermented biogenic acid can reduce the rise in blood lipids caused by lipid accumulation.

[0143] Example 9: Effects of a synbiotic preparation made from Bifidobacterium animalis subsp. lactis CCFM1363 and a glycoside fermented biogener on liver function in mice on a high-fat diet.

[0144] The methods for grouping, modeling, and treating mice are the same as in Example 3.

[0145] After the experiment, blood was collected from the mice and they were euthanized. Whole blood was allowed to stand for 1 hour, then centrifuged at 3000g for 15 minutes to obtain serum. Liver function-related indicators, including ALT (alanine aminotransferase) and AST (aspartate aminotransferase), were detected using an automated biochemical analyzer. The results are as follows: Figure 8 .

[0146] Depend on Figure 8It was found that, compared with the control group, the ALT and AST levels in the model group were significantly increased, indicating that lipid accumulation induced by the high-fat diet model caused chronic inflammation in the mouse liver. The ALT and AST levels in the serum of mice in the combined preparation and the licorice fermented emodin intervention groups were reduced to varying degrees compared with the model group, indicating that they could reduce the inflammatory response caused by liver lipid accumulation. Specifically, the combined preparation and the licorice fermented emodin reduced AST levels by 29.428% and 28.457%, respectively, and ALT levels by 42.588% and 37.825%, respectively. The experimental results show that the mixture of Bifidobacterium lactis subsp. CCFM1363 and licorice extract, as well as the prepared licorice fermented emodin, effectively reduced the serum biochemical indicators related to obesity and fatty liver in mice fed a high-fat diet, which is beneficial for alleviating obesity and fatty liver.

[0147] Example 10: Effects of a synbiotic preparation made from Bifidobacterium animalis subsp. lactis CCFM1363 and a glycoside fermented emodin on the expression of lipid metabolism-related genes in mice on a high-fat diet.

[0148] The methods for grouping, modeling, and treating mice are the same as in Example 4.

[0149] After the experiment, blood was collected from mice and they were euthanized. Total RNA was extracted from mouse liver tissue using an animal RNA extraction kit, and then the extracted RNA was reverse transcribed into cDNA using a reverse transcription kit. Using the cDNA as a template, real-time quantitative PCR was performed using a fluorescent dye intercalation method, with mouse GAPDH used as an internal control gene. -ΔΔCt The mRNA expression levels of the target genes MGL, HSL, ATGL, PPAR-α, PPAR-γ, and Fasn were calculated using a method. The expression levels of the target genes in each group are expressed relative to the blank control group (set to 1.0). Primers were synthesized by Shanghai Sangon Biotech Co., Ltd., and their sequences are shown in Table 4.

[0150] Table 4 Primer sequences for real-time quantitative PCR detection

[0151]

[0152] Depend on Figure 9 It can be seen that, compared with the control group, the relative expression levels of PPAR-γ and FASN genes in the model group were significantly increased, while the relative expression levels of PPAR-α, ATGL, HSL, and MGL genes were decreased. This indicates that the high-fat diet significantly promoted lipid synthesis in the liver of mice and inhibited lipid breakdown, which also explains the phenomenon of fat accumulation in the liver of mice on a high-fat diet.

[0153] Synbiotic preparations made from Bifidobacterium animalis subsp. lactis CCFM1363 and licorice extract, as well as licorice fermentation-derived probiotic preparations, can all increase the expression of MGL, HSL, ATGL, and PPAR-α genes and decrease the expression of PPAR-γ and Fasn genes to varying degrees, thereby reducing lipid accumulation in the body.

[0154] Compared with the model group, after intervention with the synbiotic preparation, the levels of PPAR-γ and FASN decreased by 16.732% and 39.631%, respectively, while the levels of ATGL, Mgl, Hsl, and PPAR-α genes increased by 282.479%, 55.149%, 363.935%, and 55.032%, respectively. After intervention with the licorice fermentation-derived probiotic, the levels of PPAR-γ and FASN decreased by 47.273% and 42.045%, respectively, while the levels of ATGL, Mgl, Hsl, and PPAR-α genes increased by 330.769%, 73.268%, 356.428%, and 19.600%, respectively. Moreover, the effects of the synbiotic preparation and the licorice fermentation-derived probiotic were better than those of administering Bifidobacterium lactis subsp. CCFM1363 or licorice extract alone by gavage. This indicates that the combined preparation of Bifidobacterium animalis subsp. lactis CCFM1363 and licorice extract, or the licorice fermentation precursor, can increase the expression of MGL, HSL, ATGL, and PPAR-α genes, and decrease the expression of PPAR-γ and Fasn genes, thereby reducing lipid accumulation in the body.

[0155] The comparative analysis of the above results shows that the combination of Bifidobacterium animalis subsp. lactis CCFM1363 and licorice extract can inhibit the increase in body weight under a high-fat diet, reduce the levels of aging-related secretory phenotypes IL-1β and IL-6 in mice, decrease the content of β-galactosidase in the liver, reduce the levels of TC, TG, HDL-c, and LDL-c in serum, reduce liver tissue damage caused by lipid accumulation, increase the expression of lipid metabolism-related genes MGL, HSL, ATGL, and PPAR-α, decrease the expression of lipid synthesis-related genes PPAR-γ and Fasn, and improve the blood glucose homeostasis level in mice. Moreover, the effect is significantly better than that of single strain CCFM1363 and single licorice extract. Bifidobacterium animalis subsp. lactis CCFM1363 has the effect of multi-target anti-aging and transforming licorice to enhance efficacy.

[0156] 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 Bifidobacterium animalis subsp. lactis ( Bifidobacterium animalis subsp. lactis CCFM1363, the Bifidobacterium lactis subspecies CCFM1363, was deposited at the Guangdong Provincial Center for Microbial Culture Collection on December 6, 2023, with accession number GDMCC No: 64111.

2. The postbiotic prepared from the animal bifidobacterium Bifidobacterium lactis CCFM1363 of claim 1, characterized by, The postbiotic is obtained by fermenting the Bifidobacterium animalis subsp. lactis CCFM1363 in a culture medium containing liquorice extract, inactivating the fermentation liquor, and then homogenizing; the liquorice extract is extracted by water extraction, with a material-to-water ratio of 1:10 and an extraction temperature of 90-95°C for 45 min in the first extraction and a material-to-water ratio of 1:5 and an extraction temperature of 90-95°C for 30 min in the second extraction.

3. A symbiotic preparation prepared from the animal bifidobacterium Bifidobacterium animalis subsp. lactis CCFM1363 characterised in that, The symbiotic preparation contains the Bifidobacterium animalis subsp. lactis CCFM1363 and liquorice products; the liquorice products include liquorice or liquorice extract; the liquorice extract is extracted by water extraction, with a material-to-water ratio of 1:10 and an extraction temperature of 90-95°C for 45 min in the first extraction and a material-to-water ratio of 1:5 and an extraction temperature of 90-95°C for 30 min in the second extraction.

4. A product comprising the postbiotic of claim 2 or the synbiotic formulation of claim 3, characterized in that, The product includes food, medicine or health products.

5. The product of claim 4, wherein, The product is a freeze-dried powder of the postbiotic or the symbiotic preparation, or a reconstituted product containing the freeze-dried powder of the postbiotic or the symbiotic preparation.

6. A method of degrading glycyrrhizinic acid, characterized by, The Bifidobacterium animalis subsp. lactis CCFM1363 of claim 1 is added to a culture medium containing liquorice acid for fermentation.

7. A process for the preparation of glycyrrhetinic acid, characterized in that, The Bifidobacterium animalis subsp. lactis CCFM1363 of claim 1 is added to a culture medium containing liquorice acid for fermentation.

8. A method of enhancing medicinal efficacy of liquorice, characterized in that, The method is any of the following: (1) the Bifidobacterium animalis subsp. lactis CCFM1363 of claim 1 is added to a culture medium containing liquorice products for fermentation; (2) the Bifidobacterium animalis subsp. lactis CCFM1363 of claim 1 is mixed with liquorice products; The liquorice products include liquorice or liquorice extract; the liquorice extract is extracted by water extraction, with a material-to-water ratio of 1:10 and an extraction temperature of 90-95°C for 45 min in the first extraction and a material-to-water ratio of 1:5 and an extraction temperature of 90-95°C for 30 min in the second extraction.

9. Use of the Bifidobacterium animalis subsp. lactis CCFM1363 of claim 1 and / or the postbiotic of claim 2 and / or the symbiotic preparation of claim 3 in the preparation of health products for reducing lipid.

10. Use of the Bifidobacterium animalis subsp. lactis CCFM1363 of claim 1 and / or the postbiotic of claim 2 and / or the symbiotic preparation of claim 3 in the preparation of medicine for treating fatty liver.

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