Lactobacillus crispatus based on multi-target to achieve anti-aging effect and application of its transformation of glycyrrhiza potentiation

Abstract

The application discloses a lactobacillus crispatus based on multiple targets to realize anti-aging effect and application of transformation of glycyrrhiza for efficiency enhancement, and belongs to the technical field of microorganisms and the technical field of medicine. The lactobacillus crispatus CCFM1362 provided in the application can ferment glycyrrhiza extract, can improve the content of glycyrrhizic acid, hemiglycyrrhizin isoflavone B, naringenin chalcone and other substances, can enhance the efficacy of glycyrrhiza extract, and can realize the effect of multiple targets for anti-aging, and the specific embodiments are as follows: (1) the content of beta-galactosidase in liver tissue is inhibited; (2) the level of aging-related secretory phenotype in liver tissue is reduced; (3) the Nrf2 related pathway is activated, and the level of oxidative stress is reduced; (4) the NF-kappa B related pathway is activated, and the level of chronic inflammation is reduced. Therefore, the lactobacillus crispatus CCFM1362 and the glycyrrhiza extract have great application prospect in the production of anti-aging products.

Description

Technical Field

[0001] This invention relates to a strain of Lactobacillus curvature that achieves anti-aging effects based on multiple targets and its application in enhancing the efficacy of licorice through transformation, belonging to the fields of microbial technology and pharmaceutical technology. Background Technology

[0002] With the increasing aging of the population, anti-aging research has become a hot topic in the fields of biomedicine and microbiology. Aging is a complex biological process influenced by multiple factors, including oxidative stress, chronic inflammation, metabolic decline, and immune function deterioration. It not only leads to a gradual weakening of bodily functions but also increases the risk of chronic diseases such as diabetes, cardiovascular disease, and neurodegenerative diseases. Traditional anti-aging methods often rely on chemically synthesized drugs, such as rapamycin and its derivatives, metformin, etc., but these drugs are often accompanied by certain side effects such as immunosuppression, metabolic abnormalities, potential nephrotoxicity, and gastrointestinal discomfort.

[0003] In recent years, medicinal and edible materials and their microbial metabolites have received widespread attention due to their safety and efficacy. Licorice is a commonly used medicinal and edible material with a wide range of applications. Related studies have found that licorice also exhibits good effects in antioxidation, anti-inflammation, anti-tumor, and antiviral activities. Furthermore, the application potential of licorice extract in anti-aging has attracted considerable attention. Triterpenoid saponins in licorice are considered its main pharmacologically active substances, primarily including glycyrrhizic acid (GL), glycyrrhizinic acid monoglucuronide (GAMG), and glycyrrhizinic acid (GA), with glycyrrhizic acid being the most abundant.

[0004] Studies have shown that glycyrrhizic acid is less effective than smaller molecules such as glycyrrhetinic acid and glycyrrhetinic acid in terms of absorption and physiological activity. Direct use of licorice extract is unlikely to yield immediate results, and its efficacy is often limited when ingesting products containing licorice extract. Therefore, improving the efficacy of licorice extract is crucial for its development into anti-aging products. Summary of the Invention

[0005] The technical problem to be solved by the present invention is to overcome the defects and deficiencies of the prior art and to provide the application of Lactobacillus crispatus CCFM1362 in products that enhance the anti-aging efficacy of licorice extract.

[0006] Another objective of this invention is to provide a fermented licorice extract and composition that enhances anti-aging effects based on multiple targets.

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

[0008] The above-mentioned objective of this invention is achieved through the following technical solution:

[0009] This invention provides a strain of Lactobacillus crispatus CCFM1362, which was deposited at the Guangdong Provincial Center for Microbial Culture Collection on November 9, 2023, with accession number GDMCC No: 63995, and the deposit address is 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou.

[0010] In one embodiment of the present invention, after being cultured on MRS medium for 48 hours, the *Lactobacillus curvatureii* CCFM1362 colonies are round, convex, or lenticular, slightly white, opaque, and have a smooth to mucous-like soft surface. Fermentation of licorice extract with this bacterium can significantly increase glycyrrhetinic acid content, promoting the multi-target anti-aging effects of licorice extract. Specifically, this manifests in: (1) significantly reducing the content of aging marker β-galactosidase in mouse liver tissue; (2) significantly reducing the content of aging-related secretory phenotypes such as IL-1β and IL-6 in mouse liver tissue; (3) increasing the antioxidant level of mice; (4) activating the Nrf2-related pathway in mice and reducing oxidative stress levels; and (5) activating the NF-κB-related pathway in mice and reducing chronic inflammation levels.

[0011] The present invention also provides a metabiotic prepared from the said Lactobacillus curvature CCFM1362, the metabiotic comprising inactivated bacteria and / or metabolites of Lactobacillus curvature CCFM1362.

[0012] The present invention also provides a synbiotic preparation made from the Lactobacillus curvature CCFM1362, the synbiotic preparation comprising Lactobacillus curvature CCFM1362 and licorice products.

[0013] In one embodiment of the present invention, the licorice product comprises licorice or licorice extract.

[0014] The present invention also provides a method for preparing the postgenetic agent, comprising the following steps:

[0015] (1) Activate Lactobacillus curli CCFM1362 to obtain bacterial solution;

[0016] (2) Inoculate the bacterial solution of (1) into the fermentation substrate containing licorice extract and carry out fermentation;

[0017] (3) After fermentation is completed, the fermentation liquid is heat-treated and then homogenized under high pressure to obtain the final product.

[0018] In one embodiment of the present invention, step (1) involves streaking Lactobacillus curli CCFM1362 on MRS solid medium, inverting the plate for culture (preferably inverting at 37°C for 48 hours), and then picking a single colony and inoculating it into MRS liquid medium (preferably 5 mL) for culture (preferably incubating at 37°C for 48 hours) to obtain bacterial solution.

[0019] 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).

[0020] In one embodiment of the present invention, the inoculation amount of bacterial solution in step (2) is 1-8% (v / v).

[0021] 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).

[0022] 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).

[0023] 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).

[0024] The present invention also provides products containing the said post-biotic or said synbiotic preparation, said products including food, medicine or health products.

[0025] 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.

[0026] The present invention also provides a method for enhancing the medicinal efficacy of licorice, wherein the licorice is treated with any of the following methods:

[0027] (1) Extract the active ingredients from licorice to obtain an extract, and then mix the extract with the Lactobacillus curvature CCFM1362;

[0028] (2) Extract the active ingredients from licorice to obtain an extract, and then add the extract to the fermentation medium of Lactobacillus curvature CCFM1362 for culture.

[0029] The present invention also provides a method for degrading glycyrrhizic acid or producing glycyrrhetinic acid by adding the Lactobacillus curvature CCFM1362 to a culture medium containing glycyrrhizic acid for fermentation.

[0030] This invention provides the use of Lactobacillus curvature CCFM1362, its postbiotics and / or synbiotics in the preparation of pharmaceuticals that can alleviate lipid accumulation.

[0031] This invention provides the use of Lactobacillus curvature CCFM1362, its postbiotics and / or synbiotics in the preparation of medicines that can alleviate age-related diseases.

[0032] This invention provides the use of Lactobacillus curvature CCFM1362, its postbiotics and / or synbiotics in the preparation of pharmaceuticals that can alleviate oxidative damage.

[0033] This invention provides the use of Lactobacillus curvature CCFM1362, its postbiotics and / or synbiotics in the preparation of medicaments that can alleviate inflammatory responses.

[0034] In one embodiment of the present invention, the product includes at least one of the following functions:

[0035] (1) Reduce the content of β-galactosidase in liver tissue;

[0036] (2) Reduce the levels of aging-related secretory phenotypes such as IL-1β and IL-6 in liver tissue;

[0037] (3) Enhance the body's antioxidant levels;

[0038] (4) Activate Nrf2-related pathways to reduce oxidative stress levels;

[0039] (5) Activate NF-κB-related pathways and reduce chronic inflammation levels.

[0040] In one embodiment of the present invention, the pharmaceutical product contains Lactobacillus curlis CCFM1362, a drug carrier, and / or pharmaceutical excipients.

[0041] This invention also provides a method for enhancing the efficacy of licorice extract, wherein the method involves adding *Lactobacillus curvaturei* CCFM1362 to a system containing licorice extract. This method is not for disease diagnosis or treatment purposes. Specifically, the method involves adding *Lactobacillus curvaturei* CCFM1362 to a system containing licorice extract, followed by fermentation.

[0042] This invention provides the application of Lactobacillus curvature CCFM1362, its postbiotics and / or synbiotics in the preparation of health products that can alleviate oxidative damage.

[0043] Lactobacillus curlis, a common type of lactobacillus, is an important component of the gut microbiota. It plays a vital role in maintaining intestinal balance, inhibiting the growth and reproduction of harmful microorganisms, resisting pathogen infection, and enhancing the body's immunity. Previous studies have demonstrated the numerous health benefits of Lactobacillus curlis. It is also one of the most widely used lactobacillus strains in the food industry today, such as Lactobacillus curlis VB250, Lactobacillus curlis CCFM1118, and Lactobacillus curlis W962.

[0044] Therefore, the application of Lactobacillus curlis CCFM1362 to products containing licorice extract has great application prospects and a solid foundation.

[0045] Beneficial effects:

[0046] 1. The *Lactobacillus curlis* CCFM1362 of the present invention can grow in a fermentation substrate rich in licorice extract, converting glycyrrhizic acid into small molecule active substances. The conversion rate of glycyrrhizic acid reaches 39.58%, and the active substances produced include glycyrrhetinic acid 3-O-glucuronide, glycyrrhetinic acid, hemi-glycyrrhizin isoflavone B, glycyrrhizin chalcone B, 2',4',7-trihydroxy-6'-methoxy-3'-isopentenyl isoflavone, etc.

[0047] 2. The synergistic preparation of Lactobacillus curlis CCFM1362 and licorice extract of the present invention, or the post-biotic prepared by fermentation in licorice extract, can promote the efficacy of licorice extract and achieve the effects of relieving lipid accumulation, anti-aging, oxidative damage and / or inflammatory response.

[0048] Products with enhanced anti-aging effects can be produced using Lactobacillus curlis CCFM1362 and licorice extract. The production process uses glycyrrhizic acid extract as a raw material to enhance the efficacy of licorice extract. This process has the advantages of being safe, efficient, low-cost, and having a mild reaction, making it suitable for large-scale industrial production.

[0049] Preservation of biological materials

[0050] Lactobacillus crispatus (CCFM1362), taxonomically named Lactobacillus crispatus, was deposited on November 9, 2023, at the Guangdong Provincial Center for Microbial Culture Collection (GDMCC No.: 63995), located at Building 59, No. 100 Xianlie Middle Road, Guangzhou. Attached Figure Description

[0051] Figure 1 HPLC chromatograms of licorice extract fermented by Lactobacillus curlis CCFM1362 at 0h and 48h, and HPLC chromatograms of glycyrrhizic acid and glycyrrhetinic acid standards;

[0052] Figure 2 Weight gain in mice of different groups;

[0053] Figure 3 : β-galactosidase content in mice of different groups;

[0054] Figure 4 Levels of aging-related secretory phenotypes (IL-1β, IL-6) in mice from different groups;

[0055] Figure 5 Antioxidant levels in the livers of mice in different groups;

[0056] Figure 6 : Nrf2 activation levels in the livers of mice in different groups;

[0057] Figure 7 Activation levels of the NF-κB p65-related pathway in the liver of mice from different groups;

[0058] "*" 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

[0059] The present invention will be further described below with reference to specific embodiments.

[0060] The C57BL / 6J mice used in the following examples were purchased from Beijing Vital River Company.

[0061] The culture media involved in the following examples are as follows:

[0062] 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, and Tween-80 1 mL / L, pH 6.2–6.4.

[0063] 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, and agar 20.0 g / L, pH 6.2–6.4.

[0064] 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, cysteine ​​hydrochloride 0.5g / L.

[0065] 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.

[0066] The preparation method of Lactobacillus curvature-licorice fermented biogenic agent involved in the following examples is as follows:

[0067] (1) Streaking of Lactobacillus curli CCFM1362 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;

[0068] (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;

[0069] (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).

[0070] The preparation method of the Lactobacillus curvature-licorice fermentation synergistic preparation involved in the following examples is as follows:

[0071] (1) Activate Lactobacillus curli CCFM1362 to obtain bacterial solution;

[0072] (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;

[0073] (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;

[0074] (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 combining CFU (caffeine curcuma) CCFM1362 with 78 mg / kg of licorice extract.

[0075] Example 1: Isolation, screening, identification and preservation of Lactobacillus curlis CCFM1362

[0076] The specific steps are as follows:

[0077] 1. Screening

[0078] 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 Lactobacillus curvature 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 CCFM1362. The typical colonies of Lactobacillus curvature are round, white, and smooth.

[0079] 2. Identification

[0080] The genome of strain CCFM1362 was extracted, and the 16S rDNA of strain CCFM1362 was amplified and sequenced (performed by Suzhou Genewiz Biotechnology Co., Ltd., and the nucleotide sequence of the amplified 16S rDNA of CCFM1362 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 Lactobacillus curlis, and it was named Lactobacillus curlis CCFM1362.

[0081] 3. Preservation of microbial strains

[0082] Lactobacillus curlis CCFM1362 was inoculated into 5 mL LMRS liquid medium and cultured 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.

[0083] 4. Preservation of bacterial strains

[0084] It was deposited on November 9, 2023 at the Guangdong Provincial Microbial Culture Collection Center (GDMCC), with accession number GDMCCNo: 63995, located at 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou.

[0085] Example 2: Transformation of licorice extract by Lactobacillus curvature CCFM1362

[0086] 1. Fermentation method:

[0087] (1) The *Lactobacillus curlis* CCFM1362 from Example 1 was streaked on MRS solid medium and the plate was incubated upside down at 37°C for 48 hours; a single colony was picked and inoculated into 5 mL of MRS liquid medium and incubated at 37°C for 48 hours to prepare seed culture.

[0088] (2) Add 4% (v / v) of the seed culture of Lactobacillus curlis CCFM1362 (initial 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.

[0089] Dilute the fermentation broth three times with methanol, shake well, and the resulting sample is ready for testing. Take 1 mL of the sample and filter it through a 0.22 μm organic phase filter membrane to obtain the test solution.

[0090] 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.

[0091] 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.

[0092] Table 1. Content of each component of Lactobacillus curlis CCFM1362 before and after fermentation in licorice extract-containing medium.

[0093] Content of each component (μg / mL) Fermentation 0h Fermentation for 48 hours glycyrrhizic acid 239.45±5.61 144.68±2.83 glycyrrhetinic acid 0 37.15±2.24

[0094] Note: The glycyrrhetinic acid content at 0h of fermentation was far below the detection limit of 100ppm.

[0095] The results showed that after 48 hours of fermentation, the glycyrrhizic acid content in the fermentation medium decreased by 39.58% compared to before fermentation. This indicates that by using *Lactobacillus curvatureii* of the present invention to ferment licorice extract, the licorice extract is significantly converted into glycyrrhetinic acid and other active small molecules.

[0096] Example 3: Analysis of metabolites of licorice extract before and after fermentation with Lactobacillus cristata CCFM1362

[0097] 1. Metabolite extraction

[0098] Transfer 100 μL of the liquid sample obtained in Example 2 into 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; take an equal amount of supernatant from all samples and mix them to form a QC sample for instrumental analysis.

[0099] 2. On-machine testing

[0100] 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).

[0101] 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.

[0102] Table 2. Changes in terpenoids, shikimic acid, and phenylpropionic acid detected in the fermentation broth of licorice extract before and after fermentation.

[0103] molecular weight Molecular formula name Log2FoldChange Changes after fermentation 821.3975 <![CDATA[C 42 H 62 O 16 ]]> glycyrrhizic acid -0.486 - 629.3663 <![CDATA[C 36 H 54 O 10 ]]> Glycyrrhetinic acid 3-O-glucuronide 2.492 + 471.3452 <![CDATA[C 30 H 46 O4]]> glycyrrhetinic acid 5.878 + 353.1008 <![CDATA[C 20 H 16 O6]]> Glycyrrhizin B 5.572 + 287.0908 <![CDATA[C 16 H 14 O5]]> Licorice Chalcone B 3.521 + 367.1192 <![CDATA[C 21 H 20 O6]]> 2',4',7-Trihydroxy-6'-methoxy-3'-isopentenyl isoflavone 3.361 + 405.1717 <![CDATA[C 25 H 26 O5]]> 6',8'-Diisopentenyl dydoxylin flavonoids 2.161 + 301.0697 <![CDATA[C 16 H 12 O6]]> Red clover extract 3.826 + 273.0754 <![CDATA[C 15 H 12 O5]]> 3',4',7'-Trihydroxyflavanone 3.382 + 271.0618 <![CDATA[C 15 H 12 O5]]> Naringin chalcone 6.218 + 257.08 <![CDATA[C 15 H 12 O4]]> Trihydroxychalcone 2.735 + 455.354 <![CDATA[C 30 H 48 O3]]> Ursolic acid 4.448 + 249.1864 <![CDATA[C 16 H 26 O2]]> Perilla lactone 3.199 +

[0104] Example 4: Effects of a synergistic preparation of Lactobacillus curvature CCFM1362 and a glycogenase-derived biogener on weight gain in mice on a high-fat diet.

[0105] 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 Lactobacillus curvaturei group (5 × 10⁻⁶ mcg / kg licorice extract). 9 CFU Lactobacillus curlis CCFM1362), Lactobacillus curlis-licorice extract synergistic preparation group (5×10) 9 CFU Lactobacillus curlis CCFM1362 and 78 mg / kg licorice extract), Lactobacillus curlis-licorice fermentation precursor group (78 mg / kg licorice extract, calculated based on the concentration of licorice extract before fermentation).

[0106] The experiment lasted 13 weeks: After one week of acclimatization, mice were fed a low-fat, low-sugar diet starting from the second week, 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 blank group and the model group were administered an equal volume of physiological saline as controls until the end of the experiment. All groups had free access to water and food. After the experiment, the mice were dissected, and the liver tissue was frozen for later use.

[0107] 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.

[0108] Depend on Figure 2 The results showed that the weight gain rate of mice administered Lactobacillus curlis CCFM1362 and licorice extract by gavage decreased by 4.678% and 14.152% respectively compared with the model group. In contrast, the weight gain rate of the combined preparation and the licorice fermentation preservative group decreased by 23.952% and 28.785% respectively compared with the model group. This indicates that the combined preparation prepared by Lactobacillus curlis CCFM1362 fermentation of licorice extract and the licorice fermentation preservative both have the effect of enhancing and alleviating weight gain in mice, and the licorice fermentation preservative group has a better effect.

[0109] Table 3 Grouping of experimental animals

[0110]

[0111] Example 5: Effects of a synergistic preparation of Lactobacillus curvature CCFM1362 and licorice extract and a licorice fermentation-derived biogener on β-galactosidase (β-GAL) levels in mice.

[0112] The methods for grouping, modeling, and treating mice are the same as in Example 4.

[0113] 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 aging-related β-galactosidase (β-GAL) level according to the corresponding ELISA kit instructions.

[0114] 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 3 It was found that oral administration of Lactobacillus curlis CCFM1362 and licorice extract alone reduced the β-galactosidase content in mice by 3.644% and 17.056%, respectively. In contrast, the combined preparation of Lactobacillus curlis CCFM1362 and licorice extract and the licorice fermentation-derived prebiotic group showed reductions of 22.384% and 25.214%, respectively. This indicates that both the combined preparation of Lactobacillus curlis CCFM1362 fermented with licorice extract and the licorice fermentation-derived prebiotic have the effect of reducing the β-galactosidase content in mice, and the licorice fermentation-derived prebiotic group showed the best anti-aging control effect.

[0115] Example 6: Effects of a synergistic formulation prepared from Lactobacillus curvaturei CCFM1362 and licorice extract, and a licorice fermentation-derived biotic on aging-related secretion phenotypes in mice.

[0116] The methods for grouping, modeling, and treating mice are the same as in Example 4.

[0117] 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.

[0118] Depend on Figure 4 The results showed that gavage administration of *Lactobacillus curlifron* CCFM1362 and licorice extract alone to mice resulted in a 23.877% and 19.901% decrease in IL-1β levels, respectively, compared to the model group. Furthermore, gavage administration of a synbiotic preparation of *Lactobacillus curlifron* CCFM1362 and licorice extract, and a licorice fermentation-derived prebiotic, resulted in a 33.406% and 53.024% decrease in IL-1β levels in the mouse liver, respectively. In addition, gavage administration of *Lactobacillus curlifron* CCFM1362 and licorice extract alone to mice resulted in a 16.895% and 5.129% decrease in IL-6 levels, respectively, compared to the model group. Gavage administration of the synbiotic preparation of *Lactobacillus curlifron* CCFM1362 and licorice extract resulted in a 38.063% decrease in IL-6 levels, and gavage administration of a licorice fermentation-derived prebiotic resulted in a 51.422% decrease in IL-6 levels. Following drug intervention, both the synbiotic preparation obtained from Lactobacillus curlis CCFM1362 fermented licorice extract and the licorice fermentation precursor reduced the level of age-related secretory phenotypes in mice, indicating that the drug has strong potential in the field of anti-aging.

[0119] Example 7: Effects of a synergistic preparation of Lactobacillus curvature CCFM1362 and licorice extract and a licorice fermentation-derived biogener on Nrf2-related pathways and antioxidant indicators in mice.

[0120] The methods for grouping, modeling, and treating mice are the same as in Example 4.

[0121] Weigh a portion of frozen liver tissue and add PBS (pH 7.4, 4℃) at a ratio of 1:9 (m / V). Homogenize and centrifuge at 12000×g for 30 min at 4℃. Collect the supernatant and determine the levels of Nrf2, MDA, GSH-px, and SOD according to the corresponding kit instructions. The results are as follows: Figure 5 , Figure 6 .

[0122] Depend on Figure 5It was found that, compared with the blank control group, the activities of GSH-px and SOD in the liver of mice in the model group were significantly reduced, while the content of MDA was significantly increased, indicating that lipid accumulation caused by a high-fat diet can cause oxidative damage to the liver of mice and inhibit the Nrf2-related pathway for antioxidant stress. After intervention with the synergistic preparation of Lactobacillus cruciformis CCFM1362 and licorice extract and the glycogenotropic agent after licorice fermentation, the antioxidant-related indicators of mice were significantly improved. Specifically, the GSH-px level in the liver of mice treated with licorice extract alone and Lactobacillus cruciformis CCFM1362 alone increased by 8.577% and 24.703% compared with the model group, respectively, while the synergistic preparation of Lactobacillus cruciformis CCFM1362 and licorice extract and the glycogenotropic agent after licorice fermentation increased by 27.813% and 42.276%, respectively. In terms of SOD level, the SOD level in the liver of mice treated with licorice extract alone and Lactobacillus cruciformis CCFM1362 alone increased by 47.489% and 22.87%, respectively, compared with the model group. The combined effect of Lactobacillus curlis CCFM1362 and licorice extract increased MDA levels by 91.000% and 49.793% respectively, compared to the model group. In terms of MDA levels, oral administration of licorice extract and Lactobacillus curlis CCFM1362 alone decreased MDA levels in mouse livers by 16.662% and 17.552% respectively compared to the model group, while the combined effect of Lactobacillus curlis CCFM1362 and licorice extract and the combined effect of licorice fermentation and biogenic intervention increased MDA levels by 44.918% and 47.308% respectively.

[0123] At the Nrf2 level, by Figure 6 The results showed that the Nrf2 level in the liver of mice treated with licorice extract and Lactobacillus cruciformis CCFM1362 alone increased by 2.097% and 16.170% compared with the model group, respectively. The synergistic preparation of Lactobacillus cruciformis CCFM1362 and licorice extract, and the intervention with licorice fermentation-derived biogenics, increased the levels by 24.724% and 33.186%, respectively. This indicates that both the Lactobacillus cruciformis CCFM1362-licorice extract synergistic preparation and the licorice fermentation-derived biogenics group can reduce the oxidative stress level induced by lipid accumulation in mice, and have the ability to enhance the liver's antioxidant level, thereby exerting anti-aging effects.

[0124] Example 8: Effects of a synergistic formulation prepared from Lactobacillus curvatureii CCFM1362 and licorice extract, and a licorice fermentation-derived probiotic, on chronic inflammation levels in mice.

[0125] The methods for grouping, modeling, and treating mice are the same as in Example 4.

[0126] Weigh a portion of frozen liver tissue and add PBS (pH 7.4, 4℃) at a ratio of 1:9 (m / V). Homogenize and centrifuge at 12000×g for 30 min at 4℃. Collect the supernatant and determine the levels of NF-κB p65, COX2, and iNOS according to the corresponding kit instructions. The results are as follows: Figure 7 .

[0127] Depend on Figure 7 It can be seen that, compared with the blank control group, NF-κB p65 in the liver of model group mice was activated due to chronic inflammation caused by lipid accumulation, and the content of its downstream inflammatory factors COX2 and iNOS also increased, indicating that lipid accumulation caused chronic inflammation of the liver. In mice administered licorice extract alone, NF-κB p65, COX2, and iNOS levels decreased by 4.638%, 16.414%, and 3.328% respectively compared to the model group, failing to effectively regulate inflammation-related factors. In mice administered Lactobacillus cruciformis CCFM1362 alone, these levels decreased by 25.264%, 18.307%, and 10.409% respectively compared to the model group, showing improvement compared to licorice alone, indicating that the strain has a certain effect on regulating inflammation in mice. However, after intervention with a synergistic preparation of Lactobacillus cruciformis CCFM1362 and licorice extract, and with licorice fermentation-derived probiotics, the degree of relief for multiple inflammatory indicators was stronger than in the groups administered the strain alone. After intervention with the Lactobacillus cruciformis CCFM1362-licorice extract synergistic preparation, NF-κB... The levels of p65, COX2, and iNOS decreased by 28.032%, 32.335%, and 18.174% respectively compared to the model group. However, after intervention with *Lactobacillus curvaturei* CCFM1362-licorice fermented biogener, the levels of NF-κB p65, COX2, and iNOS decreased by 39.956%, 33.184%, and 20.364% respectively compared to the model group, with all indicators returning to levels comparable to the control group. This indicates that both the combined preparation and the licorice fermented biogener can alleviate chronic inflammation in the body, thereby exerting anti-aging potential.

[0128] The comparative analysis of the above results shows that the combined use of Lactobacillus curvature CCFM1362 and licorice extract can significantly inhibit the increase of β-galactosidase content in mouse liver tissue, regulate the level of aging-related secretory phenotypes such as IL-1β and IL-6 in liver tissue, enhance the body's antioxidant level, reduce oxidative stress level, and reduce chronic inflammation level; moreover, the effect is significantly better than that of single strain CCFM1362 or single licorice extract. Lactobacillus curvature CCFM1362 has the effect of multi-target anti-aging and transforming licorice to enhance efficacy.

[0129] 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 curvature ( Lactobacillus crispatus The Lactobacillus curlis CCFM1362 was deposited at the Guangdong Provincial Center for Microbial Culture Collection on November 9, 2023, with accession number GDMCC No: 63995.

2. The metabiotic prepared from *Lactobacillus curlis* CCFM1362 as described in claim 1, characterized in that, The preparation method of the post-genetic agent is as follows: fermenting Lactobacillus curlis CCFM1362 in a culture medium containing licorice extract, and then inactivating and homogenizing the fermentation broth; the extraction method of the licorice extract is as follows: water extraction, the first extraction temperature is 90-95℃, the material-to-water ratio is 1:10, and the extraction time is 45min; the second extraction temperature is 90-95℃, the material-to-water ratio is 1:5, and the extraction time is 30min.

3. A synbiotic preparation made from *Lactobacillus curlis* CCFM1362 as described in claim 1, characterized in that, The synbiotic preparation includes Lactobacillus curvatureis CCFM1362 and licorice extract; the extraction method of the licorice extract is as follows: water extraction, first extraction temperature 90-95℃, material-to-water ratio 1:10, extraction time 45min; second extraction temperature 90-95℃, material-to-water ratio 1:5, extraction time 30min.

4. A product containing the post-biotic of claim 2 or the synbiotic of claim 3, characterized in that, The product in question is food, medicine, or health supplement.

5. The product as described in claim 4, characterized in that, 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.

6. A method for enhancing the medicinal efficacy of licorice, characterized in that, Licorice was treated in any of the following ways: (1) Extract the active ingredients from licorice to obtain an extract, and then mix the extract with the Lactobacillus curvature CCFM1362 described in claim 1; (2) Extract the active ingredients from licorice to obtain an extract, and then add the extract to the fermentation medium of Lactobacillus curlis CCFM1362 as described in claim 1 for cultivation. The extraction method of the extract is as follows: water extraction, first extraction temperature 90-95℃, material-to-water ratio 1:10, extraction time 45min; second extraction temperature 90-95℃, material-to-water ratio 1:5, extraction time 30min.

7. A method for degrading glycyrrhizic acid or producing glycyrrhetinic acid, characterized in that, The *Lactobacillus curvature* CCFM1362 of claim 1 was added to a culture medium containing glycyrrhizic acid for fermentation.

8. The use of the Lactobacillus curvature CCFM1362 of claim 1, the postbiotic of claim 2, or the synbiotic of claim 3 in the preparation of a medicament for improving inflammatory responses caused by lipid accumulation.

9. The use of the Lactobacillus curvature CCFM1362 of claim 1, the post-biotic of claim 2, or the synbiotic of claim 3 in the preparation of health products with antioxidant functions.

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