Sublatus subsp. sublatus

Abstract

The application discloses a strain of paracaseicolus glycyrrhizae capable of transforming glycyrrhizic acid to enhance the strain to relieve lipid accumulation, oxidative damage and inflammatory reaction, and belongs to the technical field of microorganisms and the technical field of medicine. The paracaseicolus glycyrrhizae CCFM1361 in the application can transform glycyrrhizic acid, increase the content of glycyrrhetinic acid, glycyrrhetinic acid 3-O-glucuronide and ursolic acid and the like, and increase the efficacy of glycyrrhiza extract, which is specifically embodied in the following aspects: (1) inhibiting the weight gain of high-fat diet mice; (2) reducing the lipid accumulation in the liver and the pathological changes caused by the lipid accumulation; (3) improving the liver antioxidant level of high-fat diet mice; (4) improving the inflammatory reaction caused by the lipid accumulation; and (4) reducing the inflammatory level of the body by activating the NF-kappa B p65 related path. Therefore, the paracaseicolus glycyrrhizae CCFM1361 and glycyrrhizic acid have great application prospects in the production of products with the efficacy of relieving lipid accumulation, oxidative damage and inflammatory reaction.

Description

Technical Field

[0001] This invention relates to a strain of *Lactobacillus paracasei* that can transform licorice to enhance its ability to alleviate lipid accumulation, oxidative damage, and inflammatory responses, belonging to the fields of microbial technology and pharmaceutical technology. Background Technology

[0002] Metabolic chronic diseases are widely recognized as a global challenge. These diseases are primarily caused by metabolic disorders of the three basic nutrients: fat, protein, and carbohydrates. Unhealthy diets and lifestyles can trigger metabolic disturbances, leading to the unhealthy accumulation of lipids and ultimately a series of diseases such as hyperlipidemia, cardiovascular disease, fatty liver, and diabetes. The occurrence and development of obesity and visceral fat accumulation are regulated by multiple signaling pathways. For example, the fatty acid synthesis pathway participates in the synthesis and breakdown of body fat; PPARs participate in adipocyte differentiation and lipid metabolism; and adipose tissue influences the body's fat metabolism through various cytokines. The active ingredients in food and medicine are mainly flavonoids, polyphenols, saponins, and terpenes. However, the "active ingredients" in herbs often suffer from low bioavailability or low activity. Their efficacy depends on the action of gut microbiota; therefore, specific bacterial transformation and modification are needed to improve the efficacy and stability of active ingredients, achieving better fat reduction effects.

[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, antitumor, and antiviral activity. Furthermore, the application potential of licorice extract in lowering lipids 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 absorbable and physiologically active than smaller molecules such as glycyrrhetinic acid monoglucuronide and glycyrrhetinic acid. Direct use of licorice extract is unlikely to yield immediate results, and its efficacy is often poor when consumed in combination with other licorice extracts. Therefore, improving the efficacy of licorice extract is crucial for its development into lipid-lowering 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 Lacticaseibacillus paracasei CCFM1361 in promoting the efficacy of licorice extract in alleviating oxidative damage and inflammatory response caused by lipid accumulation in the liver.

[0006] Another objective of this invention is to provide a fermented licorice extract and composition that enhances the relief of oxidative damage and inflammatory response caused by lipid accumulation in the liver.

[0007] Another objective of this invention is to provide a method for promoting the conversion of glycyrrhizic acid and increasing the content of glycyrrhetinic acid in licorice extract.

[0008] Specifically, this application adopts the following technical solution:

[0009] This invention provides a strain of Lactobacillus paracasei CCFM1361, which was deposited at the Guangdong Provincial Center for Microbial Culture Collection on November 9, 2023, with accession number GDMCC No: 63994.

[0010] In one embodiment of the present invention, after being cultured on MRS medium for 48 hours, the *Lactobacillus paracasei* CCFM1361 colonies are round, convex, or lenticular, slightly white, opaque, and have a smooth to mucous-like soft surface. Fermentation of licorice extract using *Lactobacillus paracasei* CCFM1361 can convert glycyrrhizic acid and significantly increase glycyrrhetinic acid content, promoting the effect of licorice extract in alleviating oxidative damage and inflammatory responses caused by lipid accumulation in the liver. Specifically, this manifests in: (1) significantly inhibiting weight gain in mice on a high-fat diet; (2) reducing lipid accumulation in the liver and the lesions it causes; (3) increasing the antioxidant level of the liver in mice on a high-fat diet; (4) improving inflammatory responses caused by lipid accumulation; and (5) reducing the level of inflammation in the body by activating the NF-κB p65-related pathway.

[0011] The present invention also provides a metabiotic prepared from the *Lactobacillus paracasei* CCFM1361, the metabiotic comprising inactivated bacteria and / or metabolites of *Lactobacillus paracasei* CCFM1361.

[0012] In one embodiment of the present invention, the metabiotic is obtained by fermenting licorice extract with Lactobacillus paracasei CCFM1361, and the fermentation method is as follows:

[0013] (1) Activate Lactobacillus paracasei CCFM1361 to obtain bacterial solution;

[0014] (2) Inoculate the bacterial solution of (1) into the fermentation substrate containing licorice extract and carry out fermentation;

[0015] (3) After fermentation is completed, the fermentation liquid is heat-treated and then homogenized under high pressure to obtain the final product.

[0016] In one embodiment of the present invention, step (1) involves streaking Lactobacillus paracasei CCFM1361 onto 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 culture.

[0017] 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).

[0018] In one embodiment of the present invention, the inoculation amount of bacterial solution in step (2) is 1-8% (v / v).

[0019] 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).

[0020] 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).

[0021] 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).

[0022] The present invention also provides a synbiotic preparation made from Lactobacillus paracasei CCFM1361, the synbiotic preparation comprising Lactobacillus paracasei CCFM1361 and licorice extract.

[0023] The present invention also provides products containing the said post-biotic or said synbiotic preparation.

[0024] In one embodiment of the present invention, the product is food, medicine, or health product.

[0025] In one embodiment of the present invention, the pharmaceutical product contains Lactobacillus paracasei CCFM1361, a drug carrier, and / or pharmaceutical excipients.

[0026] In one embodiment of the present invention, the health product contains Lactobacillus paracasei CCFM1361, a carrier, and / or excipients.

[0027] 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.

[0028] 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:

[0029] (1) Extract the active ingredients from licorice to obtain an extract, and then mix the extract with the Lactobacillus paracasei CCFM1361;

[0030] (2) Extract the active ingredients from licorice to obtain an extract, and then add the extract to the culture medium of Lactobacillus paracasei CCFM1361 for fermentation.

[0031] The present invention also provides a method for degrading glycyrrhizic acid, wherein the Lactobacillus paracasei CCFM1361 is added to a culture medium containing glycyrrhizic acid for fermentation.

[0032] The present invention also provides a method for preparing glycyrrhetinic acid, wherein the Lactobacillus paracasei CCFM1361 is added to a culture medium containing glycyrrhetinic acid for fermentation.

[0033] The present invention also provides the use of the aforementioned *Lactobacillus paracasei* CCFM1361, or the aforementioned metabiotic, or the aforementioned synbiotic preparation in the preparation of a medicament for alleviating lipid accumulation, oxidative damage, and / or inflammatory responses.

[0034] In the above applications, the product includes at least one of the following functions:

[0035] (a) Significantly inhibits weight gain in individuals on a high-fat diet;

[0036] (b) Reduce lipid accumulation in the liver and the resulting pathological changes;

[0037] (c) Enhances liver antioxidant levels in individuals with a high-fat diet;

[0038] (d) Improves the inflammatory response caused by lipid accumulation;

[0039] (e) Reduces the level of inflammation in the body by activating the NF-κB p65-related pathway.

[0040] The present invention also provides a method for increasing the glycyrrhetinic acid content in licorice extract, which involves adding Lactobacillus paracasei CCFM1361 to a system containing licorice extract.

[0041] Beneficial effects:

[0042] The *Lactobacillus paracasei* CCFM1361 provided by this invention has a novel property of degrading glycyrrhizic acid. After fermentation in a fermentation substrate rich in licorice extract, the degradation rate of glycyrrhizic acid reaches 32.93%, and it is converted into substances such as glycyrrhetinic acid, glycyrrhetinic acid 3-O-glucuronide, ursolic acid, pachymic acid A, and α-ionone.

[0043] The *Lactobacillus paracasei* CCFM1361 described in this invention can synergistically enhance the efficacy of licorice extract, including alleviating the increase in body fat and the harm to liver function caused by a high-fat diet, improving the ability of licorice extract to reduce inflammatory damage, and reducing oxidative stress in the body.

[0044] Lactobacillus paracasei CCFM1361 and licorice extract can be used to produce products that enhance the efficacy of relieving oxidative damage and inflammation caused by lipid accumulation in the liver. The production process uses glycyrrhizic acid extract as a raw material, promoting 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 Lactobacillus paracasei CCFM1361 to products containing licorice extract has significant application prospects and a solid foundation.

[0045] Preservation of biological materials

[0046] Lactobacillus paracasei (CCFM1361), taxonomically named Lacticaseibacillus paracasei, was deposited on November 9, 2023, at the Guangdong Provincial Center for Microbial Culture Collection (GDMCC No.: 63994), located at Building 59, No. 100 Xianlie Middle Road, Guangzhou. Attached Figure Description

[0047] Figure 1 HPLC chromatograms of licorice extract fermented by Lactobacillus paracasei CCFM1361 at 0h and 48h, and HPLC chromatograms of glycyrrhizic acid and glycyrrhetinic acid standards;

[0048] Figure 2 Weight gain in mice of different groups after drug intervention;

[0049] Figure 3 H&E stained liver tissue sections from different groups of mice;

[0050] Figure 4 Oil Red O stained tissue sections of mouse liver from different groups;

[0051] Figure 5 : Levels of four lipid parameters (TG, TC, LDL-c, and HDL-c) in mice from different groups;

[0052] Figure 6 Levels of MDA, SOD, and GSH-px in the livers of mice in different groups;

[0053] Figure 7 Levels of IL-1β, IL-6, NF-κB p65, iNOS, and COX2 in the livers of mice in different groups.

[0054] "*" 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

[0055] The C57BL / 6J mice used in the following examples were purchased from Beijing Vital River Company.

[0056] The culture media involved in the following examples are as follows:

[0057] 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.

[0058] 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.

[0059] 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.

[0060] 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.

[0061] The preparation method of the Lactobacillus paracasei-licorice extract synergistic formulation involved in the following examples is as follows:

[0062] (1) Activate Lactobacillus paracasei CCFM1361 to obtain bacterial solution;

[0063] (2) In MRS medium, inoculate the bacterial suspension from (1) with a viable count of 5 × 10⁶. 7 CFU / mL, cultured to the logarithmic growth phase of the strain to obtain Lactobacillus paracasei CCFM1361 bacterial culture;

[0064] (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;

[0065] (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 of Lactobacillus paracasei CCFM1361 and 78 mg / kg of licorice extract.

[0066] The following examples involve the preparation formula of Lactobacillus paracasei-licorice fermented biogenics:

[0067] (1) Streaking of Lactobacillus paracasei CCFM1361 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 post-biotic solution. The obtained post-biotic solution was freeze-dried and resuspended in physiological saline according to the required dose before gavage.

[0070] Example 1: Isolation, screening, identification and preservation of Lactobacillus paracasei

[0071] The specific steps are as follows:

[0072] 1. Screening

[0073] 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 paracasei* 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 CCFM1361. The typical colonies of *Lactobacillus paracasei* are round, white, and smooth.

[0074] 2. Identification

[0075] The genome of strain CCFM1361 was extracted, and the 16S rDNA of strain CCFM1361 was amplified and sequenced (performed by Suzhou Genewiz Biotechnology Co., Ltd., and the nucleotide sequence of the amplified 16S rDNA of CCFM1361 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 paracasei, and it was named Lactobacillus paracasei CCFM1361.

[0076] 3. Preservation of microbial strains

[0077] Lactobacillus paracasei CCFM1361 was inoculated into 5 mL of MRS 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.

[0078] 4. Preservation of bacterial strains

[0079] The Lactobacillus paracasei CCFM1361 was deposited at the Guangdong Provincial Microbial Culture Collection Center (GDMCC) on November 9, 2023, with accession number GDMCC No: 63994, located at 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou.

[0080] Example 2: Transformation of glycyrrhizic acid by Lactobacillus paracasei CCFM1361

[0081] 1. Fermentation method:

[0082] (1) The Lactobacillus paracasei CCFM1361 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.

[0083] (2) Add 4% (v / v) of the seed culture of *Lactobacillus paracasei* CCFM1361 (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.

[0084] 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.

[0085] 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.

[0086] 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.

[0087] Table 1. Contents of various components after fermentation of *Lactobacillus paracasei* CCFM1361 in licorice extract-containing medium.

[0088] Content of each component (μg / mL) Fermentation 0h Fermentation for 48 hours glycyrrhizic acid 239.45±5.61 160.60±3.29 glycyrrhetinic acid 0 27.18±1.95

[0089] Note: The glycyrrhetinic acid content at 0h of fermentation was far below the detection limit of 100ppm.

[0090] The results showed that after 48 hours of fermentation, the glycyrrhizic acid content in the fermentation medium decreased by 32.93% compared to before fermentation. This indicates that after fermenting licorice extract with *Lactobacillus paracasei* of the present invention, the licorice extract was significantly converted into glycyrrhetinic acid and other active small molecules.

[0091] Example 3: Analysis of glycyrrhizic acid metabolites before and after fermentation by Lactobacillus paracasei CCFM1361

[0092] 1. Metabolite extraction

[0093] 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.

[0094] 2. On-machine testing

[0095] 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).

[0096] 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 paracasei* CCFM1361 for 48 hours, the main metabolites of glycyrrhizic acid were terpenes, shikimic acid and phenylpropionic acid derivatives, fatty acids, amino acids, and short peptides.

[0097] Table 2. Terpenoids detected in glycyrrhizic acid fermentation broth and changes in substances before and after fermentation.

[0098] molecular weight Molecular formula name <![CDATA[Log2FoldChange]]> Changes after fermentation 821.3975 <![CDATA[C 42 H 62 O 16 ]]> glycyrrhizic acid -0.486 - 471.3452 <![CDATA[C 30 H 46 O4]]> glycyrrhetinic acid 2.278 + 629.3663 <![CDATA[C 36 H 54 O 10 ]]> Glycyrrhetinic acid 3-O-glucuronide 2.920 + 455.354 <![CDATA[C 30 H 48 O3]]> Ursolic acid 6.120 + 497.3285 <![CDATA[C 31 H 46 O5]]> Poria cocos acid A 2.068 + 193.1582 <![CDATA[C 13 H 20 O]]> α-Ionone 3.187 +

[0099] Example 4: Effects of a synergistic formulation prepared from *Lactobacillus paracasei* CCFM1361 and fermented licorice post-biotics on weight gain in mice on a high-fat diet.

[0100] 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 paracasei group (5 × 10⁻⁶ mcg / kg licorice extract). 9 CFU (Cactobacillus paracasei CCFM1361), Cactobacillus paracasei-licorice extract synergistic preparation (5×10) 9 CFU (Cactobacillus paracasei CCFM1361, 78 mg / kg licorice extract) and CFU-fermented licorice post-biotic group (78 mg / kg licorice extract, calculated based on the concentration of licorice extract before fermentation).

[0101] 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 (Table 1), 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.

[0102] 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.

[0103] Depend on Figure 2 It was found that the weight gain rate of mice treated with Lactobacillus paracasei CCFM1361 and licorice extract by gavage decreased by 18.817% and 14.152% respectively compared with the model group, which was less effective than the combined preparation and post-biotic group (36.247% and 24.414% respectively). After the drug intervention ended, both the combined preparation and post-biotic prepared from Lactobacillus paracasei CCFM1361 fermented licorice extract had the effect of alleviating weight gain in mice, and the combined preparation group showed the best weight control effect.

[0104] Table 3 Grouping of experimental animals

[0105]

[0106] Example 5: Effects of a synergistic preparation of *Lactobacillus paracasei* CCFM1361 and fermented licorice post-biotic on hepatic fat accumulation and lesions in mice on a high-fat diet.

[0107] The methods for grouping, modeling, and treating mice are the same as in Example 4.

[0108] 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 3 , Figure 4 .

[0109] Depend on Figure 3 , Figure 4 It was found that the liver tissues of mice in the blank group and the group treated with fermented licorice post-biotic prepared from *Lactobacillus paracasei* CCFM1361 showed uniform staining, less fat accumulation, and tightly packed, homogeneous hepatocytes with regular shapes. The liver tissues of mice in the other groups showed greater 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 *Lactobacillus paracasei* CCFM1361 and licorice extract, or the fermented licorice post-biotic prepared in this invention, effectively inhibited liver fat accumulation and lesions in mice on a high-fat diet. In particular, the post-biotic preparation significantly reduced the degree of liver damage and fatty lesions.

[0110] Example 6: Effects of a synergistic preparation of *Lactobacillus paracasei* CCFM1361 and fermented licorice post-biotic on blood lipid biochemical indicators in mice on a high-fat diet.

[0111] The methods for grouping, modeling, and treating mice are the same as in Example 4.

[0112] 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 5 .

[0113] Depend on Figure 5It 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 caused elevated blood lipids. Drug intervention results showed that, compared with the model group, mice administered Lactobacillus paracasei CCFM1361 and licorice extract by gavage alone had TC levels decreased by 5.411% and 9.932%, respectively, and TG levels decreased by 12.261% and 22.222%, respectively. The combined preparation group and the fermented licorice extract post-biotic group both reduced their levels to varying degrees. Specifically, the post-biotic derived from Lactobacillus paracasei CCFM1361 and licorice extract reduced TC and TG levels by 30.936% and 21.264%, respectively, restoring them to levels comparable to the control.

[0114] 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 Lactobacillus paracasei CCFM1361 by gavage decreased by 4.702% and 6.700%, 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 effects of the combined preparation and the post-biotic from fermented licorice. Among them, the synbiotic formulation reduced LDL-c and HDL-c levels by 18.760% and 5.893%, respectively, while the post-fermented licorice extract reduced LDL-c and HDL-c levels by 45.729% and 19.048%, respectively. This indicates that the synbiotic formulation of Lactobacillus paracasei CCFM1361 and licorice extract, as well as the post-fermented licorice extract, have great potential in reducing blood lipid levels. Based on the four blood lipid indicators in mice, it is further shown that the synbiotic formulation of Lactobacillus paracasei CCFM1361 and licorice extract, as well as the post-fermented licorice extract, can reduce the rise in blood lipids caused by lipid accumulation.

[0115] Example 7: Effects of a synergistic formulation prepared from *Lactobacillus paracasei* CCFM1361 and fermented licorice post-biotics on liver antioxidant levels in mice on a high-fat diet.

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

[0117] Weigh a portion of frozen liver tissue and add PBS (pH 7.4, 4℃) at a ratio of 1:9 (m / V). Homogenize the mixture and centrifuge at 12000×g for 30 min at 4℃. Collect the supernatant and determine the levels of MDA, GSH, and SOD according to the corresponding kit instructions. The results are as follows: Figure 6 .

[0118] Depend on Figure 6It was found that, compared with the blank control group, the GSH and SOD activities in the liver of the model group mice were significantly reduced, and the MDA content was significantly increased, indicating that lipid accumulation caused by a high-fat diet can cause oxidative damage to the liver of mice. GSH depletion and reduced SOD activity were significantly improved after intervention with a synergistic preparation of Lactobacillus paracasei CCFM1361 and licorice extract and post-fermented licorice. Among them, the levels of GSH-px in the livers of mice administered licorice extract or Lactobacillus paracasei CCFM1361 by gavage alone increased by 8.577% and 14.716% respectively compared with the model group, while the levels increased by 40.334% and 29.888% respectively after intervention with the combined preparation and fermented licorice. In terms of SOD level, the levels of SOD in the livers of mice administered licorice extract or Lactobacillus paracasei CCFM1361 by gavage alone increased by 47.489% and 24.553% respectively compared with the model group, while the levels increased by 54.655% and 83.541% respectively after intervention with the combined preparation and fermented licorice. In terms of MDA level, the levels of MDA in the livers of mice administered licorice extract or Lactobacillus paracasei CCFM1361 by gavage alone decreased by 16.662% and 36.695% respectively compared with the model group, while the levels increased by 48.316% and 52.878% respectively after intervention with the combined preparation and fermented licorice. The results showed that the combined preparation of Lactobacillus paracasei CCFM1361 and licorice extract, as well as the post-fermented licorice serotonin group, could reduce the level of oxidative stress caused by lipid accumulation in mice and have the ability to enhance the antioxidant level of the liver.

[0119] Example 8: Effects of a synergistic formulation prepared from *Lactobacillus paracasei* CCFM1361 and fermented licorice post-biotics on chronic inflammation levels in mice on a high-fat diet.

[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 NF-κB p65, COX2, iNOS, IL-1β, and IL-6 according to the corresponding kit instructions. The results are as follows: Figure 7 .

[0122] Depend on Figure 7It 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. At the same time, the levels of its downstream inflammatory factors COX2, iNOS, IL-1β and IL-6 also increased, indicating that lipid accumulation caused chronic inflammation in the liver. In mice administered licorice extract alone, NF-κB p65, COX2, iNOS, IL-1β, and IL-6 levels decreased by 4.638%, 16.414%, 3.328%, 19.901%, and 5.134%, respectively, compared to the model group. In mice administered Lactobacillus paracasei CCFM1361 alone, these levels decreased by 10.962%, 19.873%, 15.785%, 35.228%, and 22.726%, respectively, compared to the model group. However, after intervention with a synergistic formulation of Lactobacillus paracasei CCFM1361 and licorice extract, and post-fermented licorice biotrophic factors, the relief of multiple inflammatory markers was significantly stronger than in the single-gavage groups. After intervention with the Lactobacillus paracasei CCFM1361-licorice extract synergistic formulation, NF-κB... The levels of p65, COX2, iNOS, IL-1β, and IL-6 decreased by 23.708%, 37.555%, 20.563%, 70.043%, and 58.529%, respectively, compared to the model group. However, after intervention with *Lactobacillus paracasei* CCFM1361-fermented licorice post-biotic, the levels of NF-κB p65, COX2, iNOS, IL-1β, and IL-6 decreased by 6.238%, 19.873%, 3.527%, 15.812%, and 45.952%, respectively, compared to the model group. This indicates that both the synbiotic preparation and the fermented licorice post-biotic group can reduce the level of chronic inflammation caused by lipid accumulation.

[0123] The comparative analysis of the above results shows that the Lactobacillus paracasei CCFM1361 + licorice extract group can inhibit the weight gain of mice on a high-fat diet; reduce lipid accumulation in the liver and the lesions it causes; enhance the antioxidant level of the liver in mice on a high-fat diet; effectively improve the inflammatory response caused by lipid accumulation; and reduce the body's inflammation level by activating the NF-κB p65-related pathway. Moreover, the effect is significantly better than that of single strain CCFM1361 and single licorice extract. Lactobacillus paracasei CCFM1361 can convert glycyrrhizic acid to enhance its effect in alleviating lipid accumulation and the oxidative damage and inflammatory response it causes.

[0124] 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 paracasei ( Lacticaseibacillus paracasei The Lactobacillus paracasei CCFM1361 was deposited at the Guangdong Provincial Center for Microbial Culture Collection on November 9, 2023, with accession number GDMCCNo: 63994.

2. The metabiotic prepared from *Lactobacillus paracasei* CCFM1361 as described in claim 1, characterized in that, The post-biotic is obtained by adding the Lactobacillus paracasei CCFM1361 of claim 1 to a culture medium containing licorice extract for fermentation; 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 paracasei* CCFM1361 as described in claim 1, characterized in that, The synbiotic preparation contains *Lactobacillus paracasei* CCFM1361 and licorice products; the licorice products are licorice or 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. The method for preparing the postgenetic agent according to claim 2, characterized in that, Lactobacillus paracasei CCFM1361 was fermented in a culture medium containing licorice extract, and then the fermentation broth was inactivated and homogenized. The extraction method of the licorice extract was as follows: water extraction, the first extraction temperature was 90-95℃, the material-to-water ratio was 1:10, and the extraction time was 45 min; the second extraction temperature was 90-95℃, the material-to-water ratio was 1:5, and the extraction time was 30 min.

5. A product containing the post-biotic of claim 2 or the synbiotic of claim 3, characterized in that, The product is either food or medicine.

6. The product as described in claim 5, 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.

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

8. The product as described in claim 7, 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.

9. 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 paracasei CCFM1361 as described in claim 1; (2) Extract the active ingredients from licorice to obtain an extract, and then add the extract to the culture medium of Lactobacillus paracasei CCFM1361 as described in claim 1 for fermentation; 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.

10. A method for degrading glycyrrhizic acid or a method for preparing glycyrrhetinic acid, characterized in that, The *Lactobacillus paracasei* CCFM1361 of claim 1 was added to a culture medium containing glycyrrhizic acid for fermentation.

11. The use of the *Lactobacillus paracasei* CCFM1361 of claim 1, or the post-biotic of claim 2, or the synbiotic of claim 3, in the preparation of a medicament for relieving inflammatory responses caused by lipid accumulation in the liver.

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