Bifidobacterium animalis with both lipid-lowering and anti-aging effects and application of bifidobacterium animalis for transforming liquorice for synergism

Abstract

The invention discloses bifidobacterium animalis with both lipid-lowering and anti-aging effects and application of the bifidobacterium animalis to conversion of liquorice for synergism, and belongs to the technical field of microorganisms and medicines. The strain can ferment a licorice extract and increase the content of glycyrrhetinic acid, glycyrrhetinic acid 3-O-glucuronide, 5-O-methylgenistein and other active ingredients so as to enhance the efficacy of the licorice extract, and the specific expressions are as follows: (1) the body weight gain under high fat diet is inhibited, and the four items of blood fat are reduced; (2) the levels of organism aging-related secretory phenotypes IL-1beta and IL-6 are reduced; (3) the content of beta-galactosidase in the liver is reduced; (4) damage caused by liver lipid accumulation is reduced; and (5) the expression of lipid metabolism genes MGL, HSL, ATGL and PPAR-alpha is promoted, and the expression of lipid synthesis genes PPAR-gamma and Fasn is inhibited. Therefore, the bifidobacterium animalis subsp. Lactis CCFM1363 is combined with the licorice extract for application, and has wide development potential of lipid-lowering and anti-aging products.

Description

Technical Field

[0001] The invention relates to an animal Bifidobacterium lactis subspecies CCFM1363 and the application of anti-aging and lipid-lowering licorice fermentation postbiotics and transformed licorice synergistic enhancement prepared therefrom, belonging to the fields of microbial technology and medical technology. Background Art

[0002] As the aging population continues to intensify, anti-aging research has become a core issue in the field of biomedicine and microbial technology. Aging is a complex biological process affected by multiple factors. It not only involves multiple aspects such as oxidative stress, chronic inflammation, decreased metabolic function and immune function decline, but also directly leads to the gradual decline of body functions and significantly increases the risk of chronic diseases such as diabetes, cardiovascular disease and neurodegenerative diseases. It is worth noting that unhealthy diet and lifestyle habits play an important role in this. They cause metabolic disorders in the body and promote the accumulation of unhealthy lipids, thereby inducing a series of diseases such as hyperlipidemia, cardiovascular disease, fatty liver and diabetes. These disease states, in turn, aggravate the body's chronic inflammation, oxidative stress and DNA damage, forming a vicious circle and further accelerating the aging process.

[0003] In recent years, medicinal and edible materials and their microbial metabolites have received extensive attention due to their safety and effectiveness. Licorice is a commonly used medicinal and edible material with a wide range of uses. Related studies have found that licorice also shows good effects in anti-oxidation, anti-inflammatory, anti-tumor, and antiviral aspects. In addition, the application potential of licorice extracts in anti-aging has received widespread attention. The triterpenoid saponin compounds in licorice are considered to be its main pharmacological substances, mainly including glycyrrhizic acid (GL), monoglucuronic acid glycyrrhetinic acid (GAMG) and glycyrrhetinic acid (GA), among which glycyrrhizic acid has the highest content.

[0004] Studies have shown that glycyrrhizic acid is not as good as active small molecules such as monoglucuronic acid glycyrrhetinic acid and glycyrrhetinic acid in terms of absorption and physiological activity. It is difficult to obtain direct effects by directly using licorice extracts, and the efficacy is poor when consuming licorice extracts. Therefore, how to promote the efficacy of licorice extracts is the key to affecting the development of licorice extracts into lipid-lowering and anti-aging related products. Summary of the invention

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

[0006] Another object of the present invention is to provide a fermented licorice extract and composition that can achieve enhanced lipid-lowering and anti-aging effects.

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

[0008] The above-mentioned purpose of the present invention is achieved through the following technical solutions:

[0009] The present invention provides a strain of Bifidobacterium animalis subsp. lactis CCFM1363, which has been deposited in Guangdong Provincial Microbiological Culture Collection Center on December 6, 2023, with a deposit number of GDMCC No: 64111.

[0010] In one embodiment of the present invention, the colonies of the animal Bifidobacterium lactis subspecies CCFM1363 of the present invention after being cultured on MRS medium for 48 hours are round, convex or lens-shaped, slightly white, opaque, and have a smooth to mucous soft surface.

[0011] In one embodiment of the present invention, the use of the animal Bifidobacterium lactis subspecies CCFM1363 to ferment licorice extract can significantly increase the content of glycyrrhetinic acid, and promote the licorice extract to play a lipid-lowering and anti-aging role. Specifically, it is embodied in: (1) controlling the weight gain of obese bodies; (2) reducing the level of aging-related secretory phenotypes in the body; (3) reducing the content of β-galactosidase in the body; (4) inhibiting the accumulation of fat and lesions in the liver of the body; (5) reducing the body's blood lipid level; (6) effectively increasing the expression of lipid metabolism-related genes MGL, HSL, ATGL, PPAR-α, and reducing the expression of lipid synthesis-related genes PPAR-γ and Fasn.

[0012] The present invention also provides a postbiotic prepared by the animal Bifidobacterium lactis subspecies CCFM1363, which is obtained by fermenting a licorice extract by the animal Bifidobacterium lactis subspecies CCFM1363.

[0013] In one embodiment of the present invention, the preparation method of the postbiotics comprises the following steps:

[0014] (1) activating Bifidobacterium animalis subspecies lactis CCFM1363 to obtain a bacterial liquid;

[0015] (2) The bacterial solution of (1) was inoculated into the fermentation base containing licorice extract, and the number of live bacteria inoculated was 5×10 7 CFU / mL, anaerobic fermentation for 48h;

[0016] (3) After fermentation is completed, the number of viable bacteria is 1×10 9 CFU / mL, the fermentation broth is heat treated and then homogenized under high pressure to obtain the product.

[0017] In one embodiment of the present invention, step (1) is to streak animal Bifidobacterium lactis subsp. CCFM1363 on MRS solid culture medium, invert the plate in an anaerobic environment for culture (preferably inverted culture at 37°C for 48 hours), and then pick a single colony and inoculate it into MRS liquid culture medium (preferably 5 mL) for culture (preferably cultured at 37°C for 48 hours) to obtain a bacterial liquid.

[0018] In one embodiment of the present invention, the concentration of the licorice extract in the fermentation base containing the 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 the bacterial solution in step (2) is 1-8% (v / v).

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

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

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

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

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

[0025] (1) activating Bifidobacterium animalis subspecies lactis CCFM1363 to obtain a bacterial liquid;

[0026] (2) Inoculate the bacterial solution from (1) into MRS medium, with the number of viable bacteria being 5×10 7 CFU / mL, cultured in an anaerobic environment until the strain grew into the logarithmic growth phase, and obtained the bacterial solution of Bifidobacterium animalis subsp. lactis;

[0027] (3) collecting the bacterial cells from the liquid culture medium by centrifugation, mixing with a freeze-drying protective agent (12% skim milk powder), and freeze-drying to obtain a freeze-dried powder of the strain;

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

[0029] In one embodiment of the present invention, step (1) is to streak animal Bifidobacterium lactis subsp. CCFM1363 on MRS solid culture medium, invert the plate in an anaerobic environment for culture (preferably inverted culture at 37°C for 48 hours), and then pick a single colony and inoculate it into MRS liquid culture medium (preferably 5 mL) for culture (preferably cultured at 37°C for 48 hours) to obtain a bacterial liquid.

[0030] In one embodiment of the present invention, the concentration of the licorice extract in the fermentation base containing the 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 the bacterial solution in step (2) is 1-8% (v / v).

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

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

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

[0035] The present invention provides a product containing the animal Bifidobacterium lactis subspecies CCFM1363 and / or its postbiotics and / or its synbiotic preparation.

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

[0037] In one embodiment of the present invention, the product is a lyophilized powder of the postbiotic or synbiotic preparation, or a reconstituted product containing the lyophilized powder of the postbiotic or synbiotic preparation.

[0038] The invention provides a method for degrading glycyrrhizic acid. The method comprises adding the animal Bifidobacterium lactis subspecies CCFM1363 into a culture medium containing glycyrrhizic acid for fermentation.

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

[0040] The present invention provides a method for enhancing the medicinal efficacy of licorice, which is any of the following:

[0041] (1) adding the animal Bifidobacterium lactis subspecies CCFM1363 to a culture medium containing a licorice product for fermentation;

[0042] (2) Mixing the animal Bifidobacterium lactis subspecies CCFM1363 with a liquorice product.

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

[0044] The present invention also provides the use of the animal Bifidobacterium lactis subspecies CCFM1363 and / or its postbiotics and / or its synbiotic preparation in the preparation of lipid-lowering and / or anti-aging medicines.

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

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

[0047] In one embodiment of the present invention, the drug comprises at least one of the following functions:

[0048] (1) Reduce weight gain in response to a high-fat diet;

[0049] (2) reduce the levels of IL-1β and IL-6, the secretory phenotype associated with aging;

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

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

[0052] (5) Reduce blood lipid levels;

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

[0054] The invention also provides an anti-aging medicine, which contains animal bifidobacterium lactis subspecies CCFM1363 and liquorice extract.

[0055] The present invention also provides a medicine with anti-aging effect, which contains the product obtained by fermenting the licorice extract with the animal Bifidobacterium lactis subspecies CCFM1363.

[0056] In one embodiment of the present invention, in the product, the added amount of the animal Bifidobacterium lactis subspecies CCFM1363 is at least: 1×10 9 CFU / mL.

[0057] In one embodiment of the present invention, the medicine contains Bifidobacterium animalis lactis subspecies CCFM1363, a drug carrier and / or a pharmaceutical excipient.

[0058] Beneficial effects:

[0059] The animal Bifidobacterium lactis subspecies CCFM1363 of the present invention has the ability to transform glycyrrhizic acid. After fermentation in a fermentation base material 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 apigenin isoliquiritigenin are generated.

[0060] The animal Bifidobacterium lactis subspecies CCFM1363 described in the present invention can promote the efficacy of licorice extract, including alleviating the degree of weight gain and damage to liver function under a high-fat diet, improving the ability of licorice extract to enhance lipid metabolism, and reducing the body's anti-aging ability.

[0061] Animal Bifidobacterium lactis subspecies CCFM1363 and licorice extract can be used to produce products with enhanced anti-aging effects. The production process can use glycyrrhizic acid extract as a raw material to promote the effectiveness of licorice extract. The process has the advantages of safety, high efficiency, low cost, and mild reaction. The process is suitable for large-scale industrial production. Therefore, the use of animal Bifidobacterium lactis subspecies CCFM1363 in products containing licorice extract has great application prospects and application basis.

[0062] Biomaterial Deposit

[0063] Bifidobacterium animalis subsp. lactis CCFM1363, taxonomically named Bifidobacterium animalis subsp. lactis, was deposited in Guangdong Provincial Microbiological Culture Collection Center on December 6, 2023, with the deposit number GDMCC No: 64111, and the deposit address is Building 59, No. 100 Xianlie Middle Road, Guangzhou. BRIEF DESCRIPTION OF THE DRAWINGS

[0064] Figure 1 : Liquid chromatograms of 0h and 48h of the fermented liquorice extract of Bifidobacterium animalis subspecies lactis CCFM1363 and liquid chromatograms of the standard products of glycyrrhizic acid and glycyrrhetinic acid;

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

[0066] Figure 3 :IL-1β and IL-6 levels in aging-related secretory phenotypes of mice in different groups;

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

[0068] Figure 5 :H&E stained tissue sections of livers of mice in different groups;

[0069] Figure 6 : Oil red O stained tissue sections of liver of mice in different groups;

[0070] Figure 7 : Comparison of four blood lipid indicators of TG, TC, LDL-c, and HDL-c in mice of different groups;

[0071] Figure 8 : Comparison of ALT and AST indicators in mice of different groups;

[0072] Fig. 9 :Expression of fat metabolism-related genes in different groups of mice.

[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 DESCRIPTION

[0074] The animal Bifidobacterium lactis subsp. lactis CCFM1363 used in the present invention has been deposited in the Guangdong Provincial Microbiological Culture Collection Center on December 6, 2023, with a deposit number of GDMCC No: 64111, and the deposit address is 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou.

[0075] The C57BL / 6J mice involved in the following examples were purchased from Beijing Weitonglihua Company.

[0076] The culture medium involved in the following examples is as follows:

[0077] MRS liquid culture medium: yeast powder 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 hydrogen 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 powder 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 hydrogen 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 base 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 of licorice extract is: water extraction, primary extraction temperature 90-95℃, material-water ratio 1:10, extraction time 45min; secondary extraction temperature 90-95℃, material-water ratio 1:5, extraction time 30min. After drying, the final licorice extract yield is 12%, that is, 1g licorice extract is equivalent to 8.33g licorice raw material.

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

[0082] (1) activating Bifidobacterium animalis subspecies lactis CCFM1363 to obtain a bacterial liquid;

[0083] (2) Inoculate the bacterial solution from (1) into MRS medium, with the number of viable bacteria being 5×10 7 CFU / mL, cultured in an anaerobic environment until the strain grew into the logarithmic growth phase, and obtained the bacterial solution of Bifidobacterium animalis subsp. lactis;

[0084] (3) collecting the bacterial cells from the liquid culture medium by centrifugation, mixing with a freeze-drying protective agent (12% skim milk powder), and freeze-drying to obtain a freeze-dried powder of the strain;

[0085] (4) Adjust the dose according to the mouse weight (in kg) and mix into 5×10 9 CFU animal Bifidobacterium lactis subspecies CCFM1363 + 78 mg / kg licorice extract is obtained.

[0086] The preparation method of animal Bifidobacterium lactis subsp. lactis-licorice fermentation postbiotics involved in the following embodiments is as follows:

[0087] (1) streaking animal Bifidobacterium lactis subsp. CCFM1363 on MRS solid medium, inverting and culturing at 37° C. for 48 h, then picking a single colony and inoculating it into 5 mL MRS liquid medium and culturing it at 37° C. for 48 h to obtain a bacterial solution;

[0088] (2) The bacterial solution of (1) was added to the fermentation base containing licorice extract and fermented at 37°C for 48 h until the number of viable bacteria reached 1×10 9 CFU / mL;

[0089] (3) After fermentation, the fermentation liquid was subjected to heat treatment at 65°C for 30 min, and then subjected to high pressure homogenization at 1200 Bar for 6 times. The obtained postbiotic solution was freeze-dried and resuspended in physiological saline according to the required dose (78 mg / kg licorice extract, calculated based on the concentration of licorice extract before fermentation) before oral administration.

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

[0091] The specific steps are as follows:

[0092] 1. Screening

[0093] The samples are derived from healthy human feces. After pretreatment, the samples are stored in a -80°C refrigerator in 20% glycerol. After being taken out and thawed, they are mixed and 0.5 mL of the sample is pipetted into 4.5 mL of normal saline, and gradient dilutions are performed with normal saline. Appropriate gradient dilutions are selected and spread on MRS solid culture medium, and cultured at 37°C for 48 hours. Typical colonies of animal Bifidobacterium lactis subsp. are picked and streaked on MRS solid culture medium for purification, and single colonies are picked and transferred to MRS liquid culture medium for bacterial enrichment, and preserved in 30% glycerol to obtain a strain named CCFM1363; among them, the typical colonies of animal Bifidobacterium lactis subsp. are 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 Jinweizhi Biotechnology Co., Ltd., and the nucleotide sequence of 16S rDNA amplified from 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 animalis subsp. lactis, and was named Bifidobacterium animalis subsp. lactis CCFM1363.

[0096] 3. Preservation of strains

[0097] Animal Bifidobacterium lactis subsp. CCFM1363 was inoculated into 5 mL of MRS liquid culture medium and cultured in an anaerobic environment at 37°C for 24 h. 1 mL of bacterial solution was taken into a sterile centrifuge tube, centrifuged at 8000 r / min for 3 min, the upper culture medium was discarded, and the bacterial sludge was resuspended in 30% glycerol solution and stored at -80°C.

[0098] 4. Strain preservation

[0099] The animal Bifidobacterium lactis subspecies CCFM1363 was deposited in the Guangdong Provincial Microbiological Culture Collection Center (GDMCC) on December 6, 2023, with a deposit number of GDMCC No: 64111, and the deposit address is 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 culture medium, and the plate was inverted and cultured at 37° C. for 48 h; a single colony was picked and inoculated into 5 mL of MRS liquid culture medium and cultured at 37° C. for 48 h to prepare a seed solution.

[0103] (2) Add 4% (v / v) seed solution of Bifidobacterium animalis subspecies CCFM1363 (live bacteria inoculation concentration of 5×10 7 CFU / mL), the final concentration of licorice extract in the culture medium was 10 mg / mL, and the culture was fermented at 37°C for 48 h. The fermentation broth at 0 h and 48 h was taken into 2 mL EP tubes, respectively, and centrifuged at 8000 rpm for 5 min to remove the bacteria, and the supernatant was transferred to a 5 mL EP tube.

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

[0105] The reference solution and the sample solution were injected and tested according to the following liquid chromatography conditions: using Waters e2695 liquid chromatograph, liquid column X Bridge RC18 (250×4.6mm, 5μm); mobile phase: water (A); 0.1% phosphoric acid acetonitrile (B); detector: ultraviolet detector (UV) 237nm; column oven: 35°C; injection volume: 10μL; elution conditions: flow rate 1.0mL / min, gradient elution program: 0min, 38%B; 0-3min, 38-50%B; 3-10min, 50-52%B; 10-20min, 52-85%B; 20-30min, 85-90%B; 30-40min, 90-38%B. The peak time of glycyrrhizic acid is 6.8min, and the peak time of glycyrrhetinic acid is 22.6min.

[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 shown.

[0107] Table 1 Contents of components of Bifidobacterium animalis subsp. lactis CCFM1363 after fermentation in medium containing licorice extract

[0108] Content of each component (μg / mL) Fermentation 0h Fermentation 48h Glycyrrhizic acid 239.45±5.61 149.87±4.51 Glycyrrhetinic 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 48h of fermentation, the glycyrrhizic acid content in the fermentation medium was reduced by 37.41% compared with that before fermentation. It can be seen that after the licorice extract was fermented with the animal Bifidobacterium lactis subsp. lactis of the present invention, the licorice extract was largely converted into other active small molecules such as glycyrrhetinic acid.

[0111] Example 3: Metabolite analysis of licorice extract before and after fermentation with Bifidobacterium animalis subsp. lactis CCFM1363

[0112] 1. Metabolite extraction

[0113] Pipette 100 μL of liquid sample into an EP tube, add 400 μL of extract (methanol:acetonitrile = 1:1 (v / v)), the extract contains isotope-labeled internal standard; vortex mix for 30 seconds, ultrasonic for 10 minutes (ice water bath); let stand at -40℃ for 1 hour; centrifuge the sample at 4℃, 12000rpm (centrifugal force 13800 (×g), radius 8.6cm) for 15 minutes, and take the supernatant; centrifuge the supernatant at 4℃, 12000rpm (centrifugal force 13800 (×g), radius 8.6cm) for 15 minutes; take the supernatant into the injection bottle for detection; take an equal amount of supernatant from all samples and mix them into QC samples for detection.

[0114] 2. On-machine testing

[0115] For non-polar metabolites, this project used Vanquish (Thermo Fisher Scientific) ultra-high performance liquid chromatograph, and the target compounds were chromatographed on a Phenomenex Kinetex C18 (2.1mm×50mm, 2.6μm) liquid chromatography column. The liquid chromatography phase A was aqueous phase containing 0.01% acetic acid, and the phase B was isopropanol: acetonitrile (1:1, v / v). Column temperature: 25℃, sample plate temperature: 4℃, injection volume: 2μL. The Orbitrap Exploris 120 mass spectrometer was able to perform primary and secondary mass spectrometry data acquisition under the control of the control software (Xcalibur, version: 4.4, Thermo). The detailed parameters are as follows: Sheath gas flow rate: 50Arb, Aux gas flow rate: 15Arb, Capillary temperature: 320℃, Sweep Gas: 1Arb, Vaporizer Temp: 350℃. Full ms resolution: 60000, MS / MS resolution: 15000, Collision energy: SNCE 20 / 30 / 40, Spray Voltage: 3.8kV (positive) or -3.4kV (negative).

[0116] The information, including compound name, molecular formula, molecular weight, retention time, etc., was collected by consulting the data and combining with the online Chinese medicine database. The database was imported into CompoundDiscoverer 3.3 software, and the collected data were analyzed by using CompoundDiscoverer 3.3 software. The possible structure of the compound was inferred by studying the fragment ions, loss groups, mass spectrometry fragmentation rules, etc. of each molecular ion in the high collision energy channel. Differential metabolites were screened based on P-value<0.05 and Log2 Fold Change>2. After licorice extract was fermented by Lactobacillus crispatus CCFM1362 for 48 hours, the metabolites were mainly terpenes, shikimic acid and phenylpropionic acid, fatty acids, amino acids and short peptides.

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

[0118] Molecular weight Molecular formula name <![CDATA[log2foldchange]]> Changes after fermentation 821.3975 <![CDATA[C 42 H 62 O 16 ]]> Glycyrrhizic acid -0.956 - 629.3663 <![CDATA[C 36 H 54 O 10 ]]> Glycyrrhetinic acid 3-O-glucuronide 8.563 + 471.3452 <![CDATA[C 30 H 46 O4]]> Glycyrrhetinic acid 5.878 + 551.1751 <![CDATA[C 26 H 30 O 13 ]]> Isoliquiritigenin 3.826 + 353.1008 <![CDATA[C 20 H 16 O6]]> Licorice Isoflavone B 4.436 + 287.0908 <![CDATA[C 16 H 14 O5]]> Licoricechalcone B 3.627 + 255.067 <![CDATA[C 15 H 12 O4]]> Licorice 3.026 + 285.075 <![CDATA[C 16 H 12 O5]]> 5-O-Methylgenistein 5.572 + 195.0668 <![CDATA[C 10 H 12 O4]]> Hydroxytyrosol acetate 4.436 + 405.1717 <![CDATA[C 25 H 26 O5]]> 6',8'-Diisopentenylgenistein 2.161 + 301.0697 <![CDATA[C 16 H 12 O6]]> Trifoliol 3.627 + 455.354 <h2 style=";text-align:left;direction:ltr"><![CDATA[C <h2 style=";text-align:left;direction:ltr"> 30 <h2 style=";text-align:left;direction:ltr"> H<h2 style=";text-align:left;direction:ltr"> 48 <h2 style=";text-align:left;direction:ltr"> O3]]><h2 style=";text-align:left;direction:ltr"> Ursolic acid 3.314 +

[0119] Example 4: Effects of a synbiotic preparation prepared from Bifidobacterium animalis subsp. lactis CCFM1363 and postbiotics from licorice fermentation on body weight control in mice fed a high-fat diet

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

[0121] The experiment lasted for 13 weeks: after the mice adapted for one week, the blank group was fed with a low-fat and low-sugar diet from the second week, and the other groups were fed with a high-fat diet. The modeling period was 8 weeks. From the 10th week, each experimental group was gavaged with a mixture of strain freeze-dried powder and licorice extract or fermented freeze-dried powder (dissolved in normal saline at the corresponding dose) at a volume of 0.2 mL / mouse / day. The blank group and the model group were gavaged with the same amount of normal saline as a control until the end of the experiment. All groups had free access to water and food.

[0122] After the intervention, the mice's mental state was observed regularly every day and the mice were weighed every week. Figure 2 shown.

[0123] Depend on Figure 2 It can be seen that the weight growth rate of mice gavaged with animal Bifidobacterium lactis subsp. CCFM1363 and licorice extract alone was 14.632% and 14.152% lower than that of the model group, which was not as good as the 22.455% and 40.072% of the synbiotic preparation and licorice fermentation postbiotics groups. After the drug intervention, the synbiotic preparation made of animal Bifidobacterium lactis subsp. CCFM1363 fermented licorice extract and licorice fermentation postbiotics both had the effect of alleviating the weight gain of mice, and the licorice fermentation postbiotic group showed the best weight control effect.

[0124] Table 3 Experimental animal groups

[0125]

[0126] Example 5: Effects of a synbiotic preparation prepared from Bifidobacterium animalis subsp. lactis CCFM1363 and licorice fermentation postbiotics on the level of aging-related secretory phenotypes in mice

[0127] The mouse grouping, modeling and treatment methods were the same as in Example 4.

[0128] Part of the frozen liver tissue was weighed, added to PBS (pH 7.4, 4°C) at a ratio of 1:9 (m / V), homogenized, and centrifuged at 4°C and 12,000×g for 30 min. The supernatant was collected and the IL-1β and IL-6 levels were determined according to the instructions of the corresponding Elisa kit.

[0129] Depend on Figure 3 It can be seen that the IL-1β level of mice gavaged with animal Bifidobacterium lactis subspecies CCFM1363 and licorice extract alone decreased by 22.176% and 19.901% compared with the model group, respectively, which was not as good as the 31.220% and 57.129% of the synbiotic preparations with equal doses and licorice fermentation postbiotics. In addition, at the IL-6 level, the IL-6 level of mice gavaged with animal Bifidobacterium lactis subspecies CCFM1363 and licorice extract alone decreased by 10.644% and 5.129% compared with the model group, respectively, which was not as good as the 40.382% and 25.249% of the synbiotic preparations with equal doses and licorice fermentation postbiotics. After drug intervention, the synbiotic preparations made from animal Bifidobacterium lactis subspecies CCFM1363 fermented licorice extract and licorice fermentation postbiotics both had the effect of reducing the level of aging-related secretory phenotypes in mice, indicating that the drug has strong potential in the field of anti-aging.

[0130] Example 6: Effect of a synbiotic preparation prepared from animal Bifidobacterium lactis subspecies CCFM1363 and postbiotics from licorice fermentation on the content of β-galactosidase (β-GAL) in mice

[0131] The mouse grouping, modeling and treatment methods were the same as in Example 4.

[0132] Part of the frozen liver tissue was weighed, and PBS (pH 7.4, 4°C) was added at a ratio of 1:9 (m / V), homogenized, and centrifuged at 4°C and 12,000×g for 30 min. The supernatant was collected and the level of senescence-related β-galactosidase was determined according to the instructions of the corresponding Elisa kit.

[0133] In aging cells, the number and size of lysosomes increase, and β-galactosidase, as an enzyme directly related to the quality level of lysosomes, is an important biomarker for characterizing the level of aging. Figure 4 It can be seen that compared with the model group, the β-galactosidase content of mice gavaged with animal Bifidobacterium lactis subsp. CCFM1363 and licorice extract alone decreased by 7.782% and 17.056%, which was not as good as the 24.644% and 29.431% of the same dose of synbiotic preparation and licorice fermentation postbiotics. After the drug intervention, the synbiotic preparation made of animal Bifidobacterium lactis subsp. CCFM1363 fermented licorice extract and licorice fermentation postbiotics both had the effect of alleviating the increase of aging markers in mice, and the licorice fermentation postbiotic group showed the best anti-aging control effect.

[0134] Example 7: Effects of a synbiotic preparation prepared from Bifidobacterium animalis subsp. lactis CCFM1363 and postbiotics from licorice fermentation on fat accumulation and lesions in the liver of mice fed a high-fat diet

[0135] The mouse grouping, modeling and treatment methods were the same as in Example 4.

[0136] After the experiment, blood was collected and the mice were killed. The liver tissue was fixed in 4% paraformaldehyde solution for 24 hours and then stained with hematoxylin-eosin (H&E) to evaluate the degree of liver fatty degeneration 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 in Figure 5 , Figure 6 .

[0137] Depend on Figure 5 , Figure 6 It is known that the liver tissue of mice in the blank group and the licorice fermentation postbiotic administration group prepared by animal bifidobacterium lactis subspecies CCFM1363 is uniformly colored, with less fat accumulation, and its hepatocytes are tight and uniform, showing a regular shape. More fat accumulation occurs in the liver tissue of the remaining groups of mice, and obvious pathological changes occur, a large number of fat vacuoles around the cells, and the hepatocytes are loosely connected to each other, the cell contents flow out, the cells swell, and the cell integrity is destroyed. It can be seen from the experimental results that the licorice fermentation postbiotics prepared by animal bifidobacterium lactis subspecies CCFM1363 of the present invention and the licorice extract mixture or the prepared licorice fermentation postbiotics effectively inhibit the liver fat accumulation and lesions of mice on a high-fat diet, especially the postbiotic preparation significantly reduces the degree of liver damage and fatty lesions.

[0138] Example 8: Effects of a synbiotic preparation prepared from Bifidobacterium animalis subsp. lactis CCFM1363 and postbiotics from licorice fermentation on blood lipids in mice fed a high-fat diet

[0139] The mouse grouping, modeling and treatment methods were the same as in Example 4.

[0140] After the experiment, blood was collected and the mice were killed. The whole blood of the mice was allowed to stand for 1 hour and then centrifuged at 3000g for 15 minutes to obtain serum. The blood lipid-related indicators were tested using a fully automatic biochemical analyzer. The related indicators included: 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 can be seen that compared with the control group, the TC and TG contents of the model group increased significantly, indicating that the accumulation of lipids in the mice caused the increase of blood lipids. The results of drug intervention showed that compared with the model group, the mice group that were gavaged with animal Bifidobacterium lactis subspecies CCFM1363 and licorice extract alone had TC decreased by 6.301% and 9.032%, and TG decreased by 20.152% and 22.222%, respectively. The combined preparation and licorice fermentation postbiotic preparation groups reduced their contents to varying degrees, among which the licorice fermentation postbiotics fermented by animal Bifidobacterium lactis subspecies CCFM1363 and licorice extract decreased TC and TG indicators by 21.075% and 29.885%, respectively, and the combined preparation decreased TC and TG indicators by 8.081% and 29.502%, respectively.

[0142] At the same time, compared with the control group, the LDL-c and HDL-c levels in the model group were significantly higher than those in the blank group. The results of drug intervention showed that the LDL-c and HDL-c levels of the mice group gavaged with Bifidobacterium lactis subspecies CCFM1363 alone were reduced by 2.848% and 1.607% compared with the model group, and the LDL-c and HDL-c levels of the mice group gavaged with licorice extract alone were reduced by 15.913% and 0.952% compared with the model group, which were both inferior to the effects of the combined preparation and licorice fermentation postbiotics. Among them, the synergistic preparation had an effect of reducing LDL-c and HDL-c levels by 21.441% and 1.964%, respectively; the licorice fermentation postbiotics had an effect of reducing LDL-c and HDL-c levels by 22.111% and 18.869%, respectively, indicating that the synergistic preparation of animal Bifidobacterium lactis subspecies CCFM1363 and licorice extract and licorice fermentation postbiotics have great potential in lowering blood lipid levels. According to the four blood lipid indicators of mice, it is further shown that the synergistic preparation of animal Bifidobacterium lactis subspecies CCFM1363 and licorice extract and licorice fermentation postbiotics can reduce the increase in blood lipids caused by lipid accumulation.

[0143] Example 9: Effects of a synbiotic preparation prepared from Bifidobacterium animalis subsp. lactis CCFM1363 and postbiotics from licorice fermentation on liver function in mice fed a high-fat diet

[0144] The mouse grouping, modeling and treatment methods were the same as in Example 3.

[0145] After the experiment, blood was collected and the mice were killed. The whole blood of the mice was allowed to stand for 1 hour and then centrifuged at 3000g for 15 minutes to obtain serum. The liver function related indicators were tested using a fully automatic biochemical analyzer. The related indicators included: ALT (alanine aminotransferase) and AST (aspartate aminotransferase). The results are as follows Figure 8 .

[0146] Depend on Figure 8It can be seen that compared with the control group, the ALT and AST contents of the modeling group were significantly increased compared with the control group, indicating that the lipid accumulation caused by the high-fat model caused chronic inflammation in the mouse liver. The ALT and AST contents in the serum of the mice in the synbiotic preparation and licorice fermentation postbiotic intervention groups were reduced to varying degrees compared with the model group, indicating that the inflammatory response caused by liver lipid accumulation can be reduced, among which the synbiotic preparation and licorice fermentation postbiotic preparation reduced the AST content by 29.428% and 28.457%, respectively, and reduced the ALT content by 42.588% and 37.825%, respectively; the experimental results show that the mixture of animal Bifidobacterium lactis subspecies CCFM1363 and licorice extract of the present invention and the prepared licorice fermentation postbiotics effectively reduced the serum biochemical index levels related to obesity and fatty liver in mice on a high-fat diet, which is beneficial to alleviate obesity and fatty liver.

[0147] Example 10: Effects of a synbiotic preparation prepared from Bifidobacterium animalis subsp. lactis CCFM1363 and postbiotics from licorice fermentation on the expression of genes related to fat metabolism in mice fed a high-fat diet

[0148] The mouse grouping, modeling and treatment methods were the same as in Example 4.

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

[0150] Table 4 Real-time fluorescence quantitative PCR detection primer sequences

[0151]

[0152] Depend on Fig. 9 It can be seen that compared with the blank group, the relative gene expression levels of PPAR-γ and FASN in the modeling group were significantly increased, while the relative gene expression levels of PPAR-α, ATGL, HSL, and MGL were decreased, indicating that the high-fat diet significantly promoted lipid synthesis in the mouse liver and inhibited lipid decomposition, which also explains the phenomenon of fat accumulation in the liver of mice fed a high-fat diet.

[0153] The synbiotic preparation made from animal Bifidobacterium lactis subspecies CCFM1363 and licorice extract and the postbiotic preparation made from licorice fermentation can increase the expression of MGL, HSL, ATGL, and PPAR-α genes, and reduce the expression of PPAR-γ and Fasn genes to varying degrees, thereby reducing lipid accumulation in the body.

[0154] Among them, compared with the model group, after the intervention of the synbiotic preparation, the levels of PPAR-γ and FASN decreased by 16.732% and 39.631%, and the levels of ATGL, Mgl, Hsl, and PPAR-α genes increased by 282.479%, 55.149%, 363.935%, and 55.032%; after the intervention of licorice fermentation postbiotics, the levels of PPAR-γ and FASN decreased by 47.273% and 42.045%, and the levels of ATGL, Mgl, Hsl, and PPAR-α genes increased by 330.769%, 73.268%, 356.428%, and 19.600%, respectively. The effects of the synbiotic preparation and licorice fermentation postbiotics were better than those of gavage of animal Bifidobacterium lactis subspecies CCFM1363 or licorice extract alone. It shows that the combination of animal Bifidobacterium lactis subspecies CCFM1363 and licorice extract or licorice fermentation postbiotics can increase the expression of MGL, HSL, ATGL, and PPAR-α genes, and reduce the expression of PPAR-γ and Fasn genes to reduce lipid accumulation in the body.

[0155] The comparative analysis of the above results showed that the animal Bifidobacterium lactis subsp. CCFM1363 + licorice extract group could inhibit the weight gain under high-fat diet, reduce the levels of IL-1β and IL-6, reduce the β-galactosidase content in the liver, reduce the levels of TC, TG, HDL-c and LDL-c in serum, reduce the damage of liver tissue caused by lipid accumulation, enhance the expression of lipid metabolism-related genes MGL, HSL, ATGL and PPAR-α, reduce the expression of lipid synthesis-related genes PPAR-γ and Fasn, and improve the blood glucose homeostasis level in mice; and the effect was significantly better than that of single strain CCFM1363 and single licorice extract. Animal Bifidobacterium lactis subsp. CCFM1363 had multi-target anti-aging effect and transformed licorice to enhance the effect.

[0156] Although the present invention has been disclosed as above in the form of a preferred embodiment, it is not intended to limit the present invention. Anyone familiar with this technology can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be based on the definition of the claims.

Claims

1. A strain of Bifidobacterium animalis subsp. lactis CCFM1363, which was deposited in Guangdong Provincial Microbiological Culture Collection Center on December 6, 2023, with a deposit number of GDMCC No: 64111.

2. The postbiotic prepared by the animal Bifidobacterium lactis subspecies CCFM1363 according to claim 1, characterized in that: The postbiotics are obtained by fermenting the animal Bifidobacterium lactis subspecies CCFM1363 in a culture medium containing licorice extract, inactivating the fermentation liquid, and then homogenizing it.

3. A synbiotic preparation prepared from the animal Bifidobacterium lactis subspecies CCFM1363 according to claim 1, characterized in that: The synbiotic preparation contains the animal Bifidobacterium lactis subspecies CCFM1363 and a licorice product; the licorice product includes licorice or a licorice extract.

4. A product containing the postbiotics according to claim 2 or the synbiotic preparation according to claim 3, characterized in that: The products include food, medicine or health products.

5. The product according to claim 4, characterized in that The product is a freeze-dried powder of the postbiotic or synbiotic preparation, or a reconstituted product containing the freeze-dried powder of the postbiotic or synbiotic preparation.

6. A method for degrading glycyrrhizic acid, characterized in that: The animal Bifidobacterium lactis subspecies CCFM1363 described in claim 1 is added to a culture medium containing glycyrrhizic acid for fermentation.

7. A method for preparing glycyrrhetinic acid, characterized in that: The animal Bifidobacterium lactis subspecies CCFM1363 described in claim 1 is added to a culture medium containing glycyrrhizic acid for fermentation.

8. A method for enhancing the medicinal efficacy of licorice, characterized in that: The method is any of the following: (1) adding the animal Bifidobacterium lactis subspecies CCFM1363 described in claim 1 to a culture medium containing a licorice product for fermentation; (2) mixing the animal Bifidobacterium lactis subspecies CCFM1363 described in claim 1 with a liquorice product; The licorice product comprises licorice or licorice extract.

9. Use of the animal Bifidobacterium lactis subspecies CCFM1363 and / or its postbiotics and / or its synbiotic preparations as described in claim 1 in the preparation of lipid-lowering and / or anti-aging drugs.

10. The use according to claim 9, characterized in that: The drug has at least one of the following functions: (1) Reduce weight gain in response to a high-fat diet; (2) reduce the levels of IL-1β and IL-6, the secretory phenotype associated with aging; (3) Reduce the body's β-galactosidase content; (4) Inhibit liver fat accumulation and damage; (5) Reduce blood lipid levels; (6) Increase the expression of lipid metabolism-related genes MGL, HSL, ATGL, and PPAR-α, and reduce the expression of lipid synthesis-related genes PPAR-γ and Fasn.

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