Lactobacillus crispatus for realizing anti-aging effect based on multiple target spots and application of lactobacillus crispatus for transforming liquorice to achieve synergism

Abstract

The invention discloses lactobacillus crispatus capable of realizing an anti-aging effect based on multiple target spots and application of the lactobacillus crispatus to conversion of liquorice for synergism, and belongs to the technical field of microorganisms and the technical field of medicines. The lactobacillus crispatus CCFM1362 provided by the invention can be used for fermenting a licorice extract, increasing the contents of glycyrrhetinic acid, semi-licoflavone B, naringenin chalcone and other substances and enhancing the effect of the licorice extract, so that the multi-target anti-aging effect is realized, which is specifically reflected as follows: (1) the content of beta-galactosidase in liver tissues is inhibited; (2) reducing the level of senescence-related secretion phenotypes in liver tissues; (3) activating an Nrf2 related pathway, and reducing the oxidative stress level; (4) NF-kappa B related pathways are activated, and the chronic inflammation level is reduced. Therefore, the lactobacillus crispatus CCFM1362 and the licorice extract have a huge application prospect in the production of products with anti-aging effects.

Description

Technical Field

[0001] The invention relates to a Lactobacillus crispatus strain for achieving anti-aging effects based on multiple targets and application of the strain in transforming liquorice for enhancing synergy, belonging to the fields of microbial technology and medical technology. Background Art

[0002] With the aging of the population, anti-aging research has become a hot topic in the fields of biomedicine and microbial technology. Aging is a complex and multi-factorial biological process involving oxidative stress, chronic inflammation, decreased metabolic function, and immune function decline. It not only leads to the gradual weakening of body functions, but also increases the risk of chronic diseases such as diabetes, cardiovascular disease, and neurodegenerative diseases. Traditional anti-aging methods mostly rely on chemical synthetic drugs, such as rapamycin and its derivatives, metformin, etc., but these drugs are often accompanied by certain side effects such as immunosuppression, metabolic abnormalities, potential renal toxicity, and gastrointestinal discomfort.

[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 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 Lactobacillus crispatus CCFM1362 in a product for enhancing the anti-aging effect of licorice extract.

[0006] Another object of the present invention is to provide a fermented licorice extract and composition that achieves enhanced anti-aging efficacy based on multiple targets.

[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 Lactobacillus crispatus CCFM1362, which was deposited in Guangdong Provincial Microbiological Culture Collection Center on November 9, 2023, with a deposit number of GDMCC No: 63995, and a deposit address of 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou.

[0010] In one embodiment of the present invention, after the Lactobacillus crispatus CCFM1362 is cultured on MRS medium for 48 hours, the colonies are round, convex or lens-shaped, slightly white, opaque, and have a smooth to mucous soft surface. The bacteria can significantly increase the content of glycyrrhizic acid by fermenting licorice extract with the bacteria, and promote the licorice extract to exert a multi-target anti-aging effect. Specifically, it is embodied in: (1) significantly reducing the content of β-galactosidase, a marker of aging, in mouse liver tissue; (2) significantly reducing the content of aging-related secretory phenotypes such as IL-1β and IL-6 in mouse liver tissue; (3) improving the antioxidant level of the mouse body; (4) activating the mouse Nrf2-related pathway and reducing the level of oxidative stress; (5) activating the mouse NF-κB-related pathway and reducing the level of chronic inflammation.

[0011] The present invention also provides a postbiotic prepared by the Lactobacillus crispatus CCFM1362, wherein the postbiotic comprises the inactivated bacteria and / or metabolites of the Lactobacillus crispatus CCFM1362.

[0012] The present invention also provides a synbiotic preparation prepared by the Lactobacillus crispatus CCFM1362, and the synbiotic preparation comprises the Lactobacillus crispatus CCFM1362 and a liquorice product.

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

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

[0015] (1) activating Lactobacillus crispatus CCFM1362 to obtain a bacterial liquid;

[0016] (2) inoculating the bacterial solution of (1) into a fermentation base material containing licorice extract to carry out fermentation;

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

[0018] In one embodiment of the present invention, step (1) is to streak Lactobacillus crispatus CCFM1362 on MRS solid culture medium, culture the plate inverted (preferably cultured 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.

[0019] 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).

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

[0021] 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).

[0022] 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).

[0023] 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).

[0024] The present invention also provides products containing the postbiotics or the synbiotic preparation, and the products include food, medicine or health care products.

[0025] 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.

[0026] The present invention also provides a method for enhancing the medicinal efficacy of licorice, which comprises subjecting the licorice to any of the following treatments:

[0027] (1) extracting effective ingredients from licorice to obtain an extract, and then mixing the extract with the Lactobacillus crispatus CCFM1362;

[0028] (2) extracting effective ingredients from liquorice to obtain an extract, and then adding the extract to the fermentation medium of the Lactobacillus crispatus CCFM1362 for culturing.

[0029] The present invention also provides a method for degrading glycyrrhizic acid or producing glycyrrhetinic acid, wherein the Lactobacillus crispatus CCFM1362 is added to a culture medium containing glycyrrhizic acid for fermentation.

[0030] The present invention provides application of Lactobacillus crispatus CCFM1362, its postbiotics and / or synbiotic preparations in preparing medicines capable of alleviating lipid accumulation.

[0031] The present invention provides application of Lactobacillus crispatus CCFM1362, its postbiotics and / or synbiotic preparations in preparing medicines capable of alleviating aging-related diseases.

[0032] The present invention provides application of Lactobacillus crispatus CCFM1362, its postbiotics and / or synbiotic preparations in preparing medicines capable of alleviating oxidative damage.

[0033] The present invention provides application of Lactobacillus crispatus CCFM1362, its postbiotics and / or synbiotic preparations in preparing medicines capable of alleviating inflammatory reactions.

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

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

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

[0037] (3) Improve the body’s antioxidant level;

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

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

[0040] In one embodiment of the present invention, the medicine contains Lactobacillus crispatus CCFM1362, a drug carrier and / or a pharmaceutical excipient.

[0041] The present invention also provides a method for promoting the efficacy of licorice extract, which is to add Lactobacillus crispatus CCFM1362 to a system containing licorice extract. The method is for non-disease diagnosis and treatment purposes. The specific method is: after adding Lactobacillus crispatus CCFM1362 to a system containing licorice extract, fermentation is performed.

[0042] The invention provides application of Lactobacillus crispatus CCFM1362, its postbiotics and / or synbiotic preparations in preparing health products capable of alleviating oxidative damage.

[0043] Lactobacillus crispatus is a common lactobacillus and an important component of the intestinal flora. It plays an important role in maintaining intestinal balance, inhibiting the growth and reproduction of harmful microorganisms in the intestine, resisting pathogen infection, and enhancing the body's immunity. Past studies have shown that Lactobacillus crispatus has many benefits for human health. Lactobacillus crispatus is also one of the most widely used lactobacillus species in the food industry today, such as Lactobacillus crispatus VB250, Lactobacillus crispatus CCFM1118 and Lactobacillus crispatus W962.

[0044] Therefore, the application of Lactobacillus crispatus CCFM1362 in licorice extract-containing products has great application prospects and application basis.

[0045] Beneficial effects:

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

[0047] 2. The synbiotic preparation prepared by Lactobacillus crispatus CCFM1362 and licorice extract of the present invention, or the postbiotics prepared by fermentation in licorice extract, can promote the efficacy of licorice extract, thereby achieving the effects of alleviating lipid accumulation, anti-aging, oxidative damage and / or inflammatory response.

[0048] Products with enhanced anti-aging effects can be produced using Lactobacillus crispatus CCFM1362 and licorice extract. The production process can use glycyrrhizic acid extract as raw material to promote the efficacy 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.

[0049] Biomaterial Deposit

[0050] Lactobacillus crispatus CCFM1362, taxonomically named Lactobacillus crispatus, was deposited in Guangdong Provincial Microbiological Culture Collection Center on November 9, 2023, with the deposit number GDMCCNo: 63995, and the deposit address is Building 59, No. 100 Xianlie Middle Road, Guangzhou. BRIEF DESCRIPTION OF THE DRAWINGS

[0051] Figure 1 : Liquid chromatograms of 0h and 48h of liquorice extract fermented by Lactobacillus crispatus CCFM1362 and liquid chromatograms of glycyrrhizic acid and glycyrrhetinic acid standards;

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

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

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

[0055] Figure 5 : Antioxidant levels in liver of mice in different groups;

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

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

[0058] “*” indicates a significant difference compared with the Model group (P<0.05), and “**” indicates an extremely significant difference compared with the Model group (P<0.01). DETAILED DESCRIPTION

[0059] The present invention will be further described below in conjunction with specific embodiments.

[0060] The C57BL / 6J mice involved in the following examples were purchased from Beijing Weitonglihua Company.

[0061] The culture medium involved in the following examples is as follows:

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

[0063] MRS solid 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, Tween-80 1 mL / L and agar 20.0 g / L, pH 6.2-6.4.

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

[0065] 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.

[0066] The preparation method of Lactobacillus crispatus-licorice fermentation postbiotics involved in the following embodiments is:

[0067] (1) Streak Lactobacillus crispatus CCFM1362 on MRS solid medium, invert and culture at 37°C for 48 hours, then pick a single colony and inoculate into 5 mL MRS liquid medium and culture at 37°C for 48 hours to obtain a bacterial solution;

[0068] (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;

[0069] (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.

[0070] The preparation method of the Lactobacillus crispatus-licorice fermentation synbiotic preparation involved in the following embodiments is:

[0071] (1) activating Lactobacillus crispatus CCFM1362 to obtain a bacterial liquid;

[0072] (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;

[0073] (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;

[0074] (4) Adjust the dose according to the mouse weight (in kg) and mix into 5×10 9 CFU Lactobacillus crispatus CCFM1362 + 78 mg / kg licorice extract is obtained.

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

[0076] The specific steps are as follows:

[0077] 1. Screening

[0078] The samples were derived from healthy human feces. After pretreatment, the samples were stored in a -80°C refrigerator in 20% glycerol. After being taken out and thawed, they were mixed and 0.5 mL of the sample was pipetted into 4.5 mL of normal saline, and gradient dilutions were performed with normal saline. Appropriate gradient dilutions were selected and spread on MRS solid culture medium, and cultured at 37°C for 48 hours. Typical colonies of Lactobacillus crispatus were picked and streaked on MRS solid culture medium for purification, and single colonies were picked and transferred to MRS liquid culture medium for bacterial enrichment, and preserved in 30% glycerol to obtain a strain named CCFM1362; among them, the typical colonies of Lactobacillus crispatus were round, white and smooth.

[0079] 2. Identification

[0080] The genome of strain CCFM1362 was extracted, and the 16S rDNA of strain CCFM1362 was amplified and sequenced (performed by Suzhou Jinweizhi Biotechnology Co., Ltd., and the nucleotide sequence of 16S rDNA amplified from CCFM1362 is shown in SEQ ID NO.1). The sequence was compared with the nucleic acid sequence in NCBI, and the results showed that the strain was Lactobacillus crispatus, and was named Lactobacillus crispatus CCFM1362.

[0081] 3. Preservation of strains

[0082] Lactobacillus crispatus CCFM1362 was inoculated into 5 mL MRS liquid culture medium and cultured 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.

[0083] 4. Strain preservation

[0084] It was deposited in the Guangdong Provincial Microbiological Culture Collection Center (GDMCC) on November 9, 2023, with the deposit number GDMCCNo: 63995, and the deposit address is 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou.

[0085] Example 2: Conversion of Licorice Extract by Lactobacillus crispatus CCFM1362

[0086] 1. Fermentation method:

[0087] (1) Streak Lactobacillus crispatus CCFM1362 in Example 1 on MRS solid culture medium, and incubate the plate upside down at 37° C. for 48 h; pick a single colony and inoculate it into 5 mL of MRS liquid culture medium and culture it at 37° C. for 48 h to prepare a seed solution.

[0088] (2) Add 4% (v / v) seed solution of Lactobacillus crispatus CCFM1362 (initial inoculation concentration is 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.

[0089] The fermentation broth was diluted 3 times with methanol and shaken to obtain the sample to be tested; 1 mL of the sample was taken and passed through a 0.22 μm organic phase filter membrane to obtain the solution to be tested.

[0090] 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.

[0091] The contents of glycyrrhizic acid and glycyrrhetinic acid in the fermentation broth were calculated using the external standard method. The results are shown in Table 1. Figure 1 shown.

[0092] Table 1 Contents of components of Lactobacillus crispatus CCFM1362 before and after fermentation in medium containing licorice extract

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

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

[0095] The results showed that after 48 hours of fermentation, the glycyrrhizic acid content in the fermentation medium was reduced by 39.58% compared with that before fermentation. It can be seen that after the licorice extract was fermented with Lactobacillus crispatus of the present invention, the licorice extract was largely converted into other active small molecules such as glycyrrhetinic acid.

[0096] Example 3: Metabolite analysis of licorice extract before and after fermentation by Lactobacillus crispatus CCFM1362

[0097] 1. Metabolite extraction

[0098] Pipette 100 μL of the liquid sample obtained in Example 2 into an EP tube, add 400 μL of extract (methanol:acetonitrile=1:1 (v / v)), the extract contains an isotope-labeled internal standard; vortex mix for 30 s, ultrasonicate for 10 min (ice water bath); let stand at -40°C for 1 h; centrifuge the sample at 4°C, 12000 rpm (centrifugal force 13800 (×g), radius 8.6 cm) for 15 min, and take the supernatant; centrifuge the supernatant at 4°C, 12000 rpm (centrifugal force 13800 (×g), radius 8.6 cm) for 15 min; take the supernatant into a sample injection bottle for machine detection; take an equal amount of supernatant from all samples and mix them into QC samples for machine detection.

[0099] 2. On-machine testing

[0100] 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).

[0101] 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.

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

[0103] Molecular weight Molecular formula name Log2FoldChange Changes after fermentation 821.3975 <![CDATA[C 42 H 62 O 16 ]]> Glycyrrhizic acid -0.486 - 629.3663 <![CDATA[C 36 H 54 O 10 ]]> Glycyrrhetinic acid 3-O-glucuronide 2.492 + 471.3452 <![CDATA[C 30 H 46 O4]]> Glycyrrhetinic acid 5.878 + 353.1008 <![CDATA[C 20 H 16 O6]]> Licorice Isoflavone B 5.572 + 287.0908 <![CDATA[C 16 H 14 O5]]> Licoricechalcone B 3.521 + 367.1192 <![CDATA[C 21 H 20 O6]]> 2',4',7-Trihydroxy-6'-methoxy-3'-isopentenyl isoflavone 3.361 + 405.1717 <![CDATA[C 25 H 26 O5]]> 6',8'-Diisopentenylgenistein 2.161 + 301.0697 <![CDATA[C 16 H 12 O6]]> Trifoliol 3.826 + 273.0754 <![CDATA[C 15 H 12 O5]]> 3',4',7'-Trihydroxyflavanone 3.382 + 271.0618 <![CDATA[C 15 H 12 O5]]> Naringenin Chalcone 6.218 + 257.08 <![CDATA[C 15 H 12 O4]]> Trihydroxychalcone 2.735 + 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 4.448 + 249.1864 <![CDATA[C 16 H 26 O2]]> Sclareolide 3.199 +

[0104] Example 4: Effects of a synbiotic preparation prepared from Lactobacillus crispatus CCFM1362 and postbiotics from licorice fermentation on weight gain in mice fed a high-fat diet

[0105] 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), Lactobacillus crispatus group (5×10 9 CFU Lactobacillus crispatus CCFM1362), Lactobacillus crispatus-licorice extract combination preparation group (5×10 9 CFU Lactobacillus crispatus CCFM1362 and 78 mg / kg licorice extract), Lactobacillus crispatus-licorice fermentation postbiotic group (78 mg / kg licorice extract, calculated based on the concentration of licorice extract before fermentation).

[0106] The experiment lasted for 13 weeks: After the mice adapted for one week, the blank group was fed a low-fat and low-sugar diet from the second week, and the other groups were fed 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 an equal amount of normal saline as a control until the end of the experiment. All groups were free to drink water and eat. After the experiment, the mice were dissected and the liver tissue was frozen for later use.

[0107] After the intervention, the mice's mental state was observed regularly every day and the mice were weighed every week. Figure 2 shown.

[0108] Depend on Figure 2 It can be seen that the weight growth rate of mice gavaged with Lactobacillus crispatus CCFM1362 and licorice extract alone was 4.678% and 14.152% lower than that of the model group, while the weight growth of the synbiotic preparation and licorice fermentation postbiotics groups was 23.952% and 28.785% lower than that of the model group, respectively, indicating that the synbiotic preparation made from licorice extract fermented with Lactobacillus crispatus CCFM1362 and licorice fermentation postbiotics both have the effect of enhancing the effect of alleviating weight gain in mice, and the licorice fermentation postbiotics group has a better effect.

[0109] Table 3 Experimental animal groups

[0110]

[0111] Example 5: Effects of a synbiotic preparation prepared from Lactobacillus crispatus CCFM1362 and licorice extract and licorice fermentation postbiotics on the content of β-galactosidase (β-GAL) in mice

[0112] The mouse grouping, modeling and treatment methods were the same as in Example 4.

[0113] 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 level of senescence-related β-galactosidase (β-GAL) was determined according to the instructions of the corresponding Elisa kit.

[0114] 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 3 It can be seen that the β-galactosidase content of mice gavaged with Lactobacillus crispatus CCFM1362 and licorice extract alone decreased by 3.644% and 17.056%, respectively, while that of the synbiotic preparation prepared by gavage with Lactobacillus crispatus CCFM1362 and licorice extract and the licorice fermentation postbiotic group was 22.384% and 25.214%, indicating that the synbiotic preparation prepared by fermentation of licorice extract with Lactobacillus crispatus CCFM1362 and the licorice fermentation postbiotic group both have the effect of reducing the β-galactosidase content of mice, and the licorice fermentation postbiotic group presents the best anti-aging control effect.

[0115] Example 6: Effects of a synbiotic preparation prepared from Lactobacillus crispatus CCFM1362 and licorice extract and licorice fermentation postbiotics on the aging-related secretory phenotype level in mice

[0116] The mouse grouping, modeling and treatment methods were the same as in Example 4.

[0117] 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.

[0118] Depend on Figure 4 It can be seen that the IL-1β levels of mice gavaged with Lactobacillus crispatus CCFM1362 and licorice extract alone decreased by 23.877% and 19.901% respectively compared with the model group, while the IL-1β levels in the liver of mice gavaged with Lactobacillus crispatus CCFM1362 and licorice extract combined preparation and licorice fermentation postbiotics decreased by 33.406% and 53.024% respectively. In addition, in terms of IL-6 levels, the IL-6 levels of mice gavaged with Lactobacillus crispatus CCFM1362 and licorice extract alone decreased by 16.895% and 5.129% respectively compared with the model group, the IL-6 levels decreased by 38.063% after gavage with the combined preparation of Lactobacillus crispatus CCFM1362 and licorice extract, and the IL-6 levels decreased by 51.422% after gavage with licorice fermentation postbiotics. After drug intervention, the synbiotic preparation prepared from licorice extract fermented by Lactobacillus crispatus CCFM1362 and licorice fermentation postbiotics both had the effect of reducing the level of aging-related secretory phenotype in mice, indicating that the drug has strong potential in the field of anti-aging.

[0119] Example 7: Effects of the synbiotic preparation prepared from Lactobacillus crispatus CCFM1362 and licorice extract and licorice fermentation postbiotics on Nrf2-related pathways and antioxidant indicators in mice

[0120] The mouse grouping, modeling and treatment methods were the same as in Example 4.

[0121] Part of the frozen liver tissue was weighed and 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 levels of Nrf2, MDA, GSH-px, and SOD were determined according to the instructions of the corresponding kits. The results are shown in Figure 5 , Figure 6 .

[0122] Depend on Figure 5It can be seen that compared with the blank control group, the activity of GSH-px and SOD in the liver of mice in the model group was significantly reduced, and the MDA content was significantly increased, indicating that lipid accumulation caused by a high-fat diet can cause oxidative damage in the liver of mice and inhibit the Nrf2 anti-oxidative stress-related pathway. After intervention with the combined preparation of Lactobacillus crispatus CCFM1362 and licorice extract and licorice fermentation postbiotics, the antioxidant-related indicators of mice were significantly improved. Among them, the GSH-px levels in the livers of mice gavaged with licorice extract and Lactobacillus crispatus CCFM1362 alone increased by 8.577% and 24.703% compared with the model group, respectively, while the combined preparation of Lactobacillus crispatus CCFM1362 and licorice extract and licorice fermentation postbiotics intervention increased by 27.813% and 42.276%, respectively; in the SOD level, the SOD levels in the livers of mice gavaged with licorice extract and Lactobacillus crispatus CCFM1362 alone increased by 47.489% and 22.87, respectively, compared with the model group. 1%, while the synbiotic preparation prepared by Lactobacillus crispatus CCFM1362 and licorice extract and the intervention with licorice fermentation postbiotics increased by 91.000% and 49.793%, respectively; in terms of MDA level, the MDA levels in the liver of mice gavaged with licorice extract and Lactobacillus crispatus CCFM1362 alone were reduced by 16.662% and 17.552% compared with the model group, while the synbiotic preparation prepared by Lactobacillus crispatus CCFM1362 and licorice extract and the intervention with licorice fermentation postbiotics increased by 44.918% and 47.308%, respectively.

[0123] At the Nrf2 level, Figure 6 It can be seen that the Nrf2 levels in the liver of mice gavaged with licorice extract and Lactobacillus crispatus CCFM1362 alone increased by 2.097% and 16.170% compared with the model group, respectively, and the synbiotic preparation prepared by Lactobacillus crispatus CCFM1362 and licorice extract and licorice fermentation postbiotics intervention increased by 24.724% and 33.186%, respectively. This shows that the synbiotic preparation of Lactobacillus crispatus CCFM1362-licorice extract and the licorice fermentation postbiotic group can reduce the oxidative stress level caused by lipid accumulation in mice, have the ability to improve the antioxidant level of the liver, and thus play an anti-aging effect.

[0124] Example 8: Effects of a synbiotic preparation prepared from Lactobacillus crispatus CCFM1362 and licorice extract and licorice fermentation postbiotics on chronic inflammation levels in mice

[0125] The mouse grouping, modeling and treatment methods were the same as in Example 4.

[0126] Part of the frozen liver tissue was weighed and 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 levels of NF-κB p65, COX2, and iNOS were determined according to the instructions of the corresponding kit. The results are shown in Figure 7 .

[0127] Depend on Figure 7 It can be seen that compared with the blank control group, NF-κB p65 in the liver of the model group mice was activated due to the chronic inflammation caused by lipid accumulation, and the content of its downstream inflammatory factors COX2 and iNOS also increased, indicating that lipid accumulation triggers chronic inflammation of the liver. In the group of mice gavaged with licorice extract alone, the NF-κB p65, COX2, and iNOS indicators decreased by 4.638%, 16.414%, and 3.328% respectively compared with the model group, which made it difficult to effectively regulate inflammatory-related factors; in the group gavaged with Lactobacillus crispatus CCFM1362 alone, these indicators decreased by 25.264%, 18.307%, and 10.409% respectively compared with the model group, which was higher than that gavage with licorice, indicating that the strain had a certain effect on regulating inflammation in mice; and after intervention with the synbiotic preparation prepared with Lactobacillus crispatus CCFM1362 and licorice extract and licorice fermentation postbiotics, the relief degree of multiple inflammatory indicators was stronger than that of the group gavaged with the strain alone. After intervention with the synbiotic preparation of Lactobacillus crispatus CCFM1362-licorice extract, NF-κB Compared with the model group, the levels of p65, COX2, and iNOS decreased by 28.032%, 32.335%, and 18.174%; after the intervention of Lactobacillus crispatus CCFM1362-licorice fermentation postbiotics, the levels of NF-κB p65, COX2, and iNOS decreased by 39.956%, 33.184%, and 20.364% compared with the model group, and all indicators returned to the same level as the control group. This shows that both the synbiotic preparation and the licorice fermentation postbiotic group can alleviate the level of chronic inflammation in the body, thereby exerting the potential of anti-aging.

[0128] The comparative analysis of the above results showed that the combined use of Lactobacillus crispatus CCFM1362 and licorice extract group can significantly inhibit the increase of β-galactosidase content in mouse liver tissue, and regulate the levels of aging-related secretion phenotypes such as IL-1β and IL-6 in liver tissue, enhance the body's antioxidant level, reduce the level of oxidative stress, and reduce the level of chronic inflammation; and the effect is significantly better than that of a single strain CCFM1362 or a single licorice extract. Lactobacillus crispatus CCFM1362 has the effect of achieving anti-aging at multiple targets and converting licorice to enhance synergy.

[0129] 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 Lactobacillus crispatus CCFM1362, which was deposited in Guangdong Provincial Microbiological Culture Collection Center on November 9, 2023, with a deposit number of GDMCC No: 63995.

2. The postbiotic prepared by the Lactobacillus crispatus CCFM1362 according to claim 1, characterized in that: The postbiotics include inactivated bacteria and / or metabolites of Lactobacillus crispatus CCFM1362.

3. The synbiotic preparation prepared by the Lactobacillus crispatus CCFM1362 according to claim 1, characterized in that: The synbiotic preparation comprises Lactobacillus crispatus CCFM1362 and a liquorice product; the liquorice product comprises liquorice or a liquorice extract.

4. The method for preparing the postbiotics according to claim 2, characterized in that: Lactobacillus crispatus CCFM1362 is fermented in a culture medium containing licorice extract, and then the fermentation liquid is inactivated and homogenized.

5. 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.

6. The product according to claim 5, 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.

7. A method for enhancing the medicinal efficacy of liquorice, characterized in that: Treat the licorice with any of the following: (1) extracting active ingredients from licorice to obtain an extract, and then mixing the extract with the Lactobacillus crispatus CCFM1362 according to claim 1; (2) extracting the active ingredients from liquorice to obtain an extract, and then adding the extract to the fermentation medium of Lactobacillus crispatus CCFM1362 according to claim 1 for culturing.

8. A method for degrading glycyrrhizic acid or producing glycyrrhetinic acid, characterized in that: The Lactobacillus crispatus CCFM1362 according to claim 1 is added to a culture medium containing glycyrrhizic acid for fermentation.

9. Use of the Lactobacillus crispatus CCFM1362 of claim 1, or the postbiotics of claim 2, or the synbiotic preparation of claim 3 in the preparation of a drug for anti-aging and / or relieving lipid accumulation and / or relieving oxidative damage and / or relieving inflammatory response, characterized in that: The drug has at least one of the following functions: (a) Inhibit the body's weight gain; (b) reducing the level of β-galactosidase, a marker of aging, in liver tissue; (c) reducing the level of senescence-associated secretory phenotype in liver tissue; the senescence-associated secretory phenotype includes IL-1β and IL-6; (d) Improve the body’s antioxidant level; (e) Improve the inflammatory response caused by lipid accumulation.

10. Use of the Lactobacillus crispatus CCFM1362 according to claim 1, or the postbiotic according to claim 2, or the synbiotic preparation according to claim 3 in the preparation of a health product with antioxidant function.

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