Lactobacillus paracasei capable of converting liquorice to enhance liquorice to relieve lipid accumulation, oxidative damage and inflammatory response

Abstract

The invention discloses a Lactobacillus paracasei strain capable of converting licorice and enhancing the licorice to relieve lipid accumulation, oxidative damage and inflammatory response, and belongs to the technical field of microorganisms and the technical field of medicines. The lactobacillus paracasei CCFM1361 can convert glycyrrhizic acid, increase the content of glycyrrhetinic acid, glycyrrhetinic acid 3-O-glucuronide, ursolic acid and other substances, and increase the efficacy of the licorice extract, which is specifically embodied in (1) inhibiting the weight gain of high fat diet mice; (2) reducing liver lipid accumulation and lesion caused by the liver lipid accumulation; (3) the anti-oxidation level of the liver of the high-fat diet mouse is improved; (4) the inflammatory reaction caused by lipid accumulation is improved; and (4) reducing the inflammation level of the organism by activating an NF-kappa B p65 related pathway. Therefore, the lactobacillus paracasei CCFM1361 and glycyrrhizic acid have huge application prospects in production of products with the efficacy of resolving lipid accumulation and oxidative damage and inflammatory reaction caused by lipid accumulation.

Description

Technical Field

[0001] The invention relates to a Lactobacillus paracasei strain capable of transforming liquorice to enhance the liquorice's ability to alleviate lipid accumulation, oxidative damage and inflammatory response, and belongs to the fields of microbial technology and medical technology. Background Art

[0002] Metabolic chronic diseases are recognized as the current global problem. Metabolic chronic diseases are mainly caused by metabolic disorders of the three basic nutrients: fat, protein, and sugar. Unhealthy diet and lifestyle habits can cause metabolic disorders in the body, which in turn lead to the accumulation of lipids, and ultimately cause a series of diseases such as hyperlipidemia, cardiovascular disease, fatty liver, and diabetes. The occurrence and development of obesity and visceral fat accumulation are regulated by multiple signal pathways, such as: fatty acid synthesis pathways participate in the synthesis and decomposition of body fat; PPAR participates in adipocyte differentiation and lipid metabolism; adipose tissue affects the body's metabolism of fat through a variety of cytokines. The ingredients that play a role in medicine and food are mainly flavonoids, polyphenols, saponins, terpenes, etc., while most of the "active ingredients" in herbs have the disadvantages of low bioavailability or low activity. Their efficacy depends on the role of the flora, so the transformation and modification of specific strains are required to improve the efficacy and stability of the active ingredients to achieve better fat reduction effects.

[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 effects. In addition, the application potential of licorice extracts in lipid-lowering has received widespread attention. The triterpenoid saponin compounds in licorice are considered to be its main pharmacological substances, mainly including glycyrrhizic acid (GL), glycyrrhetinic acid monoglucuronic 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 as lipid-lowering 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 paracasei CCFM1361 in promoting the effect of licorice extract in alleviating oxidative damage and inflammatory response caused by liver lipid accumulation.

[0006] Another object of the present invention is to provide a fermented licorice extract and composition that can enhance and alleviate oxidative damage and inflammatory response caused by lipid accumulation in the liver.

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

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

[0009] The invention provides a strain of Lactobacillus paracasei CCFM1361, wherein the Lactobacillus paracasei CCFM1361 has been deposited in Guangdong Provincial Microbiological Culture Collection Center on November 9, 2023, with a deposit number of GDMCC No: 63994.

[0010] In one embodiment of the present invention, after the Lactobacillus paracasei CCFM1361 is cultured on MRS medium for 48 hours, the colony is round, convex or lens-shaped, slightly white, opaque, and has a smooth to mucous soft surface. The Lactobacillus paracasei CCFM1361 fermentation of licorice extract can convert glycyrrhizic acid and significantly increase the content of glycyrrhetinic acid, thereby promoting the licorice extract to play a role in alleviating oxidative damage and inflammatory response caused by liver lipid accumulation. Specifically, it is embodied in: (1) significantly inhibiting the weight gain of mice fed a high-fat diet; (2) reducing liver lipid accumulation and the lesions caused by it; (3) improving the liver antioxidant level of mice fed a high-fat diet; (4) improving the inflammatory response caused by lipid accumulation; (5) reducing the body's inflammation level by activating the NF-κB p65 related pathway.

[0011] The present invention also provides postbiotics prepared by the Lactobacillus paracasei CCFM1361, wherein the postbiotics include inactivated bacteria and / or metabolites of the Lactobacillus paracasei CCFM1361.

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

[0013] (1) activating Lactobacillus paracasei CCFM1361 to obtain a bacterial liquid;

[0014] (2) inoculating the bacterial solution of (1) into a fermentation base material containing licorice extract to carry out fermentation;

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

[0016] In one embodiment of the present invention, step (1) is to streak Lactobacillus paracasei CCFM1361 on MRS solid culture medium, culture the plate inverted (preferably cultured inverted 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.

[0017] 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).

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

[0019] 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).

[0020] 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).

[0021] 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).

[0022] The invention also provides a synbiotic preparation prepared by Lactobacillus paracasei CCFM1361, which comprises Lactobacillus paracasei CCFM1361 and a liquorice extract.

[0023] The present invention also provides a product containing the postbiotics or the synbiotic preparation.

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

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

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

[0027] 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.

[0028] 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:

[0029] (1) extracting effective ingredients from licorice to obtain an extract, and then mixing the extract with the Lactobacillus paracasei CCFM1361;

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

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

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

[0033] The present invention also provides the use of the Lactobacillus paracasei CCFM1361, or the postbiotics, or the synbiotic preparation in preparing a medicine for alleviating lipid accumulation, oxidative damage and / or inflammatory response.

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

[0035] (a) Significantly inhibited weight gain in subjects fed a high-fat diet;

[0036] (b) Reduce liver lipid accumulation and the lesions it causes;

[0037] (c) improve liver antioxidant levels in subjects fed a high-fat diet;

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

[0039] (e) Reduce the body's inflammation level by activating the NF-κB p65-related pathway.

[0040] The present invention also provides a method for promoting the increase of the content of glycyrrhetinic acid in a licorice extract, which comprises adding Lactobacillus paracasei CCFM1361 into a system containing the licorice extract.

[0041] Beneficial effects:

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

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

[0044] Lactobacillus paracasei CCFM1361 and licorice extract can be used to produce products that can relieve oxidative damage caused by liver lipid accumulation and improve inflammatory response. 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, mild reaction, etc. The process is suitable for large-scale industrial production. Therefore, the use of Lactobacillus paracasei CCFM1361 in products containing licorice extract has great application prospects and application basis.

[0045] Biomaterial Deposit

[0046] Lactobacillus paracasei CCFM1361, taxonomically named Lactobacillus paracasei, was deposited in the Guangdong Provincial Microbiological Culture Collection Center on November 9, 2023, with the collection number GDMCCNo: 63994, and the collection address is Building 59, No. 100 Xianlie Middle Road, Guangzhou. BRIEF DESCRIPTION OF THE DRAWINGS

[0047] Figure 1 : Liquid chromatograms of 0h and 48h of liquorice extract fermented by Lactobacillus paracasei CCFM1361 and liquid chromatograms of glycyrrhizic acid and glycyrrhetinic acid standards;

[0048] Figure 2 : The weight gain of mice in different groups after drug intervention;

[0049] Figure 3 :H&E stained tissue sections of livers of mice in different groups;

[0050] Figure 4 : Oil red O stained tissue sections of liver of mice in different groups;

[0051] Figure 5 : Levels of four blood lipids: TG, TC, LDL-c, and HDL-c in mice of different groups;

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

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

[0054] “*” indicates a significant difference compared with the Model group (P<0.05), and “**” indicates an extremely significant difference compared with the Model group (P<0.01). DETAILED DESCRIPTION

[0055] The C57BL / 6J mice involved in the following examples were purchased from Beijing Weitonglihua Company.

[0056] The culture medium involved in the following examples is as follows:

[0057] 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.

[0058] 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.

[0059] 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.

[0060] 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.

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

[0062] (1) activating Lactobacillus paracasei CCFM1361 to obtain a bacterial liquid;

[0063] (2) Inoculate the bacterial solution from (1) into MRS medium, with the number of viable bacteria being 5×10 7 CFU / mL, culture to the logarithmic growth phase of the strain to obtain the bacterial solution of Lactobacillus paracasei CCFM1361;

[0064] (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;

[0065] (4) Adjust the dose according to the mouse weight (in kg) and mix into 5×10 9 CFU Lactobacillus paracasei CCFM1361 + 78 mg / kg licorice extract is obtained.

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

[0067] (1) Streak Lactobacillus paracasei CCFM1361 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 heat treated at 65°C for 30 min, and then high-pressure homogenized at 1200 Bar for 6 times to obtain the obtained postbiotic solution. The obtained postbiotic solution was freeze-dried and resuspended in physiological saline according to the required dose before oral administration.

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

[0071] The specific steps are as follows:

[0072] 1. Screening

[0073] The sample comes from healthy human feces. After pretreatment, the sample is stored in a -80°C refrigerator in 20% glycerol. After being taken out and thawed, it is mixed and 0.5 mL of the sample is pipetted and added to 4.5 mL of normal saline. The normal saline is used for gradient dilution. The appropriate gradient dilution liquid is selected and spread on MRS solid culture medium, and cultured at 37°C for 48 hours. The typical colony of Lactobacillus paracasei is picked and streaked on MRS solid culture medium for purification. A single colony is picked and transferred to MRS liquid culture medium for bacterial enrichment, and preserved in 30% glycerol to obtain a strain named CCFM1361; wherein, the typical colony of Lactobacillus paracasei is round, white and smooth.

[0074] 2. Identification

[0075] The genome of strain CCFM1361 was extracted, and the 16S rDNA of strain CCFM1361 was amplified and sequenced (performed by Suzhou Jinweizhi Biotechnology Co., Ltd., and the nucleotide sequence of 16S rDNA amplified by CCFM1361 is shown in SEQ ID NO.1). The sequence was compared with the nucleic acid sequence in NCBI, and the results showed that the strain was Lactobacillus paracasei, and was named Lactobacillus paracasei CCFM1361.

[0076] 3. Preservation of strains

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

[0078] 4. Strain preservation

[0079] The Lactobacillus paracasei CCFM1361 was deposited in the Guangdong Provincial Microbiological Culture Collection Center (GDMCC) on November 9, 2023, with a collection number of GDMCC No: 63994, and the collection address is 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou.

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

[0081] 1. Fermentation method:

[0082] (1) The Lactobacillus paracasei CCFM1361 in Example 1 was streaked on an MRS solid 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 medium and cultured at 37° C. for 48 h to prepare a seed solution.

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

[0084] 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.

[0085] 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.

[0086] The contents of glycyrrhizic acid and glycyrrhetinic acid in the fermentation broth were calculated using the external standard method. The results are shown in Table 1. Figure 1 shown.

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

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

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

[0090] The results showed that after 48h of fermentation, the glycyrrhizic acid content in the fermentation medium was reduced by 32.93% compared with that before fermentation. It can be seen that after the licorice extract was fermented with Lactobacillus paracasei of the present invention, the licorice extract was largely converted into other active small molecules such as glycyrrhetinic acid.

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

[0092] 1. Metabolite extraction

[0093] 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.

[0094] 2. Machine testing

[0095] 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).

[0096] 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 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 48h of fermentation of glycyrrhizic acid by Lactobacillus paracasei CCFM1361, the metabolites were mainly terpenes, shikimic acid and phenylpropionic acid, fatty acids, amino acids and short peptides.

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

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

[0099] Example 4: Effects of a synbiotic preparation prepared from Lactobacillus paracasei CCFM1361 and fermented licorice postbiotics on weight gain in mice fed a high-fat diet

[0100] 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 paracasei group (5×10 9 CFU Lactobacillus paracasei CCFM1361), Lactobacillus paracasei-licorice extract combination preparation group (5×10 9 CFU Lactobacillus paracasei CCFM1361, 78 mg / kg licorice extract), Lactobacillus paracasei-fermented licorice postbiotic group (78 mg / kg licorice extract, calculated based on the concentration of licorice extract before fermentation).

[0101] 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 (control 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 (Table 1), 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.

[0102] After the intervention, the mice's mental state was observed regularly every day and the mice were weighed every week. Figure 2 shown.

[0103] Depend on Figure 2 It can be seen that the weight growth rate of mice gavaged with Lactobacillus paracasei CCFM1361 and licorice extract alone was 18.817% and 14.152% lower than that of the model group, which was not as good as the 36.247% and 24.414% of the synbiotic preparation and postbiotics groups. After the drug intervention, the synbiotic preparation and postbiotics prepared by fermenting licorice extract with Lactobacillus paracasei CCFM1361 both had the effect of alleviating the weight gain of mice, and the synbiotic preparation group showed the best weight control effect.

[0104] Table 3 Experimental animal groups

[0105]

[0106] Example 5: Effects of a synbiotic preparation prepared from Lactobacillus paracasei CCFM1361 and fermented licorice postbiotics on liver fat accumulation and pathological changes in mice fed a high-fat diet

[0107] The mouse grouping, modeling and treatment methods were the same as in Example 4.

[0108] 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 3 , Figure 4 .

[0109] Depend on Figure 3 , Figure 4 It can be seen that the liver tissue of mice in the blank group and the fermented licorice postbiotics administration group prepared by Lactobacillus paracasei CCFM1361 is evenly 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 fermented licorice postbiotics prepared by Lactobacillus paracasei CCFM1361 and the licorice extract mixture of the present invention or the preparation 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.

[0110] Example 6: Effects of a synbiotic preparation prepared from Lactobacillus paracasei CCFM1361 and fermented licorice postbiotics on blood lipid biochemical indices in mice fed a high-fat diet

[0111] The mouse grouping, modeling and treatment methods were the same as in Example 4.

[0112] 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 5 .

[0113] Depend on Figure 5It can be seen that compared with the control group, the TC and TG contents in 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 TC of the mice group that were gavaged with Lactobacillus paracasei CCFM1361 and licorice extract alone decreased by 5.411% and 9.932%, and the TG decreased by 12.261% and 22.222%, respectively; the synbiotic group and the fermented licorice postbiotic group both reduced their contents to varying degrees, among which the fermented postbiotics of Lactobacillus paracasei CCFM1361 and licorice extract decreased TC and TG indicators by 30.936% and 21.264%, respectively, and recovered to the same level as the control.

[0114] At the same time, compared with the control group, the levels of LDL-c and HDL-c in the model group were significantly higher than those in the blank group. The results of drug intervention showed that compared with the model group, the LDL-c and HDL-c of the group of mice gavaged with Lactobacillus paracasei CCFM1361 alone decreased by 4.702% and 6.700%, respectively. Compared with the model group, the LDL-c and HDL-c of the group of mice gavaged with licorice extract alone decreased by 15.913% and 0.952%, respectively, which were not as good as the effects of the combined preparation and fermented licorice postbiotics. Among them, the synergistic preparation had an effect of reducing LDL-c and HDL-c levels of 18.760% and 5.893%, and the fermented licorice postbiotics had an effect of reducing LDL-c and HDL-c levels of 45.729% and 19.048%, respectively, indicating that the synergistic preparation of Lactobacillus paracasei CCFM1361 and licorice extract and fermented licorice 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 Lactobacillus paracasei CCFM1361 and licorice extract and fermented licorice postbiotics can reduce the increase in blood lipids caused by lipid accumulation.

[0115] Example 7: Effects of a synbiotic preparation prepared by Lactobacillus paracasei CCFM1361 and fermented licorice postbiotics on liver antioxidant levels in mice fed a high-fat diet

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

[0117] 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 MDA, GSH, and SOD were determined according to the instructions of the corresponding kit. The results are shown in Figure 6 .

[0118] Depend on Figure 6It can be seen that compared with the blank control group, the GSH and SOD activities in the liver of the model group mice were significantly reduced, and the MDA content was significantly increased, indicating that lipid accumulation caused by a high-fat diet can cause oxidative damage to the liver of mice. GSH depletion and reduced SOD activity were significantly improved after the synbiotic preparation of Lactobacillus paracasei CCFM1361 and licorice extract and the intervention of fermented licorice postbiotics. Among them, the GSH-px levels in the livers of mice gavaged with licorice extract and Lactobacillus paracasei CCFM1361 alone were increased by 8.577% and 14.716% compared with the model group, respectively, and increased by 40.334% and 29.888% after the intervention of synbiotic preparations and fermented licorice postbiotics, respectively; in SOD levels, the SOD levels in the livers of mice gavaged with licorice extract and Lactobacillus paracasei CCFM1361 alone were increased by 47.489% and 24.553% compared with the model group, and increased by 54.655% and 83.541% after the intervention of synbiotic preparations and fermented licorice postbiotics, respectively; and in MDA levels, the MDA levels in the livers of mice gavaged with licorice extract and Lactobacillus paracasei CCFM1361 alone were decreased by 16.662% and 36.695% compared with the model group, respectively, and increased by 48.316% and 52.878% after the intervention of synbiotic preparations and fermented licorice postbiotics, respectively. It was shown that the combined preparation of Lactobacillus paracasei CCFM1361 and licorice extract and the fermented licorice postbiotic group can reduce the oxidative stress level caused by lipid accumulation in mice and have the ability to enhance the antioxidant level of the liver.

[0119] Example 8: Effects of a synbiotic preparation prepared from Lactobacillus paracasei CCFM1361 and fermented licorice postbiotics on chronic inflammation in mice fed a high-fat diet

[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 NF-κB p65, COX2, iNOS, IL-1β, and IL-6 were determined according to the instructions of the corresponding kits. The results are shown in Figure 7 .

[0122] Depend on Figure 7It 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 levels of its downstream inflammatory factors COX2, iNOS, IL-1β, and IL-6 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, iNOS, IL-1β, and IL-6 indicators decreased by 4.638%, 16.414%, 3.328%, 19.901%, and 5.134% respectively compared with the model group; in the group gavaged with Lactobacillus paracasei CCFM1361 alone, these indicators decreased by 10.962%, 19.873%, 15.785%, 35.228%, and 22.726% respectively compared with the model group; and after the intervention of the combined preparation of Lactobacillus paracasei CCFM1361 and licorice extract and fermented licorice postbiotics, the relief degree of multiple inflammatory indicators was stronger than that of the group gavage alone. After the intervention of the combined preparation of Lactobacillus paracasei CCFM1361-licorice extract, NF-κB Compared with the model group, the levels of p65, COX2, iNOS, IL-1β, and IL-6 decreased by 23.708%, 37.555%, 20.563%, 70.043%, and 58.529%; after the intervention of Lactobacillus paracasei CCFM1361-fermented licorice postbiotics, the levels of NF-κB p65, COX2, iNOS, IL-1β, and IL-6 decreased by 6.238%, 19.873%, 3.527%, 15.812%, and 45.952% compared with the model group. This indicates that both the combined preparation and the fermented licorice postbiotic group can reduce the level of chronic inflammation caused by lipid accumulation.

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

[0124] 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 paracasei CCFM1361, which was deposited in Guangdong Provincial Microbiological Culture Collection Center on November 9, 2023, with a deposit number of GDMCCNo: 63994.

2. The postbiotic prepared by Lactobacillus paracasei CCFM1361 according to claim 1, characterized in that The postbiotics include inactivated bacteria and / or metabolites of Lactobacillus paracasei CCFM1361.

3. The synbiotic preparation prepared by Lactobacillus paracasei CCFM1361 according to claim 1, characterized in that, The synbiotic preparation contains Lactobacillus paracasei CCFM1361 and a liquorice product; the liquorice product includes liquorice or a liquorice extract.

4. The method for preparing the postbiotics according to claim 2, characterized in that: Lactobacillus paracasei CCFM1361 was fermented in a culture medium containing licorice extract, and then the fermentation liquid was 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 licorice, characterized in that: Treat the licorice with any of the following: (1) extracting effective ingredients from licorice to obtain an extract, and then mixing the extract with the Lactobacillus paracasei CCFM1361 according to claim 1; (2) extracting effective ingredients from licorice to obtain an extract, and then adding the extract to the culture medium of Lactobacillus paracasei CCFM1361 according to claim 1 for fermentation.

8. A method for degrading glycyrrhizic acid or a method for preparing glycyrrhetinic acid, characterized in that: The Lactobacillus paracasei CCFM1361 according to claim 1 is added to a culture medium containing glycyrrhizic acid for fermentation.

9. Use of the Lactobacillus paracasei CCFM1361 according to claim 1, or the postbiotic according to claim 2, or the synbiotic preparation according to claim 3 in the preparation of a medicament for alleviating lipid accumulation, oxidative damage and / or inflammatory response.

10. The use according to claim 9, characterized in that: The drug has at least one of the following functions: (a) Inhibit weight gain in individuals who eat a high-fat diet; (b) Reduce liver lipid accumulation and the lesions it causes; (c) improve liver antioxidant levels in subjects fed a high-fat diet; (d) Improve the inflammatory response caused by lipid accumulation; (e) Reduce the body's inflammation level.

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