Lactobacillus paracasei, probiotic preparation for relieving non-alcoholic fatty liver and preparation method of probiotic preparation

Abstract

The invention relates to lactobacillus paracasei, a probiotic preparation for relieving non-alcoholic fatty liver and a preparation method of the probiotic preparation. The invention provides a Lactobacillus paracasei LG01, and the preservation number of the Lactobacillus paracasei LG01 is CGMCC (China General Microbiological Culture Collection Center) NO.32953. The Lactobacillus paracasei LG01 has the advantages that the preservation number is CGMCC NO.32953; the strain is preserved in the China General Microbiological Culture Collection Center on December 20, 2024, the code of the preservation unit is CGMCC, the address of the preservation unit is Institute of Microbiology, Chinese Academy of Sciences, No.3, No.1 Yard, Beichen West Road, Chaoyang District, Beijing, the postcode is 100101, and the preservation number is CGMCC 32953. The bacterium is viable during preservation. The invention also provides a fermentation product and a probiotic preparation of the Lactobacillus paracasei.

Description

Technical Field

[0001] The present invention belongs to the field of probiotics, and specifically relates to Lactobacillus paracasei, its probiotic preparation, probiotic fermentation preparation and their preparation methods. Background Art

[0002] Non-alcoholic fatty liver disease (NAFLD) is a chronic liver disease caused by excessive intake of high-fat diet, involving multiple aspects such as insulin resistance, glucose and lipid metabolism disorders, oxidative stress, inflammatory response, and intestinal-liver axis disorders, and is closely related to metabolic syndromes such as obesity, diabetes, hyperlipidemia, and hypertension. In recent years, with the changes in lifestyle and dietary structure, the prevalence of NAFLD has been continuously increasing and the patients are mostly getting younger. It is reported that the prevalence of NAFLD has reached 29.2%, and it is expected to reach 30% in 2030.

[0003] The main manifestations of NAFLD are the increase in the levels of triglycerides and cholesterol in the liver, fatty degeneration of liver cells, and the increase in the body's blood lipid levels. The pathogenesis is relatively complex and is closely related to the activities and expressions of various enzymes and proteins. Especially, MOGAT1 enzyme, as a key substance in the synthesis of liver triglycerides, the increase in its level will lead to the rapid synthesis and accumulation of triglycerides in the liver. Currently, the commonly used drugs for treating NAFLD are mainly insulin sensitizers, lipid-lowering drugs such as fibrates and statins, or anti-inflammatory and liver-protecting drugs. The main purpose is to balance the oxidation / antioxidation system, control metabolic disorders, and achieve the purpose of improving NAFLD. However, long-term use of these drugs has side effects such as liver function damage and gastrointestinal discomfort. At present, there is no safe and side-effect-free drug for treating NAFLD. Therefore, a safe, effective and side-effect-free treatment method for NAFLD has become a major research hotspot at present.

[0004] Some studies have shown that non-alcoholic fatty liver disease can be improved by supplementing probiotics in multiple directions and at multiple targets. For example, in patent CN103911335B, a strain of Lactobacillus johnsonii BS15 with the ability to reduce cholesterol in vitro was screened from traditional fermented yogurt. After intervention in high-fat diet mice, it was found that this strain could significantly reduce the body weight and liver weight of mice and the deposition level of lipid droplets in the liver; similarly, in patent CN118064335B, the inventor screened out a strain of Lactobacillus rhamnosus LRY12 with excellent non-alcoholic fatty liver treatment function, and its fermented traditional Chinese medicine compound composition could improve the total cholesterol and triglyceride levels in the liver tissue of high-fat mice and reduce the liver weight of high-fat mice. Therefore, a probiotic strain with excellent lipid-lowering function can be screened from natural fermented foods to explore its improvement effect on NAFLD.

[0005] In addition, many substances that are both medicine and food also have an improving effect on non-alcoholic fatty liver. Dandelion, a traditional plant that can be used both as medicine and food, contains many bioactive components such as flavonoids, lignans, volatile oils, organic acids, and polysaccharides in its roots, and has functions such as anti-inflammatory, antioxidant, and liver protection. Selecting appropriate probiotics to ferment dandelion may have the potential function of improving NAFLD. Therefore, finding functional substances that can inhibit the activity of MOGAT1 enzyme in the fermentation products may become a new effective way to improve NAFLD. Summary of the Invention

[0006] The first object of the present invention is to provide a Lacticaseibacillus paracasei LG01 that can effectively ferment dandelion and obtain a fermentation product for alleviating non-alcoholic fatty liver.

[0007] The second object of the present invention is to provide a probiotic preparation containing the above-mentioned Lacticaseibacillus paracasei.

[0008] The third object of the present invention is to provide a preparation method of the probiotic preparation containing the Lacticaseibacillus paracasei.

[0009] The fourth object of the present invention is to provide a probiotic fermentation preparation for effectively alleviating non-alcoholic fatty liver.

[0010] The fifth object of the present invention is to provide a preparation method of the probiotic fermentation preparation for effectively alleviating non-alcoholic fatty liver.

[0011] The present invention is realized through the following technical solutions:

[0012] A Lacticaseibacillus paracasei LG01 with a preservation number of CGMCC NO. 32953. This strain was preserved in the China General Microbiological Culture Collection Center on December 06, 2024. The preservation unit code is CGMCC, and the address of the preservation unit is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, with a postal code of 100101 and a preservation number of CGMCC 32953. The bacteria were alive when preserved.

[0013] A probiotic preparation comprising the above-mentioned Lacticaseibacillus paracasei and a lyophilization protectant.

[0014] The composition of the lyophilization protectant is as follows: skim milk powder 4 - 6%, whey protein 1 - 3%, trehalose 3 - 5%, sucrose 2 - 3%, sodium thiosulfate 1 - 3%, mannitol 1 - 3%, sorbitol 0.5 - 1.5%, and the balance is distilled water.

[0015] The preparation method of the probiotic preparation includes the steps of fermenting and culturing probiotics to obtain a bacterial sludge, and mixing the bacterial sludge with a freeze-drying protectant and then freeze-drying.

[0016] The culture medium used for the fermentation culture includes the following components by weight percentage: peptone 0.8-1.0%, beef extract powder 0.4-0.6%, yeast extract powder 0.3-0.5%, glucose 1.5-2.0%, Tween 0.05-0.15%, dipotassium hydrogen phosphate heptahydrate 0.1-0.2%, sodium acetate trihydrate 0.4-0.6%, magnesium sulfate heptahydrate 0.01-0.02%, manganese sulfate tetrahydrate 0.003-0.005%, dandelion extract 0.3-0.5%, and the balance is distilled water.

[0017] A fermentation product is obtained by fermenting dandelion extract with the Lactobacillus paracasei.

[0018] The fermentation product further includes a MOGAT1 enzyme inhibitor;

[0019] The structure of the MOGAT1 enzyme inhibitor is as follows:

[0020]

[0021] A probiotic fermentation preparation includes the Lactobacillus paracasei and the fermentation product of claim 6.

[0022] For the probiotic fermentation preparation, the preparation method includes the steps of fermenting and culturing probiotics to obtain a fermentation broth, and rotary evaporating and freeze-drying the fermentation broth to obtain a freeze-dried powder of the probiotic fermentation broth.

[0023] The culture medium used for the fermentation culture includes the following components by weight percentage: peptone 0.8-1.0%, beef extract powder 0.4-0.6%, yeast extract powder 0.3-0.5%, glucose 1.5-2.0%, Tween 0.05-0.15%, dipotassium hydrogen phosphate heptahydrate 0.1-0.2%, sodium acetate trihydrate 0.4-0.6%, magnesium sulfate heptahydrate 0.01-0.02%, manganese sulfate tetrahydrate 0.003-0.005%, dandelion extract 0.3-0.5%, and the balance is distilled water.

[0024] The preparation method of the dandelion extract is as follows:

[0025] The dandelion roots are crushed into a powder of 40-60 mesh, added with 70-95% ethanol according to a solid-liquid ratio of 1:10.0 (g / mL), and heated under reflux for 3 times, 2 hours each time. The extraction solutions are combined, filtered, and concentrated under reduced pressure at 0.08-0.09 MPa to obtain the dandelion extract. Compared with the prior art, the beneficial effects of the present invention are as follows:

[0026] The Lactobacillus paracasei provided by the present invention can ferment dandelion and obtain MOGAT1 enzyme inhibitor.

[0027] The probiotic preparation provided by the present invention helps to relieve non-alcoholic fatty liver.

[0028] The preparation method of the probiotic preparation provided by the present invention is simple and can be mass-produced. Description of the Drawings

[0029] Figure 1 Shows the survival rates of bacteria in the bacterial suspensions of LC01, LC02, LC03, LC04, LC05, LG01, LG02, LG03, LG04, and LG05 after treatment with artificial gastric juice.

[0030] Figure 2 Shows the survival rates of bacteria in the bacterial suspensions of LC01, LC02, LC03, LC04, LC05, LG01, LG02, LG03, LG04, and LG05 after treatment with artificial intestinal juice.

[0031] Figure 3 Shows the detection results of the cell viability of primary mouse hepatocytes AML12.

[0032] Figure 4 Shows the graph of the change in mouse body weight. Detailed Embodiments

[0033] Example 1

[0034] Isolation and screening of strain LG01, the specific steps are as follows:

[0035] (1) Strain isolation

[0036] Take 25 mL of traditional food fermentation broth, perform gradient dilution, coat it on MRS medium, place it upside down in an anaerobic incubator, and incubate at 37 °C for 48 hours. After colonies grow on the plate, pick out single colonies with different colors, sizes, and shapes for purification until a single strain is obtained, perform Gram staining, retain Gram-positive bacteria, and conduct strain screening.

[0037] (2) Strain screening

[0038] Streak and activate the retained Gram-positive bacteria on an MRS plate containing 0.8% calcium carbonate, and pick out the bacteria with a calcium dissolution circle and slightly larger size.

[0039] (3) 10 strains of bacteria with different morphologies were screened out and named LC01, LC02, LC03, LC04, LC05, LG01, LG02, LG03, LG04, and LG05 respectively, and the next artificial gastric juice and intestinal juice tolerance experiments were carried out.

[0040] (4) Artificial gastric juice tolerance test

[0041] The selected bacteria were activated, and the treated LC01, LC02, LC03, LC04, LC05, LG01, LG02, LG03, LG04, and LG05 bacterial solutions were inoculated into artificial gastric juice with a pH of 3.0 at a ratio of 1:10.0, and reacted in an incubator at 37 °C for 3 h. The viable bacteria count was measured and the survival rate was calculated. Figure 1 The survival rates of the bacteria in the LC01, LC02, LC03, LC04, LC05, LG01, LG02, LG03, LG04, and LG05 bacterial solutions after treatment with artificial gastric juice are shown.

[0042] (5) Artificial intestinal juice tolerance test

[0043] The bacterial solution treated in artificial gastric juice for 3 h was inoculated into artificial intestinal juice (containing 0.2% bile salt) with a pH of 6.8 at a ratio of 1:10.0, and reacted in an incubator at 37 °C for 3 h. The viable bacteria count was measured and the survival rate was calculated. Figure 2 The survival rates of the bacteria in the LC01, LC02, LC03, LC04, LC05, LG01, LG02, LG03, LG04, and LG05 bacterial solutions after treatment with artificial intestinal juice are shown. (6) Cholesterol and triglyceride degradation ability

[0044] After activating the highly tolerant strains LC02, LC03, LC04, LG01, and LG03, they were respectively inoculated into MRS liquid medium containing cholesterol at a ratio of 2%, cultured at 37 °C for 24 h, the cholesterol content was measured, and the cholesterol clearance rate was calculated.

[0045] After activating the highly tolerant strains LC02, LC03, LC04, LG01, and LG03, they were inoculated into MRS liquid medium containing triglyceride at a ratio of 2%, cultured at 37 °C for 24 h, the triglyceride content was measured, and the triglyceride clearance rate was calculated. The specific results are shown in Table 1.

[0046] Table 1:

[0047] Strain Cholesterol degradation rate (%) Triglyceride degradation rate (%) LC02 44.10 65.28 LC03 50.66 31.49 LC03 11.44 11.17 LG01 80.45 75.71 LG03 71.34 52.98

[0048] As can be seen from Table 1, the strains LC02, LG01, and LG03 have strong cholesterol and triglyceride degradation abilities.

[0049] (7) Antioxidant capacity determination

[0050] After activating the strains LC02, LG01, and LG03, their ABTS free radicals, DPPH free radicals, OH - free radicals, O2 - The free radical scavenging ability and reducing ability, and the specific results are shown in Table 2.

[0051] Table 2:

[0052]

[0053] As can be seen from Table 2, through comprehensive analysis, the antioxidant ability of strain LG01 is the strongest, and it is the preferred strain.

[0054] (8) Molecular biological identification of strain LG01

[0055] Pick a single colony of strain LG01 and transfer it into Lysis Buffer for Microorganism to Direct PCR, and lyse it in a water bath at 80°C for 30 min to obtain the bacterial DNA template.

[0056] The universal primers for amplifying the bacterial 16S rRNA gene are as follows:

[0057] 27F: 5’-CAGAGTTTGATCCTGGCTCAG-3’;

[0058] 1492R: 5’-GGTTACCTTGTTACGACTT-3’.

[0059] The reaction system (25 μL) is as follows: 1 μL each of the universal primers 27F and 1492R for amplifying the bacterial 16S rRNA gene, 0.5 μL of the bacterial DNA template, 12.5 μL of 2×RapidTaq Master Mix, and finally make up to 25 μL with ddH 2 O.

[0060] PCR reaction procedure: pre-denaturation at 95°C for 3 min; 30 cycles (denaturation at 94°C for 25 s, annealing at 55°C for 25 s, extension at 72°C for 25 s), then hold at 72°C for 5 min to complete the extension; store at 4°C. After the PCR is completed, the reaction mixture is electrophoresed in a 1.5% agarose gel, and the electrophoresis buffer is 1×TAE (50 mmol / L Tris-acetate, 1 mmol / L EDTA, pH 8.0). (9) Sequence LG01, and after comparing the obtained sequencing results, it is identified as belonging to Lactobacillus paracasei.

[0061] The 16S DNA sequencing results of this strain are as follows:

[0062]

[0063] Example 2

[0064] After fermenting dandelion extract with the preferred bacteria, it was unexpectedly found that there were substances in the fermentation broth that could act on the MOGAT1 enzyme. Therefore, the substances in the fermentation broth were extracted, separated, and identified. The specific steps are as follows:

[0065] (1) Fermentation

[0066] Add dandelion extract to the MRS medium, inoculate with 2% of bacteria LG01, culture at 37 °C, and centrifuge to collect the supernatant.

[0067] (2) Extraction

[0068] After the fermentation is completed, add 95% ethanol to the fermentation broth for soaking, centrifuge and discard the bacterial residue, collect the ethanol-soaked supernatant and load it onto an HP20 macroporous adsorption resin column at a speed of 0.5 BV / h for adsorption, and then elute with 3 column volumes of water and 50% ethanol-aqueous solution respectively, with an elution flow rate of 1 BV / h to remove some impurity pigments; subsequently, elute with 4 column volumes of 80% ethanol, collect the eluate, and concentrate to obtain a paste-like crude extract.

[0069] (3) Purification

[0070] Take the paste-like crude extract, add a small amount of methanol and dissolve it by ultrasonic treatment, then stir it well with G200-300 mesh silica gel and remove the solvent by vacuum drying; pack the silica gel with the crude product into the upper layer of a G200-300 mesh silica gel column, and then use petroleum ether:ethyl acetate = 10:1, petroleum ether:ethyl acetate = 5:1, petroleum ether:ethyl acetate = 2:1 as the mobile phase for gradient elution, and collect the eluate in fractions.

[0071] (4) Detection

[0072] The collected eluate was detected for the active components by HPLC. After combining the same components, it was concentrated under reduced pressure in vacuo. The collected concentrated solution was dissolved in DMSO and then loaded onto a DAC dynamic axial compression system. After detection by HPLC, the target products were combined. After drying in vacuo, the pure compound was obtained. HPLC analysis conditions: Mobile phase: Chromatographic grade acetonitrile (phase A); pure water containing 0.1% (v:v) phosphoric acid (phase B); Chromatographic column: ZORBAX SB-C18 (250×4.6 mm, 5 μm); Flow rate: 1.0 mL / min; Maximum pressure limit: 400 bar; Detector: VWD; Column temperature: 25 °C; Wavelength: 255 nm, 305 nm; Injection volume: 20 μL; Retention time: 25 min; Gradient elution conditions: Starting from A 10%, B 90%; At 5 min, it reached A 30%, B 70%; At 10 min, it reached A 50%, B 50%; At 15 min, it reached A 70%, B 30%; At 20 min, it reached A 90%, B 10%; At 21 min, A reached 100%, at 22 min, A remained 100%, at 24 min, it reached A 10%, B 90%, and at 25 min, it remained A 10%, B 90%. For the said DAC dynamic axial compression system, the packing material was C18, the mobile phase was 75% acetonitrile-aqueous solution, the flow rate was controlled at 10 ml / min, and the detection wavelength was 286 nm.

[0073] (5) Among the substances extracted by the above method, the following compounds exhibited the effect of MOGAT1 enzyme inhibitors.

[0074]

[0075] The spectral data of the compound are as follows:

[0076] 1H NMR (300 MHz, CD 3 OD, δ, ppm):

[0077] 9.68 (1H in alcohol), 9.45 (1H in alcohol), 7.28 (1H in benzene), 7.12 (1H in benzene), 7.11 (1H in benzene), 7.10 (1H in benzene), 6.86 (1H in benzene), 6.81 (1H in benzene), 3.69 (1H in methine), 2.94 (4H in methylene), 2.81 (4H in pyrrolidine), 2.53 (2H in methylene), 2.0 (1H in pyrrolidine), 1.66 (2H in pyrrolidine), 1.54 (2H in methylene), 1.49 (1H in pyrrolidine). 13C NMR (125 MHz, CD 3 OD, δ, ppm): 196.8, 155.7, 152.4, 140.8, 133.8, 131.9, 129.4, 128.9, 128.5, 124.7, 120.4, 117.7, 115.4, 58.1, 46.6, 45.3, 41.6, 41.3, 37.8, 36.5, 36.2, 35.7.

[0078] Example 3

[0079] Furthermore, the inhibitory test of the MOGAT1 enzyme activity in primary mouse hepatocytes was carried out using the above compound

[0080] (1) Materials and Instruments

[0081] Experimental cells: Primary mouse hepatocytes AML12.

[0082] Culture medium: DMEM (Dulbecco's modified eagle medium, DMEM) medium containing 10% FBS (fetal bovine serum) and 0.1% double antibiotics (penicillin, streptomycin).

[0083] Instruments: COS 2 Thermostatic incubator (Thermo Forma 3310, USA); Inverted biological microscope (Model XD - 101, Nanjing); Flexstation 3 multi-functional microplate reader (Molecular Devices, Sunnyvale, California, USA).

[0084] (2) Experimental Method

[0085] Cell model establishment and culture: After cell resuscitation, use DMEM medium containing 10% FBS and 0.1% double antibiotics, and conduct routine culture in a 37°C incubator containing 5% CO 2 2. When the cells grow to a density of 80% - 90% in the culture dish, passage them every two days, and select cells in the logarithmic growth phase for experiments. After establishing the cell NAFLD model with oleic acid, intervene with different concentrations of the sample to be tested.

[0086] Cell viability detection: Take the cell suspension and plate it at a cell density of 1×10 5 cells / mL. After the cells are completely adherent, administer drugs at different concentrations. After 24 hours of drug administration, operate and calculate according to the CCK8 kit.

[0087] Cell biochemical index detection: Detect the activities of MOGAT1 enzyme, and the contents of TC and TG according to the kit instructions.

[0088] Data analysis: Analyze the experimental results using Graphpad prism 7.0 software.

[0089] (3) Results

[0090] The results of cell viability detection are as Figure 3 shown. When the concentration of dandelion extract is 15 μM, 20 μM, and 25 μM, the cell viability does not decrease significantly. When the concentration exceeds 50 μM, the cell viability shows a significant decrease, proving that the intervention concentration of the extract is most suitable below 50 μM and will not be toxic to the cells. Therefore, in the follow-up, the cells are intervened with these 3 concentrations of 15 μM, 20 μM, and 25 μM.

[0091] The detection results of other indicators are shown in Table 3.

[0092] Table 3:

[0093]

[0094] In summary, this compound has an inhibitory effect on MOGAT1 enzyme, can also reduce the contents of cholesterol and triglyceride, and can treat or improve non-alcoholic fatty liver disease.

[0095] Example 4

[0096] The preparation method of the probiotic fermentation preparation is as follows:

[0097] LG01 Fermentation Culture: Inoculate the activated probiotic liquid into the culture medium and incubate at 37 °C for 24 h. The culture medium comprises the following components by weight percentage: peptone 0.8 - 1.0%, beef extract powder 0.4 - 0.6%, yeast extract powder 0.3 - 0.5%, glucose 1.5 - 2.0%, Tween 0.05 - 0.15%, dipotassium hydrogen phosphate heptahydrate 0.1 - 0.2%, sodium acetate trihydrate 0.4 - 0.6%, magnesium sulfate heptahydrate 0.01 - 0.02%, manganese sulfate tetrahydrate 0.003 - 0.005%, dandelion extract 0.3 - 0.5%, and the balance is distilled water;

[0098] Preparation of Freeze-dried Powder of LG01 Fermentation Broth: Rotavaporize and freeze-dry the probiotic fermentation broth to obtain the freeze-dried powder of the fermentation broth. The probiotic is Lactobacillus paracasei LG01.

[0099] Example 5

[0100] Study on the effect of the above freeze-dried powder of probiotic fermentation broth on improving NAFLD in high-fat diet mice is as follows:

[0101] In Example 3, it was found that LG01-fermented dandelion extract could significantly inhibit the activity of MOGAT1 enzyme in primary mouse hepatocytes, block the synthesis of triglycerides in primary mouse hepatocytes, reduce the lipid content in mouse hepatocytes, and have the function of improving cell lipid levels in vitro. Therefore, in this example, high-fat diet mice were used to construct a NAFLD model to explore the inhibitory effect of the freeze-dried powder of LG01 probiotic fermentation broth on the activity of MOGAT1 enzyme in high-fat diet mice in vivo and its improvement effect on NAFLD in mice.

[0102] (1) Experimental method

[0103] Select 48 SPF-grade male C57BL / 6J mice, 8 weeks old, with a body weight of 20 ± 2 g. After 1 week of adaptive feeding, randomly divide them into a normal control group (C, 12 mice), fed with normal feed, and the remaining mice were fed with a high-fat diet for 3 weeks to establish a model. Select the mice with successful modeling and group them again, namely a model group (M, 12 mice), a G1 group (LG01 freeze-dried powder, 12 mice), and a G2 group (freeze-dried powder of LG01 fermentation broth, 12 mice), and intervene for 8 weeks. During this period, the mice in different groups were fed different diets, as shown in Table 4 for details.

[0104] Table 4:

[0105]

[0106] After 8 weeks of intervention with LG01 powder and freeze-dried powder of fermentation broth, record the body weight of the mice, take blood from the eyeballs and sacrifice them, prepare mouse serum samples and liver samples, and measure the activities of MOGAT1 enzyme, and the contents of TC and TG in the serum and liver.

[0107] (2) Results

[0108] The body weight changes of the mice are as Figure 4 shown. The results show that the body weight of the M group increased significantly. After 2 weeks of intervention with the LG01 bacterial powder, the body weight of the mice was significantly lower than that of the M group, indicating that the intervention with the LG01 bacterial powder alleviated the body weight gain of the mice to a certain extent. After 1 week of intervention with the freeze-dried powder of LG01 fermentation broth, the body weight of the mice decreased significantly, and the longer the intervention time, the more obvious the effect. This indicates that the intervention with the freeze-dried powder of LG01 fermentation broth has a more obvious inhibitory effect on the body weight gain of the mice compared with the LG01 bacterial powder. The specific results of the detection of the activities of MOGAT1 enzyme, TC, and TG contents in the serum and liver of the mice are shown in Table 5.

[0109] Table 5:

[0110]

[0111] As shown in Table 5, compared with the C group, the activity of MOGAT1 enzyme in the mice of the M group increased significantly and the contents of TC and TG increased significantly. After the intervention with the LG01 bacterial powder and the freeze-dried powder of the fermentation broth, the activity of MOGAT1 enzyme in the serum and liver of the mice was significantly lower than that of the M group, and the contents of TC and TG decreased significantly, indicating that the intervention with the LG01 bacterial powder and the freeze-dried powder of the fermentation broth alleviated the synthesis of cholesterol and triglyceride in the mice to a certain extent, reduced the lipid levels in the serum and liver of the mice, improved the NAFLD of the mice, and the effect of the freeze-dried powder of the LG01 fermentation broth was more obvious.

[0112] In summary, the freeze-dried powder of the LG01 fermentation broth is helpful for the treatment of non-alcoholic fatty liver.

Claims

1. A Lactobacillus paracasei LG01, characterized in that: Its accession number is CGMCC NO.32953.

2. A probiotic preparation, characterized in that: The invention comprises the Lactobacillus paracasei as claimed in claim 1 and a freeze-drying protective agent.

3. The probiotic preparation according to claim 2, characterized in that: The freeze-drying protective agent comprises: 4-6% skim milk powder, 1-3% whey protein, 3-5% trehalose, 2-3% sucrose, 1-3% sodium thiosulfate, 1-3% mannitol, 0.5-1.5% sorbitol and the balance is distilled water.

4. The method for preparing the probiotic preparation according to claim 2, characterized in that: The method comprises the steps of fermenting and culturing probiotics to obtain bacterial mud, mixing the bacterial mud with a freeze-drying protective agent, and then freeze-drying the mixture.

5. The method for preparing the probiotic preparation according to claim 4, characterized in that: The culture medium used in the fermentation culture comprises the following components in percentage by weight: 0.8-1.0% peptone, 0.4-0.6% beef extract, 0.3-0.5% yeast extract, 1.5-2.0% glucose, 0.05-0.15% Tween, 0.1-0.2% dipotassium hydrogen phosphate heptahydrate, 0.4-0.6% sodium acetate trihydrate, 0.01-0.02% magnesium sulfate heptahydrate, 0.003-0.005% manganese sulfate tetrahydrate, 0.3-0.5% dandelion extract, and the balance distilled water.

6. A fermentation product, characterized in that: The method is obtained by fermenting the dandelion extract with the Lactobacillus paracasei according to claim 1.

7. The fermentation product according to claim 6, characterized in that: Also included are MOGAT1 enzyme inhibitors; The structure of the MOGAT1 enzyme inhibitor is as follows:

8. A probiotic fermentation preparation, characterized in that: The method comprises the Lactobacillus paracasei as claimed in claim 1 and the fermentation product as claimed in claim 6.

9. The probiotic fermentation preparation according to claim 8, characterized in that: The preparation method of the probiotic fermentation preparation comprises the steps of fermenting and culturing the probiotics to obtain a fermentation liquid, and rotary evaporating and freeze-drying the fermentation liquid to obtain a probiotic fermentation liquid freeze-dried powder.

10. The probiotic fermentation preparation according to claim 8, characterized in that: The culture medium used in the fermentation culture comprises the following components in percentage by weight: 0.8-1.0% peptone, 0.4-0.6% beef extract, 0.3-0.5% yeast extract, 1.5-2.0% glucose, 0.05-0.15% Tween, 0.1-0.2% dipotassium hydrogen phosphate heptahydrate, 0.4-0.6% sodium acetate trihydrate, 0.01-0.02% magnesium sulfate heptahydrate, 0.003-0.005% manganese sulfate tetrahydrate, 0.3-0.5% dandelion extract, and the balance distilled water.

11. The probiotic fermentation preparation according to claim 10, characterized in that: The preparation method of the dandelion extract is as follows: The dandelion root is crushed into 40-60 mesh powder, and 70-95% ethanol is added according to a solid-liquid ratio of 1:10.0 (g / mL), and heated to reflux for 3 times, each time for 2 hours; the extracts are combined, filtered, and concentrated under reduced pressure at 0.08-0.09 MPa to obtain the product.

Images