A liver-protecting probiotic composition and its use in preventing or treating acute liver injury

Abstract

The application provides a liver-protecting probiotic composition and application thereof in prevention or treatment of acute liver injury, and belongs to the technical field of microorganisms and medicines.The application provides a lactobacillus crispatus LJ003, and the preservation number is CGMCC No.30723.The application further provides a method for preparing fermented artichoke leaf powder, polyphenols and chlorogenic acid by using the lactobacillus crispatus LJ003, and fermented artichoke leaf powder prepared by using the lactobacillus crispatus LJ003.The fermented artichoke leaf powder prepared by the application can be used for preparing the liver-protecting probiotic composition, the obtained liver-protecting probiotic composition has a good protective effect on hepatocytes, can effectively improve lipid metabolism disorder of hepatocytes caused by excessive alcohol intake, and has a remarkable effect on prevention or treatment of acute liver injury.

Description

Technical Field

[0001] This invention belongs to the field of microbiology and pharmaceutical technology, specifically relating to a liver-protecting probiotic composition and its application in the prevention or treatment of acute liver injury. Background Technology

[0002] In the complex physiological system of the human body, the liver occupies a pivotal position, being the largest and most functionally diverse digestive gland. The liver is deeply involved in many key processes, including digestion, metabolism, excretion, detoxification, and immunity, playing a central role, especially in metabolic function. It also participates in hematopoiesis, undertaking the crucial tasks of storing and releasing hematopoietic factors and synthesizing various enzymes to ensure normal metabolic function. Furthermore, the liver possesses a powerful detoxification capacity for harmful substances from external sources and metabolic products. However, the liver also faces numerous threats. Many harmful factors can reach the liver via the portal vein or systemic circulation from the gastrointestinal tract and be transformed, making the liver highly susceptible to damage from toxic substances, leading to varying degrees of liver injury. Acute liver injury is relatively common and serious. If acute liver injury is not controlled promptly and effectively, it can progress rapidly, leading to liver fibrosis and further deteriorating into more serious liver diseases such as cirrhosis and liver cancer, posing a significant threat to human health.

[0003] Chemical-induced liver injury is a common cause of acute liver injury, mainly triggered by certain drugs, alcohol, and toxic substances in the environment. For example, most liver injuries clinically are caused by alcohol, and alcoholic liver injury can be further subdivided into acute alcoholic liver injury and chronic alcoholic liver injury. Acute alcoholic liver injury is caused by excessive alcohol intake, leading to liver function impairment, and often presents with symptoms of acute hepatitis such as loss of appetite, nausea, liver discomfort, and jaundice. Its pathogenesis is complex, including the direct hepatotoxicity of alcohol and its metabolite acetaldehyde, oxidative stress and reactive oxygen species damage, cytokine and endotoxin-mediated immune inflammation mechanisms, and other factors such as local tissue hypoxia, endoplasmic reticulum stress, increased iron load, and apoptosis. Although drug development for liver diseases has been continuously advancing, existing chemical drugs are expensive and have many adverse reactions, which greatly limits their clinical application. In recent years, traditional Chinese medicine has shown significant advantages in the research field of treating liver injury, especially with its low toxicity and wide range of pharmacological effects. Numerous natural plants have been proven to have hepatoprotective effects, primarily through mechanisms such as scavenging free radicals, combating lipid peroxidation, and lowering transaminase levels. However, most hepatoprotective products on the market currently use ingredients like cordyceps, pine pollen, and ganoderma lucidum, which present challenges such as high costs and unstable raw material supply, thus limiting the application of these products.

[0004] Therefore, it is urgent to develop a novel, simple, and effective product for the prevention or treatment of acute liver injury, which is of great significance for improving the current treatment of acute liver injury and enhancing the quality of life of patients. Summary of the Invention

[0005] To address the problems existing in the prior art, the primary objective of this invention is to provide a curly Lactobacillus (Lactobacillus curvatureus) Lactobacillus crispatus LJ003, deposited by China General Microbiological Culture Collection Center (CGMCC), accession number: CGMCC No.30723, deposit date: May 22, 2024.

[0006] A second objective of this invention is to provide the application of the aforementioned Lactobacillus curvature LJ003 in the fermentation preparation of polyphenols and chlorogenic acid using artichoke leaves as raw material.

[0007] A third objective of this invention is to provide a method for preparing fermented artichoke leaf powder and the fermented artichoke leaf powder obtained therefrom.

[0008] A fourth objective of this invention is to provide a method for preparing polyphenols and chlorogenic acid using the aforementioned Lactobacillus curvature LJ003.

[0009] A fifth objective of this invention is to provide a liver-protecting probiotic composition and its preparation method.

[0010] A sixth objective of the present invention is to provide the use of the above-mentioned hepatoprotective probiotic composition in the preparation of a medicament for the prevention or treatment of acute liver injury.

[0011] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0012] This invention provides a *Lactobacillus curvaturei* LJ003, wherein the *Lactobacillus curvaturei* ( Lactobacillus crispatus The accession number of LJ003 is CGMCC No.30723.

[0013] This invention also provides the application of Lactobacillus curvature LJ003 in the fermentation preparation of polyphenols and chlorogenic acid using artichoke leaves as raw material.

[0014] The present invention also provides a method for preparing fermented artichoke leaf powder, comprising the following steps: mixing artichoke leaf powder, glucose and water in a mass ratio of 20:2:80, sterilizing to obtain fermentation raw material; inoculating the above-mentioned Lactobacillus curvature LJ003 into the fermentation raw material for fermentation culture to obtain fermentation culture product; centrifuging and drying the fermentation culture product to obtain fermented artichoke leaf powder.

[0015] The present invention also provides fermented artichoke leaf powder prepared by the above method.

[0016] The present invention also provides a method for preparing polyphenols, comprising the following steps: mixing artichoke leaf powder, glucose and water in a mass ratio of 20:2:80, sterilizing to obtain fermentation raw material; inoculating the above-mentioned Lactobacillus curvature LJ003 into the fermentation raw material for fermentation culture to obtain fermentation culture product; and extracting polyphenols from the fermentation culture product.

[0017] The present invention also provides a method for preparing chlorogenic acid, comprising the following steps: mixing artichoke leaf powder, glucose and water in a mass ratio of 20:2:80, sterilizing to obtain fermentation raw material; inoculating the above-mentioned Lactobacillus curvature LJ003 into the fermentation raw material for fermentation culture to obtain fermentation culture product; and extracting chlorogenic acid from the fermentation culture product.

[0018] This invention also provides a liver-protecting probiotic composition, comprising, by weight parts: 25-50 parts sheep bone marrow oligopeptide, 20-30 parts isomaltooligosaccharide, 10-15 parts erythritol, 1-2 parts glutathione-enriched yeast, 5-8 parts milk thistle seed oil powder, 1-8 parts sheep spleen peptide, 0.05-0.08 parts Haematococcus pluvialis microcapsule powder, 3-5 parts corn oligopeptide, 2-4 parts fermented artichoke leaf powder, 1-2.5 parts sunflower lecithin, 0.08-0.1 parts lutein microcapsule powder, 0.6-1.2 parts vitamin E, 0.01-0.03 parts (3R,3'S)-dihydroxy-β-carotene, 0.25-0.5 parts *Lactobacillus plantarum* LZ015 freeze-dried powder, and *Lactobacillus rhamnosus* NKU. 0.25-0.5 parts of FL1-8 freeze-dried powder and 0.25-0.5 parts of Bifidobacterium lactis BR001 freeze-dried powder; the fermented artichoke leaf powder is the above-mentioned fermented artichoke leaf powder.

[0019] Preferably, the viable count of the *Lactobacillus plantarum* LZ015 freeze-dried powder is 5.0 × 10⁻⁶. 11 cfu / g ~7.0×10 11 The viable count of the *Lactobacillus rhamnosus* NKU FL1-8 lyophilized powder was 4.0 × 10⁻⁶ cfu / g. 11 cfu / g ~5.0×10 11 cfu / g; the viable count of the Bifidobacterium lactis BR001 lyophilized powder is 6.0 × 10⁻⁶. 11 cfu / g ~8.0×10 11 cfu / g.

[0020] The present invention also provides a method for preparing the above-mentioned liver-protecting probiotic composition, comprising the following steps: mixing the components in proportion to obtain the liver-protecting probiotic composition.

[0021] The present invention also provides the use of the above-mentioned hepatoprotective probiotic composition in the preparation of a medicament for the prevention or treatment of acute liver injury.

[0022] Compared with the prior art, the beneficial effects of the technical solution of the present invention are as follows:

[0023] This invention provides the first known strain of *Lactobacillus curvatureii* LJ003 and its application in the fermentation of artichoke leaves to prepare polyphenols and chlorogenic acid. The artichoke leaf powder fermented with *Lactobacillus curvatureii* LJ003 exhibits a significant increase in polyphenol and chlorogenic acid content, making it suitable for the preparation of polyphenols or chlorogenic acid.

[0024] This invention also provides a liver-protecting probiotic composition, which, by adding artichoke leaf powder obtained from Lactobacillus curvature LJ003 fermentation and various probiotic freeze-dried powders, can significantly reduce the inhibition of SOD activity by ethanol, reduce the level of oxidative stress in the liver, inhibit the production of pro-inflammatory factors, effectively improve the lipid metabolism disorder of hepatocytes caused by excessive ethanol intake, reduce the damage to hepatocytes caused by ethanol, reduce the inflammatory response induced by ethanol, and have a good protective effect on hepatocytes, thus exerting the effects of prevention and / or treatment of acute liver injury.

[0025] Biological Preservation Instructions

[0026] Lactobacillus curlis LJ003, classified and named Lactobacillus curlis ( Lactobacillus crispatus (), depositary institution: China General Microbiological Culture Collection Center (CGMCC), address: No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, accession number: CGMCC No. 30723, deposit date: May 22, 2024.

[0027] Lactobacillus plantarum LZ015, classified and named Lactobacillus plantarum ( Lactiplantibacillus plantarum (), deposited at: China General Microbiological Culture Collection Center (CGMCC), address: No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, accession number: CGMCCNo. 29395, deposit date: December 25, 2023.

[0028] Lactobacillus rhamnosus NKU FL1-8, classified and named Lactobacillus rhamnosus ( Lacticaseibacillus rhamnosus (), depositary institution: Guangdong Provincial Microbial Culture Collection Center (GDMCC), depositary address: 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, Guangdong Academy of Sciences Institute of Microbiology, deposit number: GDMCC No. 64053, deposit date: November 23, 2023.

[0029] Bifidobacterium lactis BR001, classified and named Bifidobacterium lactis ( Bifidobacterium lactis (), deposited at: China General Microbiological Culture Collection Center (CGMCC), address: No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, accession number: CGMCC No. 23665, deposit date: October 25, 2021. Attached Figure Description

[0030] Figure 1 Macroscopic morphology of Lactobacillus curlis LJ003 colonies in Petri dishes;

[0031] Figure 2 Microscopic morphology of Lactobacillus curvature LJ003;

[0032] Figure 3 : Chlorogenic acid standard curve;

[0033] Figure 4 The activity levels of ALT and AST in the serum of mice in each group;

[0034] Figure 5 The levels of TC and TG in the serum of mice in each group;

[0035] Figure 6 : The level of MDA in the liver tissue of mice in each group;

[0036] Figure 7 The level of SOD in the liver tissue of mice in each group;

[0037] Figure 8 The levels of IL-1β, IL-6, and TNF-α in the livers of mice in each group;

[0038] Figure 9 Results of hematoxylin-eosin staining of liver tissue from mice in each group;

[0039] Figure 10 The effect of each group on MDA content;

[0040] Figure 11 The effects of each group on inflammatory factors;

[0041] Figure 12 The effect of each group on SOD content;

[0042] Figure 13 Effects of each group on the activities of ALT and AST enzymes. Detailed Implementation

[0043] This invention provides a *Lactobacillus curvaturei* LJ003, wherein the *Lactobacillus curvaturei* ( Lactobacillus crispatusThe preservation number of LJ003 is CGMCC No. 30723. The *Lactobacillus curvature* LJ003 described in this invention was isolated and purified from artichoke leaf extract, and identification confirmed that it belongs to *Lactobacillus curvature* (…). Lactobacillus crispatus This invention provides the application of *Lactobacillus curvatureii* LJ003 in the fermentation preparation of polyphenols and chlorogenic acid using artichoke leaves as raw material. The depository was deposited on May 22, 2024, at the China General Microbiological Culture Collection Center, located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences.

[0044] This invention provides a method for preparing fermented artichoke leaf powder, comprising the following steps: mixing artichoke leaf powder, glucose, and water in a mass ratio of 20:2:80, sterilizing, and obtaining fermentation raw material; inoculating the fermentation raw material with *Lactobacillus curvatureii* LJ003 for fermentation culture to obtain fermentation culture product; centrifuging and drying the fermentation culture product to obtain fermented artichoke leaf powder. The sterilization method is preferably 121℃ for 30 min; the fermentation raw material is preferably cooled to room temperature, and *Lactobacillus curvatureii* LJ003 is inoculated at room temperature. The inoculation amount of *Lactobacillus curvatureii* LJ003 (seed liquid) is preferably 3% (v / v), and the viable count of the inoculated *Lactobacillus curvatureii* LJ003 (seed liquid) is preferably 3.0 × 10⁻⁶. 9 ~4.0×10 9 cfu / g; the fermentation temperature is preferably 36~38℃, more preferably 37℃; the fermentation time is 22~26h, more preferably 23h, 24h or 25h; the centrifugation conditions are preferably 6000r / min for 20min; the drying temperature is preferably 65~75℃, more preferably 70℃; the drying time is preferably 4~8h, more preferably 6h.

[0045] The fermented artichoke leaf powder prepared by this invention has a high content of polyphenols and chlorogenic acid, with the polyphenol content reaching 120 mg / g or more and the chlorogenic acid content reaching 28 mg / g or more.

[0046] This invention also provides a method for preparing polyphenols, comprising the following steps: mixing artichoke leaf powder, glucose, and water in a mass ratio of 20:2:80, sterilizing, and obtaining fermentation raw material; inoculating the fermentation raw material with *Lactobacillus curvatureii* LJ003 for fermentation culture to obtain fermentation culture product; and extracting polyphenols from the fermentation culture product. The sterilization method is preferably 121℃ for 30 min; the fermentation raw material is preferably cooled to room temperature, and *Lactobacillus curvatureii* LJ003 is inoculated at room temperature. The inoculation amount of *Lactobacillus curvatureii* LJ003 (seed liquid) is preferably 3% (v / v), and the viable count of the inoculated *Lactobacillus curvatureii* LJ003 (seed liquid) is preferably 3.0 × 10⁻⁶.9 ~4.0×10 9 cfu / g; the fermentation temperature is preferably 36~38℃, more preferably 37℃; the fermentation time is 22~26h, more preferably 23h, 24h or 25h; the polyphenols can be obtained by extraction using conventional extraction methods in the art.

[0047] This invention also provides a method for preparing chlorogenic acid, comprising the following steps: mixing artichoke leaf powder, glucose, and water in a mass ratio of 20:2:80, sterilizing, and obtaining fermentation raw material; inoculating the fermentation raw material with *Lactobacillus curvatureii* LJ003 for fermentation culture to obtain fermentation culture product; and extracting chlorogenic acid from the fermentation culture product. The sterilization method is preferably 121℃ for 30 min; the fermentation raw material is preferably cooled to room temperature, and *Lactobacillus curvatureii* LJ003 is inoculated at room temperature. The inoculation amount of *Lactobacillus curvatureii* LJ003 (seed liquid / activation liquid) is preferably 3% (v / v), and the viable count of the inoculated *Lactobacillus curvatureii* LJ003 (seed liquid / activation liquid) is preferably 3.0 × 10⁻⁶. 9 ~4.0×10 9 cfu / g; the fermentation temperature is preferably 36~38℃, more preferably 37℃; the fermentation time is 22~26h, more preferably 23h, 24h or 25h; the chlorogenic acid can be obtained by extraction using conventional extraction methods in the art.

[0048] This invention also provides a liver-protecting probiotic composition, comprising, by weight parts: 25-50 parts sheep bone marrow oligopeptide, 20-30 parts isomaltooligosaccharide, 10-15 parts erythritol, 1-2 parts glutathione-enriched yeast, 5-8 parts milk thistle seed oil powder, 1-8 parts sheep spleen peptide, 0.05-0.08 parts Haematococcus pluvialis microcapsule powder, 3-5 parts corn oligopeptide, 2-4 parts fermented artichoke leaf powder, 1-2.5 parts sunflower lecithin, 0.08-0.1 parts lutein microcapsule powder, 0.6-1.2 parts vitamin E, 0.01-0.03 parts (3R,3'S)-dihydroxy-β-carotene, 0.25-0.5 parts *Lactobacillus plantarum* LZ015 freeze-dried powder, and *Lactobacillus rhamnosus* NKU. 0.25-0.5 parts of FL1-8 freeze-dried powder and 0.25-0.5 parts of Bifidobacterium lactis BR001 freeze-dried powder; the fermented artichoke leaf powder is fermented artichoke leaf powder prepared by Lactobacillus curvature LJ003.

[0049] Preferably, the liver-protecting probiotic composition of the present invention comprises the following components: 30-40 parts of sheep bone marrow oligopeptide, 22-26 parts of isomaltooligosaccharide, 12-14 parts of erythritol, 1.2-1.5 parts of glutathione-enriched yeast, 6-7 parts of milk thistle seed oil powder, 2-6 parts of sheep spleen peptide, 0.06-0.07 parts of Haematococcus pluvialis microcapsule powder, 4 parts of corn oligopeptide, 3 parts of fermented artichoke leaf powder, 1.5-2 parts of sunflower lecithin, 0.09 parts of lutein microcapsule powder, 0.8-1 part of vitamin E, 0.02 parts of (3R,3'S)-dihydroxy-β-carotene, 0.3-0.4 parts of *Lactobacillus plantarum* LZ015 freeze-dried powder, 0.3-0.4 parts of *Lactobacillus rhamnosus* NKU FL1-8 freeze-dried powder, and 0.3-0.4 parts of *Bifidobacterium lactis* BR001 freeze-dried powder.

[0050] The viable count of the *Lactobacillus plantarum* LZ015 freeze-dried powder described in this invention is 5.0 × 10⁻⁶. 11 cfu / g ~7.0×10 11 cfu / g, preferably 6.0 × 10⁻⁶ 11 cfu / g. The preservation number of *Lactobacillus plantarum* LZ015 described in this invention is CGMCC No. 29395. The preparation method of the *Lactobacillus plantarum* LZ015 lyophilized powder may be as follows: *Lactobacillus plantarum* LZ015 is statically cultured to obtain a culture product; lyophilization protectant is added to the culture product at a ratio of 1:2 (culture product volume: lyophilization protectant volume), and after thorough mixing, the mixture is freeze-dried. The static culture temperature of the present invention is 36~38℃, preferably 37℃; the static culture time is 18h; the static culture medium composition can be selected from glucose 30g, bovine bone peptone 10g, yeast extract 5g, beef extract 10g, soybean peptone 5g, dipotassium hydrogen phosphate 3g, diammonium hydrogen citrate 4g, sodium acetate 5g, Tween 80 1g, magnesium sulfate 0.58g, manganese sulfate 0.25g, cysteine ​​hydrochloride 0.5g, and purified water 1000g; the freeze-drying protectant composition can be selected from β-cyclodextrin 2g, Tween 80 0.2g, trehalose 10g, skim milk 8g, L-cysteine ​​0.1g, and water 100g; the freeze-drying temperature is -30~-40℃, and the time is 64~72h.

[0051] The viable count of the *Lactobacillus rhamnosus* NKU FL1-8 lyophilized powder described in this invention is 4.0 × 10⁻⁶. 11 cfu / g ~5.0×10 11 cfu / g, preferably 4.5 × 10⁻⁶ 11cfu / g. The preservation number of *Lactobacillus rhamnosus* NKU FL1-8 described in this invention is GDMCC No. 64053. The preparation method of the lyophilized *Lactobacillus rhamnosus* NKU FL1-8 powder can be as follows: *Lactobacillus rhamnosus* NKU FL1-8 is statically cultured to obtain a culture product; a lyophilization protectant is added to the culture product at a ratio of 1:2 (culture product volume: lyophilization protectant volume), and after thorough mixing, the mixture is freeze-dried. The static culture temperature of the present invention is 36~38℃, preferably 37℃; the static culture time is 18h; the static culture medium composition can be selected from glucose 25g, bovine bone peptone 8g, yeast extract 15g, soybean peptone 10g, dipotassium hydrogen phosphate 2g, diammonium hydrogen citrate 2g, sodium acetate 5g, Tween 80 1g, magnesium sulfate 0.58g, manganese sulfate 0.25g, cysteine ​​hydrochloride 0.5g, and purified water 1000g; the freeze-drying protectant composition can be selected from skim milk 10g, Tween 80 0.2g, trehalose 10g, soybean polysaccharide 1g, collagen peptide 1g, monosodium glutamate 2g, L-cysteine ​​0.1g, and water 100g; the freeze-drying temperature is -30~-40℃, and the time is 64~72h.

[0052] The viable count of the lyophilized Bifidobacterium lactis BR001 powder described in this invention is 6.0 × 10⁻⁶. 11 cfu / g ~8.0×10 11 cfu / g, preferably 7.0 × 10⁻⁶ 11 cfu / g. The preservation number of Bifidobacterium lactis BR001 described in this invention is CGMCC No. 23665. The preparation method of the lyophilized powder of Bifidobacterium lactis BR001 can be selected as follows: anaerobic static culture of Bifidobacterium lactis BR001 to obtain a culture product; adding a lyophilization protectant to the culture product at a ratio of 1:2 (culture product volume: lyophilization protectant volume), mixing thoroughly, and then freeze-drying. The anaerobic static culture temperature of the present invention is 36~38℃, preferably 37℃; the anaerobic static culture time is 21h; the culture medium composition of the anaerobic static culture can be selected as follows: 25g glucose, 10g fructooligosaccharide, 10g tryptone, 20g yeast extract, 6g soybean peptone, 2g dipotassium hydrogen phosphate, 2g diammonium hydrogen citrate, 5g sodium acetate, 1g Tween 80, 0.58g magnesium sulfate, 0.25g manganese sulfate, 0.5g cysteine ​​hydrochloride, and 1000g purified water; the freeze-drying protectant composition can be selected as follows: 2g β-cyclodextrin, 0.1g Tween 80, 10g trehalose, 8g skim milk, 0.1g L-cysteine, 4g sucrose, and 100g water; the freeze-drying temperature is -30~-40℃, and the time is 64~72h.

[0053] The preparation method of the liver-protecting probiotic composition of the present invention includes the following steps: mixing the components in proportion to obtain the liver-protecting probiotic composition.

[0054] The present invention also provides the application of the above-mentioned hepatoprotective probiotic composition in the preparation of a drug for the prevention or treatment of acute liver injury. The hepatoprotective probiotic composition can significantly reduce the inhibition of SOD activity by ethanol, reduce the level of oxidative stress in the liver, inhibit the production of pro-inflammatory factors, effectively improve the lipid metabolism disorder of hepatocytes caused by excessive ethanol intake, reduce the damage to hepatocytes caused by ethanol, reduce the inflammatory response induced by ethanol, and have a good protective effect on hepatocytes, thus exerting the effects of prevention and / or treatment of acute liver injury.

[0055] The technical solutions of this invention will be clearly and completely described below with reference to the embodiments thereof. Obviously, the described embodiments are only a part of the embodiments of this invention, and not all of them. All other embodiments obtained by those skilled in the art based on the embodiments of this invention without creative effort are within the scope of protection of this invention.

[0056] In a specific embodiment of the present invention, sheep bone marrow oligopeptides were purchased from Dezhou Lanli Biotechnology Co., Ltd.; isomaltooligosaccharide was purchased from Baolingbao Biotechnology Co., Ltd.; erythritol was purchased from Jinan Guangfu Food Additives Co., Ltd.; glutathione-enriched yeast was purchased from Angel Yeast Co., Ltd.; milk thistle seed oil powder was purchased from Xi'an Haisifu Biotechnology Co., Ltd.; sheep spleen peptides were purchased from Dezhou Lanli Biotechnology Co., Ltd.; Haematococcus pluvialis microcapsule powder was purchased from Hubei Yashida Biotechnology Co., Ltd.; corn oligopeptides were purchased from Dezhou Lanli Biotechnology Co., Ltd.; sunflower lecithin was purchased from Hebei Desong Biotechnology Co., Ltd.; lutein microcapsule powder was purchased from Shandong Tianyin Biotechnology Co., Ltd.; vitamin E was purchased from Beijing Youyikangyuan Biotechnology Co., Ltd.; (3R,3'S)-dihydroxy-β-carotene was purchased from Shandong Tianyin Biotechnology Co., Ltd.; and artichoke leaves were purchased from Yunnan Chengjiang Xinlei Trading Co., Ltd.

[0057] Unless otherwise specified, the following embodiments are all conventional methods.

[0058] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0059] Example 1

[0060] Isolation and identification of Lactobacillus curvature LJ003:

[0061] 1. Add 200g of artichoke leaves to 20g of glucose and 800g of purified water, mix thoroughly, and soak at 37℃ for 48 hours. Use the soaking solution for bacterial isolation. According to GB4789.35-2023 National Food Safety Standard, Microbiological Examination of Food - Examination of Lactic Acid Bacteria, use commercially modified MRS solid isolation medium. After multiple serial dilutions, anaerobic culture for 72 hours is performed for bacterial isolation, and single colonies are picked.

[0062] Based on the colony characteristics of lactic acid bacteria, corresponding single colonies were selected and cultured in MRS liquid medium at 37℃ for 24 hours. All cultured strains underwent Gram staining, catalase test, gelatin liquefaction test, oxidase test, indole test, and nitrate reduction test. Strains that were Gram-positive and negative in all four tests (catalase, gelatin liquefaction, oxidase, indole, and nitrate reduction) were selected, resulting in seven strains: CX-1, CX-2, CX-3, CX-4, CX-5, CX-6, and CX-7. These strains were stored in 15% glycerol at -80℃ for later use.

[0063] Artichoke leaves were pulverized (using a 200-mesh sieve). 200g of artichoke leaf powder was mixed with 4g of glucose and 800g of purified water, and the pH was adjusted to 7.0. The mixture was then sterilized at 121℃ for 30 minutes. Seven selected bacterial strains were cultured in MRS liquid medium at 37℃ for 18 hours, activated twice, and then inoculated at 3% onto the sterilized artichoke leaf powder medium. After culturing at 37℃ for 24 hours, the pH and viable cell count of the seven strains were measured. The results are as follows:

[0064] Table 1. pH value and viable cell count of each strain

[0065]

[0066] The results showed that CX-4 grew well in artichoke leaf powder medium with the highest viable count when the pH was lowered, CX-6 grew better, CX-5, CX1, and CX-7 grew slightly worse, and CX-2 and CX-3 had the lowest viable counts in this medium.

[0067] The macroscopic morphology of Lactobacillus curvature LJ003 colonies in Petri dishes is as follows: Figure 1 The image shows that the colonies are white, round, moist, opaque, and have neat edges.

[0068] The microstructure of Lactobacillus curvature LJ003 is as follows Figure 2 The images show that the bacteria are rod-shaped, arranged singly or in pairs, and are Gram-positive.

[0069] Strain CX-4 was sent to Sangon Biotech (Shanghai) Co., Ltd. for 16S rDNA sequencing, and the result identified it as *Lactobacillus curlis* (…). Lactobacillus crispatus The strain, named Lactobacillus curlis LJ003, was deposited on May 22, 2024, at the China General Microbiological Culture Collection Center, Institute of Microbiology, Chinese Academy of Sciences, No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing.

[0070] Example 2

[0071] A method for preparing fermented artichoke leaf powder:

[0072] Activation: Take the LJ003 glycerol cryovial stored at -80℃, thaw it at room temperature, and then, under aseptic conditions, use a pipette to inoculate 3% of the inoculum into MRS liquid medium. Incubate at 37℃ for 18 hours until OD500 is reached. 600 The concentration was 8.0, and the activated Lactobacillus curlyis LJ003 was obtained.

[0073] Take 200g of artichoke leaf powder (200 mesh sieve), add 20g of glucose and 800g of purified water, mix well and sterilize at 121℃ for 30min. Inoculate with Lactobacillus curvature LJ003 at 3% (v / v) at room temperature, and let it ferment at 37℃ for 24h. After fermentation, centrifuge at 6000r / min for 20min, and dry the precipitate obtained by centrifugation at 70℃ for 6h to obtain fermented artichoke leaf powder.

[0074] Experimental Example 1

[0075] The polyphenol and chlorogenic acid contents of the fermented artichoke leaf powder prepared in Example 2 were determined:

[0076] 1. Polyphenol content detection

[0077] Weigh 100g each of artichoke leaf powder (CX) and fermented artichoke leaf powder (FJCX), add 1500mL of 75% ethanol to each, extract by ultrasonication for 20min, centrifuge at 3500r / min for 10min to obtain the supernatant.

[0078] The polyphenol content in artichoke leaves was determined using the Folin-Ciocalteu colorimetric method, with gallic acid as the standard. 0.5 g of gallic acid standard was accurately weighed, dissolved in distilled water, and diluted to 0.05 mg / mL to obtain a standard solution. 0, 0.2, 0.4, 0.6, 0.8, 1.0, and 1.2 mL of this solution were respectively pipetted into 10 mL volumetric flasks, and 0.5 mL of Folin-Ciocalteu reagent was added to each. The mixture was shaken well, allowed to stand for 1 min, and then 1.5 mL of 20% Na₂CO₃ solution was added. The solution was then diluted to 10.0 mL with distilled water, thoroughly mixed, and incubated in a 70℃ water bath for 10 min. The absorbance was measured at 765 nm using a spectrophotometer. A standard curve was plotted with gallic acid concentration as the x-axis and absorbance as the y-axis. The regression equation for the standard curve was: y = 151.07x + 0.0011(R²). 2 = 0.9998).

[0079] The polyphenol content in artichokes was determined using the method described above, and calculated using the following formula:

[0080]

[0081] In the formula: C represents the polyphenol content in the sample solution (μg / mL), V represents the volume of the sample solution (mL), DF represents the dilution factor, and W represents the mass of the artichoke leaf powder (g). The results are as follows:

[0082] Table 2. Polyphenol content in artichoke leaf powder and fermented artichoke leaf powder

[0083]

[0084] The results showed that the polyphenol content of artichoke leaf powder fermented with Lactobacillus curvature LJ003 was significantly increased, with the polyphenol content of fermented artichoke leaf powder (FJCX) increasing by 46.96% compared to artichoke leaf powder (CX). These results indicate that fermentation of artichoke leaf powder with Lactobacillus curvature LJ003 can significantly improve its polyphenol content.

[0085] The obtained supernatant was concentrated to 1 / 5 of its original volume using a rotary evaporator. The concentrate was then freeze-dried to obtain 8.39 g of artichoke leaf powder (CX) extract and 12.26 g of fermented artichoke leaf powder (FJCX) extract, which showed no difference in polyphenol content compared to that determined by the Folin-Ciocalteu colorimetric method.

[0086] 2. Chlorogenic acid content detection

[0087] Weigh 10 mg of chlorogenic acid standard, dissolve it completely in 70% (v / v) ethanol, and dilute to 100 mL. Shake well to obtain a 100 μg / mL chlorogenic acid standard stock solution. Pipette 1 mL, 1.5 mL, 2 mL, 2.5 mL, and 3 mL of the stock solution into separate 10 mL volumetric flasks, dilute to volume with 70% (v / v) ethanol, and shake well. Using the 70% (v / v) ethanol solution as a blank control, measure the absorbance of each solution at 327 nm. Plot a standard curve with the standard solution concentration as the x-axis and absorbance as the y-axis. The results are shown below. Figure 3 As shown, find its regression equation.

[0088] According to Beer-Lambert law, within a certain concentration range, the absorbance value is directly proportional to the sample concentration. Figure 3 It can be seen that chlorogenic acid concentrations show a good linear relationship in the range of 0.01 mg / mL to 0.03 mg / mL.

[0089] Weigh 0.1 g of the above-mentioned artichoke powder (CX) and fermented artichoke leaf powder (FJCX), place them in a brown volumetric flask, and dissolve them in 70% ethanol to a final volume of 100 mL. Using the 70% (V / V) ethanol solution as a blank control, measure the absorbance at a wavelength of 327 nm. Perform the measurement three times in parallel, and calculate the sample content by substituting the values ​​into the standard curve equation.

[0090] The chlorogenic acid content in two extracts, artichoke powder (CX) and fermented artichoke leaf powder (FJCX), was determined using a UV-UV-Vis spectrophotometer. The results are as follows:

[0091] Table 3. Chlorogenic acid content in artichoke leaf powder and fermented artichoke leaf powder

[0092]

[0093] The results showed that the chlorogenic acid content in fermented artichoke leaf powder was higher than that in unfermented artichoke powder (CX), with an increase of 42.73%.

[0094] 3. Comparative experiment on fermentation of artichoke leaf powder by different Lactobacillus curvature.

[0095] Activation of Lactobacillus curlis LJ003: Take a cryovial of Lactobacillus curlis LJ003 stored at -80℃, thaw it at room temperature, and then, under aseptic conditions, use a pipette to inoculate 3% (v / v) of the inoculum into MRS liquid medium. Incubate at 37℃ for 18 hours until OD500. 600 The concentration was 8.0, and the activated Lactobacillus curlyis LJ003 was obtained.

[0096] Activation of Lactobacillus curlifi LCr86 (purchased from Wecon): Take the Lactobacillus curlifi LCr86 glycerol cryovial stored at -80℃, thaw it at room temperature, and then, under aseptic conditions, use a pipette to inoculate 3% (v / v) of the inoculum into MRS liquid medium. Incubate at 37℃ for 18 hours until OD... 600 The concentration was 7.6, resulting in activated Lactobacillus curlyis LCr86.

[0097] Activation of Lactobacillus curlifron LC-G22 (purchased from Runying Biotechnology): Take the Lactobacillus curlifron LC-G22 glycerol cryovial stored at -80℃, thaw it at room temperature, and then, under aseptic conditions, use a pipette to inoculate 3% (v / v) of the inoculum into MRS liquid medium. Incubate at 37℃ for 18 hours until OD... 600 The concentration was 8.5, and the activated Lactobacillus curlyis LC-G22 was obtained.

[0098] Take 200g of artichoke leaf powder (200 mesh sieve), add 20g of glucose and 800g of purified water, mix well and divide into three portions. Sterilize at 121℃ for 30min. Inoculate Lactobacillus curvature LJ003, Lactobacillus curvature LCr86 and Lactobacillus curvature LC-G22 at 3% (v / v) at room temperature. After static fermentation at 37℃ for 24h, detect the viable cell count and pH value of the fermentation broth.

[0099] Fermented artichoke leaf powder was obtained by centrifugation. The centrifuged artichoke leaf powder was dried at 70℃ to obtain artichoke leaf powder fermented by three strains: FJCX-003, FJCX-86, and FJCX-22. Polyphenol extraction was performed on the three different fermented artichoke powders. 100g of each of the three fermented artichoke powders was added to 1500mL of 75% ethanol, and the mixture was extracted ultrasonically for 20min, centrifuged at 3500r / min for 10min, and the supernatant was obtained. The polyphenol content of the supernatant was then determined.

[0100] The supernatant was concentrated to 1 / 5 of its original volume using a rotary evaporator. The concentrate was then freeze-dried to obtain three types of fermented artichoke freeze-dried powders, which were then weighed.

[0101] The results are as follows:

[0102] Table 4. Polyphenol content in artichoke leaf powder fermented with different Lactobacillus curvature.

[0103]

[0104] Note: Compared with Lactobacillus curvature LC-G22, *: P < 0.05; **: P < 0.01.

[0105] The results showed that all three *Lactobacillus curvature* strains could ferment artichoke leaf powder. However, *Lactobacillus curvature* strains LCr86 and LC-G22 did not grow vigorously in the culture medium containing artichoke leaf powder, with low viable cell counts and low metabolic acid production capacity. *Lactobacillus curvature* strain LJ003, isolated from artichoke leaf powder, was resistant to the antibacterial components in the powder and grew vigorously. The polyphenol content of fermented artichoke leaf powder FJCX-003 was 21.93% higher than that of FJCX-86 and 35.77% higher than that of FJCX-22, indicating that *Lactobacillus curvature* strain LJ003 is more suitable for fermenting artichoke leaf powder than *Lactobacillus curvature* strains LCr86 and LC-G22.

[0106] Example 3

[0107] A method for preparing polyphenols:

[0108] Activation: Take the LJ003 glycerol cryovial stored at -80℃, thaw it at room temperature, and then, under aseptic conditions, use a pipette to inoculate 3% (v / v) of the inoculum into MRS liquid medium. Incubate at 37℃ for 18 hours until OD (Organic Dysfunction Syndrome) is reached. 600 The concentration was 8.0, and the activated Lactobacillus curlyis LJ003 was obtained.

[0109] Take 200g of artichoke leaf powder (200 mesh sieve), add 20g of glucose and 800g of purified water, mix well and sterilize at 121℃ for 30min. Inoculate with Lactobacillus curvature LJ003 at 3% (v / v) at room temperature, and let it ferment at 37℃ for 24h. After fermentation, extract polyphenols.

[0110] Example 4

[0111] A method for preparing chlorogenic acid:

[0112] Activation: Take the LJ003 glycerol cryovial stored at -80℃, thaw it at room temperature, and then, under aseptic conditions, use a pipette to inoculate 3% (v / v) of the inoculum into MRS liquid medium. Incubate at 37℃ for 18 hours until OD (Organic Dysfunction Syndrome) is reached. 600 The concentration was 8.0, and the activated Lactobacillus curlyis LJ003 was obtained.

[0113] Take 200g of artichoke leaf powder (200 mesh sieve), add 20g of glucose and 800g of purified water, mix well and sterilize at 121℃ for 30min. Inoculate with Lactobacillus curvature LJ003 at 3% (v / v) at room temperature, and let it ferment at 37℃ for 24h. After fermentation, extract chlorogenic acid.

[0114] Example 5

[0115] A liver-protecting probiotic composition (by weight): 50 parts sheep bone marrow oligopeptide, 25 parts isomaltooligosaccharide, 10 parts erythritol, 1 part glutathione-enriched yeast, 5 parts milk thistle seed oil powder, 1 part sheep spleen peptide, 0.05 parts Haematococcus pluvialis microcapsule powder, 3 parts corn oligopeptide, 2 parts fermented artichoke leaf powder prepared in Example 2, 1.5 parts sunflower lecithin, 0.08 parts lutein microcapsule powder, 0.6 parts vitamin E, 0.02 parts (3R,3'S)-dihydroxy-β-carotene, 0.25 parts *Lactobacillus plantarum* LZ015 lyophilized powder, 0.25 parts *Lactobacillus rhamnosus* NKU FL1-8 lyophilized powder, and 0.25 parts *Bifidobacterium lactis* BR001 lyophilized powder.

[0116] The preparation method includes the following steps:

[0117] (1) Preparation of freeze-dried Lactobacillus plantarum LZ015 powder:

[0118] Take the cryopreserved tube of Lactobacillus plantarum LZ015 (CGMCC No. 29395) stored at -80℃, thaw it at room temperature, and then use a pipette to inoculate 3% (v / v) of the seed solution into MRS liquid medium under aseptic conditions. Incubate at 37℃ for 18 h until the OD600 reaches 7.5 to obtain activated Lactobacillus plantarum LZ015. Lactobacillus plantarum LZ015 was inoculated at 3% (v / v) into a culture medium (the culture medium formula is: 25g glucose, 8g bovine bone peptone, 15g yeast extract, 10g soybean peptone, 2g dipotassium hydrogen phosphate, 2g diammonium hydrogen citrate, 5g sodium acetate, 1g Tween 80, 0.58g magnesium sulfate, 0.25g manganese sulfate, 0.5g cysteine ​​hydrochloride, and 1000g purified water), and cultured at 37℃ for 18h. Then, at a ratio of culture product volume to freeze-drying protectant volume of 1:2, freeze-drying protectant (the freeze-drying protectant formula is: 2g β-cyclodextrin, 0.2g Tween 80, 10g trehalose, 8g skim milk, 0.1g L-cysteine, and 100g water) was added, thoroughly mixed, and freeze-dried at -30℃ for 68h to obtain Lactobacillus plantarum LZ015 freeze-dried powder with a viable count of 6.0 × 10⁻⁶. 11 cfu / g.

[0119] (2) Preparation of freeze-dried Lactobacillus rhamnosus NKU FL1-8 powder:

[0120] Take the cryopreserved tube of Lactobacillus rhamnosus NKU FL1-8 (GDMCC No. 64053) stored at -80℃, thaw it at room temperature, and then use a pipette to inoculate 3% (v / v) of the inoculum into MRS liquid medium under aseptic conditions. Incubate at 37℃ for 18 h until the OD600 reaches 10.5 to obtain activated Lactobacillus rhamnosus NKU FL1-8. Lactobacillus rhamnosus NKU FL1-8 was inoculated at 3% (v / v) into a culture medium (the culture medium formula is: 25g glucose, 8g bovine bone peptone, 15g yeast extract, 10g soybean peptone, 2g dipotassium hydrogen phosphate, 2g diammonium hydrogen citrate, 5g sodium acetate, 1g Tween 80, 0.58g magnesium sulfate, 0.25g manganese sulfate, 0.5g cysteine ​​hydrochloride, and 1000g purified water), and cultured at 37℃ for 18h. Then, at a ratio of culture product volume to freeze-drying protectant volume of 1:2, freeze-drying protectant (the freeze-drying protectant formula is: 10g skim milk, 0.2g Tween 80, 10g trehalose, 1g soybean polysaccharide, 1g collagen peptide, 2g monosodium glutamate, 0.1g L-cysteine, and 100g water) was added, thoroughly mixed, and freeze-dried at -30℃ for 68h to obtain Lactobacillus rhamnosus NKU. FL1-8 lyophilized powder has a viable count of 4.5 × 10⁻⁶. 11 cfu / g.

[0121] (3) Preparation of lyophilized Bifidobacterium lactis BR001 powder:

[0122] Take the glycerol cryovial of Bifidobacterium lactis BR001 (CGMCC No. 23665) stored at -80℃, thaw it at room temperature, and then, under aseptic conditions, use a pipette to inoculate 3% (v / v) of the inoculum into modified MRS liquid medium. Incubate anaerobically at 37℃ for 21 h until the OD600 reaches 7.0 to obtain activated Bifidobacterium lactis BR001. Inoculate Bifidobacterium lactis BR001 at 3% (v / v) into a culture medium (the culture medium formula is: glucose 25g, fructooligosaccharides 10g, tryptone 10g, yeast extract 20g, soybean peptone 6g, dipotassium hydrogen phosphate 2g, diammonium hydrogen citrate 2g, sodium acetate 5g, Tween 80). 1g of Bifidobacterium lactis, 0.58g of magnesium sulfate, 0.25g of manganese sulfate, 0.5g of cysteine ​​hydrochloride, and 1000g of purified water were added and anaerobically cultured at 37℃ for 21h. Then, at a ratio of 1:2 (culture product volume: freeze-drying protectant volume), freeze-drying protectant (formulation: 2g β-cyclodextrin, 0.1g Tween 800, 10g trehalose, 8g skim milk, 0.1g L-cysteine, 4g sucrose, and 100g water) was added, mixed thoroughly, and freeze-dried at -30℃ for 68h to obtain Bifidobacterium lactis BR001 freeze-dried powder with a viable count of 7.0×10⁻⁶. 11 cfu / g.

[0123] (4) Mix the components in proportion to obtain the liver-protecting probiotic composition.

[0124] Example 6

[0125] The only difference from Example 5 is that:

[0126] Liver-protecting probiotic composition (by weight): 45 parts sheep bone marrow oligopeptide, 21.54 parts isomaltooligosaccharide, 14 parts erythritol, 1.7 parts glutathione-enriched yeast, 5.5 parts milk thistle seed oil powder, 2 parts sheep spleen peptide, 0.05 parts Haematococcus pluvialis microcapsule powder, 3.5 parts corn oligopeptide, 2.5 parts fermented artichoke leaf powder prepared in Example 2, 2 parts sunflower lecithin, 0.08 parts lutein microcapsule powder, 0.9 parts vitamin E, 0.03 parts (3R,3'S)-dihydroxy-β-carotene, 0.45 parts *Lactobacillus plantarum* LZ015 lyophilized powder, 0.35 parts *Lactobacillus rhamnosus* NKU FL1-8 lyophilized powder, and 0.4 parts *Bifidobacterium lactis* BR001 lyophilized powder.

[0127] Example 7

[0128] The only difference from Example 5 is that:

[0129] Liver-protecting probiotic composition (by weight): 41.07 parts sheep bone marrow oligopeptide, 20 parts isomaltooligosaccharide, 12 parts erythritol, 1.5 parts glutathione-enriched yeast, 6 parts milk thistle seed oil powder, 8 parts sheep spleen peptide, 0.06 parts Haematococcus pluvialis microcapsule powder, 4 parts corn oligopeptide, 2.5 parts fermented artichoke leaf powder prepared in Example 2, 2.5 parts sunflower lecithin, 0.09 parts lutein microcapsule powder, 1 part vitamin E, 0.03 parts (3R,3'S)-dihydroxy-β-carotene, 0.5 parts *Lactobacillus plantarum* LZ015 lyophilized powder, 0.3 parts *Lactobacillus rhamnosus* NKU FL1-8 lyophilized powder, and 0.45 parts *Bifidobacterium lactis* BR001 lyophilized powder.

[0130] Example 8

[0131] The only difference from Example 5 is that:

[0132] Liver-protecting probiotic composition (by weight): 36 parts sheep bone marrow oligopeptide, 27 parts isomaltooligosaccharide, 13 parts erythritol, 1.2 parts glutathione-enriched yeast, 7 parts milk thistle seed oil powder, 5.27 parts sheep spleen peptide, 0.07 parts Haematococcus pluvialis microcapsule powder, 3.5 parts corn oligopeptide, 3 parts fermented artichoke leaf powder prepared in Example 2, 2 parts sunflower lecithin, 0.1 parts lutein microcapsule powder, 0.8 parts vitamin E, 0.01 parts (3R,3'S)-dihydroxy-β-carotene, 0.3 parts *Lactobacillus plantarum* LZ015 lyophilized powder, 0.4 parts *Lactobacillus rhamnosus* NKU FL1-8 lyophilized powder, and 0.35 parts *Bifidobacterium lactis* BR001 lyophilized powder.

[0133] Example 9

[0134] The only difference from Example 5 is that:

[0135] Liver-protecting probiotic composition (by weight): 25 parts sheep bone marrow oligopeptide, 30 parts isomaltooligosaccharide, 15 parts erythritol, 2 parts glutathione-enriched yeast, 8 parts milk thistle seed oil powder, 7.21 parts sheep spleen peptide, 0.08 parts Haematococcus pluvialis microcapsule powder, 5 parts corn oligopeptide, 4 parts fermented artichoke leaf powder prepared in Example 2, 1 part sunflower lecithin, 0.1 parts lutein microcapsule powder, 1.2 parts vitamin E, 0.01 parts (3R,3'S)-dihydroxy-β-carotene, 0.4 parts *Lactobacillus plantarum* LZ015 lyophilized powder, 0.5 parts *Lactobacillus rhamnosus* NKU FL1-8 lyophilized powder, and 0.5 parts *Bifidobacterium lactis* BR001 lyophilized powder.

[0136] Comparative Example 1

[0137] The only difference from Example 9 is that the fermented artichoke leaf powder prepared in Example 2 is replaced with artichoke leaf powder.

[0138] Experimental Example 2

[0139] The efficacy of the liver-protecting probiotic compositions prepared in Examples 5, 9 and Comparative Example 1 was verified:

[0140] 1. Acute liver injury mouse experiment:

[0141] Sixty healthy male KM mice, aged 6-8 weeks and weighing 18-22g, SPF grade, were purchased from the Experimental Animal Center of Hebei Medical University. After one week of acclimatization, the mice were randomly divided into six groups of 10 mice each: normal group, model group, positive control group, experimental group 1, experimental group 5, and control group 1. The normal group and model group were administered an equal volume of physiological saline by gavage. The positive control group was administered Haiwang Jinzun (Haiwang Group) (85mg / kg BW) by gavage. Experimental group 1 was administered the composition prepared in Example 5 (85mg / kg BW), experimental group 5 was administered the composition prepared in Example 9 by gavage, and control group 1 was administered the composition prepared in Comparative Example 1 by gavage. These administrations were repeated for 21 days. Starting on day 14, the model group, positive control group, experimental group 1, experimental group 5, and control group 1 were administered 56° Baijiu (Beijing Hongxing Co., Ltd.) (12mL / kg BW) by gavage daily for 7 days, 30 minutes after each administration. Mice were fasted for 12 hours before the last alcohol administration but allowed free access to water. Two hours after the last alcohol administration, blood was collected from the eyeballs and serum was collected. The mice were then euthanized by cervical dislocation, and their livers were quickly removed. The excised liver tissue was divided into two parts, which were rapidly frozen in liquid nitrogen and fixed in 4% paraformaldehyde, respectively, for subsequent index determination.

[0142] 2. Testing items:

[0143] (1) Following the operating procedures and requirements of the aspartate aminotransferase (AST), alanine aminotransferase (ALT), triglyceride (TG), and total cholesterol (TC) kits (purchased from Nanjing Jiancheng Biotechnology Institute), the serum transaminase levels ALT and AST, and the lipid levels TG and TC were measured in mice. The results are as follows: Figures 4-5 As shown (compared with the control group, #P<0.05, ##P<0.01; compared with the model group, *P<0.05, **P<0.01).

[0144] Figure 4The activity levels of ALT and AST in the serum of mice in each group were measured. The results showed that the activity levels of ALT and AST in the model group were significantly higher than those in the control group (P<0.01). Compared with the model group, the activity levels of ALT and AST in the serum of the positive control group, experimental group 1, experimental group 5, and control group 1 were significantly lower (P<0.01). These results indicate that the positive control group, experimental group 1, experimental group 5, and control group 1 can effectively inhibit acute hepatocyte injury induced by excessive ethanol intake and have a protective effect on mouse hepatocytes. The figure shows that the activity levels of ALT and AST in experimental group 5 were lower than those in control group 1, indicating that the product obtained after fermentation of artichoke with Lactobacillus curvature LJ003 can enhance the protective effect of the composition on hepatocytes.

[0145] Figure 5 The levels of TC and TG in the serum of mice in each group were measured. The results showed that the levels of TC and TG in the serum of the model group were significantly higher than those in the control group (P<0.01). Compared with the model group, the levels of TC and TG in the serum of the positive control group, experimental group 1, experimental group 5, and control group 1 were significantly lower (P<0.01). The results indicate that prophylactic administration in experimental groups 1 and 5 can effectively improve hepatocyte lipid metabolism disorders caused by excessive ethanol intake.

[0146] 2. Weigh 0.1g of liver sample, add pre-cooled physiological saline at a volume ratio of 1:9, and homogenize completely in an ice-water bath. After homogenization, centrifuge and collect the supernatant (4℃, 3000r / min, 15min). Following the operating procedures and requirements of the malondialdehyde (MDA) detection kit and superoxide dismutase (SOD) kit (purchased from Nanjing Jiancheng Biotechnology Institute), determine the content of SOD enzyme and MDA in mouse liver. The results are as follows: Figures 6-7 As shown (compared with the control group, #P<0.05, ##P<0.01; compared with the model group, *P<0.05, **P<0.01).

[0147] Figure 6 The levels of MDA in the liver tissue of mice in each group were compared. The results showed that, compared with the normal group, the MDA level in the liver tissue of the model group mice was significantly increased (P < 0.01), indicating that excessive alcohol consumption led to oxidative stress and caused oxidative damage to the liver. Compared with the model group, the MDA levels in the positive control group, experimental group 1, experimental group 5, and control group 1 were significantly decreased (P < 0.01), indicating that the Haiwang Jinzun tablet and the positive control group, experimental group 1, experimental group 5, and control group 1 could all alleviate liver oxidative damage caused by excessive alcohol consumption, with no significant differences among them. Among these, compared with control group 1, the extract obtained from artichoke fermented with Lactobacillus curvature LJ003 had a better protective effect on hepatocytes in experimental group 5.

[0148] Figure 7 The levels of superoxide dismutase (SOD) in the liver tissues of mice in each group were measured. The results showed that the SOD activity level in the liver of the model group was significantly lower than that of the control group (P<0.01). Compared with the model group, the SOD activity levels in the livers of the positive control group, experimental group 1, experimental group 5, and control group 1 were significantly higher (P<0.01). These results indicate that excessive ethanol intake damages the liver's antioxidant enzyme system. Prophylactic administration to experimental groups 1 and 5 effectively reduced the inhibition of SOD activity by ethanol, decreased the level of oxidative stress in the liver, and mitigated the damage caused by ethanol to hepatocytes.

[0149] 3. Following the operating procedures and requirements of the interleukin-1β (IL-1β), interleukin-6 (IL-6), and tumor necrosis factor-α (TNF-α) detection kit (purchased from Shanghai Enzyme-Linked Biotechnology Co., Ltd.), the levels of pro-inflammatory factors IL-6, IL-1β, and TNF-α in mouse liver were measured. The results are as follows: Figure 8 As shown.

[0150] The results showed that, compared with the control group, the levels of pro-inflammatory factors such as IL-1β, IL-6, and TNF-α in the liver of mice in the model group were significantly increased (P<0.01). Compared with the model group, prophylactic administration in the positive control group, experimental group 1, experimental group 5, and control group 1 all significantly reduced the levels of IL-1β, IL-6, and TNF-α in the liver of mice (P<0.01). These results indicate that excessive ethanol intake triggers an inflammatory response in the liver, increasing the levels of pro-inflammatory factors. Experimental groups 1 and 5 can inhibit the production of pro-inflammatory factors, alleviate ethanol-induced inflammatory responses, and exert a hepatoprotective effect.

[0151] 4. Hematoxylin-eosin (HE) staining: Liver specimens were fixed in 4% paraformaldehyde for 24 hours, dehydrated with graded ethanol, embedded in paraffin, sectioned, and stained with HE. Pathological changes in the liver tissue were observed under a light microscope. The results are as follows: Figure 9 As shown.

[0152] The results showed that the hepatocytes of the control group mice had intact morphology and clear outlines, with radially arranged hepatic cords and round, clear hepatocyte nuclei. Compared with the control group, the hepatocytes of the model group had incomplete structure, disordered arrangement, blurred cell boundaries, and obvious inflammatory cell infiltration around the central vein. Compared with the model group, the positive control group, experimental group 1, experimental group 5, and control group 1 showed a significant reduction in inflammatory cell infiltration around the central vein, with hepatocytes arranged relatively neatly and hepatic cords clearly arranged. Compared with control group 1, experimental group 5 showed more inflammatory cell infiltration around the central vein, indicating that the increased chlorogenic acid content in the extract obtained from artichoke fermentation by Lactobacillus criniforme LJ033 has a protective effect on the liver.

[0153] Experimental Example 3

[0154] The effects of the combination of artichoke and probiotics on Hep G2 cells (human liver cancer cells) were verified in vitro. The fermented artichoke used in the experiment was the fermented artichoke leaf powder prepared in Example 2; the freeze-dried powders of Lactobacillus plantarum LZ015, Lactobacillus rhamnosus NKU FL1-8, and Bifidobacterium lactis BR001 were freeze-dried powders prepared in Example 5.

[0155] Group processing:

[0156] Group A: Fermented artichokes (0.2g);

[0157] Group B: Lactobacillus plantarum LZ015 (25mg, 100 billion / g), Bifidobacterium animalis subsp. lactis BR001 (25mg, 100 billion / g), Lactobacillus rhamnosus NKU FL1-8 (25mg, 100 billion / g).

[0158] Group C: Fermented artichoke (0.2g), Lactobacillus plantarum LZ015 (25mg, 100 billion / g), Bifidobacterium animalis subsp. lactis BR001 (25mg, 100 billion / g);

[0159] Group D: Fermented artichoke (0.2g), Bifidobacterium animalis subsp. lactis BR001 (25mg, 100 billion / g), Lactobacillus rhamnosus NKU FL1-8 (25mg, 100 billion / g).

[0160] Group E: Fermented artichoke (0.2g), Lactobacillus plantarum LZ015 (25mg, 100 billion / g), Lactobacillus rhamnosus NKU FL1-8 (25mg, 100 billion / g);

[0161] Group F: Fermented artichoke (0.2g), Lactobacillus plantarum LZ015 (25mg, 100 billion / g), Bifidobacterium animalis subsp. lactis BR001 (25mg, 100 billion / g), Lactobacillus rhamnosus NKU FL1-8 (25mg, 100 billion / g).

[0162] The above six compositions were dissolved in 1 ml of sterile purified water and mixed thoroughly before use.

[0163] After resuscitation, Hep G2 cells (Tongpai Biotechnology) were rapidly resuspended in DMEM medium containing 10% FBS. After resuscitation, they were transferred to T25 cell culture flasks and cultured in a constant temperature incubator at 37℃ and 5% CO2 for 24 hours. When 80% to 90% of the Hep G2 cells adhered to the culture vessel, they were gently washed 2 to 3 times with sterile phosphate-buffered saline (PBS), digested with 0.25% trypsin, and then pipetted to obtain a single-cell suspension. The cells were passaged at a ratio of 1:2, and cells in the logarithmic growth phase were collected for counting, plating, and experiments.

[0164] Hep G2 cells in the logarithmic growth phase were collected, and the concentration of the cell suspension was adjusted to 1×10⁻⁶. 5 Cells were seeded at a density of 1 / mL in 12-well plates and cultured in a cell culture incubator set at 37°C and 5% CO2 concentration. 1 mL of cell suspension was added to each well. The experiment was divided into a control group, a model group, and a culture medium treatment group, with each group being replicated three times.

[0165] Control group: Normal Hep G2 cells were cultured for 24 hours, with fresh culture medium replaced every 24 hours.

[0166] Model group: Hep G2 cells cultured for 24 h were aspirated, washed three times with pre-cooled PBS, and then 1 mL of 600 mmol / L ethanol was added. After treatment for 8 h, the culture medium was replaced with fresh medium every 24 h.

[0167] Experimental group: Hep G2 cell culture medium was aspirated after 24 h of culture, washed three times with pre-cooled PBS, and then 1 mL of 600 mmol / L ethanol was added to each well for 8 h. After that, the cell culture medium was aspirated, washed three times with pre-cooled PBS, and then 1 mL of solutions from groups A, B, C, D, E, and F were added to each well for 24 h. According to the measurement requirements, the cells were lysed or the supernatant was collected to measure the levels of MDA, IL-1β, TNF-α, and IL-6, and the activities of ALT, AST, and SOD enzymes. The results are as follows. Figures 10-13 As shown (compared with the control group, #P<0.05, ##P<0.01; compared with the model group, *P<0.05, **P<0.01).

[0168] Figure 10To investigate the effects of each group on MDA content, the results showed that 600 mmol / L alcohol damaged Hep G2 cells (P<0.01). Groups A, B, C, D, E, and F all repaired Hep G2 cells and reduced MDA production in alcohol-damaged Hep G2 cells. Groups A and B showed differences in Hep G2 cell repair compared to the model group (P<0.05). Groups C, D, E, and F showed significant differences in Hep G2 cell repair (P<0.01), with group F showing the most significant difference. This difference may be due to the maximized synergistic effect of probiotics and artichokes, with the combination of the three probiotic strains exhibiting optimal synergistic effects.

[0169] Figure 11 To investigate the effects of each group on inflammatory factors, the results showed that all six experimental groups reduced the levels of IL-1β, TNF-α, and IL-6 in Hep G2 cells. Compared with the model group, groups B, C, D, E, and F showed extremely significant effects (P<0.01), with group F showing the most significant effect; group A showed significant effects in TNF-α and IL-6 detection (P<0.05). These six experiments demonstrate that the combination and optimized pairing of artichoke and probiotics can inhibit the release of pro-inflammatory mediators IL-1β, TNF-α, and IL-6, assist in the repair of the body's immune dynamic balance, and prevent alcohol-induced cell damage.

[0170] Figure 12 To investigate the effects of each group on SOD content, the results showed that the SOD enzyme activity in alcohol-treated Hep G2 cells was significantly reduced by 24.46% compared to the control group (P<0.01), indicating that alcohol caused severe damage to Hep G2 cells. All six experimental samples repaired Hep G2 cells, increasing SOD activity in alcohol-damaged Hep G2 cells (P<0.01). Group F showed the most significant improvement, with the highest SOD activity reaching 95.18 U / mg prot, a significant increase of 29.36% compared to the model group (P<0.01). These results indicate that the combination of fermented artichoke and probiotics in group F was the optimal combination.

[0171] Figure 13The effects of each group on ALT and AST enzyme activities were investigated. Results showed that the alcohol-induced oxidative damage model of Hep G2 cells led to increased ALT and AST activity levels. In the model group, ALT and AST activities were significantly increased by 1.1-fold and 1.84-fold, respectively, compared to the control group (P<0.01). All six experimental samples, when used to repair Hep G2 cells, reduced ALT and AST activities in alcohol-damaged Hep G2 cells. Compared to the model group, the reduction rates of ALT and AST activities in all six groups were greater than 10%, with highly significant differences (P<0.01). Group F showed the best effect, with ALT and AST activities decreasing by 49.63% and 55.41%, respectively. Therefore, Group F had the best repair effect on alcohol-damaged Hep G2 cells.

[0172] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A type of Lactobacillus curvature LJ003, characterized in that, The curly lactobacillus ( Lactobacillus crispatus The accession number of LJ003 is CGMCC No.30723.

2. The application of Lactobacillus curvature LJ003 as described in claim 1 in the fermentation preparation of polyphenols and chlorogenic acid using artichoke leaves as raw material.

3. A method for preparing fermented artichoke leaf powder, characterized in that, The process includes the following steps: mixing artichoke leaf powder, glucose, and water in a mass ratio of 20:2:80, sterilizing the mixture, and obtaining fermentation raw material; inoculating the fermentation raw material with Lactobacillus curvatureus LJ003 as described in claim 1 for fermentation culture to obtain fermentation culture product; centrifuging and drying the fermentation culture product to obtain fermented artichoke leaf powder.

4. The fermented artichoke leaf powder prepared by the method of claim 3.

5. A method for preparing polyphenols, characterized in that, Includes the following steps: Artichoke leaf powder, glucose, and water were mixed in a mass ratio of 20:2:80 and sterilized to obtain fermentation raw material; Lactobacillus curvatureus LJ003 as described in claim 1 was inoculated into the fermentation raw material for fermentation culture to obtain fermentation culture product; polyphenols were extracted from the fermentation culture product.

6. A method for preparing chlorogenic acid, characterized in that, Includes the following steps: Artichoke leaf powder, glucose, and water were mixed in a mass ratio of 20:2:80 and sterilized to obtain fermentation raw material; Lactobacillus curvatureus LJ003 as described in claim 1 was inoculated into the fermentation raw material for fermentation culture to obtain fermentation culture product; chlorogenic acid was extracted from the fermentation culture product.

7. A liver-protecting probiotic composition, characterized in that, By weight, it includes the following components: 25-50 parts sheep bone marrow oligopeptides, 20-30 parts isomaltooligosaccharides, 10-15 parts erythritol, 1-2 parts glutathione-enriched yeast, 5-8 parts milk thistle seed oil powder, 1-8 parts sheep spleen peptides, 0.05-0.08 parts Haematococcus pluvialis microcapsule powder, 3-5 parts corn oligopeptides, 2-4 parts fermented artichoke leaf powder, 1-2.5 parts sunflower lecithin, 0.08-0.1 parts lutein microcapsule powder, and vitamin E. 0.6~1.2 parts, (3R,3'S)-dihydroxy-β-carotene 0.01~0.03 parts, *Lactobacillus plantarum* LZ015 lyophilized powder 0.25~0.5 parts, *Lactobacillus rhamnosus* NKUFL1-8 lyophilized powder 0.25~0.5 parts, and *Bifidobacterium lactis* BR001 lyophilized powder 0.25~0.5 parts; The fermented artichoke leaf powder is the fermented artichoke leaf powder as described in claim 4; the preservation number of Lactobacillus plantarum LZ015 is: CGMCC No. 29395; the preservation number of Lactobacillus rhamnosus NKU FL1-8 is: GDMCC No. 64053; and the preservation number of Bifidobacterium lactis BR001 is: CGMCC No. 23665.

8. The liver-protecting probiotic composition according to claim 7, characterized in that, The viable count of the *Lactobacillus plantarum* LZ015 freeze-dried powder is 5.0 × 10⁻⁶. 11 cfu / g ~7.0×10 11 The viable count of the *Lactobacillus rhamnosus* NKU FL1-8 lyophilized powder was 4.0 × 10⁻⁶ cfu / g. 11 cfu / g ~5.0×10 11 cfu / g; the viable count of the Bifidobacterium lactis BR001 lyophilized powder is 6.0 × 10⁻⁶. 11 cfu / g ~8.0×10 11 cfu / g.

9. The method for preparing the liver-protecting probiotic composition according to claim 7 or 8, characterized in that, Includes the following steps: The components are mixed in proportion to obtain the liver-protecting probiotic composition.

10. The use of the hepatoprotective probiotic composition according to claim 7 or 8 in the preparation of a medicament for treating acute alcoholic liver injury.

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