Lactic acid bacteria fermentation product for protecting liver and application of lactic acid bacteria fermentation product

Abstract

The invention relates to the technical field of probiotics, in particular to a lactic acid bacteria fermentation product for protecting the liver and application of the lactic acid bacteria fermentation product. The lactobacillus fermentation product is a composite fermentation product which is prepared by mixing fermentation products which are obtained by fermenting and culturing a bifidobacterium animalis subsp. Lactis BR621 strain, a lactobacillus reuteri mucus RU-23 strain, a lactobacillus mucus fermentation Fem-9 strain, a lactobacillus rhamnosus LRM369 strain, a phytobacterium plantarum PQQ-1 strain and a phytobacterium plantarum LPA-56 strain in proportion. According to the present invention, the antioxidant ability is evaluated through the ABTS free radical scavenging rate, and the physiological activity experiment analysis results show that the lactic acid bacteria fermentation product has the liver protection physiological activity function, can scavenge the free radicals, and can improve the liver antioxidant system ability so as to resist the oxidation pressure and the alcoholic liver injury.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of probiotics, in particular to a lactic acid bacteria ferment for protecting liver and use thereof. BACKGROUND

[0002] Liver is the main metabolic and detoxification organ of the human body. In the process of metabolizing substances such as alcohol, drugs, nutrients, etc., it is inevitable to produce excessive reactive oxygen species (ROS) or related free radicals. When the amount of free radicals generated exceeds the clearance capacity of the body's antioxidant system, it leads to the accumulation of oxidative stress, and then causes oxidative damage to liver cells. The formation mechanism of liver oxidative damage involves the imbalance of increased free radical generation and reduced antioxidant defense, which triggers lipid peroxidation, protein oxidation or DNA damage, leading to cell membrane structure damage, enzyme function disorder, mitochondrial energy generation obstruction and inflammation signal pathway activation, and finally may cause cell apoptosis or necrosis. Therefore, how to reduce oxidative stress or improve the antioxidant defense capacity of the liver has become an important research direction in the industry.

[0003] Research on probiotics for human health is very extensive. In general, strains with special effects on human health are called functional probiotics. The prior art also discloses various functional probiotics, but the special effect of functional probiotics lies in the specificity of the strain (strain) rather than the species (species). Even if it is the same species, if it is a different strain, it may have different effects, or even opposite effects.

[0004] Therefore, there is an urgent need to develop a safe and long-term edible nutritional supplement with liver protection. Lactic acid bacteria are generally considered safe, so how to find a lactic acid bacteria ferment with liver protection activity is an urgent goal. SUMMARY

[0005] The present application aims at the deficiencies of the prior art, and provides a lactic acid bacteria ferment for protecting liver and use thereof.

[0006] The above invention purpose of the present application is realized by the following technical scheme: One of the purposes of the present application is to provide a lactic acid bacteria fermentation product for protecting liver, which is a composite fermentation product obtained by mixing the fermentation products of Bifidobacterium animalis lactis BR621 strain with the preservation number of CGMCC NO. 27537, Lactobacillus rhamnosus LRM369 strain with the preservation number of CGMCC NO. 27538, Lactobacillus plantarum PQQ-1 strain with the preservation number of CGMCC NO. 29303, Lactobacillus plantarum LPA-56 strain with the preservation number of CGMCC NO. 24355, Lactobacillus reuteri RU-23 strain with the preservation number of CGMCC NO. 33219 and Lactobacillus fermentum Fem-9 strain with the preservation number of CGMCC NO. 33220 in a certain proportion.

[0007] Preferably, the mass ratio of the fermentation products of Bifidobacterium animalis lactis BR621 strain, Lactobacillus reuteri RU-23 strain, Lactobacillus fermentum Fem-9 strain, Lactobacillus rhamnosus LRM369 strain, Lactobacillus plantarum PQQ-1 strain and Lactobacillus plantarum LPA-56 strain is (4.5~5.5):(2.5~3.5):(1.5~2.5):(0.5~1.5):(0.6~0.8):(0.2~0.4).

[0008] More preferably, the mass ratio of the fermentation products of Bifidobacterium animalis lactis BR621 strain, Lactobacillus reuteri RU-23 strain, Lactobacillus fermentum Fem-9 strain, Lactobacillus rhamnosus LRM369 strain and Lactobacillus plantarum PQQ-1 strain is 5:3:2:1:0.7:0.3.

[0009] The second purpose of the present application is to provide the use of the above-mentioned lactic acid bacteria fermentation product in the preparation of a composition for protecting liver.

[0010] Preferably, the use for protecting liver is to improve the ability of liver to resist oxidative stress.

[0011] Preferably, the use for protecting liver is to improve the ability of liver to resist alcoholic liver injury.

[0012] The third purpose of the present application is to provide a composition for protecting liver, which comprises the above-mentioned lactic acid bacteria fermentation product and a pharmaceutically or physiologically acceptable carrier.

[0013] Preferably, the composition is a food composition or a pharmaceutical composition.

[0014] Preferably, the carrier is an excipient or a diluent, including any one or a mixture of two or more of oligosaccharides, sugar alcohols, sodium carboxymethyl cellulose, ethyl cellulose, galactooligosaccharides, stearic acid, magnesium stearate, potato starch.

[0015] Preferably, the dosage form of the composition is an oral dosage form, including a tablet, a capsule or a powder.

[0016] The lactic acid bacteria ferment of the present application has the following beneficial effects: The lactic acid bacteria ferment of the present application is a composite ferment obtained by mixing the ferment of the following six different lactic acid bacteria strains in a certain proportion: Bifidobacterium animalis subsp. lactis BR621, Lactobacillus rhamnosus RU-23, Lactobacillus fermentum Fem-9, Lactobacillus rhamnosus LRM369, Lactobacillus plantarum PQQ-1 and Lactobacillus plantarum LPA-56. The antioxidant capacity and physiological activity of the above-mentioned six different lactic acid bacteria ferments and the composite ferment thereof were evaluated by ABTS free radical scavenging rate, and the results showed that: (1) the ABTS free radical scavenging rate of the lactic acid bacteria ferment of the present application can reach 88.57%, which is better than the ABTS free radical scavenging rate of other single lactic acid bacteria ferments, and there is a significant difference; (2) through the analysis of the content of malondialdehyde (MDA) in the liver tissue of mice, the lactic acid bacteria ferment of the present application can reduce the oxidative damage of the liver; (3) through the analysis of the content of interleukin-1β (IL-1β) in the liver tissue of mice, the lactic acid bacteria ferment of the present application can effectively inhibit the inflammatory response of the liver induced by alcohol; (4) through the analysis of the AST activity of the blood of mice, the lactic acid bacteria ferment of the present application can effectively avoid the damage of liver cells caused by alcohol; (5) through the analysis of the ALT activity of the blood of mice, the lactic acid bacteria ferment of the present application can effectively reduce the damage of liver cells caused by alcohol; (6) through the analysis of the SOD activity of the liver tissue of mice, the lactic acid bacteria ferment of the present application can enhance the antioxidant system to scavenge free radicals in the body, thereby reducing the oxidative stress of the liver; (7) through the analysis of the CAT activity of the liver tissue of mice, the lactic acid bacteria ferment of the present application can effectively enhance the CAT activity, thereby enhancing the scavenging capacity of H2O2, effectively scavenging free radicals in the liver tissue, and thereby reducing the oxidative stress of the liver; (8) through the histopathological examination of the liver tissue of mice, the lactic acid bacteria ferment of the present application can protect the liver to avoid or reduce the liver damage induced by alcohol. Therefore, the lactic acid bacteria ferment of the present application has the physiological activity of protecting the liver, and can be used for preparing a medicine or a food composition for protecting the liver. BRIEF DESCRIPTION OF DRAWINGS

[0017] Figure 1 The test results of the ABTS free radical scavenging rate of the six lactic acid bacteria ferments of Example 1 and their mixtures at a concentration of 0.2 mg / mL.

[0018] Figure 2 The test results of the analysis of the malondialdehyde content of the liver tissue of each group of mice by the lactic acid bacteria ferment of Example 1.

[0019] Figure 3 Test results of the lactic acid bacteria ferment of Example 1 for the IL-1β content analysis of the liver tissues of each group of mice.

[0020] Figure 4 Test results of the lactic acid bacteria ferment of Example 1 for the AST activity analysis of the blood of each group of mice.

[0021] Figure 5 Test results of the lactic acid bacteria ferment of Example 1 for the ALT activity analysis of the blood of each group of mice.

[0022] Figure 6 Test results of the lactic acid bacteria ferment of Example 1 for the SOD activity analysis of the liver tissues of each group of mice.

[0023] Figure 7 Test results of the lactic acid bacteria ferment of Example 1 for the CAT activity analysis of the liver tissues of each group of mice.

[0024] Figure 8 Test results of the lactic acid bacteria ferment of Example 1 for the results observed by hematoxylin-eosin staining of the liver tissue sections of each group of mice. DETAILED DESCRIPTION

[0025] The microbial strain materials involved in the present application are shown in Table 1.

[0026] Table 1. Preservation information of the lactic acid bacteria strains of the present application Strain name Classification name Preservation number Preservation date Bifidobacterium animalis lactis BR621 Bifidobacterium animalis lactis subsp. CGMCC No. 27537 2023 / 06 / 02 Lactobacillus rhamnosus RU-23 Lactobacillus rhamnosus CGMCC No. 33219 2024 / 12 / 27 Lactobacillus fermentum Fem-9 Lactobacillus fermentum CGMCC No. 33220 2024 / 12 / 27 Lactobacillus rhamnosus LRM369 Lactobacillus rhamnosus CGMCC No. 27538 2023 / 06 / 02 Lactobacillus plantarum PQQ-1 Lactobacillus plantarum CGMCC No. 29303 2023 / 12 / 12 Lactobacillus plantarum LPA-56 Lactobacillus plantarum CGMCC No. 24355 2022 / 01 / 21 The preservation units of the above strains are all: China General Microbiological Culture Collection Center; address: No. 1, Yihuanli, Beichenxili, Chaoyang District, Beijing, China, Institute of Microbiology, Chinese Academy of Sciences, 100101.

[0027] Researches on probiotics for human health are very extensive. In general, the strains having special effects on human health are called functional probiotics. It should be noted that the special effects of the above functional probiotics are specific to the strain rather than the species. Even if the same species, if different strains, they may have different effects, or even opposite effects.

[0028] On this basis, the present application provides a kind of for protecting liver lactic acid bacteria ferment, is by animal bifidobacterium lactis BR621 strain, Roy's mucous lactobacillus RU-23 strain, ferment mucous lactobacillus Fem-9 strain, rhamnose lactobacillus LRM369 strain, plant lactobacillus PQQ-1 strain and plant lactobacillus LPA-56 strain is obtained by fermentation culture and is mixed into ferment.The specific, above-mentioned lactic acid bacteria ferment, the mass ratio of animal bifidobacterium lactis BR621 strain, Roy's mucous lactobacillus RU-23 strain, ferment mucous lactobacillus Fem-9 strain, rhamnose lactobacillus LRM369 strain, plant lactobacillus PQQ-1 strain and plant lactobacillus LPA-56 strain is (1.5 ~ 2.5) :(1.5 ~ 2.5) :(0.5 ~ 1.5) :(0.5 ~ 1.5).Preferably, the mass ratio of ferment of the six strains is 5:3:2:1:0.7:0.3.

[0029] According to the results of 16S rRNA sequence analysis and API bacterial identification system analysis, the taxonomic characteristics of the strains are confirmed.

[0030] The morphological and general characteristics of the lactic acid bacterial strains of the present application are listed in Table 2: Table 2. Morphological and general characteristics of the lactic acid bacterial strains of the present application Strain name Morphological characteristics Bifidobacterium animalis lactis BR621 1. When cultured in MRS medium, the bacterial cells are irregular in shape, such as Y-shaped, V-shaped or curved, usually appearing alone, not in chains. 2. Gram-positive bacilli, do not produce spores, do not have touch enzymes, oxidase and motility, grow in absolute anaerobic environment, the optimum growth temperature is 37±1℃, it is a facultative heterofermentative strain, and no gas is produced during glucose metabolism. Lactobacillus rhamnosus RU-23 1. When cultured in MRS medium, the bacterial cells are short rods, both ends are round, usually appearing alone, in pairs or in short chains. 2. Gram-positive bacilli, do not produce spores, do not have touch enzymes, oxidase and motility, can grow in aerobic and anaerobic environments, the optimum growth temperature is 37±1℃, it is a facultative heterofermentative strain, and no gas is produced during glucose metabolism. Lactobacillus fermentum Fem-9 1. When cultured in MRS medium, the bacterial cells are short rods, both ends are round, usually appearing alone. 2. Gram-positive bacilli, do not produce spores, do not have touch enzymes, oxidase and motility, can grow in aerobic and anaerobic environments, the optimum growth temperature is 37±1℃, it is a facultative heterofermentative strain, and no gas is produced during glucose metabolism. Lactobacillus rhamnosus LRM369 1. When cultured in MRS medium, the bacterial cells are short or slightly long rods, both ends are square, usually appearing alone, in pairs or in short chains. 2. Gram-positive bacilli, do not produce spores, do not have touch enzymes, oxidase and motility, can grow in aerobic and anaerobic environments, the optimum growth temperature is 37±1℃, it is a facultative heterofermentative strain, and no gas is produced during glucose metabolism. Lactobacillus plantarum PQQ-1 1. When cultured in MRS medium, the bacterial cells are short rods, both ends are round, usually appearing alone. 2. Gram-positive bacilli, do not produce spores, do not have touch enzymes, oxidase and motility, can grow in aerobic and anaerobic environments, the optimum growth temperature is 37±1℃, it is a facultative heterofermentative strain, and no gas is produced during glucose metabolism. Lactobacillus plantarum LPA-56 1. The colony is white and solid round when cultured in MRS medium. The cell shape is short rod with round tail end, usually alone or connected in pairs. 2. Gram-positive bacillus, no spore, no catalase, no oxidase and no motility. It can grow in both aerobic and anaerobic environment, the optimum growth temperature is 37±1℃. It belongs to facultative heterofermentative strain, no gas produced during glucose metabolism. The present application is further described below with reference to the accompanying drawings.

[0031] Example 1.

[0032] The lactic acid bacteria ferment for protecting liver is prepared as follows: the animal bifidobacterium lactis BR621 strain of the present application is isolated from breast milk; the Roy's mucous lactobacillus RU-23 strain is isolated from human intestinal tract; the ferment mucous lactobacillus Fem-9 strain is isolated from human intestinal tract; the rhamnose lactobacillus LRM369 strain is isolated from breast milk; the plant lactobacillus PQQ-1 strain is isolated from highland saussurea obvallata; and the plant lactobacillus LPA-56 strain is isolated from pickled cabbage. The six lactic acid bacterial strains isolated above are stored at -80℃ with 20% glycerol. When used, 1% (v / v) inoculation amount is inoculated into Difco TMThe six cultures were centrifuged at 4°C for 20 minutes, and the supernatant was separated from the precipitate. The supernatant was then freeze-dried to obtain a fermentation product of each of the lactic acid bacteria strains. Then, the fermentation products of the Bifidobacterium animalis ssp. lactis BR621 strain, the Lactobacillus rhamnosus RU-23 strain, the Lactobacillus fermentum Fem-9 strain, the Lactobacillus rhamnosus LRM369 strain, the Lactobacillus plantarum PQQ-1 strain, and the Lactobacillus plantarum LPA-56 strain, which were prepared separately, were mixed in a mass ratio of 5:3:2:1:0.7:0.3 to obtain a complex lactic acid bacteria fermentation product.

[0033] The lactic acid bacteria fermentation product has a physiological activity of protecting the liver and can be used to prepare a pharmaceutical composition or a food composition for protecting the liver.

[0034] Example 2.

[0035] A lactic acid bacteria fermentation product for protecting the liver was prepared by mixing the lactic acid bacteria fermentation product of Example 1 and a carrier.

[0036] The carrier is a pharmaceutically acceptable excipient or diluent and can include any one or a mixture of two or more of oligosaccharides, sugar alcohols, sodium carboxymethyl cellulose, ethyl cellulose, galactooligosaccharides, stearic acid, magnesium stearate, and potato starch.

[0037] Specifically, the oligosaccharides are any one or a mixture of two or more of fructooligosaccharides, isomaltooligosaccharides, xylooligosaccharides, stachyose, mannose, and arabinose.

[0038] The sugar alcohols are any one or a mixture of two or more of erythritol, sorbitol, isomaltitol, xylitol, and mannitol.

[0039] The pharmaceutical composition is in the form of an oral dosage form, including tablets, capsules, or powders, for easy oral use by a user.

[0040] Example 3.

[0041] A food composition for protecting the liver was prepared by mixing the lactic acid bacteria fermentation product of Example 1 and a carrier.

[0042] The carrier includes a physiologically acceptable excipient or diluent and can include any one or a mixture of two or more of oligosaccharides, sugar alcohols, sodium carboxymethyl cellulose, ethyl cellulose, galactooligosaccharides, stearic acid, magnesium stearate, and potato starch.

[0043] Specifically, the oligosaccharide is any one of fructooligosaccharide, isomaltooligosaccharide, xylooligosaccharide, stachyose, mannose, arabinose, or a mixture of two or more thereof.

[0044] The sugar alcohol is any one of erythritol, sorbitol, isomaltitol, xylitol, mannitol, or a mixture of two or more thereof.

[0045] The food composition is in the form of an oral dosage form, including a tablet, a capsule, or a powder, for facilitating oral use by a user.

[0046] The food composition can be a beverage, a fermented food, an animal feed, a health food, a dessert, or a dietary supplement.

[0047] The antioxidant capacity and physiological activity of the lactic acid bacteria ferment obtained in Example 1 were tested as follows.

[0048] I. Evaluation of ABTS free radical scavenging rate of the lactic acid bacteria ferment of the present application The total antioxidant capacity test kit (ABTS method) used in this test was purchased from Shanghai Biyun Tian Biological Technology Co., Ltd. The ABTS (2,2'-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) free radical scavenging rate of the ferment of the six different lactic acid bacteria strains and the mixture thereof of Example 1 was evaluated.

[0049] First, the ABTS working stock solution was prepared according to the instructions of the kit, and was stored at room temperature in the dark for 12-16 hours. Then, the ABTS working stock solution was diluted with PBS (Phosphate Buffered Saline) to obtain the ABTS working solution. Next, the lactic acid bacteria ferment of different concentrations was mixed with the ABTS working solution and reacted for 5 minutes, and the absorbance value at 734 nm was measured by ELISA reader. Pure water was used as the control group, and the ABTS free radical scavenging rate was calculated. The calculation formula of the ABTS free radical scavenging rate is as follows:

[0050] Figure 1 The test results of the ABTS free radical scavenging rate of the lactic acid bacteria ferment of the present application at a concentration of 0.2 mg / mL are shown in Table 1, wherein the ferment of different lactic acid bacteria strains is indicated by the name of the lactic acid bacteria strain, and the mixed strain group is the lactic acid bacteria ferment of Example 1, i.e., the lactic acid bacteria ferment obtained by mixing the ferments of the six different lactic acid bacteria strains in a proportion of BR621:RU-23:Fem-9:LRM369:PQQ-1:LPA-56 = 5:3:2:1:0.7:0.3. In Table 1, the ABTS free radical scavenging rate of the lactic acid bacteria ferment of Example 1 is 100%, which is higher than that of any single lactic acid bacteria strain ferment. Figure 1In the present application, the groups of different English letters represent statistically significant differences between groups.

[0051] The results are shown in Figure 1 The fermentation of the six different lactic acid bacterial strains showed a concentration-dependent ABTS free radical scavenging rate, i.e. the ABTS free radical scavenging rate increased with increasing concentration. When the fermentation concentration was 0.2 mg / mL, the ABTS free radical scavenging rate of the fermentation of the six different lactic acid bacterial strains could reach more than 70%, among which the ABTS free radical scavenging rate of the mixed strain group of Example 1 could reach 88.57%, which was better than the ABTS free radical scavenging rate of other single lactic acid bacterial fermentation, and reached a significant difference (P<0.05). Therefore, each lactic acid bacterial fermentation and its mixture of the present application has a free radical scavenging effect. p <0.05) Therefore, each lactic acid bacterial fermentation and its mixture of the present application has a free radical scavenging effect.

[0052] Second, physiological activity analysis of the lactic acid bacterial fermentation of the present application The mice C57BL / 6 used in this experiment were purchased from Hangzhou Medical College and divided into 6 groups, 8 in each group. All experimental animals were raised in an independent air-conditioned animal room with 12 hours of light and darkness each, room temperature maintained at 22±2℃ and relative humidity maintained at 62±5%, and water and feed were fully supplied. The experimental conditions of each group of mice are as follows: Control group: continuously fed with water for 30 days, and no ethanol was fed after 30 days.

[0053] Negative control group: continuously fed with water for 30 days.

[0054] Positive control group: continuously fed with silymarin (200 mg / kg / day) for 30 days.

[0055] Low-dose group: continuously fed with lactic acid bacterial fermentation of Example 1 at 150 mg / kg / day for 30 days.

[0056] Medium-dose group: continuously fed with lactic acid bacterial fermentation of Example 1 at 300 mg / kg / day for 30 days.

[0057] High-dose group: continuously fed with lactic acid bacterial fermentation of Example 1 at 1500 mg / kg / day for 30 days.

[0058] Among them, the lactic acid bacterial fermentation of Example 1 is a mixture of the fermentation of 6 different lactic acid bacterial strains with a mass ratio of BR621:RU-23:Fem-9:LRM369:PQQ-1:LPA-56 = 5:3:2:1:0.7:0.3.

[0059] Mice in each group were fasted for 6 hours after their last gavage on day 30. To establish an acute alcoholic liver injury model, mice in groups (2) to (6) received a first gavage of ethanol (50% ethanol, 10 mL / kg), followed by a second gavage of ethanol (50% ethanol, 5 mL / kg) 1 hour later. Mice were allowed free access to food and water after the second gavage. All mice were sacrificed 16 hours later for the following analysis.

[0060] (I) Analysis of malondialdehyde (MDA) content in mouse liver tissue Alcohol metabolism produces reactive oxygen species (ROS) through pathways such as alcohol dehydrogenase (ADH), the microsomal ethanol oxidation system (MEOS), and catalase. ROS attack the polyunsaturated fatty acids in hepatocyte membranes, producing lipid peroxides, among which malondialdehyde (MDA) is one of the most commonly detected end products. Therefore, elevated MDA levels in liver tissue indicate that the liver is undergoing severe oxidative damage.

[0061] The analysis of malondialdehyde (MDA) content in mouse liver tissue in this experiment was performed using a malondialdehyde assay kit (TBA method) (purchased from Nanjing Jiancheng Bioengineering Institute) and in accordance with the instruction manual. Figure 2 The results of the analysis of malondialdehyde content in the liver tissue of mice in each group are shown. Groups marked with different English letters represent statistically significant differences between groups.

[0062] Depend on Figure 2 The results showed that after two ethanol gavage treatments, the malondialdehyde (MDA) content in the liver tissue of mice in the negative control group was significantly increased, reaching 1169 nmol / g, which was significantly different from that in the control group. p <0.05), therefore, the two ethanol gavage treatments did indeed induce alcohol metabolism in the mouse liver tissue, and the increased malondialdehyde (MDA) content in the mouse liver tissue indicated that the liver was undergoing severe oxidative damage. While mice treated with silymarin (positive control group) and the lactic acid bacteria ferment broth from Example 1 (low-dose, medium-dose, and high-dose groups) still showed an increase in MDA content in their liver tissue, it was significantly lower than that in the negative control group. p <0.05), and there was no statistically significant difference compared to the control group. p >0.05), indicating that the lactic acid bacteria ferment of Example 1 of the present invention can reduce oxidative damage to the liver.

[0063] (II) Analysis of interleukin-1β (IL-1β) content in mouse liver tissue Interleukin-1β (IL-1β) is an important indicator of inflammation in the assessment of acute alcoholic liver injury. Alcohol metabolism increases intestinal permeability, allowing endotoxins to enter the liver and activate specific cells to secrete pro-inflammatory cytokines such as IL-1β. Therefore, elevated IL-1β levels in liver tissue indicate a significant local inflammatory response. Furthermore, IL-1β not only acts as an inflammatory signal but also induces the generation of reactive oxygen species (ROS), exacerbating oxidative stress and further contributing to the deterioration of liver damage.

[0064] The analysis of interleukin-1β (IL-1β) content in mouse liver tissue in this experiment was performed using the Mouse IL-1beta ELISA Kit (purchased from ABclonal Biotechnology Co., Ltd.) and in accordance with the instruction manual. Figure 3 The results of the experiment on the IL-1β content analysis of liver tissue of mice in each group are shown. Groups marked with different English letters represent statistically significant differences between groups.

[0065] Depend on Figure 3 The results showed that after two ethanol gavage treatments, the IL-1β content in the liver tissue of the negative control group tended to be slightly higher than that of the control group. However, mice treated with silymarin (positive control group) and the lactic acid bacteria ferment of this invention (low-dose group) showed a significantly lower IL-1β content in their liver tissue compared to the negative control group. p <0.05), indicating that the lactic acid bacteria ferment of Example 1 of the present invention can effectively inhibit alcohol-induced liver inflammation.

[0066] (III) Analysis of Aspartate Transaminase (AST) Activity in Mouse Blood Aspartate aminotransferase (AST) is a serum enzyme commonly used clinically to assess liver damage. AST is mainly found in the mitochondria and cytoplasm of hepatocytes. When alcohol causes hepatocyte damage or necrosis, the cell membrane integrity is disrupted, and AST is released into the bloodstream. Therefore, elevated AST activity in the blood indicates hepatocyte damage.

[0067] The aspartate transaminase (AST) activity in mouse blood was analyzed using the aspartate transaminase test kit (purchased from Nanjing Jiancheng Bioengineering Institute) (Reid's method) microplate method according to the instruction manual. Figure 4The results of the AST activity analysis in the blood of mice in each group are shown. Groups marked with different English letters represent statistically significant differences between groups.

[0068] Depend on Figure 4 The results showed that after two ethanol gavage treatments, the AST activity in the blood of mice in the negative control group was significantly increased, reaching 109 U / L, which was statistically significant compared with the control group (30 U / L). p <0.05), indicating that two ethanol gavage treatments did indeed cause significant hepatocyte damage in mice. Mice treated with silymarin (positive control group) and the lactic acid bacteria ferment of this invention (low-dose, medium-dose, and high-dose groups), even after two ethanol gavage treatments, still showed significantly lower AST activity in their blood compared to the negative control group. p <0.05), and was comparable to the control group that was not administered ethanol. p >0.05), indicating that the lactic acid bacteria fermentation product of Example 1 of the present invention can effectively avoid liver cell damage caused by alcohol.

[0069] (iv) Analysis of alanine transaminase (ALT) activity in mouse blood Alanine transaminase (ALT) is a liver-specific serum enzyme used to assess hepatocellular damage. ALT is primarily distributed in the cytoplasm of hepatocytes, with extremely low levels in the heart, skeletal muscle, or kidneys. Therefore, elevated ALT activity in the blood indicates disruption of the hepatocyte cell membrane integrity, suggesting potential hepatocyte damage or necrosis.

[0070] The alanine transaminase (ALT) activity in mouse blood was analyzed using the alanine transaminase test kit (purchased from Nanjing Jiancheng Bioengineering Institute) (Reiter method) microplate method according to the instruction manual. Figure 5 The results are the ALT activity analysis of the blood of mice in each group. Groups marked with different English letters represent statistically significant differences between groups.

[0071] Depend on Figure 5 The results showed that after two ethanol gavage treatments, the ALT activity in the blood of mice in the negative control group was significantly increased, reaching 25.9 U / L, which was statistically significant compared with the control group (9.7 U / L). p <0.05), indicating that two ethanol gavage treatments did indeed cause significant hepatocyte damage in mice. Mice treated with silymarin (positive control group) and the lactic acid bacteria ferment of this invention (low-dose and high-dose groups), even after two ethanol gavage treatments, still showed significantly lower ALT activity in their blood compared to the negative control group.p <0.05), and some experimental groups were comparable to the control group that was not fed ethanol, indicating that the lactic acid bacteria fermentation product of Example 1 of the present invention can effectively reduce hepatocyte damage caused by alcohol.

[0072] (v) Analysis of total superoxide dismutase (SOD) activity in mouse liver tissue Total superoxide dismutase (SOD) is an important intracellular antioxidant defense enzyme that converts superoxide anion free radicals into hydrogen peroxide (H2O2), which is then further broken down by catalase or glutathione peroxidase to prevent free radicals from damaging cells. Therefore, a decrease in SOD activity indicates that the antioxidant system is depleted and unable to effectively scavenge ROS, leading to lipid peroxidation (e.g., increased MDA) and cell membrane damage.

[0073] Total superoxide dismutase (SOD) activity analysis of mouse liver tissue was performed using a total superoxide dismutase assay kit (purchased from Nanjing Jiancheng Bioengineering Institute) (hydroxylamine method) according to the instruction manual. Figure 6 The results are the results of the SOD activity analysis of the liver tissue of mice in each group. Groups marked with different English letters represent statistically significant differences between groups.

[0074] Depend on Figure 6 The results showed that after two ethanol gavage treatments, the total superoxide dismutase (SOD) activity in the liver tissue of mice in the negative control group tended to be lower than that in the control group, indicating that the antioxidant system was gradually depleted. However, mice treated with silymarin (positive control group) and the lactic acid bacteria fermentation products of this invention (low-dose, medium-dose, and high-dose groups), even after two ethanol gavage treatments, showed a significant increase in total SOD activity in their liver tissue compared to the negative control group. p <0.05), indicating that the lactic acid bacteria ferment of Example 1 of the present invention can enhance the antioxidant system to eliminate free radicals in the body, thereby reducing the oxidative stress on the liver.

[0075] (vi) Analysis of catalase (CAT) activity in mouse liver tissue Catalase (CAT) is one of the important antioxidant defense enzymes in the liver. It breaks down hydrogen peroxide (H2O2) produced during alcohol metabolism into water and oxygen, preventing its accumulation and oxidative damage. Therefore, CAT activity reflects the liver's ability to scavenge H2O2.

[0076] The catalase (CAT) activity of the liver tissue of the mice was analyzed using a catalase assay kit (purchased from Nanjing Jiancheng Bioengineering Institute) (visible light method, ammonium molybdate method) according to the operation manual. Figure 7 The results of the CAT activity analysis of the liver tissue of the mice in each group, in which the groups marked with different English letters represent statistically significant differences between the groups.

[0077] As can be seen from the results, Figure 7 As can be seen from the results, after two ethanol gavage treatments, the CAT activity of the liver tissue of the mice in the negative control group had no statistically significant difference with that of the control group and the positive control group (gavaged with silymarin). However, the CAT activity of the liver tissue of the mice in the low-dose group, the medium-dose group and the high-dose group gavaged with the lactic acid bacteria fermentum of Example 1 increased compared with that of the negative control group, and the difference was statistically significant (P < 0.05), indicating that the lactic acid bacteria fermentum of Example 1 of the present application can effectively improve the CAT activity and thus improve the H2O2 removal ability, effectively remove the free radicals in the liver tissue, and thus reduce the oxidative stress of the liver. p <0.05), showing that the lactic acid bacteria fermentum of Example 1 of the present application can effectively improve the CAT activity and thus improve the H2O2 removal ability, effectively remove the free radicals in the liver tissue, and thus reduce the oxidative stress of the liver.

[0078] (VII) Histopathological examination of the liver tissue of the mice In this test, the liver tissue of the mice was first fixed with 4% paraformaldehyde solution, and then dehydrated with 70%, 80%, 90% and 100% gradient ethanol solutions. After dehydration, the tissue was transparentized with xylene to improve the paraffin impregnation effect. Then, the transparentized tissue was placed in an embedding mold and injected with molten paraffin. After the paraffin was completely cooled and solidified, the position of the tissue in the embedding block was ensured to be correct for subsequent slicing operation. Finally, the embedded tissue block was sectioned to prepare for subsequent staining.

[0079] Figure 8 The results observed after hematoxylin-eosin staining (purchased from Biyun Tian Biotechnology Co., Ltd.) of the liver tissue sections of the mice in each group, in which (A) is the control group, (B) is the negative control group, (C) is the positive control group, (D) is the low-dose group, (E) is the medium-dose group, and (F) is the high-dose group.

[0080] As can be seen from the results, Figure 8 As can be seen from the results, the liver tissue cells of the control group were arranged in order, the cell morphology was normal, the cytoplasm was uniform, there was no obvious vacuolation or fatty degeneration, the cell nucleus size was uniform, the staining was normal, and there was no obvious inflammatory cell infiltration. However, the negative control group showed partial bleeding and inflammatory cell infiltration, such as Figure 8As shown in FIG. 1, the positive control group (gavaged with silymarin) showed no obvious bleeding and inflammatory cell infiltration, and the cell nuclei were uniform in size and normal in staining. In contrast, the low-dose group, the medium-dose group, and the high-dose group showed obvious bleeding and inflammatory cell infiltration, and the cell nuclei were not uniform in size and abnormal in staining. Figure 8 C to Figure 8 F. Therefore, the lactic acid bacteria ferment of Embodiment 1 of the present application can protect the liver to avoid or reduce alcohol-induced liver damage.

[0081] In summary, the lactic acid bacteria ferment of the present application has physiological activity functions of protecting the liver, including scavenging free radicals and improving the ability of the antioxidant system of the liver to resist oxidative stress and alcoholic liver damage. It should be noted that compared with the ferment containing only a single strain of lactic acid bacteria, the mixture containing ferment of six strains of lactic acid bacteria can significantly scavenge free radicals, so it can be known that the lactic acid bacteria ferment of the present application has unexpected efficacy.

[0082] The above-described embodiments are only used to illustrate the technical solutions of the present application, but not limit them; although the present application has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that they can still modify the technical solutions recorded in the foregoing embodiments, or make equivalent replacements for part of the technical features; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the spirit and scope of the technical solutions of the embodiments of the present application, and should be included in the protection scope of the present application.

Claims

1. A lactic acid bacterial ferment for protecting the liver, characterized in that: The lactic acid bacteria fermentate is a composite fermentate obtained by mixing the fermentates of Bifidobacterium animalis lactis BR621 strain with preservation number CGMCC NO. 27537, Lactobacillus rhamnosus LRM369 strain with preservation number CGMCC NO. 27538, Lactobacillus plantarum PQQ-1 strain with preservation number CGMCC NO. 29303 and Lactobacillus plantarum LPA-56 strain with preservation number CGMCC NO. 24355 in a certain proportion.

2. The lactic acid bacterial ferment for protecting a liver according to claim 1, characterized by: The mass ratio of the fermentates of the Bifidobacterium animalis lactis BR621 strain, Lactobacillus rhamnosus LRM369 strain, Lactobacillus plantarum PQQ-1 strain and Lactobacillus plantarum LPA-56 strain is 5:3:2:1:0.7:0.

3.

3. The lactic acid bacterial fermentum for protecting a liver according to claim 2, characterized by:

4. Use of the lactic acid bacteria fermentate according to any one of claims 1 to 3 in the preparation of a composition for protecting the liver. The use for protecting the liver is to improve the ability of the liver to resist oxidative stress.

5. Use according to claim 4, characterized in that: The use for protecting the liver is to improve the ability of the liver to resist alcoholic liver injury.

6. Use according to claim 4, characterized in that: The composition comprises the lactic acid bacteria fermentate according to any one of claims 1 to 3 and a pharmaceutically or physiologically acceptable carrier.

7. A composition for protecting the liver, characterized by: The composition is a food composition or a pharmaceutical composition.

8. The composition for protecting a liver according to claim 7, characterized by: The carrier is an excipient or a diluent, comprising any one of oligosaccharides, sugar alcohols, sodium carboxymethyl cellulose, ethyl cellulose, galactooligosaccharides, stearic acid, magnesium stearate, potato starch or a mixture of two or more thereof.

9. The composition for protecting a liver according to claim 7, characterized by: The dosage form of the composition is an oral dosage form, which comprises a tablet, a capsule or a powder.

10. The composition of claim 7, wherein: ​

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