Application of Aradicatrata lactobacillus GXUN74705 in improvement of alcoholic liver injury

Abstract

The invention discloses an application of Aradiclat lactobacillus plantarum GXUN74705 in improvement of alcoholic liver injury, and belongs to the technical field of microorganisms. The invention proves that the application of the Aradicatrat lactobacillus plantarum GXUN74705 in treating the alcoholic liver injury, the strain can effectively improve the liver injury caused by alcohol, and the pathological process of the alcoholic liver injury is comprehensively relieved by regulating liver function indexes, correcting blood lipid metabolism disorder, relieving oxidative stress and inhibiting inflammatory response. In addition, the strain can also reduce liver lipid accumulation, repair a liver cell structure, and further improve liver health by adjusting an intestinal flora structure and intestinal bile acid metabolism. The strain has remarkable effects in the aspects of reducing blood fat, reducing cholesterol, resisting oxidation and the like, or has important clinical application and market value when being applied to foods, food additives, health care products, medicines or feeds as functional probiotics.

Description

Technical Field

[0001] The present invention relates to the technical field of microorganisms, and in particular to application of Lactobacillus argenteus GXUN74705 in improving alcoholic liver damage. Background Art

[0002] The liver is one of the important organs of the human body, responsible for multiple functions such as detoxification, metabolism and protein synthesis. When the liver is damaged, it can lead to toxin accumulation, metabolic abnormalities, energy deficiency and other problems. Alcoholic liver injury (ALI), also known as alcoholic liver disease, is a liver lesion caused by large amounts or long-term drinking of alcoholic beverages. Its pathological process includes stages such as alcoholic fatty liver, alcoholic hepatitis, liver fibrosis, cirrhosis and liver cancer. The occurrence of alcoholic liver injury is closely related to multiple mechanisms such as oxidative stress, inflammatory response, intestinal flora disorder and intestinal barrier dysfunction. Long-term alcohol intake can lead to intestinal flora imbalance, increase endotoxin release, and then activate liver Kupffer cells, promote the release of pro-inflammatory factors, and aggravate liver cell damage.

[0003] Currently, the primary treatment for alcoholic liver injury is medication, such as antioxidants, glucocorticoids, and hepatoprotective drugs. However, some drugs have toxic side effects or limited efficacy, limiting their clinical application. Therefore, exploring safe and effective intervention strategies is of great importance. Recent studies have shown that probiotics can improve liver health by regulating intestinal microecological balance and alleviating oxidative stress and inflammatory responses. Lactobacillus, a widely studied probiotic, has potential roles in regulating host metabolism and immune regulation, potentially providing new research directions for the intervention of alcoholic liver injury. Summary of the Invention

[0004] The present invention aims to provide the use of Lactobacillus argentulatus GXUN74705 in improving alcoholic liver damage, thereby overcoming the problems of the prior art. The present invention demonstrates that Lactobacillus argentulatus GXUN74705 improves alcohol-induced liver damage, exhibiting significant effects in lowering blood lipids, lowering cholesterol, and providing antioxidant benefits. Furthermore, Lactobacillus argentulatus GXUN74705 can be used as a functional probiotic in foods, food additives, health products, medicines, or feed, demonstrating its significant clinical applications and market value.

[0005] To achieve the above object, the present invention provides the following solutions:

[0006] The present invention provides application of Lactiplantibacillus argentoratensis GXUN74705 in preparing a functional product for alleviating alcoholic liver damage. The preservation number of the Lactiplantibacillus argentoratensis GXUN74705 is CGMCC NO.28711.

[0007] The Lactobacillus argenteus GXUN74705 significantly alleviates liver dysfunction caused by alcohol.

[0008] Optionally, the products include food, food additives and health products.

[0009] Optionally, the food includes yogurt, fermented fruit and vegetable juice and bread.

[0010] The present invention also provides use of Lactiplantibacillus argentoratensis GXUN74705 in preparing medicines for treating or assisting in treating alcoholic liver damage.

[0011] Optionally, the medicine further includes pharmaceutically acceptable carriers and excipients.

[0012] Optionally, the medicine includes solid bacterial powder.

[0013] Optionally, the viable count of the Lactobacillus argenteus GXUN74705 in the solid bacterial powder is 1.0×10 11 -1.0×10 12 CFU / g.

[0014] The present invention also provides a method for preparing a solid bacterial powder for improving alcoholic liver damage, comprising the following steps:

[0015] Lactobacillus argenteus GXUN74705 was cultured at a high density and the cells were collected by centrifugation.

[0016] The bacterial cells were mixed with a freeze-drying protective agent at a mass ratio of 1:5, pre-frozen, and then vacuum-freeze-dried to obtain the solid bacterial powder;

[0017] The deposit number of the Lactobacillus argenteus GXUN74705 is CGMCC NO.28711.

[0018] Optionally, the components of the lyoprotectant include 15 wt% skim milk powder, 5 wt% trehalose, 3 wt% sodium glutamate, 1 wt% glycerol and 0.5 wt% cysteine ​​hydrochloride.

[0019] The present invention also provides a solid bacterial powder obtained by the preparation method.

[0020] The present invention discloses the following technical effects:

[0021] The present invention confirms the application of Lactobacillus argenteus GXUN74705 in alleviating alcoholic liver damage. Animal experiments show that this strain can effectively improve alcohol-induced liver damage by regulating liver function indicators (such as reducing AST and ALT levels), correcting blood lipid metabolism disorders (such as reducing TC, TG and LDL-C, and increasing HDL-C), alleviating oxidative stress (such as reducing MDA content and increasing SOD and CAT activity), and inhibiting inflammatory responses (such as downregulating TNF-α, IL-1β and IL-6, and upregulating IL-10), thereby comprehensively alleviating the pathological process of alcoholic liver damage. In addition, this strain can also reduce liver lipid accumulation, repair liver cell structure, and further improve liver health by regulating intestinal bile acid metabolism.

[0022] The present invention confirms that the strain has significant effects in lowering blood lipids, lowering cholesterol and anti-oxidation effects, or can be applied as a functional probiotic in food, food additives, health products, medicines or feed, and has important clinical applications and market value. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0024] Figure 1 This is the modeling design drawing of the animal model of the present invention;

[0025] Figure 2 The amino acid decarboxylase activity assay (A) and hemolytic activity assay (B) of strain GXUN74705 were performed;

[0026] Figure 3 The effect of Lactobacillus argenteus GXUN74705 of the present invention on AST (A) and ALT (B) in mouse plasma;

[0027] Figure 4 The effects of the Lactobacillus argenteus GXUN74705 of the present invention on TC (A), TG (B), HDL-C (C) and LDL-C (D) in mouse serum;

[0028] Figure 5 The effect of Lactobacillus argenteus GXUN74705 of the present invention on MDA (A), SOD (B) and CAT (C) in mouse liver;

[0029] Figure 6 Effects of Lactobacillus argenteus GXUN74705 on inflammatory factors TNF-α(A), IL-1β(B), IL-6(C) and IL-10(D) in mouse liver;

[0030] Figure 7 This is an H&E staining image of a mouse liver section;

[0031] Figure 8 This is an Oil Red O staining image of mouse liver section;

[0032] Figure 9 The electrophoresis diagram (A) and statistical results (B) of the expression of fatty acid synthesis genes (FAS, SCD1 and ACACA) in mouse liver by Lactobacillus argenteus GXUN74705 of the present invention are shown;

[0033] Figure 10 The following are the analysis diagrams of the intestinal flora structure of each group of mice, among which A is the analysis of the intestinal flora at the phylum level of each group of mice; B is the analysis of the intestinal flora at the genus level of each group of mice;

[0034] Figure 11 This is the effect of the Lactobacillus argenteus GXUN74705 of the present invention on bile acid in mouse feces. DETAILED DESCRIPTION

[0035] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0036] It should be understood that the terms described herein are intended only to describe particular embodiments and are not intended to limit the present invention. In addition, for numerical ranges herein, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. The intermediate value within any stated value or stated range, as well as each smaller range between any other stated value or intermediate value within the stated range, is also encompassed within the present invention. The upper and lower limits of these smaller ranges may be independently included or excluded within the scope.

[0037] Unless otherwise indicated, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein may also be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of any conflict with any incorporated document, the contents of this specification shall prevail.

[0038] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments described herein without departing from the scope or spirit of the invention. Other embodiments will be apparent to those skilled in the art from the description of the invention. The description and examples are intended to be exemplary only.

[0039] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0040] The Lactiplantibacillus argentoratensis described in the present invention is a strain isolated from traditional fermented sauerkraut juice in Guangxi. The strain is named Lactiplantibacillus argentoratensis GXUN74705 and was deposited with the General Microbiology Center of the China National Center for Microbiological Culture Collection on October 23, 2023, with a deposit number of CGMCC No. 28711. This strain and its isolation and screening process are disclosed in an invention patent with an application date of December 7, 2023, and publication number CN117625494B.

[0041] The experimental methods in the following examples of the present invention are conventional methods unless otherwise specified; the biological and chemical reagents used are conventional reagents in the art and can be purchased commercially unless otherwise specified.

[0042] Example 1: Improvement of Alcoholic Liver Injury by Lactobacillus Argenteus GXUN74705

[0043] 1. Experimental Methods

[0044] 1. Experimental strains

[0045] Lactobacillus argentatus GXUN74705 (hereinafter referred to as LA.GXUN74705).

[0046] 2. Biosafety Test of Lactobacillus Argentulatus GXUN74705

[0047] 2.1 Antibiotic sensitivity analysis

[0048] A standard antimicrobial susceptibility testing system was established: MRS culture medium was pretreated using a 200 μL bacterial suspension smear method, and six commonly used clinical antibiotics (gentamicin, erythromycin, lincomycin, tetracycline, ampicillin, and cefazolin) were placed using the standard disk diffusion method. Cultures were incubated at 37°C for 24 hours, and the diameter of the inhibition zone was measured using a vernier caliper. The susceptibility of the strain to antibiotics was assessed according to the breakpoint criteria established by the Clinical and Laboratory Standards Institute (CLSI) in 2012.

[0049] 2.2 Amino acid decarboxylase activity analysis

[0050] The target strain was inoculated at a 2% (v / v) inoculum into a four-amino acid decarboxylase assay medium supplemented with tryptophan, histidine, lysine, and tyrosine, respectively. The culture was incubated at 37°C for 24 hours, and the color of the medium was observed. A purple color indicates a positive result, indicating a potential threat of amine production; a yellow color indicates a negative result, indicating no potential threat of amine production. Salmonella (ATCC 14028) was used as a positive control strain.

[0051] 2.3 Hemolysis assay analysis

[0052] Streak the target strain onto Columbia blood agar, using Staphylococcus aureus (ATCC 25923) as a positive control. Incubate at 37°C for 48 hours and observe for hemolysis. Phenotyping is performed based on the presence of hemolytic rings: α-hemolysis results in a grass-green halo (incomplete hemolysis), β-hemolysis results in a clear lysis zone (complete hemolysis), and γ-hemolysis results in no visible hemolysis.

[0053] 3. Experimental Animals

[0054] Ten-week-old C57BL / 6J mice were purchased from Yusibeifu (Beijing) Biotechnology Co., Ltd., license number SCXK (Beijing) 2024-0001. They were housed in the Experimental Animal Center of Guangxi Medical University in an SPF environment.

[0055] 4. Construction and grouping of animal models

[0056] Ten-week-old male mice, weighing 20-21g, were housed in the Animal Experimental Center of Guangxi Medical University (SPF environment, 23°C) with free access to food and water. After one week of adaptive feeding, the mice were randomly divided into three groups: control group (NC), alcohol group (EtOH), and intervention group (LA.GXUN74705).

[0057] On days 1-5, the NC and EtOH groups received 200 μL of normal saline per day by gavage, while the intervention group received 1×10 9 CFU / mouse / day of LA.GXUN74705 was gavaged for 5 days. Starting from the 6th day, the EtOH group and the intervention group began to receive alcohol gavage, while the NC group continued to receive normal saline gavage, and the intervention group continued to receive LA.GXUN74705 gavage. The interval between LA.GXUN74705 gavage and alcohol gavage was 4 hours, which lasted for 10 days. On the morning of the 16th day, the EtOH group and the intervention group: each mouse received an ethanol solution (50% (vol / vol)) gavage volume (μL) = mouse body weight (g) × 10; the NC group received maltodextrin gavage: each mouse received a maltodextrin solution (45.0% (wt / vol)) gavage volume (μL) = mouse body weight (g) × 10. After 9 hours of gavage, the mice were deeply anesthetized with isoflurane. The modeling technique is as follows. Figure 1 shown.

[0058] Blood was collected from the orbital sinus of anesthetized mice, and the blood was allowed to stand at room temperature for 2 hours. It was centrifuged at 4000 rpm for 15 minutes at 4°C, and the upper serum was collected and stored at -80°C for later use. While the mice were still under general anesthesia, they were killed by cervical dislocation. Subsequently, the heart, spleen, kidney, and liver of the mice were weighed. Part of the liver tissue was placed in tissue fixative for histopathological examination, part of it was quickly frozen with liquid nitrogen and placed at -80°C for oil red detection, and the remaining liver tissue was quickly frozen with liquid nitrogen and placed at -80°C for subsequent experiments. The cecal contents of the mice were collected in a clean bench, quickly frozen with liquid nitrogen, and placed at -80°C for subsequent detection of microbial diversity and metabolomics.

[0059] 5. Preparation of H&E sections of liver tissue

[0060] The tissues were embedded in paraffin and sections were prepared. After drying and dewaxing, they were stained with hematoxylin and eosin, respectively. Finally, they were dehydrated and sealed with neutral gum.

[0061] 6. Preparation of Liver Oil Red O Slices

[0062] The sections were protected from light and stained with oil red. After background differentiation, they were counterstained with hematoxylin, washed with pure water, differentiated with differentiation solution, and blued with blueing solution, and then sealed with glycerol gelatin mounting medium.

[0063] 7. Index determination

[0064] The detection of mouse serum AST, ALT, TG, TC, HDL-C and LDL-C was completed by the Laboratory Department of the First Affiliated Hospital of Guangxi Medical University.

[0065] The determination of the levels of inflammatory factors such as TNF-α, IL-1β, IL-6, and IL-10 in mouse liver and the determination of oxidative stress indicators such as MDA, SOD, and CAT were performed according to the instructions of the kit.

[0066] The expression of lipid synthesis-related genes in tissues was detected by qRT-PCR using 2 -ΔΔCT The relative expression level of the target gene was calculated using the formula.

[0067] The intestinal flora analysis and metabolite determination were commissioned to Shanghai WeiNa Biotechnology Co., Ltd.

[0068] 2. Results

[0069] 1. Biosafety Analysis of LA.GXUN74705

[0070] 1.1 Determination of antibiotic sensitivity

[0071] Antibiotic susceptibility is a key criterion for assessing the safety of probiotics. Six commonly used antimicrobial agents were tested against Lactobacillus argentinus GXUN74705. The results showed that Lactobacillus argentinus GXUN74705 was sensitive to gentamicin, erythromycin, ampicillin, tetracycline, and cefazolin, but was resistant to lincomycin (Table 1).

[0072] Table 1 Antibiotic sensitivity determination

[0073]

[0074] 1.2 Amino acid decarboxylase activity analysis

[0075] Amino acid decarboxylase can catalyze the decarboxylation reaction of amino acids, thereby producing biogenic amines. Biogenic amines are a class of active organic compounds that contain nitrogen and have a low molecular weight. They are highly toxic and are harmful to human health if ingested. In severe cases, they can be life-threatening. If a strain can encode and synthesize amino acid decarboxylase, it will decarboxylate amino acids to form biogenic amines. Figure 2 As shown in Figure A, the culture medium inoculated with the Salmonella ATCC 14028 strain turned purple, indicating a positive amino acid decarboxylase activity. In contrast, the culture medium inoculated with the Lactobacillus argentatus GXUN74705 strain turned yellow, indicating a negative amino acid decarboxylase activity. This indicates that these strains do not produce harmful metabolites such as tryptamine, histamine, cadaverine, and tyramine.

[0076] 1.3 Hemolytic assay

[0077] Hemolytic bacteria pose a threat to human health because the hemolysins they secrete can destroy cells, cause intrinsic defects in red blood cells, and trigger antigen-antibody reactions, leading to sepsis. Therefore, the Food and Agriculture Organization of the United Nations stipulates that probiotics in food cannot be hemolytic. Hemolysis can be divided into three categories: α-hemolysis presents a grass-green hemolysis ring and has a relatively low mortality rate; β-hemolysis presents a colorless and transparent hemolysis ring and has a strong pathogenicity; and γ-hemolysis has no hemolysis ring and will not cause harm to the human body. Figure 2 As shown in Figure B, a colorless and transparent β-hemolytic ring was formed around the positive control strain S. aureus ATCC 25923; while no hemolytic ring appeared around Lactobacillus argentatus GXUN74705, indicating that the strain is not hemolytic and will not cause harm to human health, and can be identified as a safe strain.

[0078] 2. Effect of LA.GXUN74705 on liver function in mice with alcoholic liver damage

[0079] ALT and AST are present in large quantities in the liver. They are important enzymes for liver cells and are also important indicators for diagnosing the extent of liver cell damage. Under normal conditions, the activity of these two enzymes in mouse serum is very low. When the liver is damaged, ALT and AST are released into the blood in large quantities. Therefore, serum ALT and AST are diagnostic indicators reflecting the extent of liver damage. Figure 3 Results showed that the serum AST and ALT levels in the EtOH group were significantly higher than those in the NC group (p<0.001), while the levels of these indicators in the intervention group were significantly lower than those in the EtOH group (p<0.01). This indicates that LA.GXUN74705 can significantly improve liver damage caused by excessive alcohol intake in mice.

[0080] 3. Effects of LA.GXUN74705 on blood lipids in mice

[0081] Alcohol intake can lead to liver lipid metabolism disorders and alcoholic fatty liver. When fatty liver disease occurs, blood lipid concentrations will also change. The levels of TC, TG, HDL-C and LDL-C in mouse serum were tested. The results are as follows: Figure 4 As shown in the results, the serum TC, TG, and LDL-C levels of mice in the EtOH group were significantly higher than those in the NC group (p<0.001), while the HDL-C level was lower than that in the NC group (p<0.001). LA.GXUN74705 intervention significantly improved the above indicators, indicating that LA.GXUN74705 has the ability to improve alcohol-induced dyslipidemia.

[0082] 4. Effects of LA.GXUN74705 on oxidative stress levels in mouse liver

[0083] like Figure 5As shown in the results, compared with the NC group, the lipid peroxidation marker MDA in the liver tissue of mice in the EtOH group was significantly increased, and the activities of the antioxidant enzymes SOD and CAT were significantly decreased; LA.GXUN74705 intervention reversed the changes in the above indicators, as manifested by a significant decrease in the content of liver MDA and a significant increase in the enzyme activities of SOD and CAT.

[0084] 5. Effect of LA.GXUN74705 on the levels of inflammatory factors in mouse liver

[0085] Long-term drinking can cause the release of inflammatory factors such as TNF-α, IL-1β and IL-6 in the liver, causing inflammatory lesions in the liver. Figure 6 As shown, compared with the NC group, the levels of inflammatory factors TNF-α, IL-1β, and IL-6 in the liver of mice in the EtOH group were significantly increased, and the level of the anti-inflammatory factor IL-10 was significantly reduced. LA.GXUN74705 intervention significantly reduced the levels of inflammatory factors and increased the level of the anti-inflammatory factor IL-10. These results indicate that long-term alcohol intervention induces an inflammatory response, and LA.GXUN74705 can effectively alleviate alcohol-induced liver inflammation.

[0086] 6. Effects of LA.GXUN74705 on liver pathology in mice

[0087] like Figure 7 As shown, the liver tissue of mice in the EtOH group showed significant damage compared to the NC group, with disorganized hepatocytes and cords, fatty and vacuolar degeneration, and abundant inflammatory cell infiltration. In the intervention group, liver damage was alleviated compared to the EtOH group, with hepatocyte morphology nearly restored to normal, fatty degeneration reduced to mild, and inflammatory cell infiltration reduced. This suggests that LA.GXUN74705 can significantly alleviate alcohol-induced liver pathology.

[0088] 7. Effect of LA.GXUN74705 on lipid accumulation in mouse liver

[0089] Oil Red O is a fat-soluble dye that can specifically make triglycerides in tissues appear red and cell nuclei appear blue. Figure 8 As shown, the NC group had fewer lipid droplets, while the EtOH group showed distinct red lipid droplets. The intervention group had significantly fewer red lipid droplets in the liver. This suggests that alcohol disrupts lipid metabolism in the liver, leading to significant lipid accumulation, and that LA.GXUN74705 can significantly alleviate alcohol-induced lipid accumulation in the liver.

[0090] 8. Effect of LA.GXUN74705 on the expression of fatty acid synthesis genes in mouse liver

[0091] like Figure 9 As shown in the results, compared with the intervention group, the mRNA and protein expression levels of fatty acid anabolism-related genes (FAS, SCD1, ACACA) in the liver of the EtOH group were significantly increased, indicating that LA.GXUN74705 can significantly restore the abnormal expression of fatty acid anabolism-related genes in liver cells.

[0092] 9. Structural changes in the intestinal flora of mice

[0093] The cecal contents of mice were collected, and the structural changes of the intestinal flora of mice in each group were analyzed. At the phylum level, the relative abundance of Verrucomicrobiota, Actinobacteriota, and Desulfobacterota in the intestines of mice in the EtOH group was significantly lower than that in the NC group, while the relative abundance of Bacteroidota was significantly higher than that in the NC group, indicating that alcohol intake destroyed the normal intestinal flora structure of mice. Compared with the EtOH group, the Verrucomicrobiota, Actinobacteria, Proteobacteria, and Bacteroidetes in the intestines of mice after LA.GXUN74705 intervention returned to levels similar to those of the NC group ( Figure 10 A). At the genus level, the relative abundance of beneficial bacteria Akkermansia, Dubosiella, Faecalibaculum, Lachnospiraceae, Muribaculum, and Coriobacteriaceae_UCG-002 was significantly reduced in the EtOH group. In contrast, these changes were reversed after LA.GXUN74705 treatment, indicating that LA.GXUN74705 restored the structure of the mouse intestinal microbiota to some extent.

[0094] 10. Effect of LA.GXUN74705 on intestinal bile acid levels in mice

[0095] Alcohol intake can cause disturbances in the intestinal flora, leading to abnormal levels of its metabolites and disrupting liver metabolic homeostasis. Two types of bile acids, cholic acid and chenodeoxycholic acid, form secondary bile acids including deoxycholic acid and lithocholic acid under the action of intestinal flora. Intestinal flora imbalance can lead to changes in the bile acid spectrum, exacerbating liver lipid accumulation and inflammation. Figure 11As shown in the data, the levels of cholic acid, chenodeoxycholate, taurocholic acid, 3-dehydrocholic acid, taurodeoxycholic acid, and deoxycholic acid in the EtOH group were severely decreased compared with those in the NC group, while the levels of these metabolites were restored after intervention with LA.GXUN74705.

[0096] In summary, the above results show that Lactobacillus argenteus GXUN74705 can improve alcohol-induced liver damage and lipid metabolism disorders, improve blood lipid and liver oxidative stress levels, restore intestinal bile acid metabolism levels, relieve liver inflammation, and improve the intestinal flora structure of mice, and has strong prebiotic properties.

[0097] Example 2 Preparation of bacterial powder for improving alcoholic liver damage using Lactobacillus argenteus GXUN74705

[0098] 1. Preparation of LA.GXUN74705 bacterial sludge

[0099] A single LA.GXUN74705 colony was inoculated into 50 mL of MRS liquid culture medium and incubated in a 37°C incubator for 18 hours. The cells were activated again in 250 mL of MRS liquid culture medium at a 5% inoculum size and incubated in a 37°C incubator for 24 hours. Finally, the activated LA.GXUN74705 was cultured anaerobically at high density in a 10 L fermentor at a 5% inoculum size and incubated at 37°C and pH 6.8 for 18 hours. The cells were then centrifuged at 8000 rpm and 4°C for 15 minutes. The supernatant was discarded, and the bacterial pellet was collected and rinsed twice with sterile PBS (pH 7.0) to obtain LA.GXUN74705 bacterial slurry.

[0100] 2. Preparation of protective agent

[0101] The freeze-dried protective agent contains 15 wt% skim milk powder, 5 wt% trehalose, 3 wt% sodium glutamate, 1 wt% glycerol, and 0.5 wt% cysteine ​​hydrochloride. Water is used as the solvent. Sterilize at 110°C until ready for use.

[0102] 3. Preparation of LA.GXUN74705 bacterial powder

[0103] The prepared LA.GXUN74705 bacterial pellet was thoroughly mixed with the protective agent solution at a mass ratio of 1:5. The pellet was pre-frozen at -40°C for 5 hours to allow it to freeze evenly on the inner wall of the container. The pellet was then vacuum-freeze-dried for 18-20 hours to obtain Lactobacillus argentinus GXUN74705 powder. After rehydration with physiological saline and washing twice, the viable count of the Lactobacillus argentinus GXUN74705 powder was determined to be 1.0 × 10 11 -1×10 12 CFU / g.

[0104] Example 3 Preparation of functional yogurt using LA.GXUN74705

[0105] 1. The production process of stirred yogurt:

[0106] Fresh milk → standardization → homogenization → sterilization → cooling → inoculation and fermentation → stirring → filling and sealing → refrigeration → post-ripening → yogurt

[0107] 2. Preparation steps:

[0108] (1) Ingredients: 3L fresh milk, 150g white sugar;

[0109] (2) Preheating: Place fresh milk and sugar in a container and heat to 50-60°C;

[0110] (3) Homogenization: Pour into homogenizer and homogenize at a pressure of (10-25MPa).

[0111] (4) Sterilization: Pour the mixture into an iron can and sterilize in a 90°C water bath for 10-15 minutes;

[0112] (5) Preparation: Add ingredients to milk and dissolve;

[0113] (6) Sterilization: Sterilize the sweetened milk in a water bath at 90°C for 10 min;

[0114] (7) Cooling: After sterilization, the milk is cooled to 40-50°C for later use;

[0115] (8) Preparation of starter culture: LA.GXUN74705 was inoculated into a test tube containing sterilized skim milk (12%, w / v) under a sterile environment and cultured at 37°C for 20 hours. The inoculum volume was 2-4% (v / v) per passage, and the culture was subcultured 2-3 times to restore viability. The culture was then stored in a refrigerator at 4°C.

[0116] (9) Inoculation and fermentation: Under sterile conditions, inoculate activated LA.GXUN74705 at an inoculum concentration of 2-4% (v / v). Ferment at a constant temperature of 42°C for 6-10 h.

[0117] (10) After-ripening: After fermentation, place in a refrigerator at 4°C for 12-24 hours;

[0118] (11) Filling and refrigeration: After the ripening is completed, fill it into 250 mL sterilized glass bottles and send it to cold storage for refrigeration.

[0119] Example 4 Preparation of functional fermented fruit and vegetable juice using LA.GXUN74705

[0120] 1. Processing process of fermented fruit and vegetable juice

[0121] Raw materials → cleaning → flash evaporation → beating → blending → homogenization → sterilization → cooling → inoculation → closed fermentation → post-ripening → filling → refrigeration

[0122] 2. Preparation method

[0123] (1) Raw materials: fresh peaches and dragon fruits;

[0124] (2) Cleaning and cutting: Wash, peel (peel peaches), and cut into small pieces;

[0125] (3) Flash evaporation: Inactivate the enzyme by flash evaporation for 0.5-1 min at 121°C and rapidly exhaust the air.

[0126] (4) Beating: According to the ratio of peach to water (weight ratio) = 1:1, gradually put the pumpkin and water into the colloid mill and grind them, performing coarse grinding and fine grinding once each. Beat the dragon fruit with a beater until the pulp is uniform and there are no lumps;

[0127] (5) Mixing and homogenizing: Peach juice (15%) and dragon fruit juice (30%) were added, and the soluble solid content was adjusted to 10° Brix with sucrose. 0.2% of the stabilizer CMC was added and mixed evenly. A two-stage homogenization method was used, first at low pressure (15 MPa) and then at high pressure (25 MPa), to obtain a particle size of 2 to 3 μm.

[0128] (6) Sterilization and cooling: The prepared composite fruit and vegetable juice is kept at 100°C for 10 minutes and then cooled to about 40°C;

[0129] (7) Inoculation and fermentation: Under sterile conditions, inoculate the activated LA.GXUN74705, and control the initial bacterial count to 10 7 CFU / mL. Fermented at 37°C for 24 h;

[0130] (8) Post-ripening: After fermentation, place in a refrigerator at 4°C for 3 h;

[0131] (9) Filling and refrigeration: After the ripening is completed, fill it into 250 mL sterilized glass bottles and send it to the cold storage for refrigeration.

[0132] Example 5 Preparation of lactic acid bacteria bread using LA.GXUN74705

[0133] 1. Formula

[0134] High-gluten flour (1kg), concentrated lactic acid bacteria (LA.GXUN74705), milk (580g), eggs (100g), sugar (200g), salt (10g), yeast (10g) and butter (100g);

[0135] 2. Preparation Method

[0136] Put all the ingredients except butter into the dough bowl and knead until a rough film appears;

[0137] Add the softened butter at room temperature and continue kneading until a smooth dough is formed.

[0138] Check whether the dough forms a translucent, tough, and smooth glove-like film;

[0139] After kneading the dough, divide and relax it: divide the dough evenly into several portions, roll them into balls and smooth the surface, cover with plastic wrap or a damp cloth and relax for a while;

[0140] Take the relaxed dough, make it into the desired shape, and put the shaped dough into the baking pan for fermentation;

[0141] Bake the fermented bread in the oven;

[0142] The baked bread is packaged and the finished product is ready for sale.

[0143] The embodiments described above are merely descriptions of preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Without departing from the spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by persons skilled in the art should fall within the scope of protection defined by the claims of the present invention.

Claims

1. Application of Lactiplantibacillus argentoratensis GXUN74705 in the preparation of a functional product for alleviating alcoholic liver damage, wherein the deposit number of the Lactiplantibacillus argentoratensis GXUN74705 is CGMCC NO.28711.

2. The use according to claim 1, characterized in that The Lactobacillus argenteus GXUN74705 significantly alleviates liver dysfunction caused by alcohol.

3. The use according to claim 1, characterized in that The products include food, food additives and health products.

4. The use according to claim 2, characterized in that The food includes yogurt, fermented fruit and vegetable juice and bread.

5. Use of Lactiplantibacillus argentoratensis GXUN74705 in the preparation of a medicament for treating or assisting in the treatment of alcoholic liver injury. The deposit number of the Lactiplantibacillus argentoratensis GXUN74705 is CGMCC NO.28711.

6. The use according to claim 5, characterized in that The medicine also includes pharmaceutically acceptable carriers and excipients.

7. The use according to claim 6, characterized in that The medicine comprises solid bacterial powder, wherein the number of viable bacteria of Lactobacillus argenteus GXUN74705 in the solid bacterial powder is 1.0×10 11 -1.0×10 12 CFU / g.

8. A method for preparing solid bacterial powder for improving alcoholic liver damage, characterized in that: The following steps are involved: Lactobacillus argenteus GXUN74705 was cultured at a high density and the cells were collected by centrifugation. The bacterial cells were mixed with a freeze-drying protective agent at a mass ratio of 1:5, pre-frozen, and then vacuum-freeze-dried to obtain the solid bacterial powder; The deposit number of the Lactobacillus argenteus GXUN74705 is CGMCC NO.28711.

9. The preparation method according to claim 8, characterized in that The freeze-drying protective agent comprises 15 wt% of skim milk powder, 5 wt% of trehalose, 3 wt% of sodium glutamate, 1 wt% of glycerol and 0.5 wt% of cysteine ​​hydrochloride.

10. A solid bacterial powder obtained by the preparation method according to claim 5 or 6.

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