Probiotic symbiotic biofilm and use in the preparation of a product for the prevention or adjuvant treatment of alcoholic liver disease

Abstract

This invention provides a probiotic symbiotic biofilm and its application in the preparation of products for the prevention or adjunctive treatment of alcoholic liver disease, relating to the field of biomaterials technology. The probiotic symbiotic biofilm is composed of *Lactobacillus rhamnosus* (…). Lactobacillus rhamnosus LRa05 and Bifidobacterium lactis ( Bifidobacterium lactis The above two microorganisms were prepared using BLa80. Under co-culture conditions, they can form a dense symbiotic biofilm. The two strains are evenly distributed and intertwined within the biofilm, forming a thicker and more stable three-dimensional structure than a single strain. This symbiotic biofilm structure endows the probiotics with stronger environmental tolerance and intestinal adhesion ability, laying the foundation for their colonization and sustained function in the intestine. It can not only significantly improve alcohol-induced intestinal barrier damage, but also effectively intervene in alcoholic liver disease, providing a new application scenario for the two strains.

Description

Technical Field

[0001] This invention relates to the field of biomaterials technology, and more particularly to probiotic symbiotic biofilms and their application in the preparation of drugs for the prevention or treatment of alcoholic liver disease. Background Technology

[0002] Current applications of probiotics mainly focus on single strains or simple mixtures. While single strains offer some probiotic benefits, their mechanisms of action are relatively simple, and their colonization and stability in the complex gut environment are often limited. Simple mixtures, although increasing gut microbiota diversity, may result in competition or antagonism between different strains, making synergistic effects difficult to achieve. Therefore, screening probiotic combinations that can mutually promote and synergistically enhance function has become a hot research topic in this field.

[0003] In recent years, researchers have begun to focus on the co-culture effects between probiotics. Studies have shown that certain probiotics, under co-culture conditions, can mutually promote growth, enhance environmental tolerance, and improve biofilm formation capabilities through cross-feeding of metabolites and communication of quorum sensing signaling molecules. Biofilms are structured communities formed by bacteria attaching to surfaces, possessing stronger environmental resistance and host adhesion capabilities, and are an important form for probiotics to colonize and function in the gut. Therefore, screening probiotic combinations capable of forming symbiotic biofilms is of great value in improving the efficacy of probiotic preparations.

[0004] Bifidobacterium lactis BLa80 and Lactobacillus rhamnosus LRa05 are two probiotics with promising applications. Existing research indicates that BLa80 has the potential to regulate gut microbiota and enhance immune function, while LRa05 shows outstanding performance in improving the intestinal barrier and alleviating inflammation. However, due to the characteristics of these two species, research on them has been limited to the digestive system, thus restricting their application scenarios.

[0005] Therefore, this invention is proposed. Summary of the Invention

[0006] To address the aforementioned technical problems, this invention provides a probiotic symbiotic biofilm and its application in the preparation of products for the prevention or adjunctive treatment of alcoholic liver disease. This provides a novel application scenario for known bacterial strains and is of great significance for improving the efficacy and quality control of probiotic preparations.

[0007] In order to achieve the objective of this invention, the following technical solution is adopted: This invention provides the application of a probiotic symbiotic biofilm in the preparation of a medicament for the prevention or adjunctive treatment of alcoholic liver disease, wherein the probiotic symbiotic biofilm is composed of Lactobacillus rhamnosus (… Lactobacillus rhamnosus LRa05 and Bifidobacterium lactis ( Bifidobacterium lactis BLa80 was prepared.

[0008] Furthermore, the ratio of viable Lactobacillus rhamnosus LRa05 to Bifidobacterium lactis BLa80 is 10:1 to 1:10, and the total viable count is not less than 10. 9 CFU / mL.

[0009] Furthermore, the preparation method of the probiotic symbiotic biofilm includes the following steps: S1. Lactobacillus rhamnosus LRa05 and Bifidobacterium lactis BLa80 were activated and cultured to obtain Lactobacillus rhamnosus LRa05 bacterial suspension and Bifidobacterium lactis BLa80 bacterial suspension, respectively. S2. Inoculate and culture the Lactobacillus rhamnosus LRa05 and Bifidobacterium lactis BLa80 bacterial suspensions respectively until both strains enter the logarithmic growth phase. Then prepare solutions with a concentration of not less than 10 9 CFU / mL of Lactobacillus rhamnosus LRa05 suspension and Bifidobacterium lactis BLa80 suspension; S3. Mix and culture the Lactobacillus rhamnosus LRa05 suspension and Bifidobacterium lactis BLa80 suspension until a dense biofilm is formed. S4. Wash the biofilm with buffer solution and freeze-dry to obtain the probiotic symbiotic biofilm.

[0010] Furthermore, the volume ratio of the Lactobacillus rhamnosus LRa05 suspension to the Bifidobacterium lactis BLa80 suspension is 1:2.

[0011] Furthermore, in step S4, the specific steps of drying are as follows: The probiotic symbiotic biofilm was washed with a buffer solution. The specific drying steps were as follows: quick-freezing with liquid nitrogen for 2-4 minutes, followed by freeze-drying at -80℃ to -60℃ and 0 Pa for 20-28 hours.

[0012] Furthermore, in S3, the culture temperature is 37°C, the culture environment is anaerobic, and the culture time is 96 hours.

[0013] Furthermore, the product is an oral preparation.

[0014] Furthermore, the oral preparation is a beverage or a solid dosage form.

[0015] The present invention also provides a probiotic symbiotic biofilm, comprising Lactobacillus rhamnosus LRa05 and Bifidobacterium lactis BLa80 used in the above applications.

[0016] The present invention has the following technical effects: LRa05 and BLa80 can form a dense symbiotic biofilm under co-culture conditions. The two strains are evenly distributed and intertwined within the biofilm, forming a thicker and more stable three-dimensional structure than a single strain. This symbiotic biofilm structure endows the probiotics with stronger environmental tolerance and intestinal adhesion ability, laying the foundation for their colonization and sustained function in the gut. It can not only significantly improve alcohol-induced intestinal barrier damage, but also effectively intervene in alcoholic liver disease, providing new application scenarios for both strains. Attached Figure Description

[0017] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0018] Figure 1 Six groups of samples were cultured for 3 hours and then analyzed using an ELISA reader at the OD500 level. 630 Measure absorbance at the specified location; Figure 2 Six groups of samples were cultured for 6 hours and then analyzed using an ELISA reader at OD500. 630 Measure absorbance at the specified location; Figure 3 Six groups of samples were cultured for 9 hours and then analyzed using an ELISA reader at OD500. 630 Measure absorbance at the specified location; Figure 4 Six groups of samples were cultured for 12 hours and then analyzed using an ELISA reader at OD500. 630 Measure absorbance at the specified location; Figure 5 The absorbance of the six groups of samples was measured at 570 nm using a full-wavelength microplate reader after 96 h of culture. Figure 6 Crystal violet was used to characterize the biofilm formation status of 6 groups of samples; Figure 7 Bar chart showing the bacterial motility test results of 6 groups of samples; Figure 8 Results of bacterial motility assays for 6 groups of samples; Figure 9 Results of bacterial adhesion assay for LRa05; Figure 10 Results of bacterial adhesion assay for BLa80; Figure 11 Raw data from the determination of c-di-Gmp; Figure 12 Results of c-di-GMP production determination in probiotic symbiotic biofilm systems; Figure 13A Analysis results of mouse body weight under different probiotic interventions; Figure 13B Results of serum alanine aminotransferase (ALT) analysis in mice under different probiotic interventions; Figure 13C Results of serum aspartate aminotransferase (AST) analysis in mice under different probiotic interventions; Figure 13D Results of triglyceride analysis in mouse serum under different probiotic interventions; Figure 13E Results of total cholesterol analysis in mouse serum under different probiotic interventions; Figure 13F Liver / body weight analysis results in mice under different probiotic interventions; Figure 13G Results of malondialdehyde analysis in mouse serum under different probiotic interventions; Figure 13H Results of superoxide dismutase analysis in mouse serum under different probiotic interventions; Figure 13I Results of colon length analysis in mice under different probiotic interventions; Figure 13J Diagrams of intestinal tissue in mice under different probiotic interventions; Figure 14 Immunofluorescence analysis results of liver and colon tissues. Detailed Implementation

[0019] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions of this invention will be clearly and completely described below. Obviously, the described embodiments are only a part of the embodiments of this invention, and not all of them. Based on the embodiments of this invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this invention.

[0020] In a first aspect, the present invention provides the use of a probiotic symbiotic biofilm in the preparation of a product for the prevention or adjunctive treatment of alcoholic liver disease, wherein the probiotic symbiotic biofilm is composed of *Lactobacillus rhamnosus* (… Lactobacillus rhamnosus LRa05 and Bifidobacterium lactis ( Bifidobacterium lactis BLa80 was prepared.

[0021] In some embodiments, the ratio of viable Lactobacillus rhamnosus LRa05 to Bifidobacterium lactis BLa80 is 1:10 to 10:1, and the total viable count is not less than 10. 9 CFU / mL.

[0022] In some embodiments, the method for preparing the probiotic symbiotic biofilm includes the following steps: S1. Lactobacillus rhamnosus LRa05 and Bifidobacterium lactis BLa80 were activated and cultured to obtain Lactobacillus rhamnosus LRa05 bacterial suspension and Bifidobacterium lactis BLa80 bacterial suspension, respectively. S2. Inoculate and culture the Lactobacillus rhamnosus LRa05 and Bifidobacterium lactis BLa80 bacterial suspensions respectively until both strains enter the logarithmic growth phase. Then prepare solutions with a concentration of not less than 10 9 CFU / mL of Lactobacillus rhamnosus LRa05 suspension and Bifidobacterium lactis BLa80 suspension; S3. Mix and culture the Lactobacillus rhamnosus LRa05 suspension and Bifidobacterium lactis BLa80 suspension until a biofilm is formed. S4. Wash the biofilm with buffer solution and freeze-dry to obtain the probiotic symbiotic biofilm.

[0023] In some embodiments, the volume ratio of the Lactobacillus rhamnosus LRa05 suspension to the Bifidobacterium lactis BLa80 suspension is 1:2.

[0024] In some embodiments, the drying steps in S4 are as follows: quick-freezing with liquid nitrogen for 2-4 minutes, followed by freeze-drying at -80°C to -60°C and 0 Pa for 20-28 hours.

[0025] In some embodiments, in step S3, the culture temperature is 37°C, the culture environment is anaerobic, and the culture time is 96 hours.

[0026] In some embodiments, the product is an oral dosage form.

[0027] In some embodiments, the oral dosage form is a beverage or a solid dosage form.

[0028] Secondly, the present invention also provides a probiotic symbiotic biofilm, including Lactobacillus rhamnosus LRa05 and Bifidobacterium lactis BLa80 used in the above applications.

[0029] The following is a detailed explanation using specific embodiments: Example 1: Bacterial co-culture and sample preparation Lactobacillus rhamnosus ( Lactobacillus rhamnosus LRa05 was purchased from Suzhou Weikang Probiotics Co., Ltd.; Bifidobacterium lactis ( Bifidobacterium lactis BLa80 was purchased from Suzhou Weikang Probiotics Co., Ltd.

[0030] S1. Activate Lactobacillus rhamnosus LRa05 and Bifidobacterium lactis BLa80. The specific steps are as follows: Under aseptic conditions, Lactobacillus rhamnosus LRa05 was inoculated with a loop to collect bacterial powder, and then the bacterial powder was inoculated into 15 mL of sterilized fresh MRS medium. The medium was then incubated overnight at 37°C under anaerobic conditions to obtain the revived Lactobacillus rhamnosus LRa05 bacterial solution. Bifidobacterium lactis BLa80 was activated in the same way to obtain Bifidobacterium lactis BLa80 bacterial solution.

[0031] S2. The obtained *Lactobacillus rhamnosus* LRa05 and *Bifidobacterium lactis* BLa80 bacterial suspensions were inoculated into 50 mL of sterilized fresh MRS medium at an inoculation rate of 1%, and cultured overnight at 37°C under anaerobic conditions until both strains entered the logarithmic growth phase. Subsequently, concentrations of not less than 10 were prepared. 9 CFU / mL of Lactobacillus rhamnosus LRa05 suspension and Bifidobacterium lactis BLa80 suspension.

[0032] Preparation concentration not less than 10 9 The specific steps for preparing CFU / mL Lactobacillus rhamnosus LRa05 suspension and Bifidobacterium lactis BLa80 suspension are as follows: The *Lactobacillus rhamnosus* LRa05 suspension was centrifuged at 6000 rpm for 10 min to completely separate the bacterial cells from the supernatant. The bacterial culture supernatant and bacterial pellet were then collected separately. The bacterial pellet of each strain was resuspended in an equal volume of fresh, sterile MRS medium to maintain a concentration of 10. 9 The bacterial culture supernatant was filtered twice using a 0.22µm sterile filter membrane at CFU / mL to obtain a sterile *Lactobacillus rhamnosus* LRa05 suspension. A *Bifidobacterium lactis* BLa80 suspension was prepared using the same method, ensuring a 1:1 ratio of viable bacteria between the two suspensions.

[0033] S3. Mix Lactobacillus rhamnosus LRa05 suspension and Bifidobacterium lactis BLa80 suspension at a volume ratio of 1:2 and incubate at 37°C under anaerobic conditions for 24 hours until a biofilm is formed.

[0034] S4. After gently washing twice with PBS and rapidly freezing in liquid nitrogen, place the sample in a vacuum freeze dryer and freeze-dry at -60℃ and 0Pa for 24 hours. A probiotic symbiotic biofilm was obtained after freeze-drying.

[0035] Experimental Example 1: Determination of growth status of mixed culture of two bacterial strains The sample used in the experiment was prepared by mixing the following proportions: 1.1 volume of Bifidobacterium lactis BLa80 suspension + 2 volumes of sterile fresh MRS medium; 2.1 volume of Bifidobacterium lactis BLa80 suspension + 2 volume of Bifidobacterium lactis BLa80 supernatant; 3.1 volume of Bifidobacterium lactis BLa80 suspension + 2 volumes of Lactobacillus rhamnosus LRa05 supernatant; 4.1 volumes of Lactobacillus rhamnosus LRa05 suspension + 2 volumes of sterile fresh MRS medium; 5.1 volumes of Lactobacillus rhamnosus LRa05 suspension + 2 volumes of LRa05 supernatant; 6.1 volume of Lactobacillus rhamnosus LRa05 suspension + 2 volume of Bifidobacterium lactis BLa80 supernatant.

[0036] The six groups of samples prepared above were added to 96-well plates and cultured at 37°C under anaerobic conditions for 3 h, 6 h, 9 h, and 12 h, respectively. The samples were then analyzed using an ELISA reader at OD500. 630 The absorbance was measured at the point.

[0037] Experimental results are as follows Figures 1-4 As shown in the figure, within 12 hours of incubation, it was observed that the supernatant of LRa05 promoted the growth of BLa80, and the supernatant of BLa80 also promoted the growth of LRa05. This means that the supernatants of both bacteria may contain substances that promote the growth of the other, suggesting that co-culturing BLa80 and LRa05 may have the potential to mutually promote growth. The group with the fastest growth rate was the bacterial culture + MRS experimental group. This is because fresh MRS medium contains sufficient nutrients and the pH is at the optimal conditions for bacterial growth. While the supernatant of one bacterium promotes the growth of the other, it is relatively lacking in nutrients. Furthermore, the metabolism of BLa80 and LRa05 produces acidic substances, causing the pH of the supernatant to decrease. Ultimately, the growth environment is not as suitable for bacterial growth as that of fresh MRS medium.

[0038] Crystal violet characterizes biofilm formation status: Take a sterile 12-well plate, place a 20 mm diameter circular coverslip at the bottom of the plate, and add the six experimental groups prepared above to the 12-well plate respectively. Add 1.8 mL of experimental sample to each well and incubate at 37℃ under anaerobic conditions for 96 h. Then discard the culture medium and gently wash each well twice with PBS buffer to completely remove free bacteria. Air dry the plate, then fix each well with 1.8 mL of methanol for 20 min, discard the methanol, and gently wash the well twice with PBS buffer. After air drying, add 0.1% crystal violet solution (w / v) to each well for staining for 30 min, discard the staining solution, and gently wash the well twice with PBS buffer. After air drying, add 1.8 mL of 33% glacial acetic acid to each well and shake well for 20 min to fully resuspend the stained biofilm. Finally, measure the absorbance of each well at 570 nm using a full-wavelength microplate reader.

[0039] Experimental results are as follows Figures 5-6 As shown, after 96 h of biofilm culture, the biofilm formation of the BLa80 suspension + LRa05 supernatant experimental group was significantly better than that of the BLa80 suspension + BLa80 supernatant experimental group (p < 0.001), and the biofilm formation of the LRa05 suspension + BLa80 supernatant experimental group was also significantly better than that of the LRa05 suspension + LRa05 suspension experimental group (p < 0.05). This indicates that the metabolites contained in the LRa05 supernatant can promote the formation of biofilm by BLa80, and vice versa.

[0040] Bacterial motility assay: The six groups of samples prepared above were cultured at 37℃ under anaerobic conditions for 12 hours. The bacterial suspension was then inoculated onto 1 / 2 MRS semi-solid plates (containing 0.5% agar) using a puncture method and cultured anaerobically at 37℃ for 96 hours. Colony diameters were measured and single-colony photographs were taken. The measurement method involved using vernier calipers to measure the transverse and longitudinal lengths of each single colony, and the average value was taken.

[0041] Experimental results are as follows Figures 7-8 As shown in the data analysis, there was no significant difference in exercise performance within the experimental groups.

[0042] Bacterial adhesion assay: The six groups of samples prepared above were cultured at 37°C under anaerobic conditions for 48 hours, and then the OD of the obtained bacterial culture was... 630 Adjusted to approximately 0.5, the adhesion of bacterial cells in different experimental groups was measured using a MUC2 coated plate. The experimental results are as follows: Figures 9-10 As shown.

[0043] Experimental Example 2: Determination of c-di-GMP in a Probiotic Symbiotic Biofilm System 1.3 volumes of fresh MRS medium; 2.1 volumes of BLa80 suspension + 2 volumes of fresh MRS medium; 3.1 volumes of LRa05 suspension + 2 volumes of fresh MRS medium; 4.0.5 volumes of BLa80 suspension + 0.5 volumes of LRa05 suspension + 2 volumes of fresh MRS medium.

[0044] Using the above grouping, a probiotic symbiotic biofilm was prepared using the method in Example 1. Samples were tested and data collected using an enzyme-linked immunosorbent assay (ELISA) reader. The raw data results are as follows: Figure 11 As shown.

[0045] The c-di-GMP content in the supernatant of the above groups was determined using a bacterial c-di-GMP enzyme-linked immunosorbent assay kit (competitive method) according to the manufacturer's instructions. The experimental results are as follows: Figure 12As shown, using blank MRS medium as a blank control, it can be seen that BLa80 has a higher c-di-GMP content, followed by BLa80+LRa05, and the LRa05 experimental group has the lowest c-di-GMP content.

[0046] Experiment Example 3: The ability of probiotic symbiotic biofilms to intervene in alcoholic liver disease Mice sourced from Nanjing Annocon Co., Ltd. (Nanjing, China), weighing 22g ± 2g.

[0047] Mice were randomly divided into 7 groups (n=4 / group): blank control group, ethanol model group, and Bifidobacterium breve BBr60 group (model + 5×10⁻⁶). 9 CFU / day), Lactobacillus gasseri LG08 group (model + 5×10) 9 CFU / day), Bifidobacterium lactis BLa80 group (model + 5×10) 9 CFU / day), Lactobacillus rhamnosus LRa05 group (model + 5×10) 9 CFU / day), Bifidobacterium lactis BLa80 and Lactobacillus rhamnosus LRa05 mixed group (model + 5×10) 9 CFU / day, live bacteria ratio of 1:1).

[0048] Mice were housed in a controlled environment (temperature: 25±2℃; humidity: 40-80%), with free access to food and water under a 12-hour light / dark cycle. For the first 7 days, mice in the probiotic group (specifically referring to the mixed group of Bifidobacterium breve BBr60, Lactobacillus gasseri LG08, Lactobacillus rhamnosus LRa05, Bifidobacterium lactis BLa80, and Lactobacillus rhamnosus LRa05, hereinafter referred to as the probiotic group) were administered probiotics (5 × 10⁻⁶) by gavage at 12:00 noon daily. 9 CFU / day, with equal proportions for each strain), and the control and ethanol groups were administered an equal volume of PBS (0.2 mL) by gavage.

[0049] After the last treatment, all mice were fasted for 6 hours (12:00-18:00). An acute ethanol model was established while probiotics were administered daily at 12:00. Mice in the model group were administered ethanol (50% ethanol, 5g / kg) by gavage at 16:00 daily, while the control group received isoenergy maltose for 3 consecutive days.

[0050] After a 24-hour fast following the last administration, mice were administered 70 mg / kg sodium pentobarbital (Shanghai Beizhuo Biochemical Technology Co., Ltd., Shanghai, China) via intraperitoneal injection. Blood samples were immediately collected from the eyes and left at room temperature for 40 minutes. The supernatant was then obtained by centrifugation at 3000g for 10 minutes at 4°C for biochemical analysis. Anesthetized mice were then euthanized by dislocation and immediately dissected for liver and colon tissue. All dissected mouse carcasses were sent to the Experimental Animal Center of Nanjing Normal University for standardized processing. Liver sections were preserved with 4% paraformaldehyde, colons were preserved with Carnoy's fixative, and remaining liver segments and colons were stored at -80°C for further experiments.

[0051] The ability of different probiotics and mixed probiotics to intervene in acute alcoholic liver disease was verified in mice. After a 7-day acclimatization period (day 0), probiotic intervention was administered for 7 days, meaning the probiotic group mice were given probiotics (5 × 10⁻⁶) by gavage at 12:00 PM daily. 9 CFU / day (equal proportions of each strain), and the control and ethanol groups were administered an equal volume of PBS (0.2 mL) by gavage. After the last treatment, all mice were fasted for 6 hours (12:00-18:00). An acute ethanol model was established while probiotics were administered daily at 12:00. Mice in the model group were administered ethanol (50% ethanol, 5 g / kg) by gavage at 16:00 daily, while the control group received an equal amount of maltose for 3 consecutive days. The following analyses were then performed: 2.1 Biochemical Analysis Serum ALT, AST, TG, and TC levels in mice were measured using an ALT, AST, TG, and TC activity assay kit (Elabscience, Wuhan), and analyzed using an automated biochemical analyzer (Hitachi 7020, Japan). Oxidative stress levels in mouse serum were assessed using an MDA assay kit (Elabscience, Wuhan) and a SOD assay kit (Elabscience, Wuhan). The results are shown below. Figures 13A-13J As shown, all experiments were conducted according to the respective manufacturer's instructions.

[0052] 2.2 Histological examination and immunofluorescence analysis Hematoxylin and eosin (H&E) staining was used to analyze liver and colon tissues. Liver tissue was fixed with 4% paraformaldehyde, and colon tissue was fixed with Carnoy's fixative, then embedded in paraffin and sectioned to uniform thickness. The samples were then dewaxed and hydrated with xylene and gradient concentrations of ethanol. After hematoxylin staining for 5 minutes, the sections were stained with eosin for 1 minute. Next, the tissues were dehydrated using gradient concentrations of ethanol followed by xylene. Finally, all sections were sealed and studied using a bright-field microscope (Olympus BX 51, Tokyo, Japan) at 200x magnification. The results are shown below. Figure 14 As shown.

[0053] 2.3 AB-PAS staining is used to detect glycogen, acidic mucopolysaccharides, and neutral mucus in tissues. This method combines Alcian Blue, periodic acid (PAA), and Schiff's reagent. Alcian Blue staining produces a blue stained area for acidic mucus, while PAS stains neutral mucus red. The specific steps include: dewaxing, Alcian Blue staining, washing, periodic acid oxidation, washing, Schiff's reagent staining, washing, hematoxylin staining of the nucleus, dehydration and clearing, and mounting. Acidic mucus appears blue, glycogen and neutral mucus appear purplish-red, and mixed mucus appears purplish-blue or bluish-purple. Colon tissue is fixed with Carnoy's fixative and then embedded in paraffin to create uniformly thick sections. The samples are then dewaxed and hydrated with xylene and gradient concentrations of ethanol. Next, the tissue was stained with AB-PAS staining kit (Servicebio, G1049) solution C for 15 min, then washed with tap water until colorless; acidified with staining solution B for 15 min, washed with tap water, and then washed twice with distilled water; stained with staining solution A in the dark for 1 h, then rinsed with running water for 5 min. Following this, the tissue was dehydrated using gradient concentrations of ethanol followed by xylene. Finally, all sections were sealed and studied using a bright-field microscope (Olympus BX 51, Tokyo, Japan) at 200x magnification. The experimental results are shown below. Figure 14 As shown.

[0054] 2.4 Immunofluorescence was used to analyze colon tissue. Paraffin sections were dewaxed and dehydrated using xylene and ethanol aqueous solution. Antigen retrieval was performed using a heat-mediated method with sodium citrate buffer (10 mM citrate, pH 6.0 + 0.05% Tween-20), followed by washing with PBS (0.01 m, pH 7.4) and then blocking in 1% bovine serum albumin for 1 hour. After washing with PBS and aspirating excess liquid, the sections were incubated with proportionally diluted Anti-ZO-1 Rabbit pAb (Proteintech, 121773-1-AP, 1:500), Anti-Occludin Rabbit pAb (Wanleibio, WL01996, 1:200), and Anti-MUC2Rabbit pAb (Abcam, ab272692, 1:2000) primary antibodies. The sections were then placed in a light-protected, humidified chamber and incubated overnight at 4 °C, followed by washing with PBS. Subsequently, the slides were exposed to light-protected AlexaFluor® 488 Goat anti-Rabbit secondary antibody (Abcam, ab150077, 1:400) and maintained at 37°C for 30 minutes. The nuclei of the fixed tissues were then restained with 4,6-diamino-2-phenylindole (DAPI). The degree of immunofluorescence staining was detected using a fluorescence microscope (Leica dmiled, Germany), and positive expression analysis was performed using ImageJ software. The experimental results are shown below. Figure 14 As shown.

[0055] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the technical solutions of the embodiments of the present invention.

Claims

1. The application of probiotic symbiotic biofilms in the preparation of products for the prevention or adjunctive treatment of alcoholic liver disease, characterized in that, The probiotic symbiotic biofilm is composed of Lactobacillus rhamnosus ( Lactobacillus rhamnosus LRa05 and Bifidobacterium lactis ( Bifidobacterium lactis BLa80 was used to prepare the product; The ratio of viable Lactobacillus rhamnosus LRa05 to Bifidobacterium lactis BLa80 is 10:1 to 1:10, and the total viable count is not less than 10. 9 CFU / mL.

2. The application of the probiotic symbiotic biofilm according to claim 1 in the preparation of products for the prevention or adjunctive treatment of alcoholic liver disease, characterized in that, The preparation method of the probiotic symbiotic biofilm includes the following steps: S1. Lactobacillus rhamnosus LRa05 and Bifidobacterium lactis BLa80 were activated and cultured to obtain Lactobacillus rhamnosus LRa05 bacterial suspension and Bifidobacterium lactis BLa80 bacterial suspension, respectively. S2. Inoculate and culture the Lactobacillus rhamnosus LRa05 and Bifidobacterium lactis BLa80 bacterial suspensions respectively until both strains enter the logarithmic growth phase. Then prepare solutions with a concentration of not less than 10 9 CFU / mL of Lactobacillus rhamnosus LRa05 suspension and Bifidobacterium lactis BLa80 suspension; S3. Mix and culture the Lactobacillus rhamnosus LRa05 suspension and Bifidobacterium lactis BLa80 suspension until a dense biofilm is formed. S4. Wash the biofilm with buffer solution and freeze-dry to obtain the probiotic symbiotic biofilm.

3. The application of the probiotic symbiotic biofilm according to claim 2 in the preparation of products for the prevention or adjunctive treatment of alcoholic liver disease, characterized in that, The volume ratio of the Lactobacillus rhamnosus LRa05 suspension to the Bifidobacterium lactis BLa80 suspension was 1:

2.

4. The application of the probiotic symbiotic biofilm according to claim 2 in the preparation of products for the prevention or adjunctive treatment of alcoholic liver disease, characterized in that, In step S4, the specific drying steps are as follows: quick-freezing with liquid nitrogen for 2-4 minutes, followed by freeze-drying at -80℃ to -60℃ and 0Pa for 20-28 hours.

5. The application of the probiotic symbiotic biofilm according to claim 2 in the preparation of products for the prevention or adjunctive treatment of alcoholic liver disease, characterized in that, In S3, the culture temperature is 37℃, the culture environment is anaerobic, and the culture time is 96h.

6. The application of the probiotic symbiotic biofilm according to claim 1 in the preparation of products for the prevention or adjunctive treatment of alcoholic liver disease, characterized in that, The product is an oral medication.

7. The application of the probiotic symbiotic biofilm according to claim 6 in the preparation of products for the prevention or adjunctive treatment of alcoholic liver disease, characterized in that, The oral preparation is either a beverage or a solid preparation.

8. A probiotic symbiotic biofilm, characterized in that, Includes Lactobacillus rhamnosus LRa05 and Bifidobacterium lactis BLa80 as used in any of the applications of claims 1-7.

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