Probiotic strain of high-yield biological membrane and application of probiotic strain

Abstract

The invention discloses a probiotic strain Bifidobacterium animalis subsp. Lactiss S8 of a high-yield biological membrane, the preservation number is GDMCC NO.66518, the characteristics of the stable high-yield biological membrane are obtained through directional screening and adaptive evolution, and the yield of the biological membrane is three times higher than that of a positive control. The extracellular polymeric substance (EPS) of the strain can significantly enhance the tolerance of the strain to severe environments such as gastric acid, cholate and the like, and improve the intestinal colonization ability. Experiments prove that the strain remarkably reduces two liver function indexes, namely ALT and AST, in an animal model, and shows that the strain has a certain effect of improving liver injury.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of microbial technology, and in particular to a high-biofilm-yield probiotic bacterial strain and application thereof. BACKGROUND

[0002] Currently, commercialized probiotics face significant technical bottlenecks in practical application. When passing through the digestive tract, traditional probiotics are subjected to strong stress from extreme environments such as gastric acid (pH 1.5-3.5) and bile salts (0.3-2%), resulting in a survival rate of live bacteria generally lower than 20%. More critically, the colonization ability of these strains in the intestinal tract is limited, and research shows that about 90% of ingested strains are excreted within 48 hours. This low colonization rate directly affects the regulatory efficacy of probiotics on the host microecology, especially in the treatment of diseases such as antibiotic-associated diarrhea and irritable bowel syndrome, which often require large doses (≥10 10 CFU / day) for long-term administration to show effects. In addition, the conventional freeze-drying process further damages the cell membrane structure of the bacteria, resulting in a high live bacteria decay rate of up to 1 order of magnitude / year during storage of the product.

[0003] Biofilm is a three-dimensional matrix structure formed by extracellular polymeric substances (EPS) secreted by microorganisms through quorum sensing regulation, mainly including polysaccharides, proteins, nucleic acids, and lipids. This natural barrier has multiple protective functions: (1) physical isolation effect, which can reduce the penetration rate of gastric acid, and increase the internal bacterial pH tolerance threshold from 4.5 to 2.5; (2) molecular sieve effect, which can selectively block the lytic effect of bile salts (molecular weight > 500 Da); (3) population resistance, the tolerance of dormant cells in the biofilm to antibiotics is increased by 10-1000 times. Animal experiments have confirmed that probiotics with biofilm can prolong the colonization time in the small intestinal villi from 24 hours to more than 72 hours, and can form microcolonies by specific binding of EPS adhesins (such as MucBP protein) to the intestinal mucus layer.

[0004] The current high-yield biofilm probiotic resources mainly have three major shortcomings: (1) the performance of natural strains is insufficient: the strains isolated from traditional fermented foods have large differences in biofilm formation ability (crystal violet method OD value 0.1-1.5), and phenotypic regression easily occurs during the subculture process (the biofilm yield decreases by 40-60% after the 10th generation); (2) the compliance risk of genetic modification: although CRISPR and other technologies can knock out group sensing inhibitory genes such as luxS to increase the EPS yield, according to the new regulations of EFSA in 2023, the application of genetically modified microorganisms (GMM) in food needs to complete a 90-day subchronic toxicity evaluation, and the commercialization cycle is as long as 3-5 years; (3) the contradiction of production process: the conventional centrifugal washing will damage the biofilm structure, and the bacterial agent with intact biofilm structure is faced with the problem of penetration blockage of the freeze-drying protective agent (such as glycerol), resulting in a low active bacteria recovery rate of not higher than 30%. These technical barriers seriously restrict the development of high-efficiency probiotic products. SUMMARY

[0005] The purpose of the present application is to improve the limitations of existing probiotics and provide a high-yield biofilm probiotic strain and application thereof.

[0006] Specifically, the present application provides a high-yield biofilm probiotic strain, which is classified and named as animal bifidobacterium lactobacillus subsp (Bifidobacterium animalis lactis subsp Bifidobacteria animalis subsp .lactiss ), the strain name is S8, the strain is preserved in Guangdong Microbial Culture Collection Center, the preservation number is GDMCC NO. 66518, the preservation date is June 28, 2025, and the preservation place is No. 59, Building 5, 100, Xianlie Middle Road, Guangzhou.

[0007] The present application also provides a culture method of the above-mentioned probiotic, which is specifically amplified in MRS culture medium.

[0008] The MRS liquid culture medium is prepared by adding 10.0 g / L of beef extract, 10.0 g / L of peptone, 5.0 g / L of yeast extract, 20.0 g / L of glucose, 2.0 g / L of anhydrous sodium acetate, 0.5 g / L of magnesium sulfate heptahydrate, 0.25 g / L of manganese sulfate monohydrate, 2 g / L of citric acid hydrogen diamine, 2.6 g / L of dipotassium hydrogen phosphate trihydrate, and 80 mL / L of Tween 80, and adjusting the pH value to 6.2-6.6. The sterilization is performed at 121℃ for 15 min.

[0009] The MRS solid culture medium is prepared by adding 20 g / L of agar powder into the MRS liquid culture medium.

[0010] The present application further provides a probiotic bacterial agent, which comprises the above-mentioned probiotic strain.

[0011] Preferably, the dosage form of the probiotic agent is a liquid agent or a freeze-dried powder.

[0012] Preferably, the dosage form of the probiotic strain is a liquid agent, and the probiotic agent is a live strain or a culture broth of the strain.

[0013] The present application further provides a preparation method of the probiotic agent, wherein the strain is inoculated into a culture medium at an inoculation ratio of 2%-5%, and then continuously activated for three generations, the bacteria in the logarithmic phase after amplification are centrifuged at a speed of 7000-8000 rpm / min for 5-10 minutes, the supernatant is discarded, and then PBS buffer is added for repeated washing, and the bacteria are centrifuged again at the same speed for 3-8 minutes to remove impurities, and finally the bacterial precipitate at the bottom is collected and resuspended in the buffer.

[0014] The present application further provides an application of the probiotic strain or the probiotic agent in the preparation of a lipid-lowering product.

[0015] The present application further provides an application of the probiotic strain or the probiotic agent in the preparation of a product for preventing and / or treating liver dysfunction.

[0016] The animal bifidobacterium lactobacillus subsp. S8 screened in the present application has the ability to resist gastric acid and bile salt, and has the characteristic of high biofilm production (OD600nm=0.51), which is three times higher than the positive control (OD600nm=0.176).

[0017] As a potential lipid-lowering effective substance, the probiotic must be able to survive and colonize in the host body before it can exert its effect. Oral administration is the most common administration method for probiotics. Before the strain enters the intestine to exert its lipid-lowering effect, it needs to withstand the severe test of the strong acid environment in the stomach and the high concentration of bile salt in the intestine. As the first barrier of the digestive tract, gastric acid can inactivate most acid-intolerant microorganisms; and bile salt further selects the surviving strains by damaging the bacterial cell membrane structure. This means that the acid and bile salt resistance of probiotics directly determines whether they can successfully pass through the upper digestive tract to reach the intestinal colonization site, and thus affects the exertion of their lipid-lowering effect. The probiotics obtained in the present application perform excellently in the bile salt resistance test and also exhibit a high survival rate in the previous gastric acid resistance experiment, indicating that these strains have dual resistance and can effectively resist the dual stress of gastric acid and bile salt, thereby being more likely to survive and colonize in the intestine after oral administration and exerting the potential lipid-lowering effect. In addition, the probiotics obtained in the present application exhibit a significant reducing effect on liver function indicators in mouse experiments, indicating that the bacteria have a certain effect on improving liver damage. BRIEF DESCRIPTION OF DRAWINGS

[0018] The above and / or other aspects of the present application will become apparent and more readily appreciated from the following description, taken in conjunction with the accompanying drawings, in which:

[0019] Figure 1 Survival rate of probiotic bacteria in simulated gastric juice environment; Figure 2 Survival rate of probiotic bacteria in simulated bile salt environment; Figure 3 Comparison chart of biofilm formation intensity of probiotic bacteria; Figure 4 Pathological results of mouse liver after feeding probiotic bacteria. DETAILED DESCRIPTION

[0020] The present application will be described in detail below with specific examples. The following examples will help those skilled in the art to further understand the present application, but do not constitute any limitation on the present application.

[0021] The probiotic bacteria described in the following examples are animal Bifidobacterium lactis subsp. Bifidobacteria animalis subsp .lactiss ) S8, which is preserved in Guangdong Microbial Culture Collection Center, with the preservation number GDMCC NO. 66518, the preservation date June 28, 2025, and the preservation location 5th floor, No. 59 Building, 100 Middle Martyrs Road, Guangzhou.

[0022] In the following examples, the formula of the MRS liquid medium is as follows: beef extract 10.0 g / L, peptone 10.0 g / L, yeast extract 5.0 g / L, glucose 20.0 g / L, anhydrous sodium acetate 2.0 g / L, magnesium sulfate heptahydrate 0.5 g / L, manganese sulfate monohydrate 0.25 g / L, citric acid diamine 2 g / L, dipotassium hydrogen phosphate trihydrate 2.6 g / L, Tween 80 mL / L, and the pH value is adjusted to 6.2-6.6. Sterilization at 121°C for 15 min.

[0023] The MRS solid medium is obtained by adding 20 g / L agar powder to the MRS liquid medium.

[0024] Example 1: Isolation, screening and identification of animal Bifidobacterium lactis subsp. S8

[0025] Collect the feces of healthy volunteers (9-month-old infants) as the original sample and place them in a sampling bottle. Weigh 1 g of the sample and dilute it in 9 ml of sterilized normal saline in a 10 ml centrifuge tube to obtain 7 gradients (the sample is diluted in normal saline to obtain gradients, and the dilution ratio is 10 -1 ~ 10 -7, according to the growth situation to select higher or lower dilution concentration). Then, select the appropriate diluent evenly coated on the surface of the screening medium, placed in a 37°C incubator for 36 to 48 hours. After the end of the culture, the number of screening bacteria between 30 to 300 plates, select single colonies from which the selected colonies were transferred to the MRS solid medium using a inoculation needle for streaking and incubated at 37°C for 24 hours, picking the new single colonies on the medium inoculated into MRS liquid medium, incubated at 37°C for 24 hours, to obtain the purified strain culture.

[0026] The purified bacteria were mixed well by shaking, and 1.5 ml of the bacteria were placed in a centrifuge tube. The bacteria in the centrifuge tube were centrifuged at 7000 rpm / min for 3 minutes, the supernatant was discarded, the bacterial pellet was retained, sterile water was added to 1 mL, mixed well by blowing, centrifuged at 7000 rpm / min for 3 minutes, the supernatant was discarded again, and the bacterial pellet was washed by repeating this step 3 times. Finally, 250 μL of sterile water was added to resuspend the bacteria, and the resulting final resuspension was used as the PCR template bacteria.

[0027] To identify the species information of the strain, PCR technology was used to amplify the 16S rDNA gene fragment. The PCR reaction system was 30 μL, and the specific composition was as follows: upstream primer 27F (sequence: 5'-AGAGTTTGATCCTGGCCTCA-3') 0.25 μL, downstream primer 1492R (sequence: 5'-GGTTACCTTGTTACGACTT-3') 0.25 μL, Taq enzyme 12.5 μL, bacteria template 2 μL, double distilled water 11 μL.

[0028] The PCR amplification program was set as follows: pre-denaturation: 94°C, 5 minutes, to ensure complete denaturation of the DNA template; cycle amplification (30 cycles): denaturation: 94°C, 30 seconds, to separate the DNA double strands; annealing: 55°C, 30 seconds, specific binding of primers to templates; extension: 72°C, 2 minutes, Taq enzyme catalyzing DNA strand extension; final extension: 72°C, 10 minutes, to ensure complete synthesis of all amplified fragments; storage: 12°C, to maintain the stability of the reaction system. After amplification, the PCR product was subjected to 1% agarose gel electrophoresis analysis to confirm the size of the target band (about 1500 bp). The PCR product meeting the expectations was sent to General Biological for gene sequencing.

[0029] After sequencing, the 27F and 1492R primer sequences returned were spliced to obtain the complete 16S rDNA sequence, and the sequencing results are shown in SEQ No:1: AAAGCTGCGGCGCTACATGCAGTCGACGGGATCCATCAGGCTTTGCTTGGTGGTGAGAGTGGCGAACGGGTGAGTAATGCGTGACCGACCTGCCCCATACACCGGAATAGCTCCTGGAAACGGGTGGTAATGCCGGATGCTCCAGTTGATCGCATGGTCTTCTGGGAAAGCTTTCGCGGTATGGGATGGGGTCGCGTCCTATCAGCTTGACGGCGGGGTAACGGCCCACCGTGGCTTCGACGGGTAGCCGGCCTGAGAGGGCGACCGGCCACATTGGGACTGAGATACGGCCCAGACTCCTACGGGAGGCAGCAGTGGGGAATATTGCACAATGGGCGCAAGCCTGATGCAGCGACGCCGCGTGAGGGATGGAGGCCTTCGGGTTGTAAACCTCTTTTATCGGGGAGCAAGCGTGAGTGAGTTTACCCGTTGAATAAGCACCGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGGTGCAAGCGTTATCCGGAATTATTGGGCGTAAAGGGCTCGTAGGCGGTTCGTCGCGTCCGGTGTGAAAGTCCATCGCTTAACGGTGGATCCGCGCCGGGTACGGGCGGGCTTGAGTGCGGTAGGGGAGACTGGAATTCCCGGTGTAACGGTGGAATGTGTAGATATCGGGAAGAACACCAATGGCGAAGGCAGGTCTCTGGGCCGTTACTGACGCTGAGGAGCGAAAGCGTGGGGAGCGAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGGTGGATGCTGGATGTGGGGCCCGTTCCACGGGTTCCGTGTCGGAGCTAACGCGTTAAGCATCCCGCCTGGGGAGTACGGCCGCAAGGCTAAACTCAAAGAAATTGACTGGGGGCCCGCACAATGCGGCGGAGCCATGCGGGATTAATTTCGAATGGCAACGGCGTAGATTCTTACCCTGGGACTTTAGACCATGCTTCCCCGGACGGATCGGTAGTACA The sequence was uploaded to the BLAST tool (http: / / www.ncbi.nlm.nih.gov / BLAST) of the National Center for Biotechnology Information (NCBI) website, aligned with known sequences in the database, and analyzed for similarity to determine the species classification information of the strain. The isolated strain is animal bifidobacterium, named animal bifidobacterium lactis subsp (Bifidobacterium animalis lactis subsp) Bifidobacteria animalis subsp .lactiss ) S8, with the accession number GDMCC NO. 66518.

[0030] Example 2: Determination of the biofilm formation ability of S8.

[0031] 200 μL of the activated bacterial solution of three generations was inoculated into 10 mL of MRS medium, thoroughly mixed by beating, and then 200 μL of the bacterial solution was added to each well of a 96-well plate and incubated at 37°C for 24 hours. After incubation, the plate was washed three times with 200 μL of PBS buffer per well to remove unattached planktonic bacteria. After washing, the plate was naturally dried, then 200 μL of 0.1% crystal violet solution was added to each well, and the biofilm was stained at room temperature for 30 minutes to fully color the biofilm. After staining, the plate was washed three times with 200 μL of PBS buffer per well to remove excess stain and dried at room temperature. Finally, 200 μL of 95% ethanol was added to each well to dissolve the crystal violet, and the absorbance was measured at 600 nm on a microplate reader to quantitatively evaluate the biofilm formation ability. In the experimental setup, the biofilm formation ability of Streptococcus mutans (Streptococcus ATCC25175) under the same conditions was used as a positive control group, and MRS medium was used as a blank control group. Each experimental group had three parallel wells to ensure data reliability. Among them, B. longum Q4 and B. longum M5 are strains isolated from different infant feces in the same batch, B. longum S8 represents the strain S8 of the present application. The evaluation criteria for biofilm formation ability are as follows: when OD600 nm≤1, the bacteria do not form biofilm; when 1<OD600 nm<3, the bifidobacterium forms weak biofilm; and when OD600 nm≥3, the bifidobacterium forms strong biofilm. The results are shown in Table 1 and Figure 3 Table 2.

[0032] Table 1 Biofilm intensity determination of animal bifidobacterium lactis subsp S8

[0033] Example 3: Determination of the ability of S8 to resist gastric acid.

[0034] 3 mg pepsin was dissolved in 1 mL of 0.5% pH = 2.5 sterilized saline solution to prepare simulated gastric acid solution. The test strain activated for three generations was inoculated in MRS liquid medium and incubated at 37°C for 18 hours to ensure that the strain was in the logarithmic growth phase. After the incubation, the bacterial solution was centrifuged at 10000 rpm / min for 10 minutes, and the supernatant was discarded to collect the bacterial precipitate. The collected bacterial precipitate was resuspended in phosphate buffer and simulated gastric acid solution, respectively, and incubated at 37°C for 2 hours. After the incubation, the bacterial solution was centrifuged again at 10000 rpm / min for 5 minutes, and the supernatant was discarded to collect the bacterial precipitate. To observe the recovery ability of the strain after the action of gastric acid, the bacterial precipitate was resuspended in MRS liquid medium and incubated at 37°C for 24 to 48 hours, and the growth of the bacterial precipitate was recorded. The bacterial solution resuspended in phosphate buffer and simulated gastric acid was gradient-diluted, respectively, and plated on MRS solid medium, and the number of colonies was counted after incubation at 37°C for 24 to 48 hours. The results are shown in Table 2 and Figure 1

[0035] Table 2 Survival rate of probiotic bacteria in simulated gastric acid environment 1 Biofilm strength determination of animal bifidobacterium S8

[0036] Example 4: Determination of bile salt tolerance of S8 3 mg of porcine bile salt was dissolved in 1 mL of pH 8.0 phosphate buffer to prepare a simulated bile salt solution. The test strain activated for three generations was inoculated in MRS liquid medium and incubated at 37°C for 18 hours to ensure that the strain was in the logarithmic growth phase. After the incubation, the bacterial solution was centrifuged at 10000 rpm / min for 10 minutes, and the supernatant was discarded to collect the bacterial precipitate. The collected bacterial precipitate was resuspended in phosphate buffer and simulated bile salt solution, respectively, and incubated at 37°C for 2 hours. After the incubation, the bacterial solution was centrifuged again at 10000 rpm / min for 5 minutes, and the supernatant was discarded to collect the bacterial precipitate. To observe the recovery ability of the strain after the action of bile salt, the bacterial precipitate was resuspended in MRS liquid medium and incubated at 37°C for 24 to 48 hours, and the growth of the bacterial precipitate was recorded. The bacterial solution resuspended in phosphate buffer and simulated bile salt was gradient-diluted, respectively, and plated on MRS solid medium, and the number of colonies was counted after incubation at 37°C for 24 to 48 hours. The results are shown in Table 3 and Figure 2

[0037] Table 3 Survival rate of probiotic bacteria in simulated bile salt environment

[0038] Example 5 Effect of S8 on liver function of mice ​​

[0039] The frozen bacteria were taken out from the ultra-low temperature freezer at -80°C, inoculated into the corresponding medium at a ratio of 2%, and continuously activated for three generations to ensure the viability of the strain. Then, the bacteria in the logarithmic phase after amplification were centrifuged at 7000 rpm / min for 5 minutes, and the supernatant was discarded. PBS was added for repeated washing, and the mixture was centrifuged again at the same speed for 5 minutes to remove impurities. Finally, the bacterial precipitate at the bottom was collected. Before the animal experiment, 1 mL of PBS was mixed with the bacterial precipitate for intragastrical administration.

[0040] SPF C57BL / 6J male mice weighing 19-22 g were used, and the experimental period was ten weeks. The first two weeks were the adaptation period of the mice, mainly for the adaptation of the animals to the new environment and the stabilization of the physiological state. From the third week, the probiotic intervention experiment was formally carried out. During the experiment, the blank group mice were fed with standard normal feed throughout the experiment, while the control group and probiotic intervention group mice were fed with high cholesterol feed. High cholesterol feed formula: 88% basic feed, 2% cholesterol, 10% lard.

[0041] The mice were placed on the electronic balance smoothly, ensuring that their limbs fully contacted the weighing platform. After the electronic balance reading stabilized, the mouse weight was recorded (accurate to 0.1 g). The mouse weight was measured regularly every four days, and the measurement time was consistent each time. In the final stage of the experiment, all mice underwent 12 h of fasting treatment. They were fixed on the operating table to collect blood samples by orbital blood collection. The blood was quickly transferred to a sterile blood collection tube and left to stand for 30 min. The blood collection tube after standing was placed in a pre-cooled centrifuge at 4°C, and centrifuged at 1400 g for 15 min. After centrifugation, the clear serum was carefully aspirated using a pipette and transferred to a labeled 1.5 mL centrifuge tube, which was stored in a -80°C freezer. When taken out, it was slowly thawed in a 4°C refrigerator, and the serum sample was gently mixed. The sample was placed in an automatic biochemical analyzer, and the measurement indexes were AST and ALT. The results are shown in Tables 4 and Figure 4

[0042] Table 4 Effect of probiotics on mouse liver function

[0043] ​As potential effective substances for lowering blood lipid, probiotics must survive and colonize in the host body before they can play their role. Oral administration is the most common way of probiotic administration. Before the strains exert their lipid-lowering effect in the intestine, they need to pass through the severe test of the strong acid environment in the stomach and the high concentration of bile salts in the intestine. As the first barrier of the digestive tract, gastric acid can inactivate most acid-intolerant microorganisms; while bile salts further screen the surviving strains by damaging the bacterial cell membrane structure. This means that the acid and bile salt tolerance of probiotics directly determines whether they can successfully pass through the upper digestive tract to reach the intestinal colonization site, and thus affect the exertion of their lipid-lowering effect. These two indicators are key parameters for screening functional probiotics. The three strains of animal bifidobacterium S8, animal bifidobacterium Q4 and animal bifidobacterium M5, which have good bile salt tolerance and gastric acid resistance, may be suitable as candidate strains for oral probiotics, as they can maintain high activity in the gastric acid environment and the high bile salt environment in the small intestine, thereby enhancing their colonization ability in the intestine.

[0044] As can be seen from Table 1 and Table 2, S8 not only performs excellently in the bile salt resistance test, but also shows a high survival rate in the previous gastric acid experiment, indicating that these strains have double resistance and can effectively resist the double stress of gastric acid and bile salt, so as to be more likely to survive and colonize in the intestinal tract after oral administration and exert a potential lipid-lowering effect. As can be seen from Table 1, S8 shows the strongest biofilm formation capacity (OD600nm=0.51), which is three times higher than that of the positive control (OD600nm=0.176), indicating that the strain has excellent adhesion and colonization capacity and can form a stable microecological structure in the intestinal tract, so as to more durably exert a lipid-lowering effect. Aspartate aminotransferase (AST) is a widely existing enzyme in cells, mainly distributed in the liver, myocardium, skeletal muscle, kidney and red blood cells. AST is a key enzyme in amino acid metabolism, which catalyzes the transamination reaction between aspartic acid and alpha-ketoglutarate to generate oxaloacetic acid and glutamic acid. When the liver cells are damaged, AST will enter the blood circulation of the human body due to the change of cell membrane permeability, causing the increase of related enzyme activity in the serum. In the metabolic disorder induced by high cholesterol diet, the liver as the core organ of lipid metabolism is easily damaged by lipid deposition, oxidative stress and inflammatory response, leading to liver cell damage and AST release. In this experiment, the AST level can be determined to evaluate the damage degree of high cholesterol diet to the liver and other organs, and to provide an important basis for the intervention effect of probiotics. Alanine aminotransferase (ALT) is a liver-specific enzyme mainly involved in the process of amino acid metabolism. Unlike AST, ALT has higher specificity, and the increase of its serum level usually means liver cell damage. In the metabolic disorder induced by high cholesterol diet, liver lipid deposition and oxidative stress can cause liver cell damage, and ALT will be released into the blood, leading to the increase of its serum level. By determining the ALT level, the damage degree of high cholesterol diet to the liver can be evaluated, and an important basis for the intervention effect of probiotics is provided. The AST and ALT levels of the control group increase due to the liver damage caused by high-fat diet. As can be seen from Table 4, the ALT and AST levels of S8 are significantly lower than those of the control group, which indicates that S8 can have a certain effect on reducing the AST and ALT levels or improving liver damage.

[0045] The application provides a high-biofilm-producing probiotic strain and a preparation method thereof, and the application also provides a thought and a method for the preparation method. There are many methods and approaches for specifically realizing the technical scheme, and the above description is only a preferred embodiment of the application. It should be noted that those skilled in the art can make some improvements and refinements without departing from the principle of the application, and these improvements and refinements should also be regarded as the protection scope of the application. The components not explicitly described in the embodiment can be realized by using the prior art.

Claims

1. A probiotic strain with high biofilm production, characterized in that, The strain was classified and named Bifidobacterium animalis subsp. Lactobacillus ( Bifidobacteria animalis subsp .lactiss The strain number is S8, the accession number is GDMCCNO. 66518, and the accession date is June 28, 2025.

2. The method for culturing the probiotic strain according to claim 1, characterized in that, Amplification culture was performed in MRS culture.

3. A probiotic preparation, characterized in that, It contains the probiotic strain described in claim 1.

4. The probiotic agent according to claim 3, characterized in that, The probiotic agent is in the form of a liquid agent or a freeze-dried powder.

5. The probiotic agent according to claim 3, characterized in that, The probiotic strain is in the form of a liquid bacterial agent, which contains live bacteria or cultured bacterial solution of the strain.

6. The method for preparing the probiotic agent according to any one of claims 3-5, wherein the bacterial strain is inoculated into the culture medium at an inoculation ratio of 2%-5%, and activated for three consecutive generations; the bacterial solution in the logarithmic phase after amplification is centrifuged at 7000-8000 rpm / min for 5-10 minutes; the supernatant is discarded, and PBS buffer is added for repeated washing; the solution is centrifuged again at the same speed for 3-8 minutes to remove impurities; finally, the bacterial precipitate at the bottom is collected and then resuspended in the buffer.

7. The application of the probiotic strain of claim 1 or the probiotic agent of claim 3 in the preparation of lipid-lowering products.

8. The use of the probiotic strain of claim 1 or the probiotic agent of claim 3 in the preparation of products for the prevention and / or treatment of liver dysfunction.

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