Bifidobacterium bifidum postbiotics with enhanced intestinal barrier function, preparation method and application thereof

Abstract

The present invention provides a Bifidobacterium bifidum postbiotic with enhanced intestinal barrier function, its preparation method and application. The preparation method of the postbiotic includes: centrifuging to obtain viable Bifidobacterium bifidum FL-228.1 from the fermentation broth of Bifidobacterium bifidum FL-228.1, and performing optimal heat treatment on the viable bacteria to obtain inactivated Bifidobacterium bifidum FL-228.1 postbiotic; the conditions of the optimal heat treatment are: the temperature is 60-80 °C, and the time is 15-25 min. The present invention inactivates Bifidobacterium bifidum FL-228.1 by using a specific combination of temperature and time, and the obtained Bifidobacterium bifidum postbiotic has the best effect of enhancing intestinal barrier function, and can be used for preparing drugs or health foods for preventing intestinal barrier damage and enhancing intestinal barrier function.

Description

Technical Field

[0001] The present invention relates to the fields of microbial technology and pharmaceutical technology, and in particular to postbiotics of Bifidobacterium bifidum with enhanced intestinal barrier function, and a preparation method and application thereof. Background Art

[0002] Bifidobacterium bifidum is a widely concerned probiotic. Due to its strong adaptability to human gastric acid and bile salts and its good utilization ability of host-derived mucin, it can play a better beneficial role during the use as a probiotic.

[0003] A large amount of evidence shows that Bifidobacterium bifidum or its inactivated cells can relieve multiple gastrointestinal diseases and achieve the effect of improving the gastrointestinal health of the host. For example: The patent document with the publication number CN106834187B mentions that Bifidobacterium bifidum CGMCC NO.13632 significantly improves the pathological indexes of constipation in mice and has the effect of preventing and treating constipation. The patent application document with the publication number CN107629988A discloses that Bifidobacterium bifidum GDMCC No.60255 significantly reduces the inflammatory response in the colorectal region of mice with colorectal cancer models and reduces the number of tumors. Bifidobacterium bifidum BGN4 can inhibit oxidative stress, reduce epithelial barrier damage and relieve disease symptoms in mice with ulcerative colitis (Lee et al. Journal of medicinal food, 2022, 25(2): 146-157.). In addition, the inactivated Bifidobacterium bifidum strain SYN-HI-001 can significantly improve the gastrointestinal symptoms of irritable bowel syndrome (publication number CN112312920A); Heat-inactivated Bifidobacterium bifidum B1628 alleviates intestinal injury and effectively improves DSS-induced colitis in mice by regulating the intestinal flora, regulating the amino acid metabolic pathway, promoting the secretion of anti-inflammatory cytokines and inhibiting pro-inflammatory mediators (Cuijiao F, et al. Nutrients, 2022, 14(24): 5233-5233.).

[0004] The intestinal barrier is composed of a single layer of epithelial cells, a mucus layer, intercellular junctions, the intestinal microbiota, immune cells in the lamina propria, and some physical and chemical components. The mucus layer contains mucin (MUC), which is mainly produced by goblet cells. The presence of the mucus layer can prevent intestinal microorganisms from directly contacting intestinal epithelial cells and causing a strong immune response. In addition, the presence of the mucus layer can also contribute to the colonization of probiotics in the intestine. The integrity of tight junctions is closely related to intestinal permeability. An increase in intestinal permeability will lead to an increased probability of harmful substances such as pathogens and pathogenic bacteria in the intestine entering the blood, which will trigger a systemic inflammatory response. The occurrence of various diseases is accompanied by damage to the intestinal barrier, and at the same time, the deepening of the degree of intestinal barrier damage also accelerates the progression of the disease. Therefore, preventing intestinal barrier damage is as important as enhancing intestinal barrier function. Summary of the Invention

[0005] The present invention provides a Bifidobacterium bifidum postbiotic with enhanced intestinal barrier function, its preparation method and application. This Bifidobacterium bifidum postbiotic has the effect of enhancing intestinal barrier function and can be used to prepare drugs or health foods for preventing intestinal barrier damage and enhancing intestinal barrier function.

[0006] The specific technical solutions are as follows:

[0007] The present invention provides a preparation method of a Bifidobacterium bifidum postbiotic, including: centrifuging to obtain viable Bifidobacterium bifidum FL-228.1 from the fermentation broth of Bifidobacterium bifidum FL-228.1, and performing optimal heat treatment on the viable bacteria to obtain inactivated Bifidobacterium bifidum FL-228.1 postbiotic;

[0008] The Bifidobacterium bifidum FL-228.1 was deposited at the China Center for Type Culture Collection on January 29, 2024, with the deposit number CCTCC NO: M2024274; the conditions for the optimal heat treatment are: the temperature is 60-80 °C, and the time is 15-25 min.

[0009] The present invention found through experiments that: inactivated Bifidobacterium bifidum FL-228.1 can up-regulate the expression of mucin MUC2 in LS174T cells under steady state; the expression level of MUC2 in the inactivated Bifidobacterium bifidum FL-228.1 group is 1.40 times that of the normal control group. Inactivated Bifidobacterium bifidum FL-228.1 can significantly up-regulate the expression levels of genes related to tight junction proteins Muc2, Claudin-1, Claudin-3, Occludin, and ZO-1 in LS174T cells.

[0010] Furthermore, the preparation method of the fermentation broth is as follows:

[0011] (1) Inoculate Bifidobacterium bifidum FL-228.1 into a fermentation medium, and place it under strict anaerobic conditions at 37°C for expanded culture to obtain a strain seed solution;

[0012] (2) Inoculate the strain seed solution into a fermentation medium, and ferment it under strict anaerobic conditions at 37°C to obtain a fermentation broth.

[0013] Furthermore, in the fermentation broth, the viable count of Bifidobacterium bifidum FL-228.1 is 1×10 8 CFU / mL to 5×10 8 CFU / mL.

[0014] Furthermore, the conditions for the optimal heat treatment are: the temperature is 60°C; the time is 15 min.

[0015] The present invention also provides postbiotics of Bifidobacterium bifidum prepared by the above-mentioned preparation method.

[0016] The present invention also provides the application of the postbiotics of Bifidobacterium bifidum in the preparation of products for enhancing intestinal barrier function.

[0017] Furthermore, the products include drugs or health foods.

[0018] Furthermore, the manifestations of enhancing intestinal barrier function are: up-regulating the relative expression levels of tight junction protein genes such as Claudin-1, Claudin-3, Occludin, and / or ZO-1; or, up-regulating the relative expression level and production of mucin MUC2 gene.

[0019] Compared with the prior art, the present invention has the following beneficial effects:

[0020] The present invention inactivates Bifidobacterium bifidum FL-228.1 using a specific combination of temperature and time, and the obtained postbiotics of Bifidobacterium bifidum have the best effect of enhancing intestinal barrier function, and can be used to prepare drugs or health foods for preventing intestinal barrier damage and enhancing intestinal barrier function. Description of the Drawings

[0021] Figure 1 It is a result diagram of the effects of different postbiotics of Bifidobacterium on the expression of MUC2 and tight junction proteins in LS174T cells in Example 2.

[0022] Figure 2Results of the effects of different postbiotics preparation methods on the expression and secretion of MUC2 in LS174T cells in Example 3.

[0023] Figure 3 Results of the effects of Bifidobacterium bifidum FL-228.1 at different heat-inactivated temperatures on the expression of MUC2 and tight junction proteins in LS174T cells in Example 4.

[0024] Figure 4 Results of the effects of Bifidobacterium bifidum FL-228.1 at different heat-inactivated times on the expression of MUC2 and tight junction proteins in LS174T cells in Example 5. Detailed implementation manners

[0025] The present invention will be further described below in conjunction with specific embodiments. The following are only specific embodiments of the present invention, but the protection scope of the present invention is not limited thereto.

[0026] Tryptone, yeast extract powder, glucose, and beef extract powder involved in the following embodiments are purchased from Sinopharm Group; RT-qPCR primers involved in the following embodiments are purchased from Shanghai Sangon Biotech Co., Ltd.; reverse transcription kits and Green Realtime PCR Master Mix are purchased from Toyobo Biochemical Co., Ltd. in Japan; ELISA kits involved in the following embodiments are purchased from Shanghai Jianglai Biotech Co., Ltd.; LS174T cells involved are purchased from Wuhan Procell Life Science & Technology Co., Ltd. MEM cell culture medium and trypsin involved are purchased from Wuhan Procell Life Science & Technology Co., Ltd.; penicillin-streptomycin solution and trypsin involved are purchased from Shanghai Beyotime Biotechnology Co., Ltd.; fetal bovine serum involved is purchased from Biological Industries.

[0027] The media involved in the following embodiments are as follows: Fermentation medium (g / L): 10 g peptone, 5 g liver extract powder, 3 g beef extract powder, 5 g yeast extract powder, 8 g tryptone, 0.5 g soluble starch, 1 g sodium chloride, 1 g dipotassium hydrogen phosphate, 1 g potassium dihydrogen phosphate, 10 g glucose, 0.01 g FeSO 4 ·7H 2 O, 0.005 g MnSO 4 、0.5 g L-cysteine, 1.25 g MgSO 4 ·7H 2 O, 1 mL Tween 80, distilled water: 1000 mL; pH: 7.1 - 7.3; sterilized at 116 °C for 30 min.

[0028] Bifidobacterium bifidum FL-228.1 involved in the following examples was deposited at the China Center for Type Culture Collection on January 29, 2024. The deposit address is No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province, and the deposit number is CCTCC NO: M2024274. It is classified as Bifidobacterium bifidum.

[0029] Lactobacillus casei K11 was deposited at the China Center for Type Culture Collection on December 2, 2020, with the deposit number CCTCC NO: M2020823

[0030] Lactobacillus rhamnosus MN45: Deposited in the Functional Dairy and Probiotics Engineering Laboratory, isolated from infant feces.

[0031] The method of the qRT-PCR experiment involved in the following examples is as follows: LS174T cells were cultured in MEM medium supplemented with 10% (v / v) heat-inactivated fetal bovine serum (FBS) and 1% (v / v) penicillin-streptomycin solution, and the culture conditions were 37°C and 5% CO 2 . To measure the expression of MUC2, cells were seeded in a 6-well cell culture plate (Greiner, Shanghai, China) at a density of 1×10 6 cells / well until the cells reached 70-80% confluence, and then the experimental treatment was started. After 24 h of cell intervention, LS174T cells were rinsed with pre-cooled PBS, and then the RNA of the cells was extracted according to the steps of the RNA extraction kit (R1200-100, Solarbio, Beijing, China), and reverse transcribed into cDNA using the ReverTra qPCR RT Master Mix with gDNA Remover kit (FSQ-301, Toyobo, Japan). qPCR analysis was performed on a BioRad-CFX96 Touch instrument (Bio-Rad, California, USA) using Green Realtime PCR Master Mix (QPK-201, Toyobo, Japan). The expression level of the target gene was normalized to the expression level of β-actin in the same sample, and the data were analyzed by the 2 -ΔΔct method. * represents a significant difference compared with the Control group in different groups, P<0.05; ** represents a significant difference compared with the Control group in different groups, P<0.01; *** represents a significant difference compared with the Control group in different groups, P<0.001.

[0032] The method of ELISA experiment involved in the following examples is as follows: LS174T cells were cultured in MEM medium supplemented with 10% (v / v) heat-inactivated fetal bovine serum (FBS) and 1% (v / v) penicillin-streptomycin solution, and the culture conditions were 37°C and 5% CO 2 . To determine the expression of MUC2, cells were seeded in a 6-well cell culture plate (Greiner, Shanghai, China) at a density of 1×10 6 cells / well until the cells reached 70-80% confluence, and then the experimental treatment was started. After 24 hours of cell intervention, the supernatant was centrifuged at 1000×g for 20 minutes, and the content of mucin MUC2 in the cells was detected according to the instructions of the human mucin 2 (MUC2) enzyme-linked immunosorbent assay kit.

[0033] Example 1 Preparation of Bifidobacterium bifidum postbiotics

[0034] This example provides a kind of Bifidobacterium bifidum postbiotics, and its preparation method is as follows:

[0035] (1) Inoculate Bifidobacterium bifidum FL-228.1 into 100 mL of fermentation medium at an inoculum volume fraction of 5%, and place it under strict anaerobic conditions at 37°C for 48 hours of expanded culture to obtain a strain seed solution with a viable cell count of 5×10 8 CFU / mL;

[0036] (2) Inoculate the strain seed solution into the fermentation medium at an inoculum volume fraction of 10%, and ferment it under strict anaerobic conditions at 37°C for 30 hours to obtain a fermentation broth;

[0037] (3) At 4°C, centrifuge the fermentation broth at 6000 rpm for 10 minutes, discard the supernatant, wash the cells with PBS buffer, repeat 3 times, and inactivate them by high heat treatment (95°C, water bath for 30 minutes);

[0038] (4) Then use the method of plate coating to detect whether there are still viable bacteria. After coating and culturing, when there is no growth of viable bacteria, resuspend it in MEM medium without antibiotics and adjust the bacterial concentration to 5×10 7 CFU / mL to obtain Bifidobacterium bifidum postbiotics.

[0039] Example 2 Effects of different Bifidobacterium postbiotics on the expression of MUC2 in LS174T cells

[0040] Bifidobacterium bifidum FL-228.1, Lactobacillus casei K11, and Lactobacillus rhamnosus MN45, which have the potential to enhance intestinal barrier and maintain intestinal homeostasis, were separately taken and different bifidobacterial postbiotics were prepared by the method described in Example 1, namely, Bifidobacterium bifidum FL-228.1 postbiotic, Lactobacillus casei K11 postbiotic, and Lactobacillus rhamnosus MN45 postbiotic were obtained.

[0041] Using the qRT-PCR experimental method, the effects of different postbiotics on the expression and secretion of MUC2 in LS174T cells were determined.

[0042] The following groups were set up:

[0043] Bifidobacterium bifidum FL-228.1 treatment group: Bifidobacterium bifidum FL-228.1 (228) or Bifidobacterium bifidum FL-228.1 postbiotic (H-228) was added to MEM medium to adjust the concentration to 5×10 7 CFU / mL;

[0044] Lactobacillus casei K11 treatment group: Lactobacillus casei K11 (K11) or Lactobacillus casei K11 postbiotic (H-K11) was added to MEM medium to adjust the concentration to 5×10 7 CFU / mL;

[0045] Lactobacillus rhamnosus MN45 treatment group: Lactobacillus rhamnosus MN45 (MN45) or Lactobacillus rhamnosus MN45 postbiotic (H-MN45) was added to MEM medium to adjust the concentration to 5×10 7 CFU / mL.

[0046] Normal control group (Control): Only MEM medium was used without adding other substances.

[0047] Experimental results: As can be seen from Figure 1 and Table 1, Bifidobacterium bifidum FL-228.1, Lactobacillus casei K11, and Lactobacillus rhamnosus MN45 can all up-regulate the expression levels of Muc2, claudin-1, claudin-3, occludin, and zo-1 genes; among the postbiotics of the three experimental strains, only the Bifidobacterium bifidum FL-228.1 postbiotic can specifically up-regulate the expression levels of Muc2, claudin-1, claudin-3, occludin, and zo-1 genes, which were up-regulated by 1.87-fold, 1.39-fold, 2.29-fold, 1.51-fold, and 1.57-fold respectively compared with the Control group. The Lactobacillus casei K11 postbiotic and the Lactobacillus rhamnosus MN45 postbiotic had no significant effect on the expression levels of Muc2, claudin-1, claudin-3, occludin, and zo-1 genes.

[0048] Table 1 Effects of postbiotics from different strains on the expression of MUC2 and tight junction proteins in LS174T cells

[0049]

[0050]

[0051] Note: Data with different letters in the same column are significantly different (P<0.05).

[0052] Example 3 Effects of different postbiotic preparation methods on the expression and secretion of MUC2 in LS174T cells

[0053] In this example, different inactivation methods were used to prepare postbiotics, and the specific content is as follows:

[0054] Conventional heat inactivation: Inoculate probiotics into a fermentation medium, culture at 37°C, monitor its fermentation growth status during the culture process, stop fermentation when the growth rate significantly decreases, obtain the probiotic culture solution, immediately cool it to room temperature, adjust the pH of the probiotic culture solution to 6.5, wash it three times with sterile PBS, centrifuge, collect the bacterial sludge, and inactivate it by high heat treatment (95°C water bath for 30 min). After detecting bacteria on a BS agar plate, it is proved that there are no viable bacteria.

[0055] Ultrasonic inactivation method: Inoculate probiotics into a fermentation medium, culture at 37°C, monitor its fermentation growth status during the culture process, stop fermentation when the growth rate significantly decreases, obtain the probiotic culture solution, immediately cool it to room temperature, adjust the pH of the probiotic culture solution to 6.5, wash it three times with sterile PBS, centrifuge, collect the bacterial sludge, sonicate at 40 kHz for 30 min, and after detecting bacteria on a BS agar plate, it is proved that there are no viable bacteria.

[0056] Enzymatic inactivation method: Inoculate probiotics into a fermentation medium, culture at 37°C, monitor its fermentation growth status during the culture process, stop fermentation when the growth rate significantly decreases, obtain the probiotic culture solution, immediately cool it to room temperature, adjust the pH of the probiotic culture solution to 6.5, wash it twice with sterile PBS, add 500 μL of RIPA lysis buffer (containing 1 mM PMSF) to 5 mL of the bacterial suspension, lyse on ice for 10 min, centrifuge at 12000g for 3 minutes, and take the supernatant.

[0057] Use qRT-PCR experiments and ELISA experiments to determine the effects of postbiotics obtained by different inactivation methods on the expression and secretion of MUC2 in LS174T cells.

[0058] Set the following groups:

[0059] Live bacteria group (228): Add normal live Bifidobacterium bifidum FL-228.1 to MEM medium and adjust the concentration to 5×10 7 CFU / mL;

[0060] Common heat-inactivated group (H-228): Add postbiotics of Bifidobacterium bifidum FL-228.1 after common heat-inactivated treatment to MEM medium and adjust the concentration to 5×10 7 CFU / mL;

[0061] Ultrasonic-inactivated group (C-228): Add postbiotics of Bifidobacterium bifidum FL-228.1 after ultrasonic-inactivated treatment to MEM medium and adjust the concentration to 5×10 7 CFU / mL;

[0062] Enzymatic-hydrolysis-inactivated group (M-228): Add postbiotics of Bifidobacterium bifidum FL-228.1 after enzymatic-hydrolysis-inactivated treatment to MEM medium and adjust the concentration to 5×10 7 CFU / mL.

[0063] Normal control group (Control): Only use MEM medium without adding other substances.

[0064] Experimental results: As can be seen from Figure 2 Table 2, postbiotics of Bifidobacterium bifidum FL-228.1 and its postbiotics after different inactivation methods can up-regulate the expression level of Muc2 gene, which are up-regulated by 1.69-fold, 1.58-fold and 1.15-fold respectively compared with the Control group. Through ELISA experiment, compared with the Control, it was found that the secretion amounts of MUC2 by Bifidobacterium bifidum FL-228.1, heat-inactivated group and ultrasonic-inactivated group were up-regulated from the original 46.23 ng / mL to 54.97, 51.72, 50.71 ng / mL respectively, and the common heat-inactivated group had the best effect.

[0065] Table 2 Effects of different postbiotic preparation methods on the expression and secretion of MUC2 in LS174T cells

[0066]

[0067] Note: Data with different letters in the same column are significantly different (P<0.05)

[0068] Example 4 Effects of Bifidobacterium bifidum FL-228.1 heat-inactivated at different temperatures on the expression of MUC2 and tight junction proteins in LS174T cells

[0069] In this example, different inactivated temperatures were used to treat Bifidobacterium bifidum FL-228.1 to obtain different postbiotics, and the effects of the postbiotics obtained at different inactivated temperatures on the expression and secretion of MUC2 in LS174T cells were detected by qRT-PCR experiments.

[0070] Preparation of postbiotics: Bifidobacterium bifidum FL-228.1 was inoculated into 100 mL of fermentation medium at an inoculum volume fraction of 5%, and cultured under strict anaerobic conditions at 37 °C for 48 h to obtain a strain seed solution with a viable cell count of 5×10 8 CFU / mL; the seed solution was inoculated into the fermentation medium at an inoculum amount of 10%, and fermented under strict anaerobic conditions at 37 °C for 30 h to obtain a fermentation broth; at 4 °C, the fermentation broth was centrifuged at 6000 rpm for 10 min, the supernatant was discarded, and the cells were washed with PBS buffer solution three times. Inactivation was carried out by heat treatment at different temperatures (60 °C, 80 °C, 100 °C, 120 °C) for 30 min, and then the plate coating method was used to detect whether there were still viable bacteria. After plating and culturing, when there was no viable bacteria growth, it was resuspended in MEM medium without antibiotics to adjust the bacterial concentration to 5×10 7 CFU / mL to obtain postbiotics.

[0071] Set the following groups:

[0072] Live bacteria group (228): Add normal live Bifidobacterium bifidum FL-228.1 to MEM medium to adjust the concentration to 5×10 7 CFU / mL;

[0073] 60 °C heat-inactivated group (228-60): Add the postbiotics of Bifidobacterium bifidum FL-228.1 heat-inactivated at 60 °C to MEM medium to adjust the concentration to 5×10 7 CFU / mL;

[0074] 80 °C inactivated group (228-80): Add the postbiotics of Bifidobacterium bifidum FL-228.1 heat-inactivated at 80 °C to MEM medium to adjust the concentration to 5×10 7 CFU / mL;

[0075] 100 °C inactivated group (228-100): Add the postbiotics of Bifidobacterium bifidum FL-228.1 heat-inactivated at 100 °C to MEM medium to adjust the concentration to 5×10 7 CFU / mL;

[0076] 120 °C inactivated group (228-120): Add the postbiotics of Bifidobacterium bifidum FL-228.1 heat-inactivated at 120 °C (to MEM medium to adjust the concentration to 5×10 7 CFU / mL;

[0077] Normal control group (Control): Only MEM medium was used without adding other substances.

[0078] Experimental results: As can be seen from Figure 3 and Table 3, Bifidobacterium bifidum FL-228.1 and its postbiotics after heat inactivation at 60 °C, 80 °C, and 100 °C can all up-regulate the expression level of the Muc2 gene, which are up-regulated by 2.31-fold, 2.17-fold, 1.47-fold, and 1.233-fold respectively compared with the Control group; while the postbiotics after heat inactivation at 120 °C have no significant effect on the expression level of the Muc2 gene.

[0079] For the genes related to tight junction proteins, Bifidobacterium bifidum FL-228.1 and its postbiotics after heat inactivation at 60 °C, 80 °C, and 100 °C can all up-regulate the expression levels of the three types of tight junction protein genes, namely claudin-1, claudin-3, occludin, and zo-1. Among them, the postbiotics after heat inactivation at 60 °C have the most significant effect, and the expressions of claudin-1, claudin-3, occludin, and zo-1 genes are up-regulated by 1.40-fold, 1.85-fold, 2.16-fold, and 2.53-fold respectively; the postbiotics after heat inactivation at 120 °C have no significant effect on the expression levels of the genes related to tight junction proteins.

[0080] Table 3 Effects of Bifidobacterium bifidum FL-228.1 with different heat inactivation temperatures on the expression of MUC2 and tight junction proteins in LS174T cells

[0081]

[0082] Note: Data with different letters in the same column are significantly different (P < 0.05)

[0083] Example 5 Effects of Bifidobacterium bifidum FL-228.1 heat-inactivated at 60 °C for different times on the expression of MUC2 and tight junction proteins in LS174T cells

[0084] In this example, Bifidobacterium bifidum FL-228.1 was treated with different inactivation times to obtain different postbiotics, and the effects of the postbiotics obtained at different inactivation times on the expression and secretion of MUC2 in LS174T cells were detected by qRT-PCR experiments.

[0085] Preparation of postbiotics: Bifidobacterium bifidum FL-228.1 was inoculated into 100 mL of fermentation medium at an inoculum volume fraction of 5%, and cultured under strict anaerobic conditions at 37 °C for 48 h to obtain a strain seed solution with a viable cell count of 5×10 8CFU / mL; The seed liquid was inoculated into the fermentation medium at an inoculation amount of 10%, and fermented at 37°C under strict anaerobic conditions for 30 h to obtain a fermentation broth. At 4°C, the fermentation broth was centrifuged at 6000 rpm for 10 min, and the supernatant was discarded. The cells were washed with PBS buffer three times, and inactivated by heat treatment at 60°C for different times (15 min, 20 min, 25 min, 30 min). Then, the plate coating method was used to detect the presence of viable bacteria. After coating and culturing, when no viable bacteria grew, it was resuspended in MEM medium without antibiotics to adjust the bacterial concentration to 5×10 7 CFU / mL to obtain postbiotics.

[0086] The following groups were set up:

[0087] Viable bacteria group (228): Normal Bifidobacterium bifidum FL-228.1 viable bacteria were added to MEM medium to adjust the concentration to 5×10 7 CFU / mL;

[0088] 15-minute inactivation group (228-15): Postbiotics of Bifidobacterium bifidum FL-228.1 after 15-minute heat inactivation were added to MEM medium to adjust the concentration to 5×10 7 CFU / mL;

[0089] 20-minute inactivation group (228-20): Postbiotics of Bifidobacterium bifidum FL-228.1 after 20-minute heat inactivation were added to MEM medium to adjust the concentration to 5×10 7 CFU / mL;

[0090] 25-minute inactivation group (228-25): Postbiotics of Bifidobacterium bifidum FL-228.1 after 25-minute heat inactivation were added to MEM medium to adjust the concentration to 5×10 7 CFU / mL;

[0091] 30-minute inactivation group (228-30): Postbiotics of Bifidobacterium bifidum FL-228.1 after 30-minute heat inactivation were added to MEM medium to adjust the concentration to 5×10 7 CFU / mL;

[0092] Normal control group (Control): Only MEM medium was used without adding other substances.

[0093] Experimental results: From Figure 4As shown in Table 4, Bifidobacterium bifidum FL-228.1 and its postbiotics after heat inactivation at 60°C for 15 min, 20 min, 25 min, and 30 min can upregulate the expression levels of Muc2, claudin-1, claudin-3, occludin, and zo-1 genes.

[0094] Table 4 Effects of Bifidobacterium bifidum FL-228.1 with different heat inactivation times on the expression of MUC2 and tight junction proteins in LS174T cells

[0095]

[0096]

[0097] Note: Data with different letters in the same column are significantly different (P<0.05).

Claims

1. An application of a Bifidobacterium bifidum postbiotic in the preparation of a drug for enhancing intestinal barrier function, characterized in that: The preparation method of Bifidobacterium bifidum postbiotics comprises: obtaining live bacteria of Bifidobacterium bifidum FL-228.1 from a fermentation liquid of Bifidobacterium bifidum FL-228.1 by centrifugation, performing optimal heat treatment on the live bacteria, and obtaining inactivated Bifidobacterium bifidum FL-228.1 postbiotics; The Bifidobacterium bifidum FL-228.1 was deposited in the China Center for Type Culture Collection on January 29, 2024, with a deposit number of CCTCC NO: M2024274; the optimal heat treatment conditions are: temperature of 60-80°C, and time of 15-25min.

2. The use according to claim 1, characterized in that The preparation method of the fermentation liquid is: (1) inoculating Bifidobacterium bifidum FL-228.1 into a fermentation medium, placing it under strict anaerobic conditions at 37° C., and expanding the culture to obtain a strain seed solution; (2) The strain seed liquid is inoculated into a fermentation medium and fermented under strictly anaerobic conditions at 37° C. to obtain a fermentation liquid.

3. The use according to claim 1, characterized in that In the fermentation broth, the number of viable bacteria of Bifidobacterium bifidum FL-228.1 was 1×10 8 CFU / mL~5×10 8 CFU / mL.

4. The use according to claim 1, characterized in that The optimal heat treatment conditions are: temperature of 60°C and time of 15 minutes.

5. The use according to claim 1, characterized in that The enhancement of intestinal barrier function is manifested by: up-regulating the relative expression level of Claudin-1, Claudin-3, Occludin and / or ZO-1 tight junction protein genes; or, 2) up-regulating the relative expression level and production of the mucin Muc2 gene.

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