Lactobacillus taiwanensis with enhanced intestinal barrier function and application thereof

Abstract

The application discloses a lactobacillus taiwanensis with enhanced intestinal barrier function and application thereof, and belongs to the technical field of microorganisms and medicine. The lactobacillus taiwanensis is preserved in the Guangdong Microbial Culture Collection Center, and the preservation number is GDMCC No.63714. The intestinal barrier enhancement and protection of the lactobacillus taiwanensis CCFM1318 are embodied in the following aspects: (1) the lactobacillus taiwanensis CCFM1318 has good in-vitro gastrointestinal tolerance, strong adhesion to intestinal epithelial cells and strong colonization in the gastrointestinal tract; (2) the lactobacillus taiwanensis CCFM1318 significantly up-regulates the expression level of tight junction protein related genes in Caco-2 cells after DSS stimulation; (3) the bacterial-free culture medium supernatant of the lactobacillus taiwanensis CCFM1318 can promote the expression and secretion of MUC2 of the LS 174T goblet cell line, and can improve the expression of MUC2 after endoplasmic reticulum stress, thereby enhancing and protecting the intestinal barrier. The lactobacillus taiwanensis CCFM1318 has great application prospect in products (such as food or medicine) for enhancing and protecting the intestinal barrier.

Description

Technical Field

[0001] This invention relates to a probiotic Lactobacillus taiwanensis strain that enhances the intestinal barrier function and its application, belonging to the fields of microbial technology and pharmaceutical technology. Background Technology

[0002] The intestine is not only a vital organ for the digestion and absorption of nutrients, but also an innate barrier for maintaining the body's internal homeostasis. Intestinal mucus is the first physical line of defense for intestinal health, preventing antigens, toxins, and bacteria from directly contacting epithelial cells, and playing a crucial role in maintaining the dynamic homeostasis of the intestinal environment. Intestinal mucins are mainly synthesized and secreted by goblet cells and have gel-like properties. Mucin 2 (MUC2) is a major component of the mucus layer's framework. Tight junction proteins are another important class of proteins maintaining the intestinal barrier. The most common tight junction proteins in intestinal epithelial cells include four types: zonal occludens (ZOs), ocludin, claudins, and junctional adhesion molecules (JAMs). Intestinal mucus and epithelial tight junctions work together to establish a highly unified mucosal barrier system, collectively restricting the contact of luminal contents with intestinal epithelial cells. Numerous animal experiments and clinical studies have found that damage to the intestinal barrier leads to increased intestinal permeability, increasing the translocation of bacterial antigens, thereby allowing various harmful substances and pathogens to enter the bloodstream, causing the development of a variety of metabolic diseases. Common diseases associated with intestinal barrier dysfunction include gastrointestinal diseases, celiac disease, food allergies, and some extraintestinal diseases such as obesity, osteoporosis, alcoholic liver disease, and fatty liver.

[0003] *Lactobacillus taiwanensis* is a Gram-positive, catalase-negative, non-motile facultative anaerobic bacterium. It was first isolated and identified from silage and is most closely related to *Lactobacillus gasseri* and *Lactobacillus johnsonii*. *Lactobacillus taiwanensis* possesses potential probiotic properties; administering 2×10⁻⁶ *Lactobacillus taiwanensis* to Balb / c mice... 8CFU / mL of *Lactobacillus taiwanensis* in water significantly upregulated the number of Treg cells in the mouse gut (published in Reynolds LA, Smith KA, Filbey KJ, Harcus Y, Hewitson JP, Redpath SA, Valdez Y, Yebra MJ, Finlay BB, Maizels RM. Commensal-pathogen interactions in the intestinal tract: lactobacilli promote infection with, and are promoted by, heminthparasites. Gut Microbes. 2014; 5(4):522-32.). Existing technology discloses that in an acute enteritis mouse model, intervention with *Lactobacillus taiwanensis* CLG01 can improve intestinal immune regulation pathways by promoting the secretion of intestinal immunoglobulin A and downregulating the expression of the inflammatory factor type II interferon (IFN-γ), thereby maintaining intestinal immune homeostasis (published in Li Xiaoyu. Whole genome sequencing and probiotic characteristics of *Lactobacillus taiwanensis* CLG01 [D]. Shandong University 2021.). In addition to improving intestinal immune dysregulation, the intestinal mucus layer and intestinal mechanical barrier also play a crucial role in maintaining the integrity of the intestinal barrier. Currently, there is no Lactobacillus taiwanensis that can improve the intestinal mucus layer and intestinal mechanical barrier. Summary of the Invention

[0004] [Technical Issues]

[0005] The technical problem to be solved by the present invention is the lack of *Lactobacillus taiwanensis* in the prior art that can enhance the intestinal barrier.

[0006] [Technical Solution]

[0007] To address the aforementioned technical problems, this invention aims to provide a probiotic *Lactobacillus taiwanensis* (L. taiwanensis) strain CCFM1318 that enhances the intestinal barrier. This strain exhibits good intestinal tolerance in vitro, strong adhesion to intestinal epithelial cells, and strong colonization within the intestine. Furthermore, it significantly upregulates the expression levels of Claudin-1, Claudin-3, Occludin, and ZO-1 tight junction protein-related genes in Caco-2 cells after DSS stimulation, thus protecting intestinal barrier function. The supernatant of this strain can alleviate endoplasmic reticulum stress induced by tunicamycin and upregulate the expression of mucin MUC2 in LS174T cells under homeostatic conditions.

[0008] This invention provides a strain of Lactobacillus taiwanensis CCFM1318, which is deposited at the Guangdong Provincial Center for Microbial Culture Collection with accession number GDMCC No. 63714 and deposit date of August 4, 2023.

[0009] The Lactobacillus taiwanensis CCFM1318 was derived from fecal samples of healthy individuals. Sequencing analysis revealed that the 16S rDNA sequence of this strain is shown in SEQ ID NO.1. The sequenced sequence was compared with the nucleic acid sequence in NCBI's BLAST database, and the results showed that the strain was Lactobacillus taiwanensis, and it was named Lactobacillus taiwanensis CCFM1318.

[0010] The Lactobacillus taiwanensis CCFM1318 has the following biological characteristics:

[0011] (a) Growth characteristics: Colonies on MRS solid medium are translucent with irregular edges.

[0012] (b) It exhibits good in vitro gastrointestinal tolerance, strong adhesion to intestinal epithelial cells, and strong colonization within the gastrointestinal tract.

[0013] (c) Significantly upregulated the expression levels of Claudin-1, Claudin-3, Occludin and ZO-1 tight junction protein-related genes in Caco-2 cells after DSS stimulation, protecting intestinal barrier function.

[0014] (d) Bacterial supernatant can improve endoplasmic reticulum stress induced by tunicamycin and upregulate the expression of mucin MUC2 in LS174T cells under homeostasis.

[0015] The present invention also provides a microbial preparation containing the above-mentioned Lactobacillus taiwanensis CCFM1318 or its fermentation broth.

[0016] In one embodiment of the present invention, the microbial preparation contains the above-mentioned Lactobacillus taiwanensis CCFM1318 or its lyophilized powder.

[0017] In one embodiment of the present invention, the viable count of *Lactobacillus taiwanensis* in the microbial preparation is not less than 10. 8 CFU / mL or 10 8 CFU / g.

[0018] The present invention also provides a product containing the above-mentioned Lactobacillus taiwanensis CCFM1318 or the above-mentioned microbial preparation, the product including food, medicine or health product.

[0019] In one embodiment of the present invention, the viable count of *Lactobacillus taiwanensis* in the product is not less than 10. 8 CFU / mL or 10 8 CFU / g.

[0020] In one embodiment of the present invention, the food includes fermented food or health food.

[0021] In one embodiment of the present invention, the fermented food includes solid food, liquid food, and semi-solid food.

[0022] In one embodiment of the present invention, the food is a dairy product, soy product, or fruit and vegetable product produced using a fermentation agent containing the aforementioned Lactobacillus taiwanensis CCFM1318, or the food is a beverage or snack containing the aforementioned Lactobacillus taiwanensis CCFM1318.

[0023] In one embodiment of the present invention, the method for preparing the fermenting agent is as follows:

[0024] (1) Culture Lactobacillus taiwanensis CCFM1318 and collect bacterial cells;

[0025] (2) Freeze-dry the bacterial cells described in step (1) to obtain a starter culture.

[0026] In one embodiment of the present invention, a freeze-drying protectant is added to the freeze-drying process, the freeze-drying protectant comprising (100-160) g / L of skim milk powder, preferably 130 g / L.

[0027] In one embodiment of the present invention, the freeze-drying protectant has a mass ratio of protectant to bacterial cells of (1-4):1, preferably 2:1.

[0028] In one embodiment of the present invention, the culture medium comprises: peptone accounting for (1-5)% of the total mass of the culture medium, yeast extract accounting for (0.5-2)% of the total mass of the culture medium, and glucose accounting for (1-4)% of the total mass of the culture medium, with the remainder made up with water. Preferably, the culture medium comprises: peptone accounting for 2% of the total mass of the culture medium, yeast extract accounting for 1% of the total mass of the culture medium, and glucose accounting for 2% of the total mass of the culture medium, with the remainder made up with water.

[0029] In one embodiment of the present invention, the drug is a drug carrier and / or pharmaceutical excipients containing the aforementioned Lactobacillus taiwanensis CCFM1318.

[0030] In one embodiment of the present invention, the drug carrier comprises one or more of microcapsules, microspheres, nanoparticles, and liposomes.

[0031] In one embodiment of the present invention, the pharmaceutical excipient comprises excipients and / or additives.

[0032] In one embodiment of the present invention, the excipient comprises one or more of the following: binder, filler, disintegrant, and lubricant.

[0033] In one embodiment of the present invention, the additive comprises one or more of a solubilizer, co-solvent, latent solvent, and preservative.

[0034] In one embodiment of the present invention, the dosage form of the medicine includes powder, granules, capsules, tablets, pills, or oral liquid.

[0035] The present invention also provides the use of the above-mentioned Lactobacillus taiwanensis CCFM1318 or the above-mentioned microbial preparation or the above-mentioned product in enhancing and / or protecting the intestinal barrier without the purpose of diagnosing and treating diseases.

[0036] In one embodiment of the present invention, the enhancement and protection of the intestinal barrier includes at least one of the following functions:

[0037] (a) Upregulate the expression levels of Claudin-1, Claudin-3, Occludin and / or ZO-1 tight junction protein genes;

[0038] (b) Upregulates the expression of mucin MUC2.

[0039] Beneficial effects:

[0040] This invention screened out a strain of Lactobacillus taiwanense CCFM1318 with good in vitro probiotic properties, which has a probiotic effect of enhancing the intestinal barrier, specifically manifested in:

[0041] 1. The supernatant of *Lactobacillus taiwanensis* CCFM1318 bacteria upregulated the expression of mucin MUC2 in LS174T cells under homeostatic conditions. The expression level of MUC2 in the *Lactobacillus taiwanensis* CCFM1318 group was 1.40 times that of the normal control group. There was no significant difference in the expression level of MUC2 between the blank culture medium control group, the *Lactobacillus johnsonii* FGSYC6MS group, and the *Lactobacillus johnsonii* FAHBZ615 group and the normal control group.

[0042] 2. Lactobacillus Taiwan CCFM1318 showed good in vitro gastrointestinal tolerance, with a 3-hour survival rate of 96.28% in simulated gastric juice and a 4-hour survival rate of 44.12% in simulated intestinal juice; it also showed strong adhesion to intestinal epithelial cells, with an adhesion rate of 8.95% to HT-29 cells.

[0043] 3. Lactobacillus taiwanensis CCFM1318 significantly upregulated the expression levels of Claudin-1, Claudin-3, Occludin, and ZO-1 tight junction protein-related genes in Caco-2 cells after DSS stimulation: The relative mRNA expression levels of Claudin-1, Claudin-3, Occludin, and ZO-1 in the Lactobacillus taiwanensis CCFM1318 group were upregulated by 1.91-fold, 2.76-fold, 2.64-fold, and 2.28-fold, respectively, compared with the model group.

[0044] Preservation of biological materials

[0045] A strain of Lactobacillus taiwanensis (CCFM1318), taxonomically named Lactobacillus taiwanensis, was deposited on August 4, 2023, at the Guangdong Provincial Center for Microbial Culture Collection (GDMCCNO: 63714), located at 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, Guangdong Academy of Sciences, Institute of Microbiology. Attached Figure Description

[0046] Figure 1 The morphology of Lactobacillus taiwanensis CCFM1318, including:

[0047] A. Colony morphology;

[0048] B cell morphology;

[0049] C growth curve

[0050] Figure 2 Effects of Lactobacillus taiwanensis CCFM1318 on MUC2 transcription in LS174T cells.

[0051] Figure 3 Adhesion of Lactobacillus taiwanensis CCFM1318 to HT-29 cells

[0052] Figure 4 The effect of Lactobacillus taiwanensis CCFM1318 on the expression level of tight junction protein in Caco-2 cells after DSS stimulation, including:

[0053] Relative expression level of AClaudin-1 mRNA;

[0054] B. Relative expression level of Claudin-3 mRNA;

[0055] Relative expression level of COccludin mRNA;

[0056] Relative expression level of D ZO-1 mRNA;

[0057] Figure 5 Effects of Lactobacillus taiwanensis CCFM1318 on MUC2 secretion in LS174T cells Detailed Implementation

[0058] The tryptone, yeast extract, glucose, and agar powder used in the following examples were purchased from Sinopharm Group; the dextran sulfate sodium (DSS) used in the following examples was purchased from MP Company, USA; the RT-qPCR primers used in the following examples were purchased from Shanghai Sangon Biotech Co., Ltd.; the reverse transcription kit and RNA extraction kit used in the following examples were purchased from Nanjing Novizan Biotech Co., Ltd.; and the Anti-MUC2 antibody (ab90007) and goat anti-rabbit IgG H&L (Alexa) used in the following examples were purchased from Nanjing Novizan Biotech Co., Ltd. 488)(ab150077), purchased from Abcam.

[0059] The Caco-2 cells and LS174T cells used in the following examples were purchased from the Shanghai Cell Bank, Chinese Academy of Sciences. The DMEM cell culture medium, fetal bovine serum, and trypsin used in the following examples were purchased from Gibco, Inc. (USA).

[0060] The culture media involved in the following examples are as follows:

[0061] MRS solid medium (g / L): 10g peptone, 10g beef extract, 5g yeast extract, 20g glucose, 5g sodium acetate, 1mL Tween 80, 2g dipotassium hydrogen phosphate, 2g diammonium citrate, 0.1g magnesium sulfate heptahydrate, 0.05g manganese sulfate monohydrate, distilled water: 1000mL; pH: 6.2-6.4; agar 20g / L; sterilize at 115℃ for 20min.

[0062] MRS liquid culture medium (g / L): 10g peptone, 10g beef extract, 5g yeast extract, 20g glucose, 5g sodium acetate, 1mL Tween 80, 2g dipotassium hydrogen phosphate, 2g diammonium citrate, 0.1g magnesium sulfate heptahydrate, 0.05g manganese sulfate monohydrate, distilled water: 1000mL; pH: 6.2-6.4; sterilize at 115℃ for 20min.

[0063] The preparation method of the Lactobacillus taiwani suspension involved in the following examples is as follows:

[0064] Streaks were applied to MRS solid medium and cultured at 37°C for 48 hours to obtain single colonies. Single colonies were picked and inoculated into MRS liquid medium and cultured at 37°C for 13-18 hours for activation. This activation process was repeated for two generations to obtain the activated solution. The activated solution was then inoculated into MRS liquid medium at a 4% (v / v) inoculation rate and cultured at 37°C for 13-18 hours to obtain the bacterial suspension. The bacterial suspension was centrifuged at 5000g for 10 minutes at 4°C to obtain *Lactobacillus taiwanensis* cells. The *Lactobacillus taiwanensis* cells were washed with physiological saline and resuspended in a 200g / L glycerol solution until the bacterial concentration reached 1×10⁻⁶. 8 The bacterial suspension was obtained by dispersing CFU / mL and stored at -80℃ for later use.

[0065] Example 1: Screening, isolation and identification of Lactobacillus taiwanense strain CCFM1318

[0066] The strain involved in this invention is a lactobacillus screened from the feces of healthy humans, and identified as *Lactobacillus taiwanensis* based on molecular biological identification of its 16S rDNA gene. Figure 1 These are colony and cell morphology diagrams and growth curves of the Lactobacillus Taiwan CCFM1318 strain in embodiments of the present invention.

[0067] The specific steps are as follows:

[0068] (1) Diluting and coating

[0069] Aseptically, 1 mL of sample (stool suspension from a healthy person) was added to 9 mL of sterile water to form a 1:10 homogeneous dilution. This dilution process was repeated to obtain 10... -2 10 -3 10 -4 10 -5 and 10 -6 Diluent. Using a sterile pipette tip, pipette 100 μL of the above 10... -4 10 -5 and 10 -6 The dilution solution was spread onto MRS solid medium using a spreader and incubated at 37°C for 48 hours to obtain dilution plates.

[0070] (2) Purification culture

[0071] Diluted platings with colony counts between 30 and 300 were prepared. For each sample, 10-20 single colonies of varying sizes (milky or white) were randomly selected and streaked onto MRS solid medium using a three-region streak method. The plates were then incubated at 37°C for 48 hours to obtain single colonies. Colony morphology was observed and recorded, and pure colonies were obtained and preserved.

[0072] Single colonies from the above streak plates were inoculated into MRS liquid medium and incubated at 37°C for 12 hours to obtain secondary purified culture solutions.

[0073] (3) Preservation and identification of strains

[0074] Mix the secondary purified culture medium well, take the bacterial cells (incubated at 37℃ for 13h) into 2mL clean bacterial culture preservation tubes, make 5 parallel aliquots, add 1mL of bacterial culture to 4 of them, centrifuge and discard the supernatant, take the bacterial cells, resuspend them with 1mL of 30% glycerol, and put them in a -80℃ freezer.

[0075] Add 50 μL of bacterial lysis buffer to a sterilized 1.5 mL EP tube. After picking up a single colony with an inoculation loop and placing it into the EP tube, heat it at 98 °C for 10 min in a metal water bath. Then add 100 μL of sterile water (twice the volume) for use as a PCR template.

[0076] The 16S rDNA PCR system is as follows:

[0077] Table 1. PCR reaction system for bacterial identification (40 μL)

[0078]

[0079] Primers: ① 16S universal primers (27F; 1492R)

[0080] PCR program: 94℃ pre-denaturation for 5 min; 94℃ denaturation for 2 min; 55℃ annealing for 30 s; 72℃ extension for 1 min; 30 cycles, 72℃ extension for 10 min, 12℃ incubation until removed.

[0081] Sequencing and Analysis: After confirmation by nucleic acid electrophoresis, the PCR products were sent to Shanghai Sangon Biotech for sequencing. The obtained spliced ​​sequence SEQ ID NO.1 was then used for species confirmation via NCBI BLAST (https: / / blast.ncbi.nlm.nih.gov / Blast.cgi). The BLAST results showed that the isolated strain was *Lactobacillus taiwanensis*, and it was named *Lactobacillus taiwanensis* CCFM1318.

[0082] The colonies of *Lactobacillus taiwanensis* CCFM1318 on MRS solid medium were translucent with irregular edges. Cold field emission scanning electron microscopy showed that CCFM1318 cells were rod-shaped of varying lengths. *Lactobacillus taiwanensis* CCFM1318 reached its growth plateau phase at 13-14 hours. Figure 1 As shown.

[0083] The bacteria obtained during the same screening were Lactobacillus johnsonii FGSYC6MS and FAHBZ615.

[0084] Example 2: Effects of Lactobacillus taiwanensis CCFM1318 culture supernatant on MUC2 expression and secretion in LS174T cells

[0085] Numerous studies have confirmed that probiotic metabolites contain a large number of bioactive molecules, such as peptidoglycans, extracellular polysaccharides, secretory proteins, bacteriocins, and short-chain fatty acids, which mediate antibacterial, anti-inflammatory, immunomodulatory, antitumor, and barrier protective effects on the host. Probiotic metabolites are an important class of metabiotics; compared to probiotics, their effects are independent of bacterial cell activity, exhibiting better stability and higher safety. In cell experiments, the effects of sterile culture supernatants from *Lactobacillus taiwanensis* CCFM1318, *Lactobacillus johnsonii* FGSYC6MS, and FAHBZ615 on MUC2 expression and secretion in LS174T cells were determined using qPCR and immunofluorescence staining.

[0086] I. Experimental Procedure

[0087] Preparation of sterile culture medium supernatant: *Lactobacillus taiwanensis* CCFM1318 was cultured in MRS medium at 37°C for 13-18 h. After culture, the culture was centrifuged at 5000×g at 4°C for 10 min. The supernatant was collected, and the pH was adjusted to 7. The culture medium was then filtered through a 0.22 μm sterile syringe filter and aliquoted into sterile centrifuge tubes to obtain the sterile culture medium supernatant. It was stored at -80°C. The same method was used to prepare sterile culture medium supernatants for *Lactobacillus johnsonii* FGSYC6MS and FAHBZ615.

[0088] qPCR experiment: LS174T cells were cultured in DMEM medium supplemented with 10% (v / v) heat-inactivated fetal bovine serum (FBS) and 1% (v / v) penicillin-streptomycin solution at 37°C and 5% CO2. To determine muc2 expression, cells were cultured at 7 × 10⁻⁶ cells / well in 6-well cell culture plates (Greiner, Shanghai, China). 5Cells were seeded at a rate of 1 cell / well until 85% confluence was achieved, at which point experimental treatments began. The groups were as follows: the experimental treatment groups were cultured in FBS-free and antibiotic-free DMEM medium supplemented with 20% supernatant of *Lactobacillus taiwanensis* CCFM1318, *Lactobacillus johnsonii* FGSYC6MS, and FAHBZ615; the blank control group was cultured in 20% sterile MRS medium (pH=7); and the normal control group received no treatment. After 24 hours of cell culture, LS174T cells were washed with ice-cold PBS, and RNA was extracted from the cells according to the instructions of the RNA extraction kit (RC112-01, Vazyme, Nanjing, China). RNA was then reverse transcribed into cDNA using a qPCR kit (R333, Vazyme, Nanjing). qPCR analysis was performed on a BioRad-CFX384 instrument (Bio-Rad, California, USA) using a SYBR Green Supermix (Q711-02, Vazyme, Nanjing). The expression levels of the target gene were normalized to the expression levels of GAPDH in the same sample, and the data were analyzed using a 23... -ΔΔct The method is used for analysis.

[0089] Immunofluorescence staining experiment: LS174T cells were cultured and seeded in laser confocal culture dishes (Beyotime, Shanghai, China). After the cells reached 85% confluence, the experimental group was treated with sterile culture supernatant of Lactobacillus Taiwan CCFM1318, the modeling group was treated with 2 μM endoplasmic reticulum (ER) stress inducer tunicamycin (TM) (#HY-A0098, MCE, Shanghai, China), and the modeling plus CCFM1318 group was treated with sterile culture supernatant of CCFM1318 + TM. Untreated cells served as the normal control group. After 12 h, the cell samples were fixed with Carnoy's solution, then washed three times with ice-cold PBS, permeabilized with 0.1% Triton X-100 / PBS, and washed again with PBS. Non-specific binding of antibodies was blocked with 1% bovine serum albumin. MUC2 antibody (Abcam, China, ab90007) was added and incubated overnight at 4°C in a humidified incubator. Unbound antibodies were washed away with PBS containing 0.1% Tween-2, and then fluorescently labeled secondary antibody (Alexa Fluor 488nm) was added and incubated at room temperature for 1 hour. Cells were then rinsed with PBS and stained with 4',6-diamidinyl-2-phenylindole (DAPI) for 1 min. Finally, imaging was performed using a laser confocal microscope (Zeiss LSM710, Germany) at a resolution of 1024 × 1024 and a magnification of 20x.

[0090] II. Experimental Results:

[0091] like Figure 2As shown, compared with the normal control group, the supernatant of sterile culture medium of Lactobacillus Taiwan CCFM1318 significantly upregulated the expression of MUC2 at the transcriptional level to 1.40 times that of the normal control group, while there was no significant change in MUC2 expression in the blank culture medium control group, Lactobacillus johnsonii FGSYC6MS group and Lactobacillus johnsonii FAHBZ615 group. Figure 5 Immunofluorescence staining results showed that the supernatant of sterile culture medium containing *Lactobacillus taiwanensis* CCFM1318 could alleviate the reduction in MUC2 secretion caused by TM. Compared with untreated cells, the supernatant of sterile culture medium containing *Lactobacillus taiwanensis* CCFM1318 could directly stimulate MUC2 production.

[0092] Example 3: Gastrointestinal tolerance of Lactobacillus taiwanense strain CCFM1318

[0093] Lactobacillus taiwanensis CCFM1318 exhibits high survival rate in simulated human gastrointestinal fluid and high adhesion to human intestinal epithelial cells.

[0094] (1) Gastrointestinal tolerance test:

[0095] The low pH value in the host's gastric juice and the high bile salt content and various enzymes in the intestinal juice are important natural barriers affecting the activity of probiotics after entering the gastrointestinal tract. Probiotics must cross the gastrointestinal environment to enter the intestine and exert their effects on the host. Therefore, the first thing to explore is the tolerance of probiotics to gastrointestinal juice.

[0096] I. Experimental Procedure

[0097] Pepsin (1:10000) was dissolved in sterile PBS buffer (pH adjusted to 3.0 with hydrochloric acid) to a final concentration of 3 g / L, which is the simulated gastric juice. Trypsin (1:250) was dissolved in sterile PBS buffer (pH adjusted to 8.0 with NaOH) to a final concentration of 1 g / L, and bile salts were added to a final concentration of 0.3%, which is the simulated intestinal juice. Both simulated gastrointestinal juices were filtered through a 0.22 μm filter membrane before use.

[0098] 2% (v / v) *Lactobacillus taiwanensis* CCFM1318 was inoculated into sterile MRS liquid medium and cultured at 37°C for 13 h. After two generations of activation, it was used in experiments. The cultured *Lactobacillus taiwanensis* fermentation broth was centrifuged at 5000×g for 10 min, and the bacterial pellet was collected. The pellet was washed three times with PBS buffer (pH = 7.4) and resuspended in fresh PBS buffer to prepare the original bacterial culture. After serial dilution, the initial viable count (CFU / mL) was determined using the pour plate method. 500 μL of the original culture was resuspended in 4.5 mL of simulated gastric fluid and cultured at 37°C for 3 h. Viable counts were then performed. Another 500 μL of the cultured culture from the simulated gastric fluid was transferred to simulated intestinal fluid and cultured at 37°C for 4 h. Viable counts were then performed again. The survival rate of the yeast strain was calculated using the following formula:

[0099] Strain survival rate (%) = [log N1 / log N0] × 100, where: N1 refers to the number of viable bacteria (CFU / mL) of the strain after treatment with simulated gastric or intestinal fluid; N0 refers to the initial number of viable bacteria (CFU / mL) of the strain.

[0100] II. Experimental Results

[0101] The results are shown in Table 2. Lactobacillus taiwanense CCFM1318 showed strong gastric juice tolerance, with a survival rate of 96.28%; the survival rate of Lactobacillus taiwanense was 44.12%.

[0102] Table 2. Survival rate and adhesion rate of Lactobacillus taiwanensis CCFM1318 to HT-29 cells in simulated gastrointestinal fluid.

[0103]

[0104] (2) Cell adhesion experiment:

[0105] Exploring the ability of probiotics to colonize the gut by adhering to intestinal epithelial cells is an important criterion for selecting probiotic strains. The human colon cancer cell line HT-29 is considered a good model for elucidating the mechanisms of host-microbe interactions.

[0106] I. Experimental Procedure

[0107] HT-29 cells were cultured in DMEM complete medium containing 10% (v / v) heat-inactivated fetal bovine serum and 1% penicillin-dextrose antibiotics at 37°C and 5% CO2. After the cells adhered and grew into a monolayer, they were digested with trypsin, resuspended in DMEM complete medium, and cell concentration was determined using a hemocytometer. The cell concentration was then adjusted to 102. 5 Cells / mL were inoculated into 12-well cell culture plates and cultured until they grew into a monolayer.

[0108] Lactobacillus Taiwani CCFM1318 was cultured on MRS medium at 37°C for 13-18 hours. After centrifugation at 5000×g at 4°C for 10 minutes, the bacterial pellet was collected, washed three times with PBS buffer, and then the bacterial suspension concentration was adjusted to approximately 10% using DMEM blank medium. 9 CFU / mL was added to the bacterial suspension, and CFDA-SE stock solution was added to bring the final concentration to 20 μmol / L. The suspension was incubated at 37°C in the dark for 15 min, then centrifuged at 5000×g, 4°C, for 10 min. The suspension was then washed three times with PBS to remove excess fluorescent dye. Finally, 500 μL of the bacterial suspension was resuspended in 500 μL of basal DMEM medium.

[0109] HT-29 cells were washed twice with sterile PBS buffer to remove unattached cells. Then, 10% PBS buffer was added to each well. 8 Lactic acid bacteria (CFU / mL) were added to each well (i.e., 100 μL of bacterial culture and 900 μL of basic DMEM were added to each well); the 12-well plate was transferred to a 5% CO2 incubator and cultured at 37°C for 4 h; the monolayer of cells in each well of the 12-well plate was washed with PBS solution at least 3 times to remove non-adhered lactic acid bacteria and metabolic secretions; then 250 μL of 0.25% trypsin-EDTA was added to each well and incubated for 10 min; then 750 μL of basic DMEM was added to terminate digestion; the cells were pipetted off, and 200 μL was added to a 96-well plate, and the fluorescence intensity was measured using a multi-functional microplate reader (excitation wavelength 488 nm) as V1; another 20 μL of the original stained bacterial culture and 180 μL of basic DMEM were measured to measure the fluorescence intensity as V2. The adhesion rate of Lactobacillus Taiwanis CCFM1318 was calculated according to the following formula:

[0110]

[0111] Fluorescent staining imaging: After 4 hours of incubation and 3 washes with PBS, 1 mL of methanol was added to each well of a 12-well plate. After fixation for 10 minutes, the methanol was removed, and the plate was air-dried before fluorescence imaging. The results are shown below. Figure 3 As shown.

[0112] II. Experimental Results

[0113] The adhesion results of Lactobacillus taiwanensis strain CCFM1318 to HT-29 cells are shown in Table 2 and Figure 3 As shown, the adhesion rate reached 8.95%, indicating that Lactobacillus taiwanensis CCFM1318 has strong colonization ability in the intestine.

[0114] Example 4: DSS (sodium dextran sulfate) induced Caco-2 cell experiment:

[0115] The DSS-induced Caco-2 cell model is closely related to the early inflammatory response in colitis. Stimulation of Caco-2 cells with a certain concentration of DSS causes cell damage. Damage to tight junctions in Caco-2 cells leads to intestinal barrier injury and increased intestinal permeability, accompanied by other inflammatory damage. Therefore, this model is often used as an in vitro colitis model to explore its anti-inflammatory activity.

[0116] I. Experimental Procedure

[0117] Caco-2 cells were cultured in DMEM complete medium containing 10% (v / v) heat-inactivated fetal bovine serum and 1% (v / v) penicillin-streptomycin solution in an incubator at 37°C and 5% CO2. Once the cell layering rate reached 85%, the cells were digested with trypsin containing 0.02% EDTA, resuspended in DMEM complete medium, and cell concentration was determined using a hemocytometer. The cell concentration was then adjusted to 2 × 10⁶ cells / mL. 5 Cells / mL were inoculated into 6-well cell culture plates and cultured until a monolayer of cells was formed. *Lactobacillus taiwanense* CCFM1318 was cultured in MRS medium at 37°C for 13 h. After centrifugation at 5000×g for 10 min, the bacterial pellet was collected, washed three times with PBS buffer, and then the bacterial suspension concentration was adjusted to approximately 10% using DMEM blank medium. 9 CFU / mL. After washing the cells that had grown into a monolayer twice with DMEM blank medium, the normal control group was added with DMEM blank medium, the model group was added with DSS medium (DMEM containing 3% (w / v) DSS), and the experimental treatment group (Lactobacillus taiwanensis CCFM1318 group) was added with 100 μL of Lactobacillus taiwanensis CCFM1318 bacterial suspension per well. The cells were co-incubated for 12 h. After incubation, the cells were washed twice with PBS. Total RNA was extracted from the cells using an RNA extraction kit, and the extracted RNA was reverse transcribed using an RNA reverse transcription kit. The primer template for RT-qPCR was the cDNA obtained after reverse transcription, and the internal reference gene was β-actin. Table 3 shows the sequences of the relevant primers. The relative changes in the target gene were analyzed by 2... -△△CT It was calculated using the method.

[0118] Table 3 Primer design for RT-qPCR

[0119]

[0120]

[0121] II. Experimental Results

[0122] Depend on Figure 4It was found that DSS stimulation significantly downregulated the expression levels of three tight junction protein-related genes (claudin-1, occludin, and zo-1) in Caco-2 cells, while *Lactobacillus taiwanensis* CCFM1318 significantly upregulated the expression levels of these three tight junction protein-related genes and enhanced the expression of claudin-3. Compared with the model group, the expression of claudin-1, occludin, zo-1, and claudin-3 genes in the *Lactobacillus taiwanensis* CCFM1318 group was upregulated by 1.91-fold, 2.76-fold, 2.64-fold, and 2.28-fold, respectively.

[0123] The above results indicate that Lactobacillus taiwanensis CCFM1318 can protect the intestinal barrier by enhancing the expression of claudin-1, occludin, zo-1 and claudin-3 genes.

[0124] Example 5: Application of Lactobacillus taiwanensis CCFM1318 in the preparation of capsules

[0125] Lactobacillus Taiwani CCFM1318 can be used to prepare capsule products. The specific preparation process for capsule products is as follows:

[0126] Lactobacillus Taiwani CCFM1318 was streaked onto MRS solid medium and cultured at 37°C for 48 h to obtain single colonies. Single colonies were picked and inoculated into MRS liquid medium and cultured at 37°C for 18 h for activation. This activation was repeated for two generations to obtain an activated solution. The activated solution was inoculated into the medium at a rate of 2% (v / v) and cultured at 37°C for 18 h to obtain a bacterial suspension. The bacterial suspension was centrifuged at 6000 rpm for 10 min to obtain bacterial sludge. The bacterial sludge was washed three times with physiological saline and then resuspended in a cryoprotectant to a concentration of 1×10⁻⁶. 10 CFU / mL was used to obtain a bacterial suspension; the bacterial suspension was added to a 30 g / L sodium alginate solution to a concentration of 2 × 10⁻⁶. 9 After reaching CFU / mL, the solution was stirred thoroughly to ensure that the Lactobacillus taiwanense CCFM1318 cells were evenly dispersed in the sodium alginate solution, resulting in a mixture. The mixture was then extruded into a 20 g / L calcium chloride solution to form granules. After the granules were allowed to stand and solidify for 30 minutes, they were filtered and collected. The collected granules were then freeze-dried for 48 hours to obtain a powder. The powder was then filled into pharmaceutical capsules to obtain the capsule product.

[0127] The culture medium is prepared by dissolving 10% enzyme hydrolyzed fat-free emulsion, 0.5% glucose, 1.5% tryptone and 0.3% yeast extract in water at 87.7% of the total weight of the culture medium, and then adjusting the pH to 6.8 to obtain the culture medium.

[0128] Example 6: Application of Lactobacillus taiwanensis in tablet preparation

[0129] Lactobacillus taiwanensis CCFM1318 can be used to prepare tablets. The specific tablet preparation process is as follows:

[0130] Lactobacillus taiwanense CCFM1318 was streaked onto MRS solid medium and cultured at 37°C for 48 h to obtain single colonies. Single colonies were picked and inoculated into MRS liquid medium and cultured at 37°C for 13 h for activation. This activation was repeated for two generations to obtain an activated solution. The activated solution was inoculated into the medium at a rate of 2% (v / v) and cultured at 37°C for 13 h to obtain a bacterial suspension. The bacterial suspension was centrifuged at 5000g for 10 min to obtain bacterial sludge. The bacterial sludge was washed three times with physiological saline and then resuspended in a cryoprotectant to a concentration of 1×10⁻⁶. 10 CFU / mL was used to obtain a bacterial suspension; the bacterial suspension was pre-cultured at 37℃ for 60 min and then freeze-dried to obtain Lactobacillus taiwanense CCFM1318 bacterial powder.

[0131] The culture medium is prepared by dissolving 10% enzyme hydrolysate, 0.5% glucose, 1.5% tryptone and 0.3% yeast extract in water at 87.7% of the total weight of the culture medium, and then adjusting the pH to 6.8 to obtain the culture medium.

[0132] The protective agent contains 130g / L of skim milk powder.

[0133] Weigh out 25.7 parts by weight of Lactobacillus Taiwan CCFM1318 bacterial powder, 55.0 parts by weight of starch, 4.5 parts by weight of cellulose derivative, 12.0 parts by weight of sodium carboxymethyl starch, 0.8 parts by weight of talc, 1.0 part by weight of sucrose, and 1.0 part by weight of water to obtain raw materials; mix the raw materials to obtain wet granules; compress the wet granules into tablets using a tableting machine from Zhongnan Pharmaceutical Machinery Factory, and then dry them using a small pharmaceutical dryer from Qingzhou Yikang Pharmaceutical Machinery Co., Ltd. to obtain tablets.

[0134] Example 7: Application of Lactobacillus taiwanensis in the preparation of fermented milk

[0135] Lactobacillus Taiwani CCFM1318 can be used to prepare fermented milk. The specific preparation process for fermented milk is as follows:

[0136] Lactobacillus taiwanense CCFM1318 was streaked onto MRS solid medium and cultured at 37°C for 48 h to obtain single colonies. Single colonies were picked and inoculated into MRS liquid medium and cultured at 37°C for 13 h for activation. This activation was repeated for two generations to obtain an activated solution. The activated solution was inoculated into the medium at a rate of 2% (v / v) and cultured at 37°C for 13 h to obtain a bacterial suspension. The bacterial suspension was centrifuged at 5000g for 10 min to obtain bacterial sludge. The bacterial sludge was washed three times with physiological saline and then resuspended in a cryoprotectant to a concentration of 1×10⁻⁶. 10 CFU / mL was used to obtain a bacterial suspension; the bacterial suspension was pre-cultured at 37℃ for 60 min and then freeze-dried to obtain Lactobacillus taiwanense CCFM1318 bacterial powder.

[0137] The culture medium is prepared by dissolving 10% enzyme hydrolysate, 0.5% glucose, 1.5% tryptone and 0.3% yeast extract in water at 87.7% of the total weight of the culture medium, and then adjusting the pH to 6.8 to obtain the culture medium.

[0138] The protective agent contains 130g / L of skim milk powder.

[0139] Lactobacillus Taiwani CCFM1318 was mixed with commercial dry powder starter cultures Lactobacillus bulgaricus and Streptococcus thermophilus in a mass ratio of 1:1:1 to obtain the starter culture. Sugar was added to fresh milk to a concentration of 50 g / L to obtain a mixture. The mixture was homogenized at 65°C and 20 MPa and then sterilized at 95°C for 5 min to obtain the fermentation raw material. The fermentation raw material was cooled to 35°C and the starter culture was inoculated into the fermentation raw material at an inoculation rate of 0.03% (v / v). Fermentation was carried out at 35°C for 16 h to obtain fermented milk. The fermented milk was placed at 42°C for 4 h to coagulate and then refrigerated at 4°C for 24 h to undergo post-ripening to obtain the finished fermented milk product.

[0140] Example 8: Application of Lactobacillus taiwanensis in the preparation of soy milk

[0141] Lactobacillus Taiwani CCFM1318 can be used to prepare soy milk. The specific preparation process for soy milk is as follows:

[0142] Lactobacillus taiwanense CCFM1318 was streaked onto MRS solid medium and cultured at 37°C for 48 h to obtain single colonies. Single colonies were picked and inoculated into MRS liquid medium and cultured at 37°C for 13 h for activation. This activation was repeated for two generations to obtain an activated solution. The activated solution was inoculated into the medium at a rate of 2% (v / v) and cultured at 37°C for 13 h to obtain a bacterial suspension. The bacterial suspension was centrifuged at 5000g for 10 min to obtain bacterial sludge. The bacterial sludge was washed three times with physiological saline and then resuspended in a cryoprotectant to a concentration of 1×10⁻⁶. 10CFU / mL was used to obtain a bacterial suspension; the bacterial suspension was pre-cultured at 37℃ for 60 min and then freeze-dried to obtain Lactobacillus taiwanense CCFM1318 bacterial powder.

[0143] The culture medium is prepared by dissolving 10% enzyme hydrolysate, 0.5% glucose, 1.5% tryptone and 0.3% yeast extract in water at 87.7% of the total weight of the culture medium, and then adjusting the pH to 6.8 to obtain the culture medium.

[0144] The protective agent contains 130g / L of skim milk powder.

[0145] Soybeans are soaked at 80℃ for 2 hours, then the soybean skins are removed to obtain peeled soybeans. The peeled soybeans are drained of soaking water and ground with boiling water to obtain soy milk. The soy milk is kept at a temperature above 80℃ for 12 minutes to obtain cooked soy milk. The cooked soy milk is filtered through a 150-mesh sieve and centrifuged to obtain crude soy milk. The crude soy milk is heated to 140-150℃ and then quickly introduced into a vacuum cooling chamber for vacuuming, allowing off-flavors to be rapidly expelled with steam, resulting in cooked soy milk. The cooked soy milk is cooled to approximately 37℃, and then Lactobacillus Taiwan CCFM1318 bacterial powder is added to the cooked soy milk to a concentration of not less than 1×10⁻⁶. 6 CFU / mL yields soy milk (soy milk must be refrigerated at 4℃).

[0146] Example 9: Application of Lactobacillus taiwanensis in the preparation of fruit and vegetable beverages

[0147] Lactobacillus taiwanensis CCFM1318 can be used to prepare fruit and vegetable beverages. The specific preparation process for fruit and vegetable beverages is as follows:

[0148] Lactobacillus Taiwani CCFM1318 was streaked onto MRS solid medium and cultured at 37°C for 48 h to obtain single colonies. Single colonies were picked and inoculated into MRS liquid medium and cultured at 37°C for 13 h for activation. This activation was repeated for two generations to obtain an activated solution. The activated solution was inoculated into the medium at a rate of 2% (v / v) and cultured at 37°C for 13 h to obtain a bacterial suspension. The bacterial suspension was centrifuged at 5000g for 10 min to obtain bacterial sludge. The bacterial sludge was washed three times with physiological saline and then resuspended in a cryoprotectant to a concentration of 1x10⁻⁶. 10 CFU / mL was used to obtain a bacterial suspension; the bacterial suspension was pre-cultured at 37℃ for 60 min and then freeze-dried to obtain Lactobacillus taiwanense CCFM1318 bacterial powder.

[0149] The culture medium is prepared by dissolving 10% enzyme hydrolysate, 0.5% glucose, 1.5% tryptone and 0.3% yeast extract in water at 87.7% of the total weight of the culture medium, and then adjusting the pH to 6.8 to obtain the culture medium.

[0150] The protective agent contains 130g / L of skim milk powder.

[0151] Fresh fruits and vegetables are washed and juiced to obtain fruit and vegetable juice; the juice is then heat-sterilized at 140℃ for 2 seconds to obtain sterilized fruit and vegetable juice; after cooling the sterilized juice to approximately 37℃, Lactobacillus Taiwan CCFM1318 bacterial powder is added to the sterilized juice to a concentration of not less than 1×10⁻⁶. 6 CFU / mL yields a fruit and vegetable beverage (which must be refrigerated at 4°C).

[0152] Example 10: Application of Lactobacillus taiwanensis in the preparation of dairy beverages

[0153] Lactobacillus taiwanensis CCFM1318 can be used to prepare dairy beverages. The specific preparation process for dairy beverages is as follows:

[0154] Lactobacillus taiwanense CCFM1318 was streaked onto MRS solid medium and cultured at 37°C for 48 h to obtain single colonies. Single colonies were picked and inoculated into MRS liquid medium and cultured at 37°C for 18 h for activation. This activation was repeated for two generations to obtain an activated solution. The activated solution was inoculated into the medium at a rate of 2% (v / v) and cultured at 37°C for 13 h to obtain a bacterial suspension. The bacterial suspension was centrifuged at 5000g for 10 min to obtain bacterial sludge. The bacterial sludge was washed three times with physiological saline and then resuspended in a cryoprotectant to a concentration of 1×10⁻⁶. 10 CFU / mL was used to obtain a bacterial suspension; the bacterial suspension was pre-cultured at 37℃ for 60 min and then freeze-dried to obtain Lactobacillus taiwanense CCFM1318 bacterial powder.

[0155] The culture medium is prepared by dissolving 10% enzyme hydrolysate, 0.5% glucose, 1.5% tryptone and 0.3% yeast extract in water at 87.7% of the total weight of the culture medium, and then adjusting the pH to 6.8 to obtain the culture medium.

[0156] The protective agent contains 130g / L of skim milk powder.

[0157] Skim milk was heat-sterilized at 95℃ for 20 minutes and then cooled to 4℃ to obtain the raw material; Lactobacillus Taiwan CCFM1318 bacterial powder was added to the raw material to a concentration of not less than 1×10⁻⁶. 6 CFU / mL yields a milk beverage (which must be refrigerated at 4°C).

[0158] Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Anyone skilled in the art can make various modifications and alterations without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be determined by the claims.

Claims

1. A strain of Lactobacillus taiwanensis ( Lactobacillus taiwanensis CCFM1318, characterized in that, The Lactobacillus taiwanensis is deposited at the Guangdong Provincial Center for Microbial Culture Collection, with accession number GDMCC No. 63714, deposit date of August 4, 2023, and deposit address of Building 59, No. 100 Xianlie Middle Road, Guangzhou.

2. A microbial preparation, characterized in that, The microbial preparation contains the *Lactobacillus taiwanensis* as described in claim 1. Lactobacillus taiwanensis CCFM1318 or its fermentation broth, or Lactobacillus taiwanensis ( Lactobacillus taiwanensis CCFM1318 freeze-dried powder.

3. The microbial preparation according to claim 2, characterized in that, The Taiwan Lactobacillus ( Lactobacillus taiwanensis The viable count of CCFM1318 is not less than 10. 8 CFU / mL or 10 8 CFU / g.

4. A product containing the Lactobacillus taiwanensis of claim 1 ( Lactobacillus taiwanensis CCFM1318 or a product containing the microbial preparation of claim 2 or 3, characterized in that, The product in question is a pharmaceutical product.

5. The product according to claim 4, characterized in that, The drug contains the aforementioned Lactobacillus taiwanensis ( Lactobacillus taiwanensis The drug carrier and / or pharmaceutical excipients of CCFM1318.

6. The product according to claim 4, characterized in that, The dosage forms of the medicine include powder, granules, capsules, tablets, pills, or oral liquid.

7. The product according to any one of claims 4-6, characterized in that, The Taiwan Lactobacillus ( Lactobacillus taiwanensis The viable count of CCFM1318 is not less than 10. 8 CFU / mL or 10 8 CFU / g.

8. A type of Lactobacillus taiwanensis ( Lactobacillus taiwanensis The preparation method of CCFM1318 fermentation agent is characterized by, Includes the following steps: (1) The Lactobacillus taiwanensis described in claim 1 ( Lactobacillus taiwanensis Cultivate in CCFM1318 and collect bacterial cells; (2) Freeze-dry the bacterial cells described in step (1) to obtain the fermentation agent.

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