Culture of lactic acid bacteria strain and use thereof for inhibiting the growth of gastrointestinal pathogens and promoting intestinal health

Abstract

The present invention relates to a culture of a lactic acid bacteria strain and its use in inhibiting the growth of gastrointestinal pathogens and promoting intestinal health. The present invention discloses a culture of a lactic acid bacteria strain and its use in inhibiting the growth of gastrointestinal pathogens and promoting intestinal health.

Description

Technical Field

[0001] The present invention relates to a culture of a lactic acid bacteria strain and to using the culture of the lactic acid bacteria strain to inhibit the growth of gastrointestinal pathogenic bacteria and promote intestinal health. Background Art

[0002] Gastrointestinal pathogenic bacteria are a group of bacteria found in the gastrointestinal tract of humans and animals, as well as in water and spoiled food. Common gastrointestinal pathogens include Salmonella enterica, Listeria monocytogenes, Escherichia coli, and Vibrio parahaemolyticus.

[0003] Infection with gastrointestinal pathogens is closely related to the integrity and function of the intestinal mucosa (i.e., the intestinal epithelium). The barrier formed by an intact intestinal epithelium (i.e., the intestinal epithelial barrier) can prevent the invasion of gastrointestinal pathogens and their secretions, thereby maintaining the sterility of the deep intestinal tissues. Tight junctions (TJs) between intestinal epithelial cells play a crucial role in the intestinal epithelial barrier, ensuring the impermeability of the intestinal epithelium and preventing the entry of commensal bacteria and pathogens in the gastrointestinal tract. However, when gastrointestinal pathogens overgrow, they can disrupt the barrier function by regulating the expression of TJ component proteins (e.g., tight junction proteins claudins, occludins, and junctional adhesion molecules (JAMs)) and damaging TJ tissue, thereby invading the host and causing a series of gastrointestinal diseases, including irritable bowel syndrome (IBS), gastroenteritis (e.g., gastritis and enteritis), ulcerative colitis (UC), typhoid fever, and bacteremia.

[0004] Currently, antibiotics are commonly used to treat gastrointestinal infections caused by pathogenic bacteria. However, antibiotics can cause antibiotic resistance in gastrointestinal pathogens and lead to serious side effects and adverse reactions. Therefore, researchers in this field are committed to developing drugs that can effectively prevent and / or treat gastrointestinal infections without causing undesirable side effects.

[0005] Lactic acid bacteria (LAB) are generally recognized as safe (GRAS) and are well-known and widely used probiotics. Common lactic acid bacteria include Lactobacillus, Lactococcus, Pediococcus, Enterococcus, Streptococcus, Bifidobacterium, Bacillus, and Leuconostoc.

[0006] Currently, many lactic acid bacteria strains have been found to have the ability to maintain intestinal epithelial function. For example, in R. Jariwala et al. (2017), Microbiology (Reading, Engl.), 163(9): 1263–1272, R. Jariwala et al. tested the efficacy of multiple strains of Lactobacillus rhamnosus, Lactobacillus fermentum, Lactobacillus helveticus, Lactobacillus salivarius, and Lactobacillus plantarum in restoring enteropathogenic E. coli (EPEC)-induced epithelial barrier dysfunction. In vitro experimental results showed that these lactic acid bacteria strains were able to effectively restore the impermeability of the human colon cancer cell line Caco-2 cell monolayer infected with EPEC. In particular, Lactobacillus rhamnosus GG (LGG) was able to restore the impermeability by approximately 93.8%. In addition, some lactic acid bacteria strains can also significantly increase the mRNA expression level of TJ component proteins and restore the normal distribution of TJ component proteins.

[0007] In addition, M. Jungersen et al. (2014), Microorganisms. 2(2): 92–110, M. Jungersen et al. reviewed the biological activity of B. animalis subsp. lactis. The known beneficial effects on the human body include inhibiting the growth of various pathogens (including Escherichia coli, Listeria monocytogenes, Salmonella enteric ser. Typhimurium, Bacillus cereus, etc.), improving TJ strength and intestinal epithelial barrier function, and inducing anti-inflammatory responses of immune cells. However, the results of multiple studies have found that animal Bifidobacterium lactis subsp. It has limited adsorption capacity for human intestinal cells and can only transiently colonize in the intestine.

[0008] Despite the aforementioned benefits of lactic acid bacteria, they are susceptible to destruction by gastric acid and bile salts after oral administration and may not readily adhere to the intestines, resulting in their inability to exert their intended effects in the intestines. Therefore, there remains a need in the art to develop lactic acid bacteria with excellent gastric acid and bile salt tolerance, as well as intestinal adsorption capacity, that can effectively inhibit the growth of gastrointestinal pathogens and promote intestinal health, for industrial use. Summary of the Invention

[0009] In the present invention, the applicant found that Lactobacillus rhamnosus BioF-115 (CGMCC No. 21684), Lactobacillus paracasei BioF-126 (CGMCC No. 21686), Bifidobacterium animalis subsp. lactis BioF-202 (CGMCC No. 21683), Lactobacillus plantarum BioF-208 (CGMCC No. 21682), Lactococcus lactis BioF-224 (CGMCC No. 21685), Streptococcus thermophilus BioF-278 (CGMCC No. 21687) and Bifidobacterium longum subsp. infantis BioF-206 (CGMCC No. 21688) were the main active ingredients of the bacterial strain. subsp. infantis) BioF-402 (CGMCC No. 21681) has excellent gastric acid and bile salt tolerance and adsorption capacity to human intestinal cells, effectively enabling these lactic acid bacteria to survive in the human digestive tract environment and reach the intestines to exert their effects in combating gastrointestinal pathogens and promoting intestinal health.

[0010] Thus, in a first aspect, the present invention provides a culture of a lactic acid bacteria strain, which is prepared by culturing a combination of lactic acid bacteria strains selected from the following in a culture medium suitable for the growth of the lactic acid bacteria strain combination:

[0011] a lactic acid bacteria strain combination comprising Bifidobacterium animalis subsp. lactis BioF-202 (CGMCC No. 21683), Lactococcus lactis BioF-224 (CGMCC No. 21685), Streptococcus thermophilus BioF-278 (CGMCC No. 21687), and Bifidobacterium longum subsp. infantis BioF-402 (CGMCC No. 21681); and

[0012] The product contains a combination of lactic acid bacteria strains including Lactobacillus rhamnosus BioF-115 (CGMCC No. 21684), Lactobacillus paracasei BioF-126 (CGMCC No. 21686), Lactobacillus plantarum BioF-208 (CGMCC No. 21682), Lactococcus lactis BioF-224, and Streptococcus thermophilus BioF-278.

[0013] Preferably, the lactic acid bacteria strain combination comprises Lactobacillus rhamnosus BioF-115, Lactobacillus paracasei BioF-126, Bifidobacterium animalis subsp. lactis BioF-202, Lactobacillus plantarum BioF-208, Lactococcus lactis BioF-224, Streptococcus thermophilus BioF-278, and Bifidobacterium longum subsp. infantis BioF-402.

[0014] Preferably, the Lactobacillus rhamnosus BioF-115, Lactobacillus paracasei BioF-126, Bifidobacterium animalis subsp. lactis BioF-202, Lactobacillus plantarum BioF-208, Lactococcus lactis BioF-224, Streptococcus thermophilus BioF-278, and Bifidobacterium longum subsp. infantis BioF-402 are cultured at a bacterial count ratio of 1:1:1:1:1:1:1:1.

[0015] In a second aspect, the present invention provides a use of a culture of the lactic acid bacteria strain described above for preparing a composition for inhibiting the growth of gastrointestinal pathogens.

[0016] Preferably, the gastrointestinal pathogen is selected from the group consisting of Salmonella enterica subsp. enterica, Listeria monocytogenes, Escherichia coli, Vibrio parahaemolyticus, Staphylococcus aureus, Shigella boydii, Shigella dysenteriae, Klebsiella pneumoniae, Yersinia enterocolitica, Proteus vulgaris, and combinations thereof.

[0017] In a third aspect, the present invention provides a use of a culture of the lactic acid bacteria strain described above for preparing a composition for promoting intestinal health.

[0018] Preferably, the promotion of intestinal health includes at least one of the following: inhibiting the growth of gastrointestinal pathogens, restoring healthy intestinal flora, promoting the repair of intestinal epithelium, and maintaining the function of intestinal epithelium.

[0019] Preferably, the composition is a pharmaceutical composition.

[0020] More preferably, the pharmaceutical composition is in a dosage form for parenteral or oral administration. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 A and Figure 1 B shows the surviving bacterial counts (log CFU / mL) of Bifidobacterium animalis subsp. lactis BB-12 culture and seven-bacteria mixed culture measured from hour 0 to hour 7 of the gastric acid and bile salt tolerance test;

[0022] Figure 2 The number of lactic acid bacteria adsorbed on Caco-2 cells in each group after co-culture with cultures of different lactic acid bacteria strains is shown; and

[0023] Figure 3 The relative mRNA expression levels of the CLDN1 gene in Caco-2 cells of each group treated with different lactic acid bacteria strains are shown. "****" indicates p < 0.0001 when compared with the seven-bacteria experimental group. DETAILED DESCRIPTION

[0024] For the purposes of this specification, it will be expressly understood that the word "comprising" means "including, but not limited to," and that the word "comprises" has a corresponding meaning.

[0025] Unless otherwise defined, all technical and scientific terms used herein have the same meanings as those commonly understood by those skilled in the art. Those skilled in the art will recognize many methods and materials similar or equivalent to those described herein that can be used to practice the present invention. Of course, the present invention is in no way limited to the methods and materials described.

[0026] The present invention provides a culture of a lactic acid bacteria strain, which is prepared by culturing a combination of lactic acid bacteria strains selected from the following in a culture medium suitable for the growth of the lactic acid bacteria strain combination:

[0027] a lactic acid bacteria strain combination comprising Bifidobacterium animalis subsp. lactis BioF-202 (CGMCC No. 21683), Lactococcus lactis BioF-224 (CGMCC No. 21685), Streptococcus thermophilus BioF-278 (CGMCC No. 21687), and Bifidobacterium longum subsp. infantis BioF-402 (CGMCC No. 21681); and

[0028] The product contains a combination of lactic acid bacteria strains including Lactobacillus rhamnosus BioF-115 (CGMCC No. 21684), Lactobacillus paracasei BioF-126 (CGMCC No. 21686), Lactobacillus plantarum BioF-208 (CGMCC No. 21682), Lactococcus lactis BioF-224, and Streptococcus thermophilus BioF-278.

[0029] According to the present invention, the culture of the lactic acid bacteria strain can be obtained by 4 to 10 12 CFU / mL of these lactic acid bacteria strains are cultured in a medium suitable for growth and obtained. In a preferred embodiment of the present invention, these lactic acid bacteria strains have a bacterial concentration in the range of 10 5 to 10 11 Bacterial concentration in CFU / mL.

[0030] As used herein, the terms "culturing," "fermentation," and "cultivation" are used interchangeably.

[0031] The culture operation process and parameter conditions fall within the professional qualities and routine skills of those skilled in the art. In this regard, reference may be made to, for example, Hsieh P S et al. (2013), New Microbiol., 36: 167-179.

[0032] According to the present invention, the culture medium suitable for culturing these lactic acid bacteria strain combinations can be prepared by those skilled in the art, or can be commercially available products, including, but not limited to: MRS broth and MRS broth supplemented with cysteine. In a preferred embodiment of the present invention, the culture medium suitable for culturing these lactic acid bacteria strain combinations is MRS broth supplemented with cysteine ​​(BD Biosciences, Difco).

[0033] Preferably, the lactic acid bacteria strain combination comprises Bifidobacterium animalis subsp. lactis BioF-202, Lactococcus lactis BioF-224, Streptococcus thermophilus BioF-278 and Bifidobacterium longum subsp. infantis BioF-402, and the culture of the lactic acid bacteria strain is prepared by culturing these lactic acid bacteria strains at a bacterial ratio of 1:1:1:1.

[0034] Preferably, the lactic acid bacteria strain combination includes Lactobacillus rhamnosus BioF-115, Lactobacillus paracasei BioF-126, Lactobacillus plantarum BioF-208, Lactococcus lactis BioF-224 and Streptococcus thermophilus BioF-278, and the culture of the lactic acid bacteria strain is prepared by culturing these lactic acid bacteria strains at a bacterial count ratio of 1:1:1:1:1.

[0035] More preferably, the lactic acid bacteria strain combination includes Lactobacillus rhamnosus BioF-115, Lactobacillus paracasei BioF-126, Bifidobacterium animalis subsp. lactis BioF-202, Lactobacillus plantarum BioF-208, Lactococcus lactis BioF-224, Streptococcus thermophilus BioF-278, and Bifidobacterium longum subsp. infantis BioF-402. In a preferred embodiment of the present invention, the culture of the lactic acid bacteria strains is prepared by culturing the lactic acid bacteria strains at a bacterial ratio of 1:1:1:1:1:1:1:1.

[0036] According to the present invention, the culture of the lactic acid bacteria strain can be a liquid culture, and the liquid culture can be further subjected to a drying process selected from the group consisting of freeze-drying, spray-drying, and fluidized bed drying to form a powder. In a preferred embodiment of the present invention, the liquid culture of the lactic acid bacteria strain is further subjected to freeze-drying to form a powder.

[0037] According to the present invention, before the drying process, the liquid culture may be subjected to a solid-liquid separation process selected from the group consisting of centrifugation (e.g., multi-stage centrifugation), filtration, gravity settling, and combinations thereof to collect the bacterial cells.

[0038] The present invention also provides a use of the culture of the lactic acid bacteria strain described above for preparing a composition for inhibiting the growth of gastrointestinal pathogenic bacteria.

[0039] According to the present invention, the gastrointestinal pathogen is selected from the group consisting of: Salmonella enterica subsp. enterica, Listeria monocytogenes, Escherichia coli, Vibrio parahaemolyticus, Staphylococcus aureus, Shigella boydii, Shigella dysenteriae, Klebsiella pneumoniae, Yersinia enterocolitica, Proteus vulgaris, and combinations thereof.

[0040] The present invention also provides a use of the culture of the lactic acid bacteria strain described above for preparing a composition for promoting intestinal health.

[0041] As used herein, the term "improving gut health" means that the individual ingesting the composition exhibits healthy gut flora, which is beneficial to human or animal health and is suitable for maintaining and / or improving digestion in the individual. Such healthy gut flora ultimately contributes to proper nutrient absorption, proper growth, less colic, less infection, less diarrhea, and optimal gut health.

[0042] According to the present invention, the promotion of intestinal health includes at least one of the following: inhibiting the growth of gastrointestinal pathogens, restoring a healthy intestinal flora, promoting intestinal epithelial repair (or maintaining the integrity of the intestinal epithelium), and maintaining the function of the intestinal epithelium.

[0043] According to the present invention, the composition may be a pharmaceutical composition.

[0044] According to the present invention, the pharmaceutical composition may be in a dosage form suitable for parenteral administration or oral administration.

[0045] According to the present invention, the pharmaceutical composition may further include a pharmaceutically acceptable carrier widely used in pharmaceutical manufacturing technology. For example, the pharmaceutically acceptable carrier may include one or more agents selected from the following: solvent, buffer, emulsifier, suspending agent, decomposer, disintegrating agent, dispersing agent, binding agent, excipient, stabilizer, chelating agent, diluent, gelling agent, preservative, wetting agent, lubricant, absorption delaying agent, liposome, and the like. The selection and amount of these agents fall within the professional knowledge and routine skills of those skilled in the art.

[0046] According to the present invention, the pharmaceutical composition can be manufactured into a dosage form suitable for parenteral administration (including injections, e.g., sterile aqueous solutions or dispersions) using techniques well known to those skilled in the art, and administered via intraperitoneal injection or sublingual administration. Preferably, the pharmaceutical composition is manufactured into a dosage form suitable for sublingual administration.

[0047] According to the present invention, the pharmaceutical composition can be manufactured into a dosage form suitable for oral administration using techniques well known to those skilled in the art, including, but not limited to, sterile powders, tablets, troches, lozenges, pellets, capsules, dispersible powders or granules, solutions, suspensions, emulsions, syrups, elixirs, slurries, and the like. Preferably, the pharmaceutical composition is manufactured into a capsule dosage form.

[0048] The present invention also provides a method for inhibiting the growth of gastrointestinal pathogens in a subject, comprising administering the composition described above to the subject.

[0049] As used herein, the terms "administering" and "administration" are used interchangeably and mean introducing, providing or delivering a predetermined active ingredient to a subject by any appropriate route to perform its intended effect.

[0050] As used herein, the term "subject" means any mammal of interest, such as humans, monkeys, cows, sheep, horses, pigs, goats, dogs, cats, mice, and rats.

[0051] According to the present invention, the dosage and frequency of administration of the composition will vary depending on the severity of the disease to be improved, the route of administration, and the weight, age, physical condition, and response of the individual to be improved. Generally speaking, the composition can be administered parenterally or orally in a single dose or divided into multiple doses.

[0052] The present invention will be further described with reference to the following examples. However, it should be understood that these examples are for illustration only and should not be construed as limitations on the implementation of the present invention.

[0053] <Example>

[0054] General experimental materials:

[0055] 1. Lactic acid bacteria strains:

[0056] The 7 lactobacilli strains used in the following examples are voluntarily separated by the applicant and commissioned by the Food Industry Research and Development Institute (FIRDI) of Taiwan, China to carry out strain identification by bioresource collection and research center (BCRC) (300 No. 331, Food Road, Hsinchu City, Taiwan, China), and have been deposited in the China General Microbiological Culture Collection Center (CGMCC) according to the regulations of the Budapest Treaty. For clarity, the relevant information of these lactobacilli strains (including: scientific name, separation source, deposit number and preservation date etc.) has been integrated in Table 1 below.

[0057] Table 1. Information about each lactic acid bacteria strain

[0058]

[0059]

[0060] For comparison, the following lactic acid bacteria strains known to promote intestinal health were also used in the following examples: Bifidobacterium animalis subsp. lactis BB-12 and Lactobacillus rhamnosus GG purchased from Chr. Hansen A / S, Denmark.

[0061] 2. Source and culture of gastrointestinal pathogenic bacteria strains:

[0062] The gastrointestinal pathogen strains used in the following examples were purchased from BCRC, FIRDI. For clarity, the scientific names and accession numbers of the various gastrointestinal pathogens are summarized in Table 2 below, and their suitable culture media and culture conditions are shown in Table 3 below.

[0063] Table 2. Information about various gastrointestinal pathogen strains

[0064]

[0065]

[0066] Table 3. Culture media and culture conditions for various gastrointestinal pathogen strains

[0067]

[0068] 3. Preparation of lactic acid bacteria culture:

[0069] First, the lactic acid bacteria strains described in item 1 above were inoculated into MRS broth (Difco, Cat. No. 288130) supplemented with 0.05% cysteine ​​and cultured in an incubator (37°C, 5% CO2) for 24 hours to activate these lactic acid bacteria strains. Then, the activated strains were inoculated into MRS broth at an inoculum size of 2% (v / v) and subcultured in an incubator (37°C, 5% CO2) for 24 hours. Then, the obtained bacterial solution was serially diluted 10 times with MRS broth, and after counting the number of bacteria by the plate counting method using MRS agar medium, freeze-dried to obtain cultures of each lactic acid bacteria strain in the form of dry powder (each having 10 5 to 10 11 CFU / g) and stored at 4°C until use.

[0070] The applicant also mixed the Lactobacillus rhamnosus BioF-115, Lactobacillus paracasei BioF-126, Bifidobacterium animalis subsp. lactis BioF-202, Lactobacillus plantarum BioF-208, Lactococcus lactis BioF-224, Streptococcus thermophilus BioF-278, and Bifidobacterium longum subsp. infantis BioF-402 described in item 1 above at a bacterial count ratio of 1:1:1:1:1:1:1 to obtain a seven-bacterium combination. This seven-bacterium combination was then prepared into a dry powdered seven-bacterium mixed culture according to the above steps and stored at 4°C until ready for use.

[0071] Before the following experiments, the cultures of the various lactic acid bacteria strains and the seven-bacteria mixed culture were preliminarily dissolved in appropriate culture media, and each had a concentration of 1×10 9 Bacterial concentration in CFU / mL.

[0072] 4. Human colon adenocarcinoma cell line

[0073] Source and culture of Caco-2:

[0074] The human colon adenocarcinoma cell line Caco-2 used in the following examples was purchased from BCRC (BCRC60182) at FIRDI. Caco-2 cells were cultured in 10 cm Petri dishes containing Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin in a 37°C, 5% CO2 incubator. Fresh medium was replaced approximately every 2-3 days. When the cells reached approximately 80-90% confluence, subculture was performed as follows: the medium was removed and the cells were washed with phosphate-buffered saline (PBS) (pH 7.4). Trypsin-EDTA was then added to detach the cells from the bottom of the dish. Afterwards, fresh culture medium was added to neutralize the activity of trypsin and the culture medium was repeatedly aspirated with a pipette to fully disperse the cells. The resulting cell suspension was then distributed into a new culture dish and cultured in an incubator set at 37° C. and 5% CO 2 .

[0075] General experimental methods:

[0076] 1. Statistical analysis:

[0077] In the following examples, the experiments of each group were repeated 3 times, and the obtained experimental data were expressed as “mean ± standard deviation (SD)”.

[0078] Example 1. Evaluation of the effectiveness of cultures of lactic acid bacteria strains against gastrointestinal pathogens

[0079] Experimental Materials:

[0080] 1.MRS agar plate:

[0081] 1.5% (w / v) agar powder was added to the MRS broth medium, and MRS agar plates were prepared according to the techniques well known and commonly used by those skilled in the art for subsequent experiments.

[0082] 2. Top agar medium:

[0083] 1.5% (w / v) agar powder was added to NB medium, BHI medium, and TSB medium supplemented with 2.5% sodium chloride, as shown in Table 4 above, and then sterilized at 121°C for 15 minutes. These thawed media were then placed in a 45°C water bath and used as top agar in subsequent experiments.

[0084] 3. Preparation of gastrointestinal pathogenic bacteria solution:

[0085] Each gastrointestinal pathogen described in item 2 of "General Experimental Materials" above was cultured for 20 hours using the appropriate culture medium and culture conditions shown in Table 3 above to activate the strains. Subsequently, the activated strains were subcultured using the culture medium and culture conditions shown in Table 3 at an inoculum size of 2% (v / v) to obtain a bacterial concentration of approximately 10 5 to 10 11 CFU / mL of bacterial suspension of various gastrointestinal pathogens.

[0086] Experimental methods:

[0087] In this example, a double agar overlay method was performed generally with reference to the method described in YTChen et al. (2020), Lett. Appl. Microbiol., 70(4): 310-317 to evaluate the effectiveness of each lactic acid bacteria strain culture in inhibiting the growth of intestinal pathogens.

[0088] First, the cultures of Lactobacillus rhamnosus BioF-115, Lactobacillus paracasei BioF-126, Bifidobacterium animalis subsp. lactis BioF-202, Lactobacillus plantarum BioF-208, Lactococcus lactis BioF-224, Streptococcus thermophilus BioF-278, and Bifidobacterium longum subsp. infantis BioF-402, as well as the seven-bacteria mixed culture obtained in item 3 of the “General Experimental Materials” above, were used as different groups (i.e., BioF-115 group, BioF-126 group, and BioF-208 group). The culture medium (approximately 1 mL) was taken from each group using a sterilized cotton swab and then drawn on an MRS agar plate along a diameter of approximately 2 cm. The plates were then cultured at 37°C for 48 hours, allowing each group of lactic acid bacteria to form a growth zone approximately 2 cm wide on the surface of the MRS agar plate. A blank control group was also used, using an MRS broth culture without any lactic acid bacteria to draw a line on the MRS agar plate.

[0089] Next, melted NB agar (i.e., top agar) was poured into each culture dish and evenly spread onto an MRS agar plate. After the NB agar solidified, a sterilized cotton swab was used to smear approximately 1 mL of E. coli culture solution onto the surface of each NB agar plate, ensuring a roughly uniform number of E. coli on each plate. The plates were then incubated in an incubator (37°C, 5% CO₂) for 24 hours. The effectiveness of each lactic acid bacteria culture in inhibiting E. coli growth was assessed by measuring the width of the inhibition zone formed on the double-layer agar plate.

[0090] The inhibitory efficacy of each group of lactic acid bacteria strain cultures against Salmonella enterica ssp. enterica, Listeria monocytogenes, and Vibrio enteritidis was evaluated generally in the same manner as described above for Escherichia coli, except that Salmonella enterica ssp. enterica, Listeria monocytogenes, and Vibrio enteritidis bacterial cultures were used instead of E. coli cultures, and melted NB agar, BHI agar, and TSB agar supplemented with 2.5% sodium chloride were used as the top agar culture media, respectively.

[0091] result:

[0092] The inhibitory effects of each group of lactic acid bacteria strain cultures on various gastrointestinal pathogens are shown in Table 4 below.

[0093] Table 4. Inhibitory effect of each group of lactic acid bacteria strain cultures on gastrointestinal pathogens

[0094]

[0095]

[0096] Note: The inhibitory efficacy is measured by the width of the inhibition zone formed, where “-” indicates an inhibition zone width of 0 cm, “+” indicates >0 to <2 cm, “++” indicates 2 to <3 cm, “+++” indicates 3 to <4 cm, “++++” indicates 4 to <5 cm, and “++” indicates 5 to <9 cm.

[0097] As shown in Table 4, the BioF-115, BioF-126, BioF-202, BioF-208, BioF-224, BioF-278, and BioF-402 groups all exhibited inhibitory effects against various gastrointestinal pathogens. In comparison, the seven-bacteria group exhibited significantly enhanced inhibitory effects. This indicates that the mixed culture of Lactobacillus rhamnosus BioF-115, Lactobacillus paracasei BioF-126, Bifidobacterium animalis subsp. lactis BioF-202, Lactobacillus plantarum BioF-208, Lactococcus lactis BioF-224, Streptococcus thermophilus BioF-278, and Bifidobacterium longum subsp. infantis BioF-402 exhibits a synergistic effect in inhibiting the growth of gastrointestinal pathogens.

[0098] Example 2. Gastric acid and bile salt tolerance assay of cultures of lactic acid bacteria strains

[0099] Based on the excellent effectiveness of a mixed culture of Lactobacillus rhamnosus BioF-115, Lactobacillus paracasei BioF-126, Bifidobacterium animalis subsp. lactis BioF-202, Lactobacillus plantarum BioF-208, Lactococcus lactis BioF-224, Streptococcus thermophilus BioF-278, and Bifidobacterium longum subsp. infantis BioF-402 in inhibiting the growth of gastrointestinal pathogens, the applicant further conducted the following experiments to confirm the survival ability of the lactic acid bacteria in the mixed culture in the digestive tract environment.

[0100] Experimental methods:

[0101] In this example, the gastric acid and bile salt tolerance test of lactic acid bacteria strains was conducted in accordance with the method described in PSHsieh et al. (2021) Exp. Ther. Med., 21(3): 188. First, the seven bacterial mixed cultures obtained in Item 3 of the "General Experimental Materials" above were divided into eight experimental groups (i.e., experimental groups 1 to 8) (10 mL per group). At the beginning of the test (i.e., the 0th hour of culture), 5 mL of MRS broth culture medium with a pH of 6.5 was added to experimental group 1 and mixed evenly. The number of surviving bacteria in the mixed culture of experimental group 1 was then calculated using the plate counting method and MRS agar medium. As for experimental groups 2 to 8, 5 mL of MRS broth culture medium with a pH of 3.5 was added and mixed evenly, and then placed in an incubator (37°C, 5% CO2) for culture. 1, 2, and 3 hours after the start of the culture, the mixed cultures of experimental groups 2, 3, and 4 were taken out, respectively, and the number of surviving bacteria therein was calculated by the plate colony counting method.

[0102] Three hours after the start of incubation, mixed cultures of experimental groups 5 to 8 were removed and centrifuged at 4,000 rpm for 10 minutes at 4°C. The supernatant was removed, and the pellets from experimental groups 5 to 8 were washed twice with 5 mL of RO water and centrifuged again to remove the remaining RO water. The pellets were then suspended in 5 mL of 1.5% bovine bile (Sigma Aldrich, Cat. No. B3883) and incubated in an incubator (37°C, 5% CO2) for further culture. At 4, 5, 6, and 7 hours after the start of incubation, mixed cultures from experimental groups 5, 6, 7, and 8 were removed, respectively, and the number of surviving bacteria was counted using the plate colony count method.

[0103] For comparison, the applicant also used the culture of Bifidobacterium animalis subsp. lactis BB-12 obtained in item 3 of the “General Experimental Methods” above and performed gastric acid and bile salt tolerance tests generally referring to the methods described above.

[0104] The obtained experimental data were then analyzed according to the method described in item 1 “Statistical analysis” of “General experimental methods” above.

[0105] result:

[0106] Figure 1 A and 1B show the surviving bacterial counts (expressed in log CFU / mL) of Bifidobacterium animalis subsp. lactis BB-12 culture and seven-bacteria mixed culture measured from hour 0 to hour 7 in the gastric acid and bile salt tolerance test, respectively.

[0107] Depend on Figure 1 As shown in Figures A and 1B, the number of viable cells of the Bifidobacterium animalis subsp. lactis BB-12 culture in a pH 3.5 medium gradually decreased over time, and the number of viable cells decreased significantly after being transferred to a medium containing ox bile. In contrast, the number of viable cells in the seven-bacteria mixed culture remained unchanged throughout the experimental period. This indicates that the mixed culture of Lactobacillus rhamnosus BioF-115, Lactobacillus paracasei BioF-126, Bifidobacterium animalis subsp. lactis BioF-202, Lactobacillus plantarum BioF-208, Lactococcus lactis BioF-224, Streptococcus thermophilus BioF-278, and Bifidobacterium longum subsp. infantis BioF-402 can effectively withstand the effects of gastric acid and bile salts. It is believed that after ingestion, the lactic acid bacteria can survive in the human digestive tract and reach the intestine to exert their effects.

[0108] Example 3. Intestinal Adhesion Assay of Cultures of Lactic Acid Bacteria

[0109] Experimental methods:

[0110] In this embodiment, the adsorption capacity of lactic acid bacteria strains to intestinal cells was determined generally according to the method described in PSHsieh et al. (2021) Exp. Ther. Med., 21(3): 188.

[0111] First, several sterilized cover slips were placed in each well of a 6-well culture plate. Then, the Caco-2 cells subcultured according to item 4 of the "General Experimental Materials" were divided into one experimental group and two comparative experimental groups (i.e., comparative experimental groups 1 and 2). The Caco-2 cells in each group were cultured at a rate of 3×10 5Cells / well were cultured in each well of a 6-well plate containing 5 μL of MEM medium and a coverslip and incubated in an incubator (37°C, 5% CO₂) until the cells reached 100% confluence, i.e., a cell monolayer was formed. The cell cultures in each group were then replaced with fresh medium and incubated in an incubator (37°C, 5% CO₂) for 1 hour. The medium was then removed and the cells washed twice with PBS. 1.5 mL of each of the seven-bacteria mixed culture, Lactobacillus rhamnosus GG culture, and Bifidobacterium animalis subsp. lactis BB-12 culture obtained in Section 3 of the "General Experimental Materials" section above were added to the cell cultures in the experimental, comparative groups 1, and 2, respectively. 1.5 mL of fresh medium was then added to each group, and the cells were co-cultured in an incubator (37°C, 5% CO₂) for 1 to 4 hours.

[0112] After washing the cell cultures in each group with PBS, the cells were fixed with 10% methanol solution for 10 minutes. Next, the coverslips with attached cell cultures were removed from each group and Gram-stained. The cells were then observed at 1,000x magnification using an upright microscope (Carl Zeiss MicroImaging, Inc.), and the number of lactic acid bacteria attached to the Caco-2 cells in a single field of view was counted.

[0113] The obtained experimental data were then analyzed according to the method described in item 1 “Statistical analysis” of “General experimental methods” above.

[0114] result:

[0115] Figure 2 The numbers of lactic acid bacteria adsorbed on Caco-2 cells in each group of cell culture are shown.

[0116] Depend on Figure 2 As can be seen, the lactic acid bacteria count measured in the experimental group was significantly higher than that in comparative experimental groups 1 and 2, and was even nearly twice as high as that in comparative experimental groups 1 and 2. This indicates that the lactic acid bacteria in the mixed culture of Lactobacillus rhamnosus BioF-115, Lactobacillus paracasei BioF-126, Bifidobacterium animalis subsp. lactis BioF-202, Lactobacillus plantarum BioF-208, Lactococcus lactis BioF-224, Streptococcus thermophilus BioF-278, and Bifidobacterium longum subsp. infantis BioF-402 have excellent adsorption capacity for intestinal cells and are significantly superior to those of commercially available lactic acid bacteria strains.

[0117] Example 4. Effect of Lactic Acid Bacteria Culture on the Expression of Tight Junction Proteins (claudins) in Intestinal Cells

[0118] Tight junctions (TJs) play an important role in the intestinal epithelial barrier. Claudins are the most important components of the TJ structure. Imbalanced expression of claudins is also one of the causes of various inflammatory bowel diseases.

[0119] In this example, the applicant evaluated the effects of lactic acid bacteria culture on intestinal epithelial structure and barrier function by measuring the expression of tight junction proteins claudins in Caco-2 cells.

[0120] Experimental Materials:

[0121] 1. Preparation of four-bacteria mixed culture and five-bacteria mixed culture:

[0122] First, the lactic acid bacteria strains in item 1 of the "General Experimental Materials" above were mixed according to the ratios shown in Table 5 below to obtain two lactic acid bacteria strain combinations (i.e., a four-strain combination and a five-strain combination). Next, these strain combinations were prepared into four-strain mixed cultures and five-strain mixed cultures (both with a concentration of 1×10 9 CFU / mL).

[0123] Table 5. Ratio of bacterial count used in each strain combination

[0124]

[0125] Experimental methods:

[0126] A. Treating human colon adenocarcinoma Caco-2 cells with cultures of lactic acid bacteria strains:

[0127] The Caco-2 cells subcultured according to item 4 of the “General Experimental Materials” above were divided into 11 groups, including 7 single-bacteria comparison groups (i.e., single-bacteria comparison groups 1 to 7), 1 four-bacteria experimental group, 1 five-bacteria experimental group, 1 seven-bacteria experimental group, and 1 control group. Each group of cells was cultured at 3×10 5 The number of cells / well was cultured in each well of a 6-well culture plate containing 3 mL of DMEM medium (supplemented with 10% FBS and 1% penicillin-streptomycin) and cultured in an incubator (37°C, 5% CO2) for 11 days, during which the medium was replaced every 3 days.

[0128] Next, the culture medium in each well was removed, and 3 mL of each of the cultures of Lactobacillus rhamnosus BioF-115, Lactobacillus paracasei BioF-126, Bifidobacterium animalis subsp. lactis BioF-202, Lactobacillus plantarum BioF-208, Lactococcus lactis BioF-224, Streptococcus thermophilus BioF-278, and Bifidobacterium longum subsp. infantis BioF-402 obtained in item 3 of the "General Experimental Materials" above, and the seven-bacterial mixed culture, as well as the four-bacterial mixed culture and the five-bacterial mixed culture obtained in the "Experimental Materials" above, were added to each group of cell cultures according to the following Table 6. A control group was added with DMEM broth without any lactic acid bacteria culture, and then co-cultured in an incubator (37°C, 5% CO2) for 6 hours. The resulting cell cultures were then used for analysis in item B below.

[0129] Table 6. Lactic acid bacteria strains cultured in each group of cells

[0130] Group Lactic acid bacteria strain culture Single bacteria comparison group 1 Lactobacillus rhamnosus BioF-115 culture Single bacteria comparison group 2 Lactobacillus paracasei BioF-126 culture Single bacteria comparison group 3 Bifidobacterium animalis subsp. lactis BioF-202 culture Single bacteria comparison group 4 Lactobacillus plantarum BioF-208 culture Single bacteria comparison group 5 Lactococcus lactis BioF-224 culture Single bacteria comparison group 6 Streptococcus thermophilus BioF-278 culture Single bacteria comparison group 7 Bifidobacterium longum subsp. infantis BioF-402 culture Four-bacteria experimental group Four-bacteria mixed culture Five-bacteria experimental group Five-bacteria mixed culture Seven bacteria experimental group Seven-bacteria mixed culture control group －

[0131] B. Determination of CLDN1 gene mRNA expression level:

[0132] First, the total RNAs (total RNAs) of each cell culture were extracted using the Total RNA Extraction Miniprep System (VIOGENE, Cat. No. GR1001) according to the manufacturer's instructions. The total RNAs of each group were respectively transcribed using cDNA reverse transcription reagents (GoScript TM Reverse Transcriptase (Promega, Cat. No. A5003) was used to perform a reverse transcription reaction according to the manufacturer's instructions to synthesize first-strand cDNA.

[0133] Next, the obtained first-strand cDNA was used as a template and reference was made to AM et al. (2005), Breast Cancer Res. 7(2): R296-305. Quantitative real-time polymerase chain reaction (qRT-PCR) was performed using a specific primer pair designed for the CLDN1 gene. In addition, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene expression was used as an internal control. Information regarding these primer pairs (including target genes, nucleotide sequences of each primer, and amplified PCR product sizes) is summarized in Table 7 below.

[0134] Table 7. Primers used for quantitative real-time PCR

[0135]

[0136] Quantitative real-time PCR using StepOnePlus TM Real-time PCR system (StepOnePlus TM Real-Time PCRSystem) (Applied Biosystems TM ) and were performed according to the manufacturer's operating instructions. The operating conditions and reaction conditions of quantitative real-time PCR are shown in Table 8 below.

[0137] Table 8. Reaction conditions for quantitative real-time PCR

[0138]

[0139]

[0140] Each PCR product thus obtained was detected by fluorescence of SYBR Green (double-stranded DNA binding dye), and the cycle threshold (C t The relative mRNA expression level of CLDN1 gene in each group was compared using C t Method (comparative C tThe CLDN1 gene cycle threshold values ​​obtained were normalized with the GAPDH gene cycle threshold values, and then the normalized CLDN1 gene cycle threshold values ​​obtained in the control group were subtracted to obtain the value.

[0141] The data were then analyzed according to the methods described in "Statistical Analysis" in Section 1 of "General Experimental Methods" above. The Student's t-test was used to assess differences between the groups compared to the seven-bacteria experimental group. A p < 0.05 result indicated statistical significance.

[0142] result:

[0143] Figure 3 The relative mRNA expression levels of CLDN1 gene measured in each group of Caco-2 cells are shown. Figure 3 As can be seen, the relative mRNA expression levels of CLDN1 in each experimental group were significantly higher than those in the single-bacteria comparison groups 1 to 7, and the relative mRNA expression level in the seven-bacteria experimental group was even more significantly higher than that in the four-bacteria and five-bacteria experimental groups. This experimental result shows that the mixed cultures of the four-bacteria, five-bacteria, and seven-bacteria combinations can synergistically stimulate the expression of tight junction proteins claudins, thereby contributing to the barrier function of the intestinal epithelium.

[0144] Although the present invention has been described with reference to the specific embodiments above, it is apparent that many modifications and variations can be made without departing from the scope and spirit of the invention. It is therefore intended that the present invention be limited only as indicated by the appended claims.

[0145] Information on the deposit of biological materials

[0146] Deposit number: CGMCC No.21684

[0147] Classification name: Lactobacillus rhamnosus

[0148] Deposit date: January 20, 2021 Depositary: China Culture Collection Administration Committee General Microbiology Center Depositary address: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing

[0149] Accession number: CGMCC No.21686

[0150] Classification name: Lactobacillus paracasei

[0151] Deposit date: January 20, 2021 Depositary: China Culture Collection Administration Committee General Microbiology Center Depositary address: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing

[0152] Accession number: CGMCC No.21683

[0153] Classification name: Bifidobacterium animalis subsp.lactis

[0154] Deposit date: January 20, 2021 Depositary: China Culture Collection Administration Committee General Microbiology Center Depositary address: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing

[0155] Accession number: CGMCC No.21682

[0156] Classification name: Lactobacillus plantarum

[0157] Deposit date: January 20, 2021 Depositary: China Culture Collection Administration Committee General Microbiology Center Depositary address: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing

[0158] Deposit number: CGMCC No.21685

[0159] Classification name: Lactococcus lactis

[0160] Deposit date: January 20, 2021 Depositary: China Culture Collection Administration Committee General Microbiology Center Depositary address: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing

[0161] Deposit number: CGMCC No.21687

[0162] Classification name: Streptococcus thermophilus

[0163] Deposit date: January 20, 2021 Depositary: China Culture Collection Administration Committee General Microbiology Center Depositary address: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing

[0164] Deposit number: CGMCC No.21681

[0165] Classification name: Bifidobacterium longum subsp. infantis

[0166] Deposit date: January 20, 2021 Deposit unit: General Microbiology Center of China Culture Collection Administration of Microorganisms Deposit unit address: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing. Sequence Listing <110> Jinqiao Biotechnology Co., Ltd. <120> Culture of lactic acid bacteria strain and use thereof for inhibiting the growth of gastrointestinal pathogens and promoting intestinal health <160> 4 <170> PatentIn version 3.5 <210> 1 <211> twenty two <212> DNA <213> Artificial sequence <220> <223> Forward primer for quantitative real-time PCR of CLDN1 <400> 1 gcgcgatatt tcttcttgca gg 22 <210> 2 <211> twenty one <212> DNA <213> Artificial sequence <220> <223> Reverse primer for quantitative real-time PCR of CLDN1 <400> 2 ttcgtacctg gcattgactg g 21 <210> 3 <211> 18 <212> DNA <213> Artificial sequence <220> <223> Forward primer for quantitative real-time PCR of GAPDH <400> 3 gaaggtgaag gtcggagt 18 <210> 4 <211> 19 <212> DNA <213> Artificial sequence <220> <223> Reverse primer for quantitative real-time PCR of GAPDH <400> 4 gaagatggtg atggatttc 19

Claims

1. A culture of lactic acid bacteria strains, which is prepared by culturing the following lactic acid bacteria strain combination in a culture medium suitable for the growth of the lactic acid bacteria strain combination: The bacterial count ratio was 1:1:1:1:1:1:1:1:1, comprising Lactobacillus rhamnosus BioF-115 with a preservation number of CGMCC No. 21684, Lactobacillus paracasei BioF-126 with a preservation number of CGMCC No. 21686, Bifidobacterium animalis subsp. lactis BioF-202 with a preservation number of CGMCC No. 21683, Lactobacillus plantarum BioF-208 with a preservation number of CGMCC No. 21682, Lactococcus lactis BioF-224 with a preservation number of CGMCC No. 21685, and Streptococcus thermophilus with a preservation number of CGMCC No. 21687. The invention relates to a lactic acid bacteria strain combination consisting of Bifidobacterium thermophilus BioF-278 and Bifidobacterium longum subsp. infantis BioF-402 with a deposit number of CGMCC No. 21681.

2. A use of the culture of the lactic acid bacteria strain according to claim 1 for preparing a pharmaceutical composition for inhibiting the growth of gastrointestinal pathogens, characterized in that: The gastrointestinal pathogens are selected from the group consisting of: Salmonella enterica subsp. enterica, Listeria monocytogenes, Escherichia coli, Vibrio parahaemolyticus, and combinations thereof.

3. Use of the culture of the lactic acid bacteria strain according to claim 1 for preparing a pharmaceutical composition for promoting intestinal health.

4. The use according to claim 3, characterized in that: The promotion of intestinal health includes at least one of the following: inhibiting the growth of gastrointestinal pathogens, restoring healthy intestinal flora, promoting the repair of intestinal epithelium, and maintaining the function of intestinal epithelium, wherein the gastrointestinal pathogens are selected from the group consisting of: Salmonella enterica subsp. enterica, Listeria monocytogenes, Escherichia coli, Vibrio parahaemolyticus, and combinations thereof.

5. The use according to claim 2 or 3, characterized in that: The pharmaceutical composition is in a dosage form for parenteral administration or oral administration.

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