CN112442457B

CN112442457B - Bacillus coagulans or metabolite thereof and application thereof in stomach health care

Info

Publication number: CN112442457B
Application number: CN202010880538.1A
Authority: CN
Inventors: 黄琡涵; 林咏翔
Original assignee: TCI Co Ltd
Current assignee: TCI Co Ltd
Priority date: 2019-08-27
Filing date: 2020-08-27
Publication date: 2022-07-22
Anticipated expiration: 2040-08-27
Also published as: TWI757850B; CN114099586A; TW202108759A; TWI749734B; TWI774046B; TW202108758A; CN112442458A; CN112442457A; CN114099586B; CN112442458B; TW202108124A

Abstract

The invention provides bacillus coagulans TCI803 or a metabolite thereof and application thereof, and the bacillus coagulans TCI803 is deposited in the institute of development of food industry by the treasury legal system, with the deposit number being BCRC910946, and the Germany collection of microorganisms with the deposit number being DSM 33486. The invention further provides application of the bacillus coagulans TCI803 in preparing a composition for stomach health care.

Description

Bacillus coagulans or metabolite thereof and application thereof in stomach health care

Technical Field

The invention relates to Bacillus coagulans, in particular to Bacillus coagulans TCI803(Bacillus coagulans TCI803) and a metabolite thereof, and application thereof in preparing a composition for stomach health care.

Background

The Digestive System (Digestive System) is a combination of vital organs used by the human body to perform, digest, harvest energy and nourish. At present, almost people have various digestive system problems due to abnormal relationship between diet and work and rest.

Among them, the stomach is particularly important and has important functions such as storage, secretion, chemical digestion, etc. The stomach is also typically the organ in the digestive system that is the earliest condition, commonly seen as gastric bloating, stomach cramps, hyperacidity, gastroesophageal reflux, gastroptosis, gastric ulcers, and the like.

Further, it was found that since the discovery of helicobacter pylori in 1983, many clinical and scientific studies have confirmed the association of helicobacter pylori infection with gastritis, gastric ulcer and duodenal ulcer, gastric cancer and gastric mucosa-associated lymphoma. Moreover, the prevalence of H.pylori infection is between about 20% and 80%.

However, the stomach is usually not acute, such as slight epigastric distending pain, burning sensation, hiccups, etc., so most people are accustomed to enduring their pain and tend to get worse.

Disclosure of Invention

In view of the above, how to more simply perform stomach health care is an important object of the inventor. The invention provides bacillus coagulans TCI803 and a metabolite thereof, and application thereof in preparing a composition for stomach health care.

In some embodiments, one Bacillus coagulans TCI803 or metabolite thereof is Bacillus coagulans deposited under accession number BCRC910946 and under accession number DSM 33486.

In some embodiments, the use of Bacillus coagulans or a metabolite thereof for the preparation of a composition for gastric health, wherein the Bacillus coagulans is Bacillus coagulans TCI803(Bacillus coagulousns TCI803), deposited under BCRC910946 and deposited under DSM 33486.

In some embodiments, bacillus coagulans TCI803 is used to inhibit proliferation of helicobacter pylori.

In some embodiments, bacillus coagulans TCI803 is used to reduce helicobacter pylori antibody IgG in blood.

In some embodiments, bacillus coagulans TCI803 is used to protect the stomach wall.

In some embodiments, bacillus coagulans TCI803 is used to enhance gastric mucin production.

In some embodiments, the bacillus coagulans TCI803 is used to reduce the inflammatory response in the stomach.

In some embodiments, bacillus coagulans TCI803 reduces an inflammatory response in the stomach by modulating an inflammatory response-associated gene, wherein the inflammatory response-associated gene includes at least one of a VEGF gene, an IL17A gene, an IL18 gene, an IL16 gene, a TNF- α gene, an IL12A gene, or an IL3 gene.

In some embodiments, Bacillus coagulans TCI803 is used to increase the pepsinogen PGI/PGII ratio.

In some embodiments, the effective dose of bacillus coagulans TCI803 is 5x10⁹CFU/day.

In summary, the Bacillus coagulans TCI803 of any of the examples is deposited in the institute for food industry development (deposited under the accession number BCRC 910946) and the German Collection of microorganisms (deposited under the accession number DSM 33486). In addition, the bacillus coagulans TCI803 and its metabolites according to any embodiment of the present invention can inhibit proliferation of gastric helicobacter pylori, reduce IgG antibodies of helicobacter pylori in blood, promote production of gastric mucin, and reduce expression of genes related to inflammatory reaction, thereby reducing inflammatory reaction in stomach. The bacillus coagulans TCI803 and metabolites thereof in any embodiment of the present invention can be used to prepare compositions for stomach health care, and the compositions can be pharmaceutical compositions or food compositions.

The invention is described in detail below with reference to the drawings and specific examples, but the invention is not limited thereto.

Drawings

FIG. 1 is a graph showing the results of the test for inhibiting helicobacter pylori by Bacillus coagulans TCI 803.

FIG. 2 is a graph showing the results of an experiment in which Bacillus coagulans TCI803 promotes production of gastric mucin.

FIG. 3 is a graph showing the results of experiments on the inhibition of inflammation-associated genes by Bacillus coagulans TCI 803.

FIG. 4 is a graph showing the results of the PGI/PGII ratio of the proproteases in blood in human experiments.

FIG. 5 is a graph showing the results of expression of VEGF gene in blood in human experiments.

FIG. 6 is a graph showing the results of expression of IL17A gene in blood in human experiments.

FIG. 7 is a graph showing the results of human experiment of IgG antibody against helicobacter pylori.

FIG. 8 is a graph showing the results of carbon 13 concentration in human breath.

Wherein, the reference numbers:

a is a round hole

B, bacteriostatic zone

Detailed Description

Bacillus coagulans TCI803(Bacillus coagulans TCI803) is a strain isolated from the highest peak in Albizzia (alpine soil). Bacillus coagulans TCI803 is deposited at the institute for the development of the food industry, by the treasury firm, under the accession number BCRC910946, and at the German Collection of microorganisms, under the accession number DSM 33486.

The Bacillus coagulans TCI803 is a gram-positive Bacillus which can produce lactic acid in Bacillus (Bacillus), can grow in an anaerobic environment, and belongs to facultative anaerobes. Endospores are produced in an environment where growth is not appropriate, and growth stops. For example, spores are produced when the living environment exceeds 50 ℃. When the spore passes through gastric acid to the small intestine, the spore can recover from the spore state and continue to grow and reproduce. Thus, the Bacillus coagulans TCI803 is acid and heat resistant.

In some embodiments, bacillus coagulans TCI803 is used to inhibit proliferation of helicobacter pylori. In some embodiments, bacillus coagulans TCI803 is used to reduce helicobacter pylori antibody IgG in blood.

In some embodiments, bacillus coagulans TCI803 is used to protect the stomach wall. In some embodiments, bacillus coagulans TCI803 is used to enhance gastric mucin production.

In some embodiments, bacillus coagulans TCI803 is used to reduce the inflammatory response of the stomach. In some embodiments, the inflammatory response-associated Gene comprises at least one of a VEGF Gene (Gene ID 7422), an IL17A Gene (Gene ID 3605), an IL18 Gene (Gene ID 3606), an IL16 Gene (Gene ID 3603), a TNF-alpha Gene (Gene ID 7124), an IL12A Gene (Gene ID 3592), or an IL3 Gene (Gene ID 3562).

Among them, the VEGF gene is a VEGF-related gene, which can increase vascular permeability and promote angiogenesis, and the VEGF is often over-secreted along with inflammatory symptoms.

Among them, the IL17A gene is related to IL17A cytokine, and the overexpression of IL-17A cytokine is known to cause the stimulation of many autoimmune inflammations, and is involved in inflammatory reactions, and is related to inflammatory diseases such as ulcer.

Among them, IL18 gene is related to interleukin IL18(interleukin-18) secretion, and interleukin IL18 clinical studies have found that it is related to many inflammatory reactions, such as: allergic, autoimmune or neurological disorders.

Wherein the IL16 gene is related to secretion of interleukin IL16(interleukin-16), and interleukin IL16 mainly acts on auxiliary T cells and is a chemoattractant of CD4 +.

Wherein, the IL12A gene is related to secretion of interleukin IL12(interleukin-12), and the interleukin IL12A mainly acts on activated T cells and natural killer cells and can also promote secretion of interferon gamma (IFN-gamma).

Among them, IL3 gene is related to secretion of interleukin IL3(interleukin-3), and interleukin IL3 mainly stimulates stem cells (stem cells) of bone marrow, and can mature immature leukocyte, increase the amount of Macrophage (Macrophage), and release histamine.

Among them, TNF-alpha gene (tumor necrosis factor-alpha) has the function of regulating immune cells, is more involved in cytokines of systemic inflammation, and is also a member of a plurality of cytokines causing acute phase reaction.

In some embodiments, bacillus coagulans TCI803 is used to increase the pepsinogen PGI/PGII ratio. In some embodiments, the effective dose of bacillus coagulans TCI803 is 5x10⁹CFU/day.

In some embodiments, the aforementioned composition comprises a specific amount of bacillus coagulans TCI 803.

In some embodiments, the composition may be a pharmaceutical. In other words, the pharmaceutical comprises an effective amount of bacillus coagulans TCI 803.

In some embodiments, the aforementioned medicaments may be formulated in a form suitable for enteral, parenteral (parenterally), oral, or topical (topically) administration using techniques well known to those skilled in the art.

In some embodiments, the dosage form for enteral or oral administration may be, but is not limited to, a lozenge (tablet), a tablet (troche), a buccal tablet (dosage), a pill (pill), a capsule (capsule), a dispersible powder (dispersible powder) or fine granules (granules), a solution, a suspension (suspension), an emulsion (emulsion), a syrup (syrup), an elixir (elixir), a slurry (syrup), or the like. In some embodiments, parenteral or topical administration dosage forms may be, but are not limited to, injectables (injections), sterile powders (sterile powders), external preparations (external preparations), or the like. In some embodiments, the administration of the injectate can be subcutaneous (subcutaneous), intradermal (intraepithelial injection), or intralesional (intrafocal injection).

In some embodiments, the aforementioned pharmaceutical may comprise a pharmaceutically acceptable carrier (pharmaceutical acceptable carrier) that is widely used in pharmaceutical manufacturing technology. In some embodiments, the pharmaceutically acceptable carrier can be one or more of the following carriers: solvents (solvent), buffers (buffer), emulsifiers (emulsifying agent), suspending agents (suspending agent), disintegrating agents (decongestant), disintegrating agents (disintegrating agent), dispersing agents (dispersing agent), binding agents (binding agent), excipients (excipient), stabilizers (stabilizing agent), chelating agents (chelating agent), diluents (diluent), gelling agents (gelling agent), preservatives (preserving agent), wetting agents (wetting agent), lubricants (lubricating agent), absorption delaying agents (absorption delaying agent), liposomes (liposome) and the like. The type and amount of carrier selected for use is within the skill and routine skill of one skilled in the art. In some embodiments, the solvent as a pharmaceutically acceptable carrier may be water, physiological saline (normal saline), Phosphate Buffered Saline (PBS), or an aqueous solution containing an alcohol (aqueous dissolution stabilizing alcohol).

In some embodiments, the aforementioned composition may comprise a food product comprising a specific content of bacillus coagulans TCI 803.

In some embodiments, the food product may be a general food, a health food, or a dietary supplement. In other words, the general food, health foods (health foods) or dietary supplements contain an effective dose of Bacillus coagulans TCI 803.

In some embodiments, the food product can be manufactured into a dosage form suitable for oral administration using techniques well known to those skilled in the art. In some embodiments, the aforementioned generic food product may be the food product itself or another food additive (food additive). In some embodiments, the generic food product may be, but is not limited to: beverages (leafages), fermented foods (fermented foods), bakery products (bakery products) or sauces.

The characteristics, mode of use and microbiological properties of the strain of Bacillus coagulans TCI803, which differ from the same known strain, are further described below.

The first embodiment is as follows: strain identification

First, the isolated strain isolated from the highest peak soil in Albizzia was subjected to strain identification. The 16S ribosomal gene (16SrDNA) sequence (SEQ ID NO:1) of this isolate was obtained by Polymerase Chain Reaction (PCR). Then, the sequence of SEQ ID NO. 1 was aligned with the 16S ribosomal gene (16SrDNA) of other Bacillus coagulans (shown in Table I) by the website of National Center for Biotechnology Information (NCBI), and the similarity of the 16SrDNA sequence of the isolate and other Bacillus coagulans (shown in Table I) was 96.86% to 96.78%. Therefore, this isolated strain was named Bacillus coagulans TCI 803.

Watch 1

Example two: bacillus coagulans TCI803 cultivation experiment

First, frozen Bacillus coagulans TCI803 was inoculated in MRS medium (BD Difco)^TMLactobacillus MRS Broth, DF 0881-17-5). Herein, bacillus coagulans TCI803 solution with an inoculation ratio of 10% was mixed with 90% medium.

MRS culture after completion of inoculation was carried out at 37 ℃ for 16 hours and single colony formation was confirmed to obtain activated Bacillus coagulans TCI 803.

And centrifuging the MRS culture medium containing the bacteria for 15 minutes at the rotating speed of 5000RMP, and filtering the bacteria by a filter screen of 0.22 micrometer (mum) after centrifugation to obtain the supernatant of the bacillus coagulans TCI 803. Here, the bacillus coagulans TCI803 supernatant contains the culture medium and the metabolites of bacillus coagulans TCI 803.

Example three: growth experiment of gastric helicobacter pylori

Further, the effect of Bacillus coagulans TCI803 on helicobacter pylori was confirmed. In this case, the medium used in the activation step was BD medium prepared from BHI broth plus 5% Sheep Blood.

First, the activation step of helicobacter pylori of the stomach was performed. The activation was accomplished by inoculating 10% of Helicobacter pylori solution to 90% of the BD medium, which was purchased from the institute of food industry development (BCRC 910946), and culturing the mixture at 37 ℃ for 72 hours.

Next, evaluation of the growth effect of bacillus coagulans TCI803 on helicobacter pylori of the stomach was performed. The medium used in the evaluation step was BD Trypticase Soy Agar II plus 5% Sheep Blood (Sheep Blood)

In the experimental group, 100 μ L of the activated helicobacter pylori was applied to a BD culture medium, a part of the medium was removed from the BD culture medium to form a round hole a, 10 μ L (μ L) of the supernatant of TCI803 of experimental two activated bacillus coagulans was filled in the round hole a, and the round hole a was cultured at 37 ℃ for 72 hours to observe whether a zone of inhibition occurred.

The control group is prepared by coating activated helicobacter pylori on BD culture medium, removing part of the culture medium from BD culture medium to form a 5mm round hole A, culturing at 37 deg.C for 72 hr, and observing whether the inhibition zone appears.

Please refer to fig. 1. In the figure, the experiment group shows an obvious inhibition zone B, while the control group does not show the inhibition zone. And the diameter of the inhibition zone B of the actual measurement experiment group can reach 23mm, which represents that the inhibition zone B has obvious effect of inhibiting the helicobacter pylori in the stomach. Therefore, the bacillus coagulans TCI803 can effectively inhibit the growth of the helicobacter pylori and achieve the stomach health care capability by inhibiting the growth of the helicobacter pylori.

Example four: gastric mucin secretion test

Sufficient gastric mucin can protect the stomach wall. Human gastric epithelial cells (hereinafter abbreviated as gastric epithelial cells) were used in this experiment, purchased from the American Type Culture Collection (American Type Culture Collection,

) AGS cell line of (ATCC CRL-1739)^TM)。

First, a 96-well plate was inoculated with 1X 10 cells per well⁴Gastric epithelial cells and 0.2mL of REE Medium (REE Medium) were cultured for 16 hours. Here, the re medium contained 10% fetal bovine serum, 1% penicillin/streptomycin and the rest RPMI 1640 basal medium.

The cultured gastric epithelial cells are divided into two groups: experimental and control groups.

Control group: without any treatment, the cells were incubated at 37 ℃ for 48 hours.

Experimental groups: the supernatant of Bacillus coagulans TCI803 prepared in experiment two was added to the REE medium at 0.5%, and cultured at 37 ℃ for 48 hours.

After each set of cells was cultured for 48 hours, 100. mu.l of the medium (liquid) was taken from each set and placed in a 1.5mL microcentrifuge tube for use. Next, the gastric mucin content was determined using an ELISA enzyme immunoassay kit (available from Elapscience, Inc., model Cat. E-EL-H2280).

It should be noted that the plots shown in the figures mentioned below are presented in relative terms, where the standard deviation is calculated using the STDEV formula of Excel software, and analyzed in the Excel software for statistically significant differences using the single Student t-test (Student t-test). In the drawings, the term "indicates a p value of less than 0.05, the term" indicates a p value of less than 0.01, and the term "indicates a p value of less than 0.001. When "more", the representation is statistically significantly different.

Refer to fig. 2. When the content of gastric mucin measured by the control group was regarded as 100 (i.e., 100%), the content of gastric mucin in the experimental group relative to the control group was 188.13 (i.e., 188%), which represents that the gastric mucin secretion amount of the experimental group was 1.8 times that of the control group. That is, after the treatment of the Bacillus coagulans TCI803, gastric mucin secretion of gastric cells can be promoted to 88%. And statistically, the p value of the experimental group relative to the control group is less than 0.0001, which represents a very significant difference.

From this, it was found that the secretion amount of gastric mucin in gastric epithelial cells was significantly increased when the gastric epithelial cells were treated with Bacillus coagulans TCI 803. Gastric mucins include water, electrolytes, lipids, proteins and polypeptide hormones. Besides, gastric mucin lubricates food, and forms a protective film by the jelly-like property of the gastric mucin to protect cells on the gastric wall from being eroded by an acidic environment, so that the important effect of stomach health care is achieved.

Example five: inhibition of genes associated with inflammation

Human gastric epithelial cells (hereinafter abbreviated as gastric epithelial cells) were used in this experiment, purchased from the American Type Culture Collection (American Type Culture Collection,

) AGS cell line of (ATCC CRL-1739)^TM). In this example, Lipopolysaccharide (LPS) is used to induce the inflammatory response of the cell.

First, 6-well plates were inoculated at 1X 10-positions per well⁵Gastric epithelial cells and 2mLREE Medium (REE Medium) and cultured for 24 hours. Here, the re medium contained 10% fetal bovine serum, 1% penicillin/streptomycin, and the remainder RPMI 1640 basal medium.

The cultured gastric epithelial cells were divided into two groups: experimental and control groups.

And (3) controlling the groups: 0.025mg/ml lipopolysaccharide was added to the REE medium, followed by culture at 37 ℃ for 24 hours.

Experimental groups: 0.025mg/ml lipopolysaccharide and 0.5% of TCI803 supernatant of Bacillus coagulans prepared in experiment two were added, followed by culture at 37 ℃ for 24 hours.

After each group of cells was cultured for 24 hours, the cells of the experimental group and the control group after the culture were disrupted with cell lysate to form two groups of cell solutions, respectively. Next, RNA in the two cell solutions was collected using RNA extraction reagent kits (purchased from Geneaid, taiwan, Lot No. fc24015-G). Then, 2000 nanograms (ng) of RNA extracted from each group was used as a template by

III reverse transcriptase (from Invitrogene, USA, No. 18080-051) was used to perform reverse transcription with primer binding to generate the corresponding cDNA. Then, the two sets of reverse transcription products were subjected to quantitative Real-Time reverse transcription polymerase chain reaction (quantitative Real-Time reverse transcription polymerase chain reaction) using ABI StepOnePlusTM Real-Time PCR system (Thermo Fisher Scientific, USA) and KAPA SYBR FAST (available from Sigma, USA, No. 38220000000) as primers in the combinations of Table II to observe the expression of genes of PBMC cells of the experimental group and the control group. The quantitative real-time reverse transcription polymerase chain reaction instrument sets the conditions of reaction at 95 ℃ for 1 second and 60 ℃ for 20 seconds for a total of 40 cycles, and performs gene quantification by using the 2-delta Ct method. In this case, the quantitative real-time RT-PCR using cDNA can indirectly quantify the mRNA expression level of each gene, and the expression level of the protein encoded by each gene can be estimated, as shown in FIG. 3.

Watch 2

Refer to fig. 3. When the expression level of the IL-18 gene in the control group is regarded as 1 (i.e., 100%), the expression level of the IL-18 gene in the experimental group relative to the control group is 0.79 (i.e., 79%), i.e., the expression level of the IL-18 gene in the experimental group is significantly reduced compared to the control group.

When the expression level of the TNFa gene in the control group is regarded as 1 (i.e., 100%), the expression level of the TNFa gene in the experimental group relative to that in the control group is 0.81 (i.e., 81%), i.e., the expression level of the TNFa gene in the experimental group is significantly reduced compared to that in the control group.

When the expression level of the IL-16 gene in the control group is regarded as 1 (i.e., 100%), the expression level of the IL-16 gene in the experimental group relative to the control group is 0.80 (i.e., 80%), i.e., the expression level of the IL-16 gene in the experimental group is significantly reduced compared to the control group.

When the expression level of IL-12a gene in the control group is regarded as 1 (i.e., 100%), the expression level of IL-12a gene in the experimental group relative to that in the control group is 0.77 (i.e., 77%), i.e., the expression level of IL-12a gene in the experimental group is significantly reduced compared to that in the control group.

When the expression level of the IL-3 gene in the control group is regarded as 1 (i.e., 100%), the expression level of the IL-3 gene in the experimental group relative to the control group is 0.86 (i.e., 86%), i.e., the expression level of the IL-3 gene in the experimental group is significantly reduced compared to the control group.

It should be noted that the plots shown in the figures mentioned below are presented in relative terms, where the standard deviation is calculated using the STDEV formula of Excel software, and analyzed in the Excel software for statistically significant differences using the single Student t-test (Student t-test). In the figures, "x" represents a p value of less than 0.05, "x" represents a p value of less than 0.01, and "x" represents a p value of less than 0.001. As more "x", the more significant the statistical difference.

And statistically, the p value of the gene expression of each group is less than 0.05, which means that each group has significant difference. Among them, the expression level of IL-16 gene and the expression level of IL-12a gene have higher difference.

From this, it was found that when gastric epithelial cells were simulated by inflammation, expression of the inflammatory response-associated gene was significantly reduced by treatment with Bacillus coagulans TCI 803. That is, the Bacillus coagulans TCI803 can effectively reduce the excessive reaction of nonspecific immunity generated by gastric epithelial cells. Thereby avoiding the damage of the stomach caused by chronic inflammation for a long time and achieving the health care capability of the stomach.

Example six: human body test

Further, the direct effect of the bacillus coagulans TCI803 on human body was confirmed.

One tablet containing 5X10 is administered to 6 subjects per day⁹CFU (i.e. 5x 10)⁹CFU/cap) live bacteria capsule of bacillus coagulans TCI803 (50 mg/day, meaning that one capsule weighing 50 mg per day is taken) and continuously for 4 weeks. Blood of each subject was collected before the first administration (hereinafter, referred to as "control group" at week 0) and after 4 weeks administration (hereinafter, referred to as "experimental group" at week 4) to perform blood tests (test by clinical trial), and each subject was asked to perform questionnaire filling and breath test.

In this regard, 6 subjects had a condition of long-term gastric distress, including one diagnosed with H.pylori infection.

(I) Propepsinogen PGI/PGII ratio in blood

The Pepsinogen (Pepsinogen) in blood includes two kinds of Pepsinogen I (Pepsinogen I) and Pepsinogen II (Pepsinogen II). The detection of the amounts of pepsinogen PGI and pepsinogen PGII in the blood can reflect the number of gastric mucosa glands and cells, and also provide the secretion function status of the gastric mucosa, which is an index for monitoring the health status of the gastric mucosa. For example, a PGI/PGII ratio of <3, and a pepsinogen PGI amount of <70ug/ml generally indicates a chronically inflamed state of the stomach. That is, generally speaking, the lower the PGI/PGII ratio, the higher the gastric inflammatory condition.

Here, the blood of 6 subjects collected at week 0 and week 4 was subjected to a clinical laboratory to determine the values of pepsinogen PGI and pepsinogen PGII in the blood, and the ratio of pepsinogen PGI/PGII was calculated.

The average values of the values detected for pepsinogen PGI and pepsinogen PGII in six subjects are shown in table three below.

Watch III

	Control group (week 0)	Experimental group (week 4)
			Pepsinogen PGI	68.37	80.13
Pepsinogen PGII	6.65	6.26

The PGI/PGII ratio was plotted according to the above values as shown in FIG. 4. As shown in FIG. 4, the mean ratio of PGI/PGII in blood at week 0 was 12.12 and the mean ratio of PGI/PGII in blood at week 4 was 14.02 among the 6 subjects. Among them, the p-value of the experimental group was less than 0.05 relative to the control group, meaning that there was a significant difference.

It can be seen that the average PGI/PGII ratio of the subjects significantly increased by 1.91 after taking the capsules containing the TCI803 of bacillus coagulans. Therefore, the bacillus coagulans TCI803 can reduce the inflammation state of the stomach, improve the health degree of the gastric mucosa and effectively achieve the health care of the stomach.

Expression of VEGF Gene and IL17A Gene

In this case, the blood of 6 subjects collected at week 0 and week 4 was assayed for VEGF value and IL17A value, and RNA in two sets of cell solutions was collected using an RNA extraction reagent kit (purchased from Geneaid, taiwan, Lot No. fc24015-G). Then, 2000 nanograms (ng) of RNA extracted from each group was used as a template by

III reverse transcriptase (from Invitrogene, USA, No. 18080-051) was used to perform reverse transcription with primer binding to generate the corresponding cDNA. Then, the two sets of reverse transcription products were subjected to quantitative Real-Time reverse transcription polymerase chain reaction (quantitative Real-Time reverse transcription polymerase chain reaction) using ABI StepOnePlusTM Real-Time PCR system (Thermo Fisher Scientific, USA) and KAPA SYBR FAST (available from Sigma, USA, No. 38220000000) as primers in the combinations of Table II to observe the expression of genes of PBMC cells of the experimental group and the control group. The quantitative real-time reverse transcription polymerase chain reaction instrument sets the conditions of reaction at 95 ℃ for 1 second and 60 ℃ for 20 seconds for a total of 40 cycles, and performs gene quantification by using the 2-delta Ct method. Here, the quantitative real-time reverse transcription polymerase chain reaction (RT-PCR) using cDNA can indirectly quantify the mRNA expression level of each gene, andthe relative expression amounts of the proteins encoded by the VEGF gene and the IL17A gene were estimated.

Refer to fig. 5. Fig. 5 shows that when the relative expression amount of the VEGF gene of the control group is regarded as 1 (i.e., 100%), the relative expression amount of the VEGF gene of the experimental group with respect to the control group is 0.56 (i.e., 56%), that is, the relative expression amount of the VEGF gene of the experimental group is significantly reduced with respect to the control group.

It should be noted that the plots shown in the figures mentioned below are presented in relative terms, where the standard deviation is calculated using the STDEV formula of Excel software, and analyzed in the Excel software for statistically significant differences using the single Student t-test (Student t-test). In the drawings, the term "indicates a p value of less than 0.05, the term" indicates a p value of less than 0.01, and the term "indicates a p value of less than 0.001. As more "x", the more significant the statistical difference.

The expression of VEGF in the blood is often considered as the index of inflammation of the digestive tract (intestinal inflammation), meaning that VEGF is an inflammatory factor of the digestive tract. From the above results, it was found that the expression of inflammatory response factors in digestive tracts was significantly reduced after administration of Bacillus coagulans TCI803, and the p-value was statistically less than 0.01, which was significantly different. That is, the bacillus coagulans TCI803 can effectively avoid the damage of the stomach caused by chronic inflammation for a long time, so as to achieve the stomach health care capability.

Refer to fig. 6. The graph shows that when the relative expression amount of IL17A gene of the control group is regarded as 1 (i.e., 100%), the relative expression amount of the experimental group with respect to IL17A gene of the control group is 0.71 (i.e., 71%), that is, the expression amount of IL17A gene of the experimental group is significantly reduced with respect to the control group.

The expression of IL17A in blood is often considered as the index of gastric inflammation, meaning that IL17A is a gastric inflammatory factor. From the above results, it was found that the expression level of the gastric inflammatory response factor was significantly reduced after administration of Bacillus coagulans TCI 803. That is, the bacillus coagulans TCI803 can effectively avoid the damage of the stomach due to chronic inflammation for a long time, so as to achieve the stomach health care capability.

(III) antibodies against blood helicobacter pylori

The test is directed to the test subject who has confirmed the diagnosis of gastric helicobacter pylori among the test subjects, and detects the variation of the antibody IgG of the helicobacter pylori in the blood, and is one of non-invasive helicobacter pylori detection methods. The amount of H.pylori antibody IgG in the blood is detected in response to the amount of H.pylori in vivo. This means that if there is a high correlation between H.pylori and antibody IgG in the stomach, the amount of H.pylori antibody IgG in the blood will generally decrease significantly if the patient is treated with an antibacterial agent.

In this experiment, subjects with confirmed gastric helicobacter pylori collected blood of subjects with confirmed gastric helicobacter pylori at week 0 and week 4, and were assigned to clinical tests to examine IgG values (in EIU) of helicobacter pylori antibody in all blood samples.

As shown in FIG. 7, the subjects diagnosed with gastric H.pylori had an IgG H.pylori antibody value of 61.4EIU in blood at week 0 and an IgG H.pylori antibody value of 55.7EIU in blood at week 4.

It was found that after taking the capsule containing the TCI803 of Bacillus coagulans, the IgG value of the antibody against helicobacter pylori in the blood of the subjects who confirmed the diagnosis of helicobacter pylori in the stomach was significantly reduced by 5.7. Therefore, the bacillus coagulans TCI803 is known to inhibit the proliferation of helicobacter pylori in human body, and effectively achieve stomach health care.

(IV) carbon 13 concentration test

The experiment aims at the test subject who confirms the diagnosis of the gastric helicobacter pylori, detects the variation of carbon 13(C-13) in the expired air, and is one of non-invasive helicobacter pylori detection methods. The execution steps are that the subject drinks the water solution containing the carbon 13 isotope, the gas exhaled by the subject is collected after 20 minutes, and the content of the carbon 13 in the exhaled gas is detected. That is, the higher the carbon-13 content in the exhaled breath, the higher the number of H.pylori in the body.

The test subject for determining gastric helicobacter pylori in the experiment entrusts the test of the medical examination of the human beings at week 0 and week 4 to detect the content of carbon 13 in the exhaled air according to the same steps.

As shown in FIG. 8, subjects confirmed to diagnose H.pylori detect the carbon 13 content in exhaled breath at week 0 at 48.7 and 27.8 at week 4.

It is thus clear that the results of the carbon 13 test in subjects with established diagnosis of H.gastrodiae are significantly reduced by 20.9 after taking the capsules containing the TCI803 of B.coagulans. Therefore, the bacillus coagulans TCI803 is known to inhibit the proliferation of helicobacter pylori in human body, and effectively achieve stomach health care.

(V) gastric-related questionnaires

First, the questionnaire was used to investigate the gastrointestinal tract status, and the following table four was used for the survey and scoring. Wherein each score in the first part represents the severity of each symptom, 0 is normal, 1 is slightly severe, 2 is slightly severe, 3 is medium, 4 is slightly severe, 5 is severe, and 6 is very severe.

Watch four

The same questionnaire content (as the first part of table four above) was used for the present survey on 6 subjects, and the above symptoms were scored at week 0 and week 4, respectively, as follows for epigastric fullness and eructation: the average of the scores given for 6 recipients of five items of hiccup, flatulence (gas), halitosis, and abdominal distension are given in table five below.

Watch five

	Control group (week 0)	Experimental group (week 4)
			Feeling of upper abdominal fullness	1.2	0.5
Belching: hiccup	1.2	0.7
			Flatulence (gas)	1.8	0.7
Halitosis	0.8	0.2
			Abdominal distention	1.2	0.5

As can be seen from the fifth table, the average score of each item decreased, meaning that each subject, on average, had a significant improvement in the symptoms. The feeling of discomfort of the whole stomach is reduced, so as to achieve the health care capability of the stomach.

In addition, the final excretion state of the digestive tract usually represents the complete function of the digestive tract. Therefore, the second part of the questionnaire is two items of frequency of defecating and time required for defecating of 6 subjects, and the above symptoms are selected at week 0 and week 4, and the statistical conditions of 6 subjects for the frequency of defecating are shown in table six below.

Watch six

	Control group (week 0)	Experimental group (week 4)
			Is not provided with	2 persons	2 persons
Is rarely used	1 person	3 persons
			Sometimes in times	2 persons	1 person
Often times, the heat exchanger is not used for heating	1 person	0 person
			Always is	0 person	0 person

From the sixth table, 50% of the subjects who have failed to successfully defecate before taking the TCI803 of bacillus coagulans are sometimes or frequently defecated, while 80% or more of the subjects who have taken the TCI803 of bacillus coagulans can be found to be not or rarely defecated. Meaning that on average, each subject had a significant improvement in the state of defecation, improving overall digestive function of the digestive tract.

The statistics for the time required to defecate for the 6 recipients are given in table seven below.

Watch seven

	Control group (week 0)	Experimental group (week 4)
			Less than 5 minutes	1 person	4 persons
5 to 10 minutes	3 persons	2 persons
			10 to 20 minutes	2 persons	0 person
20 to 30 minutes	0 person	0 person
			More than 30 minutes	0 person	0 person

As can be seen from the above-mentioned TABLE VII, with respect to the effect of the change in defecation time, more than 30% of the subjects required a longer defecation time before the administration of the Bacillus coagulans TCI803, while 100% of the subjects achieved complete defecation within a short time after the administration of the Bacillus coagulans TCI 803. Meaning that each subject has obvious improvement on defecation state on average, and the digestion condition of the digestive tract is improved as a whole.

In conclusion, the bacillus coagulans TCI803 according to any one embodiment of the present invention can be used for preparing a composition for stomach health care. Moreover, the bacillus coagulans TCI803 can inhibit the proliferation of helicobacter pylori in the stomach, and reduce the IgG antibody of the helicobacter pylori in blood. In some embodiments, bacillus coagulans TCI803 may promote production of gastric mucin, thereby protecting the stomach wall. In some embodiments, bacillus coagulans TCI803 may reduce the expression of inflammatory response-associated genes, thereby reducing the inflammatory response in the stomach. In some embodiments, the effective dose of bacillus coagulans TCI803 is 5x10⁹The PGI/PGII ratio of pepsinogen can be improved under CFU/day. Thus, in some embodiments, bacillus coagulans TCI803 and metabolites thereof may be used to prepare a composition for stomach wellness, and the composition may be a pharmaceutical composition or a food composition.

Although the present invention has been described with reference to the preferred embodiments, it will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention as defined by the appended claims.

The present invention is capable of other embodiments, and various changes and modifications can be made by one skilled in the art without departing from the spirit and scope of the invention.

[ deposit of biological Material ]

The institute for food industry development of the treasury corporation (taiwan); 24 days 10 and 2019; register number: BCRC 910946.

German collection of microorganisms (germany); year 2020, 3, 27; with the registration number DSM 33486.

[ sequence listing ]

<110> Dajiang biomedical corporation Ltd

<120> Bacillus coagulans or metabolite thereof and use thereof for preparing composition for stomach health care

<130> N/A

<150> 62/892081

<151> 2019-08-27

<160> 11

<170> PatentIn version 3.5

<210> 1

<211> 1176

<212> DNA

<213> Bacillus coagulans TCI803

<400> 1

accggggcgg ggtgcctaat acatgcaagt cgtgcggacc ttttaaaagc ttgcttttaa 60

aaggttagcg gcggacgggt gagtaacacg tgggcaacct gcctgtaaga tcgggataac 120

gccgggaacc ggggctaata ccggatagtt ttttcctccg catggaggaa aaaggaaaga 180

cggcttttgc tgtcacttac agatgggccc gcggcgcatt agctagttgg tggggtaacg 240

gctcaccaag gcaagatgcg tagccgacct gagagggtga tcggccacat tgggactgag 300

acacggccca aactcctacg ggaggcagca gtagggaatc ttccgcaatg gacgaaagtc 360

tgacggagca acgccgcgtg atgaagaagg ccttcgggtc gtaaaactct gttgccgggg 420

aagaacaagt gccgttcgaa cagggcggcg ccttgacggt acccggccag aaagccacgg 480

ctaactacgt gccagcagcc gcggtaatcg taggtggcaa gcgttgtccg gaattattgg 540

gcgtaaagcg cgcgcaggcg gcttcttaag tctgatgtga aatcttgcgg ctcaaccgca 600

agcggtcatt ggaaactggg aggcttgagt gcagagagga gagtggaatt ccacgtgtag 660

cggtgaaatg cgtagagatg tggaggaaca ccagtggcga aggcggctct ctggtctgta 720

actgacgctg aggcgcgaaa gcgtggggag caaacaggat taataccctg gtagtccacg 780

ccgtaaacga tgagtgctaa gtgttagagg gtttccgccc tttagtgctg cagctaacgc 840

attaagcact ccgcctgggg agtacggccg caaggctgaa actcaaagga ttgacggggg 900

cccgcacaag cggtggagca tgtggtttaa ttcggaagca acgcgaagaa ccttaccagg 960

tcttgacatc tctgacctcc tggagacagg cttccccttc gggggacaga gtgacaggtg 1020

gtgcatgatg tcgtcagctc gtgtcgtgag atgtggtagt cccgcacgag cgcaccctga 1080

ctagtggcag catcagtggg cactctaagg tactgcgtga cacgagaagg tgggatgacg 1140

tcacatcatg gccttatgac tgggctacca ctgcta 1176

<210> 2

<211> 23

<212> DNA

<213> Artificial Sequence

<220>

<223> IL18-F

<400> 2

tcttcattga ccaaggaaat cgg 23

<210> 3

<211> 21

<212> DNA

<213> Artificial Sequence

<220>

<223> IL18-R

<400> 3

tccggggtgc attatctcta c 21

<210> 4

<211> 20

<212> DNA

<213> Artificial Sequence

<220>

<223> IL16-F

<400> 4

gccgaagacc cttgggttag 20

<210> 5

<211> 19

<212> DNA

<213> Artificial Sequence

<220>

<223> IL16-R

<400> 5

gctggcattg ggctgtaga 19

<210> 6

<211> 22

<212> DNA

<213> Artificial Sequence

<220>

<223> TNF-α-F

<400> 6

cctctctcta atcagccctc tg 22

<210> 7

<211> 21

<212> DNA

<213> Artificial Sequence

<220>

<223> TNF-α-R

<400> 7

gaggacctgg gagtagatga g 21

<210> 8

<211> 21

<212> DNA

<213> Artificial Sequence

<220>

<223> IL12A-F

<400> 8

cacctcagtt tggccagaaa c 21

<210> 9

<211> 19

<212> DNA

<213> Artificial Sequence

<220>

<223> IL12A-R

<400> 9

ggcctccact gtgctggtt 19

<210> 10

<211> 18

<212> DNA

<213> Artificial Sequence

<220>

<223> IL3-F

<400> 10

cctgccgatc caaacatg 18

<210> 11

<211> 22

<212> DNA

<213> Artificial Sequence

<220>

<223> IL3-R

<400> 11

ccccattgag gttgttgaag tc 22

Claims

1. A Bacillus coagulans TCI803 or a metabolite thereof, wherein the Bacillus coagulans TCI803 is a Bacillus coagulans deposited under the accession number DSM 33486.

2. Use of Bacillus coagulans TCI803 or a metabolite thereof for the preparation of a composition for gastric health, wherein the Bacillus coagulans TCI803 is Bacillus coagulans TCI803, deposited under the accession number DSM 33486.

3. The use of claim 2, wherein the bacillus coagulans TCI803 is used to inhibit proliferation of helicobacter pylori.

4. The use of claim 3, wherein the Bacillus coagulans TCI803 is used for reducing IgG helicobacter pylori antibodies in blood.

5. The use according to claim 2, wherein the bacillus coagulans TCI803 is used to protect the stomach wall.

6. The use of claim 5, wherein the Bacillus coagulans TCI803 is used for promoting gastric mucin production.

7. The use of claim 2, wherein the bacillus coagulans TCI803 is used to reduce the inflammatory response of the stomach.

8. The use of claim 7, wherein the Bacillus coagulans TCI803 reduces the inflammatory response of stomach by modulating inflammatory response-related genes comprising at least one of VEGF gene, IL17A gene, IL18 gene, IL16 gene, TNF- α gene, IL12A gene or IL3 gene.

9. The use of claim 2, wherein the bacillus coagulans TCI803 is used to increase the pepsinogen PGI/PGII ratio.

10. The use according to any one of claims 2 to 9, wherein the effective dose of bacillus coagulans TCI803 is 5x10⁹CFU/day.