KR102629519B1 - Bacillus amyloliquefaciens strain for improving blood levels of TMAO and TMA elevated by high protein and high choline intake - Google Patents

Abstract

The present invention relates to the novel Bacillus amyloliquefaciens Bacillo221003 strain and its use. The novel Bacillus amyloliquefaciens Bacillo221003 strain of the present invention exhibits excellent TMA and TMAO decomposition ability. Therefore, the strain of the present invention can effectively reduce TMA and TMAO concentrations in a subject. The new strains of the present invention include, for example, TMA-related diseases such as trimethylaminuria; or for the prevention, improvement or treatment of TMAO-related diseases, such as, for example, atherosclerosis and chronic kidney disease.

Description

Bacillus amyloliquefaciens strain for improving blood levels of TMAO and TMA elevated by high protein and high choline intake}

The present invention relates to a novel Bacillus amyloliquefaciens strain with high TMA and TMAO decomposition ability and its use. Specifically, Bacillus amyloliquefaciens has TMA and TMAO decomposition ability and has an accession number of KCTC 15313BP. Faciens ( Bacillus amyloliquefaciens ) Bacillo221003 strain and its uses.

Recently, as it has been reported that high-protein diets are helpful for weight loss (Clifton, P. British Journal of Nutrition, 108(S2), S122-S129, (2012)), the number of people trying high-protein diets has increased. A high choline diet has also been reported to have beneficial effects such as improving cognitive function and promoting liver health (Wiedeman AM, et al., Nutrients. 16;10(10):1513. doi: 10.3390/nu10101513. PMID: 30332744; PMCID: PMC6213596, Oct (2018)). However, if you consume a lot of high-choline, high-protein diets, TMA and TMAO may accumulate excessively.

Trimethylamine (TMA) is a volatile tertiary aliphatic amine that can be consumed directly by eating foods containing TMA or by eating foods containing TMA precursors such as trimethylamine-N-oxide (TMAO), choline, and L-carnitine (proteins, choline-containing foods). ) is a substance produced in the body when ingested. TMA causes a 'fishy' smell at low concentrations, and when present at high concentrations, it smells like ammonia and can cause necrosis of the mucous membrane ("Trimethylamine [MAK Value Documentation, 1983]", The MAK-Collection for Occupational Health and Safety, Wiley-VCH Verlag GmbH & Co. KGaA, 27 October 2014, pp. 1-9). It is known that trimethylaminuria can be caused when TMA decomposition enzyme is deficient due to a genetic defect or when TMA accumulates in the body due to excessive intake of choline or L-carnitine (Falony G, et al., Annu. Rev . Microbiol. 69: 305-321 (2015); Gaci N, et al., World J. Gastroenterol. 20 (43): 16062-16078 (November 2014)).

TMAO (Trimethylamine N-oxide) is a small organic compound of the amine oxide series with a molecular weight of 75.1 daltons. TMAO has been known to marine biologists for a long time, but recently began to receive attention when its association with human cardiovascular disease was reported (Koeth, R.A., et al., Nat. Med. 2013, 19, 576-585). TMAO is known to be produced by metabolizing TMA in the liver, and has been reported to be a proatherogenic substance that can accelerate atherosclerosis (Gaci N, et al., World J. Gastroenterol. 20 (43): 16062-16078 (November 2014).

Meanwhile, WHO defines probiotics as live microorganisms that are beneficial to the health of the host when administered in an appropriate amount (Nat Rev Gastroenterol Hepatol 11, 506-514 (2014)). Probiotics act as a supplement to the host's gut microflora and have been reported to be effective in improving intestinal barrier function and regulating the host's immune system (Microb Ecol Health Dis 26, 25877 (2015)).

Meanwhile, various strains such as Bacillus amyloliquefaciens and Bacillus subtilis have been reported as strains that can be used as probiotics (Bai, J., et al., J Animal Sci Biotechnol 13, 7 (2022 )). The Bacillus amyloliquefaciens has been reported to be the same species as Bacillus velezensis and Bacillus methylotrophicus (Ministry of Food and Drug Safety Raw Material List Annex 1-368p.; Wang LT, et al. ., Int J Syst Evol Microbiol. 2008 Mar;58(Pt 3):671-5. doi: 10.1099/ijs.0.65191-0. PMID: 18319476; KCTC Biological Resources Research Center velezensis synonym, https://kctc.kribb .re.kr/collection/view?sn=13012).

Under this technical background, the present inventors made diligent efforts to find a method to prevent the accumulation of TMA and TMAO in the body. As a result, they identified a novel Bacillus amyloliquefaciens strain with excellent TMA or TMAO decomposition ability and transferred it to the Korea Center for Biological Resources at KCTC. It was deposited with the deposit number of 15313BP. In addition, when the new strain Bacillus amyloliquefaciens KCTC 15313BP was administered to a subject, it was confirmed that the concentration of TMA and TMAO in blood and feces was effectively reduced, and the present invention was completed.

The above information described in this background section is only for improving the understanding of the background of the present invention, and therefore does not include information that constitutes prior art already known to those skilled in the art to which the present invention pertains. It may not be possible.

The purpose of the present invention is to provide a novel strain with excellent TMA and TMAO degrading ability.

Another object of the present invention is to provide a use of the above strain for preventing, improving, or treating TMA-related diseases or TMA0-related diseases.

In order to achieve the above object, the present invention provides Bacillus amyloliquefaciens Bacillo221003 strain having an accession number of KCTC 15313BP.

The present invention also provides a composition for decomposing TMA or TMAO containing the above strain as an active ingredient.

The present invention also provides a food composition containing the above strain as an active ingredient.

The present invention also provides a pharmaceutical composition for preventing or treating TMA-related diseases or TMAO-related diseases containing the above strain as an active ingredient.

The present invention also provides a method for preventing or treating a TMA-related disease or TMAO-related disease, comprising administering the strain to a subject.

The present invention also provides the use of the above strain for preventing or treating TMA-related diseases or TMAO-related diseases.

The present invention also provides use of the strain for producing a pharmaceutical composition for preventing or treating TMA-related diseases or TMAO-related diseases.

The new Bacillus amyloliquefaciens Bacillo221003 strain of the present invention exhibits excellent TMA and TMAO decomposition ability. Therefore, the strain of the present invention can effectively reduce TMA and TMAO concentrations in a subject. The new strains of the present invention include, for example, TMA-related diseases such as trimethylaminuria; or for the prevention, improvement or treatment of TMAO-related diseases, such as, for example, atherosclerosis and chronic kidney disease.

Figure 1 is a schematic diagram showing the absorption and biosynthetic pathways of TMA and TMAO, and the prevention of accumulation of TMAO and TMA in the body through the Bacillo221003 strain of the present invention.
Figure 2 is a schematic diagram showing the decomposition path of TMA and TMAO.
Figure 3 is a graph showing the quantitative measurement results (in vitro) of TMA resolution. 5mM of TMA was added to the medium, cultured with the strain, and the TMA concentration was quantitatively measured by measuring the absorbance at a wavelength of 410 nm using an enzyme-linked immunosorbent analyzer.
Figure 4 is a graph showing the results of quantitatively measuring (in vitro) the TMA decomposition ability of other representative strains, including Bacillus amyloliquefaciens and belegensis, and comparing them with the Bacillo221003 strain of the present invention. The strains used in the experiment were Bacillus belegensis CR-502 strain (accession number KCTC 1301), Bacillus belegensis FZB42 strain (accession number KCTC 1341), and Bacillus amyloliquefaciens NRRL B-14393 strain (accession number KCTC 1660). ), Bacillus amyloliquefaciens Bs-10 strain (accession number KCTC 1104), and Bacillus amyloliquefaciens Bacillo221003 strain of the present invention (accession number KCTC 15313BP). Bacillus amyloliquefaciens and Bacillus velegensis can be understood to refer to the same species (Ministry of Food and Drug Safety Raw Material List Annex 1-368p.; Wang LT, et al., Int J Syst Evol Microbiol. 2008 Mar;58 (Pt 3):671-5. doi: 10.1099/ijs.0.65191-0. PMID: 18319476.; KCTC Biological Resources Research Center velezensis synonym, https://kctc.kribb.re.kr/collection/view?sn= 13012).
Figure 5 is a graph showing TMA and TMAO levels in feces and blood measured for 6 weeks while feeding a high choline diet (containing 1% choline) to 5-week-old female C57BL/6J mice. Figure 5A shows TMA and TMAO levels in feces, and Figure 5B shows TMA and TMAO levels in blood.
Figure 6 is a graph showing TMA and TMAO levels in feces after 6 weeks when a high choline diet and strains were simultaneously fed. The strains used in the experiment were Bacillus belegensis CR-502 strain (accession number KCTC 1301), Bacillus belegensis FZB42 strain (accession number KCTC 1341), and Bacillus amyloliquefaciens NRRL B-14393 strain (accession number KCTC 1660). ), Bacillus amyloliquefaciens Bs-10 strain (accession number KCTC 1104), and B. amyloliquefaciens Bacillo221003 strain of the present invention (accession number KCTC 15313BP).
Figure 7 is a graph showing blood TMA and TMAO levels after 6 weeks when a high choline diet and strains were simultaneously fed. The strains used in the experiment were Bacillus belegensis CR-502 strain (accession number KCTC 1301), Bacillus belegensis FZB42 strain (accession number KCTC 1341), and Bacillus amyloliquefaciens NRRL B-14393 strain (accession number KCTC 1660). Bacillus amyloliquefaciens Bs-10 strain (accession number KCTC 1104), and Bacillus amyloliquefaciens Bacillo221003 strain of the present invention (accession number KCTC 15313BP).
Figure 8 is a graph showing TMA and TMAO levels in feces and blood measured for 6 weeks while feeding a high-protein diet (containing 60% fat) to 5-week-old female C57BL/6J mice. Figure 8A shows TMA and TMAO levels in feces, and Figure 8B shows TMA and TMAO levels in blood.
Figure 9 is a graph showing TMA and TMAO levels in feces and blood after 6 weeks when a high-protein diet and the B. amyloliquefaciens Bacillo221003 strain of the present invention were simultaneously fed. Figure 9A shows TMA and TMAO levels in feces, and Figure 9B shows TMA and TMAO levels in blood.

Unless otherwise defined, all technical and scientific terms used in this specification have the same meaning as commonly understood by a person skilled in the art to which the present invention pertains. In general, the nomenclature used herein is well known and commonly used in the art.

Recently, there has been increasing interest in high-protein, high-choline diets, but if a high-choline diet or high-protein diet is consumed more than necessary, TMA and TMAO may accumulate excessively, and the accumulated TMA and TMAO can cause symptoms such as trimethylaminuria, respectively. There is a risk of causing arteriosclerosis or chronic kidney disease. Therefore, measures are needed to prevent the accumulation of TMA and TMAO in the body.

In one embodiment of the present invention, the present inventors identified a novel Bacillus amyloliquefaciens Bacillo221003 strain with excellent TMA and TMAO decomposition ability from human feces and deposited it with the Korea Biological Resources Center (deposit) Number: KCTC 15313BP).

Therefore, in one aspect, the present invention relates to the Bacillus amyloliquefaciens Bacillo221003 strain having an accession number of KCTC 15313BP.

In one embodiment of the present invention, it was confirmed that the concentration of TMA was significantly reduced when the Bacillo221003 strain was cultured in a TMA-containing medium, and that it had a significantly high level of TMA decomposition ability even when compared to other Bacillus amyloliquefaciens strains. Confirmed.

Therefore, in the present invention, the strain can be characterized as having the ability to degrade TMA and TMAO.

In the present invention, 'Bacillus amyloliquefaciens ( Bacillus amyloliquefaciens )' can be understood as a term referring to the Amyloliquefaciens species belonging to the genus Bacillus. The terms ' Bacillus velezensis ' and ' Bacillus methylotrophicus ' refer to the same species of microorganism as the term 'Bacillus amyloliquefaciens' and can be used interchangeably. (Ministry of Food and Drug Safety Raw Material List Annex 1-368p.; Wang LT, et al., Int J Syst Evol Microbiol. 2008 Mar;58(Pt 3):671-5. doi: 10.1099/ijs.0.65191-0. PMID: 18319476 .; KCTC Biological Resources Research Center velezensis synonym, https://kctc.kribb.re.kr/collection/view?sn=13012).

In one embodiment of the present invention, in an animal model administered with the above strain, the TMA concentration and TMAO concentration in blood and feces were significantly reduced, making it superior to other B. amyloliquefaciens strains not only in vitro but also in vivo (animal model). It was confirmed that TMA and TMAO resolution were observed.

In the present invention, the B. amyloliquefaciens Bacillo221003 strain may be characterized by reducing blood TMA and TMAO concentrations when administered to a subject, and in particular, reducing blood concentrations of TMA and TMAO increased due to a high-choline, high-protein diet. It may be characterized as being ordered, but is not limited to this.

Additionally, in the present invention, the B. amyloliquefaciens Bacillo221003 strain may be characterized by reducing fecal TMA and/or TMAO concentration when administered to a subject.

In the present invention, "TMA (Trimethylamine)" is a compound represented by the following formula 1, and its chemical composition is (CH ₃ ) ₃ N, and is composed of natural gas, synthetic fragrance (fish) component, photochemical substance, choline salt, flotation It is a substance used as a warning agent in the synthesis of chemicals, dyes, pesticides, ion exchange resins, cationic starches, and sweeteners (HSDB 2006).

[Formula 1]

It is known that TMA can cause trimethylaminuria if it is not broken down in the body (Falony G, et al., Annu. Rev. Microbiol. 69: 305-321 (2015)).

In the present invention, “TMAO (Trimethylamine-N-Oxide)” is a compound represented by the following formula 2, its chemical composition is (CH ₃ ) ₃ NO, and it is a white, water-soluble solid.

[Formula 2]

TMAO is also a substance produced by oxidation of trimethyl, a metabolite, when common animals consume choline (Baker, J.R.; J. Biol. Chem. 237 (4): 1309-13. doi:10.1016/S0021-9258( 18)60325-4.PMID 13864146.). In addition, TMAO has been reported as a proatherogenic substance that can accelerate atherosclerosis (Gaci N, et al., World J. Gastroenterol. 20 (43): 16062-16078 (November 2014)) .

From another aspect, the present invention relates to a composition for decomposing TMA or TMAO containing the above strain as an active ingredient.

From another aspect, the present invention relates to a method for degrading TMA or TMAO comprising administering the strain to a subject.

From another aspect, the present invention relates to the use of the above strain to degrade TMA or TMAO.

From another aspect, the present invention relates to the use of the strain for producing a composition for degrading TMA or TMAO.

In the present invention, the composition containing the B. amyloliquefaciens Bacilo221003 strain is preferably prepared in an oral dosage form, but is not limited thereto. In the present invention, the composition may be used in a composition form suitable for administration to humans or animals.

The B. amyloliquefaciens Bacilo221003 strain, which is the active ingredient of the composition of the present invention, may be provided in lyophilized or encapsulated form, culture suspension, fermentation broth, dry powder, or granule form when manufactured as an oral dosage form.

The B. amyloliquefaciens Bacilo221003 strain of the present invention can be cultured using a conventional Bacillus strain culture method. The medium can be a natural medium or a synthetic medium. For example, glucose, sucrose, dextrin, glycerol, etc. can be used as the carbon source of the medium, and peptone, meat extract, yeast, soybean, ammonium salt, nitrate, and other organic or inorganic nitrogen-containing compounds can be used as the nitrogen source. , but is not limited to this. The mineral salt contained in the medium is magnesium. Manganese, calcium. Iron, potassium, etc. may be used, but are not limited thereto. In addition to the carbon source, nitrogen source, and inorganic salt components, amino acids, vitamins, nucleic acids, etc. may be added.

In the present invention, the terms “TMA decomposition” or “TMAO decomposition” mean converting TMA or TMAO into other substances, respectively.

The TMA decomposition or TMAO decomposition may be preferably performed through an oxidation/reduction reaction, more preferably a demethylation reaction, etc., but is not limited thereto.

In certain embodiments, the oxidation/reduction reactions include: oxidation of TMA by the enzyme trimethylamine monooxygenase; Alternatively, in decomposing TMAO, it may be a reduction reaction of TMAO by the TMAO reductase enzyme, but is not limited thereto.

In certain embodiments, the demethylation reaction includes demethylation of TMA by TMADH (Trimethylamine dehydrogenase) enzyme; Alternatively, it may be a demethylation reaction of TMAO by TMAO demethylase enzyme, but is not limited thereto.

In the TMA decomposition or TMAO decomposition, formaldehyde is mainly produced as a by-product, but is not limited thereto.

In certain embodiments, the TMA decomposition or TMAO decomposition may mean that TMO or TMAO is transformed into another material in the decomposition path shown in FIG. 2, but is not limited thereto.

It will be obvious to those skilled in the art that whether decomposition of TMA or TMAO has occurred can be determined by whether or not the concentration of TMA or TMAO has decreased in the sample.

In the present invention, the composition containing the above strain as an active ingredient is preferably used for decomposing TMA or TMAO.

In the present invention, the composition for decomposing TMA or TMAO may be characterized as a probiotic composition.

The term probiotics of the present invention refers to live microorganisms that are beneficial to the health of the host when administered in an appropriate amount ( Nat Rev Gastroenterol Hepatol 11, 506-514 (2014)). Probiotics act as supplements to the host's gut microflora and, in certain embodiments, may be characterized by preventing the accumulation of TMA or TMAO.

In the present invention, the probiotic composition may further include prebiotics.

In the present invention, the composition for decomposing TMA or TMAO can be used as a food composition or pharmaceutical composition for the purpose of prevention, improvement or treatment, and the amount, form and method of use can be appropriately adjusted depending on the purpose. .

In the present invention, the composition for decomposing TMA or TMAO can be used in combination with other food compositions or pharmaceutical compositions or as an adjuvant. For example, the other food compositions may be fermented foods, fermentation starters for producing fermented foods, and health functional foods such as nutritional supplements, but are not limited thereto. For example, the other pharmaceutical compositions include, but are not limited to, those used to prevent or treat cardiovascular disease and treat trimethyluria.

In the present invention, the composition for decomposing TMA or TMAO may further include other probiotic strains, compounds, auxiliaries, additives, carriers, excipients, etc. in addition to the B. amyloliquefaciens Bacilo221003 strain of the present invention.

It has recently been reported that accumulation of TMA and TMAO produced by high-choline, high-protein diets can cause trimethylaminuria, arteriosclerosis, or chronic kidney disease (Koeth RA, Wang Z, et al., Nat Med.; 19(5):576-85, (2013) May), in one embodiment of the present invention, it was confirmed that ingestion of the strain of the present invention can prevent the accumulation of TMA and TMAO.

Therefore, from another perspective, the present invention relates to a food composition containing the above strain as an active ingredient.

The food composition of the present invention may be characterized by decomposing TMA and TMAO upon ingestion.

The food composition of the present invention may be characterized by reducing the blood concentration of TMA and TMAO upon ingestion.

The food composition of the present invention may be characterized by reducing the blood concentration of TMA or TMAO increased due to a high-choline, high-protein diet, but is not limited thereto.

Additionally, the food composition of the present invention may be characterized by decomposing TMA and TMAO upon ingestion and preventing their accumulation in the body.

The food composition of the present invention may be characterized by preventing accumulation of TMA or TMAO in the body due to a high-choline, high-protein diet, but is not limited thereto.

Therefore, in the present invention, the food composition may be characterized as having an effect of preventing or improving TMA-related diseases or TMAO-related diseases.

The food composition of the present invention may be characterized as a health functional food that shows the effect of preventing or improving TMA-related diseases or TMAO-related diseases.

The term “prevention” used in the present invention refers to all actions that suppress a disease or delay the onset of a disease by administering the food composition or pharmaceutical composition according to the present invention.

The term “improvement” used in the present invention refers to a subject suffering from a disease having the symptoms or severity of the disease reduced due to various factors including treatment. For example, a decrease in high blood pressure or a return to normal blood sugar levels in diabetes due to treatment or lifestyle improvement can be seen as examples of disease improvement, but are not limited to this. In certain embodiments of the present invention, when the food composition of the present invention is fed to a subject, the symptoms or severity of a TMA-related disease or a TMAO-related disease may be reduced and an improvement effect may be exhibited.

The term "food" in the present invention refers to meat, sausages, bread, chocolate, candies, snacks, confectionery, pizza, ramen, other noodles, gum, dairy products including ice cream, various soups, beverages, tea, drinks, alcoholic beverages, There are vitamin complexes, prebiotics, probiotics, postbiotics, health functional foods, and health foods, and include all foods in the conventional sense.

The above-mentioned functional food (functional food) is the same term as food for special health use (FoSHU), and is a medicine processed to efficiently exhibit bioregulatory functions in addition to nutritional supply, with high medical effects. It means food. Here, “function” means adjusting nutrients to the structure and function of the human body or obtaining useful effects for health purposes, such as physiological effects. The food of the present invention can be manufactured by methods commonly used in the industry, and can be manufactured by adding raw materials and ingredients commonly added in the industry. In addition, the formulation of the food can be manufactured without limitation as long as it is a formulation recognized as a food, and the health functional food according to the present invention may be in the form of powder, granules, tablets, capsules, or beverages.

The above-mentioned health food refers to food that has a more active effect of maintaining or promoting health compared to general food, and health supplement food refers to food for the purpose of health supplementation. In some cases, the terms health functional food, health food, and health supplement are used interchangeably.

The food composition may further include a physiologically acceptable carrier. The type of carrier is not particularly limited, and any carrier commonly used in the art can be used.

Additionally, the composition may contain additional ingredients that are commonly used in food compositions to improve odor, taste, vision, etc. For example, it may include vitamins A, C, D, E, B1, B2, B6, B12, niacin, biotin, folate, pantothenic acid, etc. Additionally, it may contain minerals such as zinc (Zn), iron (Fe), calcium (Ca), chromium (Cr), magnesium (Mg), manganese (Mn), copper (Cu), and chromium (Cr). Additionally, it may contain amino acids such as lysine, tryptophan, cysteine, and valine.

In addition, the composition contains preservatives (potassium sorbate, sodium benzoate, salicylic acid, sodium dehydroacetate, etc.), disinfectants (bleaching powder, high bleaching powder, sodium hypochlorite, etc.), and antioxidants (butylhydroxyanisole (BHA), butylhydroxyanisole). toluene (BHT), etc.), colorants (tar color, etc.), coloring agents (sodium nitrite, sodium nitrite, etc.), bleaching agents (sodium sulfite), seasonings (MSG, etc.), sweeteners (dulcine, cyclemate, saccharin, sodium, etc.) ), flavorings (vanillin, lactones, etc.), leavening agents (alum, D-potassium hydrogen tartrate, etc.), strengtheners, emulsifiers, thickeners (grease), coating agents, gum bases, foam suppressants, solvents, improvers, etc., food additives additives) may be included. The additives can be selected depending on the type of food and used in an appropriate amount.

The B. amyloliquefaciens Bacillo221003 strain of the present invention may further contain foodologically acceptable food supplements, may be used with other foods or food ingredients, and may be used appropriately according to conventional methods. The mixing amount of the active ingredient can be appropriately determined depending on the purpose of use (prevention, health, or therapeutic treatment).

As used herein, the term “TMA-related disease” refers to a disease whose pathological cause or symptom is excessive accumulation of trimethylamine (TMA) in the body.

Trimethylaminuria is a metabolic disease that is generally caused by genetic metabolic abnormalities. When the processing function of TMA is impaired, TMA is not eliminated and accumulates in the body, causing symptoms of body odor.

The terms trimethylaminuria and fish odor syndrome can be used interchangeably as terms referring to the same disease.

Therefore, in the present invention, the TMA-related disease may be characterized as trimethylaminuria.

As used herein, the term “TMAO-related disease” refers to a disease whose pathological cause or symptom is excessive accumulation of trimethylamine-N-oxide (TMAO) in the body.

Coronary artery disease (CAD) is a disease that occurs when the arteries around the heart become narrowed or blocked. When arteries are narrowed or blocked, blood flow is reduced, which reduces oxygen supply to the heart muscle, damaging heart muscle cells, and symptoms such as chest pain, pressure, difficulty breathing, and heart attack may appear. Recently, using a metabolomics approach, it has been reported that three phospholipid-related molecules, choline, betaine, and TMAO, are associated with atherosclerosis (Wang, Z., et al., Gut flora metabolism of phosphatidylcholine promotes cardiovascular disease . Nature 2011, 472, 57-63).

Systemic sclerosis (SSc) is a disease that causes thickening or hardening of the skin, blood vessels, and internal organs. The systemic sclerosis can be chronic and progress over a long period of time. Symptoms of systemic sclerosis appear in various forms, including hardening of the skin, digestive problems, and muscle weakness. The causes of systemic sclerosis are diverse, but accumulation of TMAO is also considered one of the causes of systemic sclerosis (Allanore, Y., et al., Systemic sclerosis. Nat Rev Dis Primers, 2015. 1: p. 15002.; Ma, G. , et al., Biosci Rep, 2017. 37(2)).

Chronic renal disease refers to a disease in which the renal function to remove waste is reduced and cannot be restored to normal. Remaining kidney function gradually declines over time, eventually leading to end-stage renal disease requiring renal replacement therapy (dialysis or renal transplantation). The most common cause of chronic kidney disease is diabetes, but it has been reported that increased concentrations of TMAO in the body are also related to chronic kidney disease (Stubbs, J., et al., J. Am. Soc. Nephrol., 2016 27: p. 305-313).

Colorectal cancer refers to a malignant tumor that occurs in the colon mucosa. Colon cancer can develop due to a variety of causes, such as genetic factors and environmental factors. In particular, environmental factors such as excessive intake of animal fat and meat are known to act as factors promoting the development of colon cancer. In addition, TMAO has been reported to increase the risk and severity of colon cancer by promoting cancer cell proliferation and angiogenesis (Yang S, et al., J Immunol Res. 2022 Jul 4;2022: 7043856. doi: 10.1155/2022/7043856. PMID: 35832644; PMCID: PMC9273394).

Therefore, in the present invention, the TMAO-related disease is selected from the group consisting of atherosclerosis, systemic sclerosis, chronic renal disease, and colorectal cancer. It can be characterized.

From another aspect, the present invention relates to a pharmaceutical composition for preventing or treating TMA-related diseases or TMAO-related diseases containing the above strain as an active ingredient.

From another aspect, the present invention relates to a method for preventing or treating a TMA-related disease or TMAO-related disease, comprising administering the strain to a subject.

From another aspect, the present invention relates to the use of the above strain for the prevention or treatment of TMA-related diseases or TMAO-related diseases.

From another aspect, the present invention relates to the use of the strain for producing a pharmaceutical composition for preventing or treating TMA-related diseases or TMAO-related diseases.

In the present invention, the TMA-related disease may be characterized as trimethylaminuria.

In the present invention, the TMAO-related disease is selected from the group consisting of atherosclerosis, systemic sclerosis, chronic renal disease, and colorectal cancer. can do.

The term “prevention” used in the present invention refers to all actions that suppress a disease or delay the onset of a disease by administering the pharmaceutical composition according to the present invention.

The term “treatment” used in the present invention refers to any action in which disease symptoms are improved or beneficially changed by administration of the pharmaceutical composition according to the present invention.

The pharmaceutical composition of the present invention exhibits a preventive or therapeutic effect on TMA-related diseases or TMAO-related diseases through the above-described TMA or TMAO decomposition activity.

Administration of the pharmaceutical composition of the present invention can prevent accumulation of TMA and TMAO in the body due to intake of a high choline diet or a high protein diet.

In addition to containing the B. amyloliquefaciens Bacillo221003 strain of the present invention, the pharmaceutical composition may further include appropriate carriers, excipients, and diluents commonly used in pharmaceutical compositions.

Carriers, excipients and diluents that may be included in the composition include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum acacia, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, These include microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxy benzoate, propylhydroxy benzoate, talc, magnesium stearate and mineral oil. When formulating the composition, it is prepared using diluents or excipients such as commonly used fillers, extenders, binders, wetting agents, disintegrants, and surfactants.

The pharmaceutical composition according to the present invention can be formulated and used in various forms according to conventional methods. Suitable dosage forms include, but are not limited to, oral dosage forms such as tablets, pills, powders, granules, dragees, hard or soft capsules, solutions, suspensions or emulsions, and suppositories.

The pharmaceutical composition according to the present invention can be prepared in a suitable dosage form using a pharmaceutically inert organic or inorganic carrier. That is, if the dosage form is a tablet, coated tablet, dragee, or hard capsule, it may contain lactose, sucrose, starch or a derivative thereof, talc, calcium carbonate, gelatin, stearic acid, or a salt thereof. Additionally, when the dosage form is a soft capsule, it may contain vegetable oil, wax, fat, semi-solid and liquid polyol. Additionally, when the formulation is in the form of a solution or syrup, it may include water, polyol, glycerol, and vegetable oil.

Additionally, the pharmaceutical composition of the present invention can be prepared as an enteric-coated preparation using methods known in the art to pass through the stomach and then reach the small intestine so that the active ingredient, the microorganism, is quickly released into the intestine.

Additionally, the composition of the present invention can be prepared as a composition in capsule form using conventional encapsulation methods. For example, a pellet containing the microorganism of the present invention can be prepared by freeze-drying using a standard carrier and then filled into a hard gelatin capsule. Alternatively, a suspension or dispersion may be prepared using the strain of the invention and any suitable pharmaceutical carrier, such as aqueous gum, cellulose, silicate or oil, and then filled into a soft gelatin capsule. .

In the present invention, the composition may be provided as an enteric-coated enteric preparation, particularly as an oral unit dosage form. “Enteric coating” within the present specification refers to all types of pharmaceutically acceptable known coatings that are not decomposed by stomach acid and remain coated, but are sufficiently decomposed in the small intestine so that the active ingredient can be released into the small intestine. Includes. The material of the enteric coating may be appropriately selected from known polymer materials, and suitable polymer materials can be found in a number of known documents (L. Lachman et al., The Theory and Practice of Industrial Pharmacy, 3rd edition, 1986, pp. 365-373; H. Sucker et al., Pharmazeutische Technologie, Thieme, 1991, pp. 355-359; Hagers Handbuch der pharmazeutischen Praxis, 4th edition, Vol. 7, pp. 739 - 742, and 766 - 778, (SpringerVerlag, 1971); and Remington's Pharmaceutical Sciences, 13th edition, pp. 1689 - 1691 (Mack Publ., Co., 1970), cellulose ester derivatives, cellulose ethers, methyl acrylate copolymers of acrylic resins and copolymers of maleic acid and phthalic acid derivatives. Combinations may be included in these, but are not limited thereto.

In addition to the above carrier, the pharmaceutical composition according to the present invention may further include preservatives, stabilizers, wetting agents, emulsifiers, solubilizers, sweeteners, colorants, osmotic pressure regulators, antioxidants, etc.

The pharmaceutical composition according to the present invention is administered in a pharmaceutically effective amount. In the present invention, "pharmaceutically effective amount" means an amount sufficient to treat a disease with a reasonable benefit/risk ratio applicable to medical treatment, and the effective dose level is determined by the type, severity, and activity of the patient's disease. , can be determined based on factors including sensitivity to the drug, time of administration, route of administration and excretion rate, duration of treatment, drugs used simultaneously, and other factors well known in the field of medicine. The pharmaceutical composition according to the present invention may be administered as an individual therapeutic agent or in combination with other therapeutic agents, may be administered sequentially or simultaneously with conventional therapeutic agents, and may be administered singly or multiple times. Considering all of the above factors, it is important to administer an amount that can achieve maximum effect with the minimum amount without side effects, and this can be easily determined by a person skilled in the art.

The administration method of the pharmaceutical composition according to the present invention can be easily selected depending on the dosage form, and may be administered orally or parenterally. Dosage may vary depending on the patient's age, gender, weight, severity of disease, and route of administration.

The compositions of the present invention may be administered in combination with other treatment regimens or therapeutic agents. When used for the prevention or treatment of cardiovascular diseases, it may preferably be used in combination with other treatments or treatments for cardiovascular diseases, but is not limited thereto and may be used in various combinations according to the judgment of the clinician. .

Additionally, the composition for decomposing TMA or TMAO of the present invention may be a quasi-drug composition.

“Quasi-drugs” refer to products that have a milder effect than pharmaceuticals among products used for the purpose of diagnosing, treating, improving, alleviating, treating, or preventing diseases in humans or animals. For example, according to the Pharmaceutical Affairs Act, quasi-drugs refer to the purpose of medicines. This excludes products used as food, and may include products used to treat or prevent diseases in humans and animals, and products that have a mild or no direct effect on the human body.

Quasi-drugs refer to preparations used for the purpose of preventing or improving disease, other than preparations used for the purpose of pharmacologically affecting the structure or function of humans or animals.

In some cases, it may be used together with other quasi-drug ingredients and may be used appropriately according to conventional methods. The mixing amount of the active ingredient can be appropriately determined depending on the purpose of use (prevention, health, or therapeutic treatment).

Example

Hereinafter, the present invention will be described in more detail through examples. These examples are only for illustrating the present invention, and it will be apparent to those skilled in the art that the scope of the present invention is not to be construed as limited by these examples.

Example 1: B. amyloliquefaciens Isolation and identification of Bacillo221003 strain

Example 1-1. Preparation of media

Bacillus amlyloliquefaciens Bacillo221003 strain was isolated from human feces. Add 1.5 g of KH ₂ PO ₄ , 7.9 g of Na ₂ HPO ₄ ·2H ₂ O, 0.8 g of NH ₄ Cl, 0.1 g of MgSO ₄ ·7H ₂ O, and 15 g of agar powder to 1 L of distilled water to prepare minimal medium. media) was created.

In 1 L of distilled water, 50 g of ethylenediaminetetraacetic acid (EDTA), 11 g of NaOH, 5 g of ZnSO ₄ ·7H ₂ O, 7.34 g of CaCl ₂ ·2H ₂ O, 2.5 g of MnCl ₂ ·6H ₂ O, 0.5 g of CoCl ₂ · 6H ₂ O, 0.5 g of (NH ₄ ) ₆ Mo ₇ O _{24.4H 2} O, 5 g of FeSO _{4.7H 2} O, and 0.2 g of CuSO _{4.5H 2} O were added to form a trace element solution. After composition, it was passed through a 0.2μm pore size filter to sterilize.

After high-pressure sterilization of the medium at 121 degrees for 15 minutes, when the medium cools to 50°C, add methanol at a concentration of 1% and trace element solution at 10 mL per 1 L of the medium, mix well, and add 20 mL each to a 9 mm Petri dish. Plate medium was prepared by dispensing.

Example 1-2. Isolation and selection of single bacteria from human feces

에서 2~10일 동안 배양하였다. 그 후, 육안으로 확인되는 순수분리 가능한 콜로니를 니들로 수득하여 새로운 평판배지에 계대 배양하고 메틸영양성 균주임을 확인하였다.1 g of human feces was added to 9 mL of sterilized phosphate buffered saline (PBS), strongly suspended, and centrifuged at 1,000 rpm for 5 minutes to settle the solid matter. Afterwards, 100 μL of the supernatant was serially diluted 1/10 by adding it to 900 μL of sterilized PBS. Dispense 100 μL of the diluted solution prepared by the above method onto the plate medium prepared by the method of Example 1-1 and spread it with a spreader, maintaining aerobic conditions 37 until colonies are visible.

were cultured for 2 to 10 days. Afterwards, colonies that were visually identifiable as pure and separable were obtained with a needle, subcultured on a new plate medium, and confirmed to be methylotrophic strains.

에서 16시간 배양하여 배양액을 제조하였다. 이후 5mM TMA가 함유된 LB 배지에 상기 배양액을 1/100 부피로 접종하여 호기, 30

조건에서 24시간 180rpm 진탕배양하였다(혐기 조건이라면, 60mM 질산나트륨이 전자수용체로 첨가된 배지에서 48시간 정치배양함). 1차 비교를 위하여 다른 속의 균주들에 대해서도 동일한 과정을 수행하였다(Escherichia coli, Lactobacillus sp., Enterococcus sp., Salmonella sp., 및 Streptococcus sp.).Analysis of TMA concentration was conducted using an enzyme immunoassay method using the TMA standard curve. After forming a single colony for each isolated strain, it was inoculated into Luria-Bertani (LB) liquid medium and incubated for 30 minutes.

Culture medium was prepared by culturing for 16 hours. Afterwards, the culture medium was inoculated at 1/100 volume in LB medium containing 5mM TMA and incubated for 30 minutes.

Culture was performed under shaking conditions at 180 rpm for 24 hours (if anaerobic conditions, culture was performed for 48 hours in a medium supplemented with 60mM sodium nitrate as an electron acceptor). For primary comparison, the same process was performed on strains of other genera ( Escherichia coli , Lactobacillus sp., Enterococcus sp., Salmonella sp., and Streptococcus sp.).

A standard solution was prepared by diluting TMA in distilled water to 0, 1.25, 2.5, 5.0, and 10.0mM, respectively. After centrifuging the liquid sample to be analyzed, 250 μL of the supernatant was placed in a sterilized Eppendorf tube. 50 μL of 20% formaldehyde, 1 mL of anhydrous toluene, and 150 μL of 50% potassium carbonate were added to the tube in that order. The tube was vortexed for 10 seconds and shaken for 5 minutes, then placed in a tube rack and left to stand for 5 minutes. When the toluene layer was separated on top, 100 μL each was loaded three times into a 96-well plate made of polypropylene. 100 μL of TMA standard solution was also loaded into each well (at this time, 100 μL of pure toluene was used as a blank). 100 μL of 0.2% picric acid-toluene solution was placed in each well. Absorbance was measured at a wavelength of 410 nm using an enzyme-linked immunosorbent analyzer.

Each strain was cultured with shaking in a medium containing TMA for 24 hours and the TMA concentration was measured. As a result, the strain isolated in Example 1-2 showed excellent TMA decomposition ability (FIG. 3), and the strain was deposited at the Korea Biological Resources Center. (Accession number: KCTC 15313BP) and was named B. amyloliquefaciens Bacillo221003.

Example 1-3. Identification of strains

To obtain 16s rRNA for identification of the isolated strain, a master mix was prepared by mixing 20 μl of polymerase, 1 μl each of primers F/R diluted to 10 pmol, and 5 μl of D.W. The base sequences of the primers used are shown in Table 1 below.

primer base sequence 27F GAG TTT GAT CAT GGC TCA G (SEQ ID NO: 1) 1492R GGT TAC CTT GTT ACG ACT T (SEQ ID NO: 2)

After dispensing 27 μl of the Master mix into each PCR tube and not performing pretreatment, 1 or 2 colonies were collected with a yellow tip and released into the Master mix; When performing pretreatment, 100 μl of 0.02 mol of NaOH solution was placed in each Ep tube, 1 or 2 colonies were collected with a yellow tip, placed in the Ep tube, vortexed, and boiled for 10 minutes. Master mix was used to prepare the pretreated sample. 3μl was dispensed into each PCR tube. Afterwards, PCR amplification was performed using a PCR device under the conditions of Enlogation time 50s and Annealing temp 52°C.

Pour 1.5% Agarose gel (1.5g of agarose + 100ml of 0.5 Dispense 2 μl of sample and 1 kb DNA ladder each and operate the device. After 15 to 20 minutes, gene amplification was confirmed using the DaiHanMaXidoc program, and gene amplification was confirmed through electrophoresis.

As a result of DNA purification of the amplified PCR product and analysis of the base sequence, the 16s rRNA of the bacillo221003 strain of the present invention included the base sequence represented by SEQ ID NO: 3, and as a result of comparing this through BLAST, the strain of the present invention was Bacillus It was confirmed to belong to amyloliquefaciens .

Example 1-4. different Bacillus velezensis and Bacillus amyloliquefaciens Comparison experiment between strains and TMA resolution

The TMA decomposition ability of strains belonging to the genus Bacillus (including Bacillus velezensis (= amyloliquefaciens ) strains belonging to the same species as the strain of the present invention) was confirmed in the same manner as in Example 1-2. As a result, the untreated group showed a TMA concentration of 5mM, and while other strains reduced the TMA concentration only to 4.5 to 3.5mM, the Bacillo221003 strain of the present invention was confirmed to reduce the TMA concentration to less than 2mM (Figure 4 ). This suggests that the strain of the present invention has significantly better TMA decomposition ability than other Bacillus amyloliquefaciens strains.

Example 2: In vivo changes in TMA and TMAO levels B. amyloliquefaciens Confirmation of effect of Bacillo221003 strain (In vivo)

Example 2-1. Experimental methods and materials

After feeding the strain to mice along with a regular diet, high-choline diet, and high-fat diet, the TMA or TMAO decomposition ability of the strain was evaluated by measuring TMA and TMAO concentrations in feces and blood. The experimental method is as follows.

및 습도 50~60%, 12시간 명암 주기가 유지되는 조건에서 사육되도록 하였다. 1주의 적응기간을 거친 후 마우스 그룹별로 44주간 일반 식이, 고콜린 식이, 고지방 식이를 제공하였으며, 식이와 물은 자유롭게 섭취할 수 있게 하였다. 45주차에 한 그룹 내 마우스 7마리를 3마리 및 4마리로 나누고, 각 소그룹에 5일 동안 하기의 처리를 하였다:Prepare 21 5-week-old female C57BL/6J mice. Seven animals were divided into three groups, and each group was provided with a normal diet (containing 0.08% choline), a high-choline diet (containing 1% choline), and a high-fat diet (containing 60% fat). At this time, all mice were kept at a temperature of 22 to 24

and were reared under conditions in which humidity of 50-60% and a 12-hour light/dark cycle were maintained. After an adaptation period of one week, each group of mice was provided with a normal diet, a high-choline diet, and a high-fat diet for 44 weeks, and food and water were allowed to be consumed freely. At week 45, 7 mice in one group were divided into 3 and 4, and each subgroup was given the following treatments for 5 days:

1) Orally administer 100 μL distilled water to 4 animals daily;

2) Three animals were orally administered 100 μL of 10 ¹¹ CFU/mL BM109 strain suspension every day (at this time, the strain was cultured for 24 hours and suspended in PBS).

Afterwards, feces and blood were collected 24 hours after the last administration of distilled water and strains. Blood was centrifuged at 13,000 rpm for 5 minutes to separate plasma, and feces were mixed with PBS at a ratio of 1:4 (w/v), pulverized, and then centrifuged at 13,000 rpm for 5 minutes to separate the supernatant. Afterwards, the supernatants of plasma and fecal suspension were mixed with methanol at a ratio of 1:4 (v/v) and centrifuged at 6,000 rpm for 10 minutes. Finally, 300 μL of the supernatant was taken and the concentrations of TMA and TMAO were analyzed using liquid chromatography mass spectrometry (LC-MS).

Example 2-2. Check the effect of each strain on changes in TMA and TMAO levels when consuming a high choline diet

When mice were fed a high choline diet without the strain (control group), it was confirmed that TMA and TMAO levels gradually increased over time (Figure 5). The TMA and TMAO levels in feces were approximately 110000 (ng/ml) and 60000 (ng/ml), respectively, and the TMA and TMAO levels in blood were approximately 1800 (ng/ml) and 3600 (ng/ml), respectively.

The B. amyloliquefaciens Bacillo221003 strain selected in Example 1 and strains belonging to the Bacillus genus (including Bacillus velezensis (= amyloliquefaciens ) strains belonging to the same species as the strain of the present invention) were fed with a high choline diet and administered TMA and TMAO. As a result of measuring the concentration, when the B. amyloliquefaciens Bacillo221003 strain was fed, the levels of TMA and TMAO in blood and feces were significantly lower than when other strains were fed (Figures 6 and 7). Specifically, there was little change in TMA and TMAO levels when feeding other strains, but when feeding B. amyloliquefaciens Bacillo221003 strain, TMA and TMAO levels in feces decreased to about 20%, respectively, and TMA and TMAO levels in blood respectively. It decreased to 25% level.

Example 2-3. Check the effect of each strain on changes in TMA and TMAO levels when consuming a high-protein diet

When mice were fed a high-protein diet that did not contain the strain (control group), it was confirmed that TMA and TMAO levels gradually increased over time (Figure 8). The TMA and TMAO levels in feces were approximately 45,000 (ng/ml) and 30,000 (ng/ml), respectively, and the TMA and TMAO levels in blood were approximately 800 (ng/ml) and 1,500 (ng/ml), respectively.

As a result of feeding the B. amyloliquefaciens Bacillo221003 strain selected in Example 2 with a high-protein diet and measuring TMA and TMAO concentrations, when feeding the B. amyloliquefaciens Bacillo221003 strain, TMA and TMAO in blood and feces were significantly lower than in the control group. The numerical values were shown (Figure 9).

Example 2-4. In animal models B. amyloliquefaciens Confirmation of the effect of Bacillo 221003 strain on preventing TMA/TMAO accumulation

The results of Examples 2-2 and 2-3 suggest that the B. amyloliquefaciens Bacillo221003 strain of the present invention has the ability to degrade TMA and TMAO in vivo, and when fed with the strain, the It can be seen that the bioaccumulation of TMA and TMAO can be effectively prevented.

As above, specific parts of the content of the present invention have been described in detail, and those skilled in the art will understand that these specific techniques are merely preferred embodiments and do not limit the scope of the present invention. It will be obvious. Accordingly, the substantial scope of the present invention will be defined by the appended claims and their equivalents.

Korea Research Institute of Bioscience and Biotechnology Biological Resources Center (KCTC) KCTC15313BP 20230202

Claims (10)

Bacillus amyloliquefaciens Bacillo221003 strain with the accession number of KCTC 15313BP.
The Bacillus amyloliquefaciens Bacillo221003 strain according to claim 1, wherein the strain has the ability to degrade TMA and TMAO.
A reagent composition for in vitro TMA or TMAO degradation containing the strain of claim 1 as an active ingredient.
A food composition containing the strain of claim 1 as an active ingredient.
The food composition according to claim 4, wherein the food composition has an effect of preventing or improving TMA-related diseases or TMAO-related diseases.
The food composition according to claim 5, wherein the TMA-related disease is trimethylaminuria.
The method of claim 5, wherein the TMAO-related disease is selected from the group consisting of atherosclerosis, systemic sclerosis, chronic renal disease, and colorectal cancer. A food composition made of.
Prevention of trimethylaminuria, atherosclerosis, systemic sclerosis, chronic renal disease or colorectal cancer containing the strain of paragraph 1 as an active ingredient. or therapeutic pharmaceutical compositions.
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