Bifidobacterium breve, composition containing bifidobacterium breve or culture thereof, and use of bifidobacterium breve or culture thereof

Abstract

The invention provides Bifidobacterium breve and a probiotic composition containing the Bifidobacterium breve, the Bifidobacterium breve and the probiotic composition containing the Bifidobacterium breve are deposited in the biological resource preservation and research center of Institute of Food Industry Development of Taiwan in China on August 8, 2022, and the deposit number BCRC 911145 is obtained. The invention also provides application of the strain or the culture thereof in preparation of a composition for inhibiting fat cell maturation, a composition for reducing fat accumulation and / or promoting fat metabolism, a composition for reducing body weight, a composition for regulating blood fat or a composition for regulating blood sugar.

Description

Bifidobacterium breve, composition containing Bifidobacterium breve or its culture, and use of Bifidobacterium breve or its culture Technical Field

[0001] The present disclosure relates to a probiotic and its application, and in particular to a Bifidobacterium breve, a composition containing Bifidobacterium breve or a culture thereof, and the use of Bifidobacterium breve or a culture thereof. Background Art

[0002] Being overweight, having high blood sugar and high blood lipids are all major risk factors for cardiovascular disease, and a healthy diet and regular exercise are one of the ways to avoid these risk factors.

[0003] Glucagon-like peptide-1 (GLP-1), a type of incretin, is known to inhibit gastric motility, delay gastric emptying, suppress appetite, promote insulin secretion from pancreatic β-cells, and inhibit glucagon secretion from pancreatic α-cells. It is a popular research target for appetite regulation, blood sugar regulation, and weight control.

[0004] Probiotics are now widely accepted as a health food ingredient. Probiotics are defined as containing sufficient live microorganisms to alter the host's gastrointestinal flora to produce beneficial health effects. Bacteria belonging to the genera Lactobacillus and Bifidobacterium are the most widely used. Probiotics typically stimulate lactic acid, lowering intestinal pH. This leads to the production of products that promote the growth of more beneficial microorganisms and enhance the host's immune response to pathogens.

[0005] As mentioned earlier, a healthy diet is one way to avoid cardiovascular disease risk factors such as overweight, high blood sugar, and high blood lipids. Therefore, there is an urgent need for research on the effects of probiotics as health foods in appetite regulation, blood sugar regulation, and weight control, in order to achieve effective health management through the consumption of probiotics.

[0006] SUMMARY OF THE INVENTION

[0007] The present disclosure provides a novel Bifidobacterium breve, which is deposited at the Bioresource Conservation and Research Center of the Food Industry Development Institute and has a deposit number of BCRC 911145.

[0008] The present disclosure also provides a use of the aforementioned novel Bifidobacterium breve or a culture thereof in preparing a composition for reducing appetite and / or promoting the secretion of glucagon-like peptide-1 (GLP-1).

[0009] The present disclosure also provides a use of the aforementioned novel Bifidobacterium breve or a culture thereof in preparing a composition for inhibiting adipocyte maturation.

[0010] The present disclosure further provides a use of the aforementioned novel Bifidobacterium breve or a culture thereof in preparing a composition for reducing fat accumulation and / or promoting fat metabolism.

[0011] Furthermore, the present disclosure provides a use of the aforementioned novel Bifidobacterium breve or a culture thereof in preparing a composition for reducing body weight.

[0012] The present disclosure further provides a use of the aforementioned novel Bifidobacterium breve or a culture thereof in preparing a composition for regulating blood lipids.

[0013] The present disclosure provides a use of the aforementioned novel Bifidobacterium breve or a culture thereof in preparing a composition for regulating blood sugar.

[0014] In addition, the present disclosure further provides a probiotic composition comprising the aforementioned novel Bifidobacterium breve or a culture thereof; and a prebiotic.

[0015] In order to make the aforementioned and other purposes, features, and advantages of the present disclosure more clearly understood, preferred embodiments are specifically described below in detail with reference to the accompanying drawings. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] FIG1A shows the relative amount of glucagon-like peptide-1 (GLP-1) secreted into the culture medium by cells treated with the culture supernatant of Bifidobacterium breve BCRC 911145 (the final concentration of the culture supernatant of Bifidobacterium breve BCRC 911145 in the culture medium of the cells was 0.25%).

[0017] Figure 1B shows the relative expression of the PYY gene in cells treated with the culture supernatant of Bifidobacterium breve BCRC 911145 (cells were cultured for 48 hours, the final concentration of the culture supernatant of Bifidobacterium breve BCRC 911145 in the cell culture medium was 0.5%). Compared with the Mock group, **: p < 0.01.

[0018] Figure 2A shows the relative amount of glucagon-like peptide-1 (GLP-1) secreted into the culture medium by cells treated with a suspension of killed cells of Bifidobacterium breve BCRC 911145 (the final concentration of the suspension of killed cells of Bifidobacterium breve BCRC 911145 in the culture medium was 0.125%). *: p < 0.05 compared to the mock group.

[0019] Figure 2B shows the relative expression of the PYY gene in cells treated with a suspension of killed cells of Bifidobacterium breve BCRC 911145 (cells were cultured for 24 hours, the final concentration of the suspension of killed cells of Bifidobacterium breve BCRC 911145 in the culture medium was 0.125%). Compared with the Mock group, ***: p<0.001.

[0020] Figure 3A shows the relative expression of the C / EBPA gene in cells treated with the culture supernatant of Bifidobacterium breve BCRC 911145 (cell culture for 6 hours, final concentration of the culture supernatant of Bifidobacterium breve BCRC 911145 in the cell culture medium was 0.25%). ***: p<0.001 compared to the Mock group.

[0021] Figure 3B shows the oil red (lipid) content in cells treated with the culture supernatant of Bifidobacterium breve BCRC 911145. Compared with the Mock group, ***: p<0.001.

[0022] Figure 4A shows the relative expression of UCP1 and UCP2 genes in cells treated with the culture supernatant of Bifidobacterium breve BCRC 911145 (cells were cultured for 24 hours, and the final concentration of the culture supernatant of Bifidobacterium breve BCRC 911145 in the cell culture medium was 0.125%). Compared with the Mock group, **: p < 0.01, ***: p < 0.001.

[0023] Figure 4B shows the glycerol content in the culture medium of cells treated with the culture supernatant of Bifidobacterium breve BCRC 911145 (the final concentration of the culture supernatant of Bifidobacterium breve BCRC 911145 in the culture medium was 0.25%). Compared with the Mock group, *: p < 0.05.

[0024] Figure 5 shows the relative expression of UCP1 and UCP2 genes in cells treated with a suspension of killed cells of Bifidobacterium breve BCRC 911145 (cells were cultured for 24 hours, and the final concentration of the suspension of killed cells of Bifidobacterium breve BCRC 911145 in the cell culture medium was 0.25%). ***: p<0.001 compared to the mock group.

[0025] Figure 6A shows the relative expression of the ATGL gene in cells treated with the culture supernatant of Bifidobacterium breve BCRC 911145 (cells were cultured for 48 hours, the final concentration of the culture supernatant of Bifidobacterium breve BCRC 911145 in the cell culture medium was 0.125%). **: p<0.01 compared to the Mock group.

[0026] Figure 6B shows the relative expression of the PLIN1 gene in cells treated with the culture supernatant of Bifidobacterium breve BCRC 911145 (cell culture for 6 hours, the final concentration of the culture supernatant of Bifidobacterium breve BCRC 911145 in the cell culture medium was 0.25%). Compared with the Mock group, ***: p<0.001.

[0027] Figure 6C shows the relative expression of the PPARG2 gene in cells treated with the culture supernatant of Bifidobacterium breve BCRC 911145 (cells were cultured for 24 hours, the final concentration of the culture supernatant of Bifidobacterium breve BCRC 911145 in the cell culture medium was 0.125%). Compared with the Mock group, ***: p<0.001.

[0028] Figure 6D shows the relative expression of LIPE (HSL) genes in cells treated with the culture supernatant of Bifidobacterium breve BCRC 911145 (cells were cultured for 48 hours, the final concentration of the culture supernatant of Bifidobacterium breve BCRC 911145 in the cell culture medium was 0.125%). *: p < 0.05 compared to the Mock group.

[0029] Figure 7A shows the relative expression of the ATGL gene in cells treated with a suspension of killed cells of Bifidobacterium breve BCRC 911145 (cells were cultured for 24 hours, the final concentration of the suspension of killed cells of Bifidobacterium breve BCRC 911145 in the culture medium was 0.25%). *: p<0.05 compared to the Mock group.

[0030] Figure 7B shows the relative expression of the PLIN1 gene in cells treated with a suspension of killed cells of Bifidobacterium breve BCRC 911145 (cells were cultured for 24 hours, and the final concentration of the suspension of killed cells of Bifidobacterium breve BCRC 911145 in the culture medium was 0.25%). Compared with the Mock group, ***: p<0.001.

[0031] Figure 7C shows the relative expression of the PPARG2 gene in cells treated with a suspension of killed cells of Bifidobacterium breve BCRC 911145 (cells were cultured for 24 hours, the final concentration of the suspension of killed cells of Bifidobacterium breve BCRC 911145 in the culture medium was 0.25%). Compared with the Mock group, ***: p<0.001.

[0032] Figure 7D shows the relative expression of the CEBPA gene in cells treated with a suspension of killed cells of Bifidobacterium breve BCRC 911145 (cells were cultured for 24 hours, and the final concentration of the suspension of killed cells of Bifidobacterium breve BCRC 911145 in the culture medium was 0.125%). Compared with the Mock group, ***: p<0.001.

[0033] Figure 8A shows the relative expression of CETP gene in cells treated with culture supernatant of Bifidobacterium breve BCRC 911145 (cell culture for 6 hours, final concentration of culture supernatant of Bifidobacterium breve BCRC 911145 in the cell culture medium was 0.5%). Compared with the Mock group, ***: p<0.001.

[0034] Figure 8B shows the relative expression of the SCARB1 gene in cells treated with the culture supernatant of Bifidobacterium breve BCRC 911145 (cell culture for 6 hours, the final concentration of the culture supernatant of Bifidobacterium breve BCRC 911145 in the cell culture medium was 0.5%). Compared with the Mock group, *: p < 0.05.

[0035] Figure 8C shows the relative expression of the ABCA1 gene in cells treated with the culture supernatant of Bifidobacterium breve BCRC 911145 (cell culture for 6 hours, the final concentration of the culture supernatant of Bifidobacterium breve BCRC 911145 in the cell culture medium was 0.5%). Compared with the Mock group, ***: p<0.001.

[0036] Figure 8D shows the relative expression of the LDLR gene in cells treated with the culture supernatant of Bifidobacterium breve BCRC 911145 (cells were cultured for 48 hours, and the final concentration of the culture supernatant of Bifidobacterium breve BCRC 911145 in the cell culture medium was 0.5%). Compared with the Mock group, **: p < 0.01.

[0037] Figure 9 shows the relative expression of the CETP, SCARB1, and ABCA1 genes in cells treated with a suspension of killed cells of Bifidobacterium breve BCRC 911145 (cells were cultured for 6 hours, and the final concentration of the suspension of killed cells of Bifidobacterium breve BCRC 911145 in the cell culture medium was 0.125%). Compared with the mock group, *: p < 0.05, **: p < 0.01, ***: p < 0.001.

[0038] Figure 10 shows the expression of glucose transporter type 4 (GLUT4) in cells treated with the culture supernatant of Bifidobacterium breve BCRC 911145. Compared with the Mock group, **: p<0.01.

[0039] Figure 11 shows the relative expression of glucose transporter type 4 (GLUT4) in cells treated with a suspension of killed bacteria of Bifidobacterium breve BCRC 911145. Compared with the Mock group, ***: p<0.001.

[0040] Figure 12 shows the results of a questionnaire survey on the subjects' appetite during the efficacy test of live bacteria powder. Compared with week 0, *: p < 0.05, **: p < 0.01.

[0041] Figure 13 shows changes in total cholesterol in subjects during an experiment testing the efficacy of live bacterial powder. *: p < 0.05 compared to week 0. Total cholesterol reference value: < 200 mg / dL.

[0042] Figure 14A shows changes in high-density lipoprotein cholesterol (HDL-C) in subjects during an experiment testing the efficacy of live bacterial powder. **p<0.01 compared to week 0. HDL-C reference value: >40 mg / dL.

[0043] Figure 14B shows the changes in low-density lipoprotein cholesterol (LDL-C) in subjects during the experiment to test the efficacy of live bacterial powder. The reference value for low-density lipoprotein cholesterol (LDL-C) is <100 mg / dL.

[0044] Figure 14C shows changes in the LDL / HDL ratio (LDL / HDL) of subjects during the experiment testing the efficacy of live bacterial powder. Reference values ​​for the LDL / HDL ratio are: men: <3.55; women: <3.22.

[0045] Figure 15 shows changes in fasting blood glucose levels in subjects during an experiment testing the efficacy of live bacteria powder. *: p < 0.05 compared to week 0. Fasting blood glucose reference value: 74-109 mg / dL.

[0046] FIG16A shows the changes in body weight of subjects in an experiment testing the efficacy of a composition containing live bacteria and soybean extract.

[0047] FIG16B shows the changes in body fat percentage of the subjects in the experiment of testing the efficacy of the composition containing live bacteria and soybean extract.

[0048] Figure 17 shows changes in serum glucagon-like peptide-1 (GLP-1) levels in subjects in an experiment (Method 1) testing the blood sugar-regulating efficacy of a composition containing live bacteria, soy extract, and apple extract. Blood samples were collected at identical time intervals from the same group of subjects without taking the composition (control group) and after taking the composition (group taking the composition). Compared to baseline (fasting blood test values ​​(hour 0) before taking the composition (without taking the composition), *: p < 0.05).

[0049] Figure 18 shows changes in blood glucose levels in subjects during an experiment (Method 1) testing the blood glucose-regulating efficacy of a composition containing live bacteria, soy extract, and apple extract. Blood samples were collected from the same group of subjects at equal time intervals before and after taking the composition (control group). Compared to baseline (fasting blood test values ​​(hour 0) before and after taking the composition (not taking the composition), *: p < 0.05, **: p < 0.01, ***: p < 0.001).

[0050] Figure 19 shows changes in serum glucagon-like peptide-1 (GLP-1) levels in subjects in an experiment (Method 2) examining the blood sugar-regulating efficacy of a composition containing live bacteria, soy extract, and apple extract. Blood samples were collected from the same group of subjects at identical time intervals, without taking the composition (control group) and after taking the composition (composition-taking group).

[0051] Figure 20A shows changes in blood glucose levels in subjects during an experiment (Method 2) testing the blood glucose-regulating efficacy of a composition containing live bacteria, soy extract, and apple extract. Blood samples were collected from the same group of subjects at equal intervals before and after taking the composition (control group). Compared to baseline (fasting blood test values ​​(hour 0) before and after taking the composition), ***: p < 0.001. #: p < 0.05 compared to the control group.

[0052] FIG20B shows changes in blood insulin concentrations in subjects during an experiment (Method 2) testing the blood glucose-regulating efficacy of a composition containing live bacteria, soy extract, and apple extract. Blood samples were collected from the same group of subjects at the same time intervals, without taking the composition (control group) and after taking the composition (group taking the composition). Compared to baseline (fasting blood test values ​​(hour 0) before taking the composition (without taking the composition), **: p < 0.01).

[0053] FIG21 shows the changes in fasting blood glucose levels in subjects on the 5th and 8th days (after taking the composition for 3 consecutive days (5th to 7th day)) of the experiment (Method 2) to examine the blood glucose regulating efficacy of the composition containing live bacteria, soy extract, and apple extract.

[0054] FIG22A shows the results of a questionnaire survey on the hunger of subjects in an experiment (method 2) to examine the blood sugar regulating effect of a composition containing live bacteria, soybean extract, and apple extract.

[0055] FIG22B shows the results of a questionnaire survey on the appetite of subjects in an experiment (Method 2) to test the blood sugar regulating effect of a composition containing live bacteria, soybean extract, and apple extract.

[0056] FIG22C shows the results of a questionnaire survey on changes in overall appetite of subjects in an experiment (Method 2) to examine the blood sugar regulating effect of a composition containing live bacteria, soybean extract, and apple extract.

[0057] Detailed Description of the Invention

[0058] The present disclosure may provide a probiotic, which may be an isolated probiotic. The isolated probiotic disclosed herein may be Bifidobacterium breve. In one embodiment, the isolated probiotic disclosed herein may be a novel Bifidobacterium breve, which was deposited at the Bioresource Collection and Research Center (BCRC) of the Food Industry Research and Development Institute (FIRDI) in Taiwan, China on August 8, 2022, and obtained the deposit number BCRC 911145. It was also deposited at the German Collection of Microorganisms and Cell Cultures GmbH (DSMZ) on September 7, 2022, and obtained the deposit number DSM 34383. For the sake of brevity, this novel Bifidobacterium breve will be referred to as Bifidobacterium breve BCRC 911145 in the following disclosure.

[0059] The efficacy of the Bifidobacterium breve BCRC 911145 disclosed herein may include, but is not limited to, reducing appetite, promoting glucagon-like peptide-1 (GLP-1) secretion, inhibiting adipocyte maturation, reducing fat accumulation, promoting fat metabolism, reducing body weight, regulating blood lipids (e.g., lowering total cholesterol, promoting high-density lipoprotein cholesterol (HDL-C) synthesis, promoting low-density lipoprotein cholesterol (LDL-C) metabolism), regulating blood sugar, etc., or any combination thereof.

[0060] The applications of the Bifidobacterium breve BCRC 911145 disclosed herein are not particularly limited. For example, the Bifidobacterium breve BCRC 911145 disclosed herein can be applied in health management and / or medical related fields, particularly, but not limited to, appetite control, weight control, body fat regulation, blood lipid regulation, and blood sugar regulation.

[0061] In one embodiment, the aforementioned Bifidobacterium breve BCRC 911145 of the present disclosure can be prepared into an oral dosage form. Examples of such oral dosage forms include, but are not limited to, powders, granules, tablets, troches, pills, capsules, lozenges, solutions, suspensions, emulsions, syrups, elixirs, or slurries.

[0062] In one specific embodiment, the aforementioned Bifidobacterium breve BCRC 911145 of the present disclosure can be prepared as a powder for oral administration. In another specific embodiment, the Bifidobacterium breve BCRC 911145 of the present disclosure can be prepared as a capsule for oral administration. In yet another specific embodiment, the Bifidobacterium breve BCRC 911145 of the present disclosure can be prepared as a solution for oral administration.

[0063] The present disclosure may also provide a probiotic culture, which is a culture of any of the aforementioned probiotics disclosed herein, wherein the probiotics described herein may include Bifidobacterium breve. In one embodiment, the probiotics described herein may be the aforementioned Bifidobacterium breve BCRC 911145.

[0064] The probiotic culture disclosed herein may include, but is not limited to, cultured probiotics (i.e., cultured probiotics alone), a culture medium containing the cultured probiotics (i.e., cultured probiotics plus a culture medium), or a culture supernatant from which the cultured probiotics have been removed (i.e., cultured culture medium alone).

[0065] The method for separating the cultured probiotics from the culture medium to obtain the cultured probiotics and the culture supernatant (cultured culture medium) is not particularly limited, as long as the two can be separated. For example, the cultured probiotics and the culture medium can be separated by centrifugation, filtration, etc., but are not limited thereto.

[0066] In addition, the short-chain fatty acids (SCFAs) contained in the culture medium or culture supernatant of the probiotic culture disclosed herein may include at least one of the following, but are not limited to: formic acid, acetic acid, propinoic acid, butyric acid, isobutyric acid, 3-methylbutyric acid, etc.

[0067] Regarding the aforementioned probiotic culture disclosed herein, in one embodiment, the cultured probiotic bacteria may be active or maintain their activity, or may be live bacteria.

[0068] Furthermore, with respect to the aforementioned probiotic culture disclosed herein, in another embodiment, the aforementioned cultured probiotics may have their own physiological activity removed (the bacteria have lost their physiological functions), or may be dead bacteria. The method for removing the aforementioned cultured probiotics' own physiological activity or rendering them dead is not particularly limited, as long as the aforementioned cultured probiotics can remove their own physiological activity or render them dead. In one embodiment, the activity of the aforementioned cultured probiotics may be removed or rendered dead by physical means, for example, by subjecting the aforementioned cultured probiotics to high temperature and / or high pressure to remove their own physiological activity (the bacteria have lost their physiological functions) or render them dead, but is not limited thereto.

[0069] In one embodiment, the probiotic culture of the present disclosure may be the cultured probiotic bacteria described above (i.e., the cultured probiotic bacteria alone). In this embodiment, in a specific embodiment, the cultured probiotic bacteria may be active or maintain their activity, or may be live bacteria. However, in another specific embodiment, the cultured probiotic bacteria may have their physiological activity removed (the bacteria have lost their physiological functions) or may be dead bacteria.

[0070] In another embodiment, the probiotic culture of the present disclosure may be a cultured medium containing one of the aforementioned probiotics (i.e., a cultured probiotic plus a cultured medium). In this embodiment, in a specific embodiment, the cultured probiotic may be active or maintain its activity, or may be a live bacterium. However, in another specific embodiment, the cultured probiotic may be depleted of its physiological activity (the bacterium has lost its physiological function) or may be a dead bacterium.

[0071] In yet another embodiment, the probiotic culture disclosed herein may be a culture supernatant from which the aforementioned probiotics have been cultured (i.e., the culture medium alone). In this embodiment, in a specific embodiment, the culture supernatant may be disinfected and / or sterilized, whereas in another specific embodiment, the culture supernatant may not be disinfected and / or sterilized. There are no particular limitations on the method for disinfecting and / or sterilizing the culture supernatant, as long as the culture supernatant can be rendered sterile and / or toxins that may be contained therein can be removed. For example, the culture supernatant may be rendered sterile and / or toxins that may be contained therein can be removed by high temperature and / or high pressure, but is not limited thereto.

[0072] Furthermore, with respect to the aforementioned probiotic culture disclosed herein, the culture medium used to culture the aforementioned probiotics to produce the aforementioned probiotic culture disclosed herein is not particularly limited, as long as it can allow the aforementioned probiotics to grow. Examples of culture media that can be used to culture the aforementioned probiotics to produce the aforementioned probiotic culture disclosed herein include, but are not limited to, MRS broth (DeMan-Rogosa-Sharpe broth), lysogeny broth (LB), skim milk medium, and the like. With respect to the aforementioned probiotic culture disclosed herein, in one embodiment, the culture medium used to culture the aforementioned probiotics to produce the aforementioned probiotic culture disclosed herein can be MRS broth.

[0073] In one embodiment, the probiotic culture disclosed herein may be prepared into an oral dosage form. Examples of such oral dosage forms may include, but are not limited to, powders, granules, tablets, troches, pills, capsules, lozenges, solutions, suspensions, emulsions, syrups, elixirs, or slurries.

[0074] In one specific embodiment, the probiotic culture of the present disclosure can be prepared as a powder for oral administration. In another specific embodiment, the probiotic culture of the present disclosure can be prepared as a capsule for oral administration. In yet another specific embodiment, the probiotic culture of the present disclosure can be prepared as a solution for oral administration.

[0075] Furthermore, for any of the aforementioned probiotic cultures disclosed herein, in a specific embodiment, the probiotic may be the aforementioned Bifidobacterium breve BCRC 911145.

[0076] The probiotic cultures disclosed herein may have the effects of reducing appetite, promoting glucagon-like peptide-1 (GLP-1) secretion, inhibiting adipocyte maturation, reducing fat accumulation, promoting fat metabolism, reducing body weight, regulating blood lipids (e.g., reducing total cholesterol, promoting high-density lipoprotein cholesterol (HDL-C) synthesis, promoting low-density lipoprotein cholesterol (LDL-C) metabolism, etc.), regulating blood sugar, or any combination thereof, but are not limited thereto.

[0077] Furthermore, the applications of the probiotic cultures disclosed herein are not particularly limited. For example, the probiotic cultures disclosed herein can be applied in health management and / or medical fields, particularly, but not limited to, appetite control, weight control, body fat regulation, blood lipid regulation, and blood sugar regulation.

[0078] Based on the above, the present disclosure may also provide the following uses of any of the aforementioned probiotics disclosed herein or any of the aforementioned cultures thereof, but are not limited thereto:

[0079] (i) Use of any of the aforementioned probiotics disclosed herein or any of the aforementioned cultures thereof in the preparation of a composition for reducing appetite and / or promoting the secretion of glucagon-like peptide-1 (GLP-1);

[0080] (ii) Use of any of the aforementioned probiotics disclosed herein or any of the aforementioned cultures thereof in the preparation of a composition for inhibiting adipocyte maturation;

[0081] (iii) Use of any of the aforementioned probiotics disclosed herein or any of the aforementioned cultures thereof in the preparation of a composition for reducing fat accumulation and / or promoting fat metabolism;

[0082] (iv) Use of any of the aforementioned probiotics disclosed herein or any of the aforementioned cultures thereof in the preparation of a composition for reducing body weight;

[0083] (v) Use of any of the aforementioned probiotics disclosed herein or any of the aforementioned cultures thereof in the preparation of a composition for regulating blood lipids. The regulating blood lipids may include, but is not limited to, lowering total cholesterol, promoting the synthesis of high-density lipoprotein cholesterol (HDL-C), promoting the metabolism of low-density lipoprotein cholesterol (LDL-C), etc.; and / or

[0084] (vi) Use of any of the aforementioned probiotics disclosed herein or any of the aforementioned cultures thereof in preparing a composition for regulating blood sugar.

[0085] Furthermore, based on the above, the present disclosure may also provide the following methods, but is not limited thereto:

[0086] (i) A method of reducing appetite and / or promoting the secretion of glucagon-like peptide-1 (GLP-1), comprising, but not limited to, administering any of the aforementioned probiotics of the present disclosure or any of the aforementioned cultures thereof to a subject in need thereof;

[0087] (ii) A method of inhibiting adipocyte maturation, comprising, but not limited to, administering any of the aforementioned probiotics disclosed herein or any of the aforementioned cultures thereof to a subject in need thereof;

[0088] (iii) A method for reducing fat accumulation and / or promoting fat metabolism, comprising, but not limited to, administering any of the aforementioned probiotics disclosed herein or any of the aforementioned cultures thereof to a subject in need thereof;

[0089] (iv) A method of reducing body weight, comprising, but not limited to, administering any of the aforementioned probiotics of the present disclosure or any of the aforementioned cultures thereof to a subject in need thereof;

[0090] (v) A method for regulating blood lipids, comprising, but not limited to, administering any of the aforementioned probiotics disclosed herein or any of the aforementioned cultures thereof to a subject in need thereof. The regulating blood lipids may include, but is not limited to, lowering total cholesterol, promoting high-density lipoprotein cholesterol (HDL-C) synthesis, promoting low-density lipoprotein cholesterol (LDL-C) metabolism, etc.; and / or

[0091] (vi) A method of regulating blood sugar, comprising, but not limited to, administering any of the aforementioned probiotics disclosed herein or any of the aforementioned cultures thereof to a subject in need thereof.

[0092] The individual may include, but is not limited to, a vertebrate. Furthermore, the vertebrate may include, but is not limited to, fish, amphibians, reptiles, birds, or mammals. Examples of mammals include, but are not limited to, humans, gorillas, monkeys, horses, donkeys, llamas, goats, pigs, dogs, cats, rabbits, guinea pigs, rats, and mice. In one embodiment, the individual may be a human.

[0093] In addition, the present disclosure further provides a probiotic composition. The probiotic composition of the present disclosure may include, but is not limited to, any of the aforementioned probiotics of the present disclosure or any of the aforementioned cultures thereof and a prebiotic.

[0094] In the probiotic composition of the present disclosure, the weight ratio of the probiotics or their cultures to the prebiotics may be about 1-6:1-12, such as about 1-5.5:1.5-11, about 1-5:1.5-10, about 1-4:2-9, about 1-3:3-5, about 1:1, about 1:1.5, about 1:2, about 1:2.5, about 1:3, about 1.5:1, about 2:1, about 2:1.5, about 2:3, about 3:1, about 3:8, about 4:7, about 5:6, about 6:5, about 6:7, etc., but is not limited thereto. In one embodiment, in the probiotic composition of the present disclosure, the weight ratio of the probiotics or their cultures to the prebiotics may be about 1:1. In another embodiment, in the probiotic composition of the present disclosure, the weight ratio of the probiotics or their cultures to the prebiotics may be about 1:1.5. In yet another embodiment, in the probiotic composition of the present disclosure, the weight ratio of the probiotics or their cultures to the prebiotics may be about 1:2.

[0095] The prebiotics may include at least one of the following ingredients, but are not limited thereto: soybean extract, oat extract, barley extract, apple extract, banana extract, tomato extract, chicory extract, asparagus extract, beet extract, garlic extract, onion extract, seaweed extract, pectin extract, tea extract, etc.

[0096] In one embodiment, the prebiotic composition of the present disclosure may include a soy extract. In this embodiment, the weight ratio of the probiotics or their cultures to the soy extract may be about 1-6:1-6, such as about 1-5:1-5, about 1.5-4:1.5-4, about 2-5:2-5, about 2.5-6:2.5-6, about 1:1, about 1:1.5, about 1:2, about 1:2.5, about 1:3, about 1:4, about 1.5:1, about 2:1, about 2.5:1, about 3:1, about 4:1, about 2:3, about 3:2, about 5:6, about 6:5, etc., but is not limited thereto. Furthermore, in a specific embodiment of this embodiment, the weight ratio of the probiotics or their cultures to the soy extract may be about 1:1. In another specific embodiment of this embodiment, the weight ratio of the probiotics or culture thereof disclosed herein to the soybean extract may be about 1:1.5.

[0097] In another embodiment, in the probiotic composition of the present disclosure, the prebiotic may include soybean extract and apple extract. In this embodiment, the weight ratio of the probiotics or culture thereof, the soybean extract, and the apple extract may be about 1-6:1-6:1-6, such as about 1-5:1-5:1-5, about 1.5-4:1.5-4:1.5-4, about 2-5:2-5:2-5, about 2.5-6:2.5-6:2.5-6, about 1:1:1, about 1:1.5:1.5, about 1:1.5:1, about 1:1:1.5, about 1.5:1:1 , about 1:2:2, about 1:2:1, about 1:1:2, about 2:2:1, about 2:1:1, about 1:2.5:2.5, about 1:2.5:1, about 1:1:2.5, about 2.5:1:1, about 1:3:3, about 1:3:1, about 1:1:3, about 3:1:1, about 1:4:4, about 1:4:1, about 1:1:4, about 4:1:1, about 2:3:3, about 3:2:2, about 5:6:6, about 6:5:5, etc., but not limited thereto. In a specific embodiment of this embodiment, the weight ratio of the probiotics or culture thereof, the soybean extract, and the apple extract can be about 1:1:1. In another specific embodiment of this embodiment, the weight ratio of the probiotics or culture thereof disclosed herein, the soybean extract, and the apple extract may be about 1:1.5:1.5.

[0098] In addition, the probiotic composition disclosed herein may be a food composition, a pharmaceutical composition, etc., but is not limited thereto.

[0099] In one embodiment, the probiotic composition of the present disclosure may be a food composition. In this embodiment, the probiotic composition of the present disclosure may be prepared as an oral dosage form, but is not limited thereto.

[0100] In another embodiment, the probiotic composition disclosed herein can be a pharmaceutical composition. In this embodiment, the probiotic composition disclosed herein can also be prepared as an oral dosage form, but is not limited thereto.

[0101] The oral dosage forms of the probiotic composition disclosed herein may include, but are not limited to, powders, granules, tablets, pills, capsules, lozenges, solutions, suspensions, emulsions, syrups, elixirs, or concentrates.

[0102] In one embodiment, the probiotic composition of the present disclosure is prepared as an oral dosage form in the form of a powder. In another embodiment, the probiotic composition of the present disclosure is prepared as an oral dosage form in the form of a capsule. In yet another embodiment, the probiotic composition of the present disclosure is prepared as an oral dosage form in the form of a solution.

[0103] Furthermore, in the embodiment where the probiotic composition of the present disclosure is a food composition, the probiotic composition of the present disclosure may further include a food-acceptable additive in addition to the probiotic culture and prebiotics of the present disclosure.

[0104] The additives acceptable to the aforementioned foods may include, but are not limited to, preservatives, bactericides, antioxidants, bleaching agents, colorants, expanders, coloring agents, flavorings, sweeteners, thickeners (pastes), binders, emulsifiers, carriers, etc., or any combination thereof.

[0105] Furthermore, in the embodiment where the probiotic composition of the present disclosure is a pharmaceutical composition, the probiotic composition of the present disclosure may further include a pharmaceutically acceptable carrier or salt in addition to the probiotic culture and prebiotics of the present disclosure.

[0106] The aforementioned pharmaceutically acceptable carriers may include, but are not limited to, solvents, dispersion media, coatings, antibacterial and antifungal agents, and agents that maintain osmotic pressure and delay absorption, etc., compatible with pharmaceutical administration. For different routes of administration, the pharmaceutical composition can be formulated into dosage forms using conventional methods.

[0107] The aforementioned pharmaceutically acceptable salts may include inorganic cations, such as alkali metal salts, such as sodium, potassium, or amine salts; alkaline earth metal salts, such as magnesium and calcium salts; and salts containing divalent or tetravalent cations, such as zinc, aluminum, or zirconium salts. Furthermore, they may include organic salts, such as dicyclohexylamine salts and methyl-D-glucamine; and amino acid salts, such as arginine, lysine, histidine, and glutamine, but are not limited thereto. Example

[0108] A. Materials and Methods

[0109] 1. Identification of bacterial species

[0110] A strain isolated from the human intestine was subjected to genome-wide average nucleotide identity analysis based on the GenomesDB database using the JSpeciesWS online web server, a software / tool ​​for average nucleotide identity (ANI) calculation. The results are shown in Table 1 below.

[0111] Table 1

[0112] According to the results of whole genome average nucleotide identity analysis, the whole genome sequence of this isolate is 97.74% to 98.43% similar to that of Bifidobacterium breve subsp. breve (as shown in Table 1). Therefore, this isolate is confirmed to be a novel Bifidobacterium breve.

[0113] This novel Bifidobacterium breve was deposited with the Bioresource Conservation and Research Center, Food Industry Development Research Institute, Taiwan, China on August 8, 2022, and was assigned the accession number BCRC 911145. This novel Bifidobacterium breve was also deposited with the German Collection of Microorganisms and Cell Cultures GmbH (DSMZ) on September 7, 2022, and was assigned the accession number DSM 34383. This novel Bifidobacterium breve will subsequently be referred to as Bifidobacterium breve BCRC 911145.

[0114] 2. Cultivation of Bifidobacterium breve BCRC 911145 and Obtaining Cultures

[0115] 2-1. Cultivation of Bifidobacterium breve BCRC 911145

[0116] 2-1-1. Preparation of 5L Fermentation Tank

[0117] Prepare 3 L of MRS broth (55 g / L) in a 5 L fermenter using commercially available food-grade MRS broth powder (manufacturer: STBIO MEDIA, INC.). Ensure that the powder is completely dissolved.

[0118] The 5 L fermenter containing the culture medium was sterilized (121°C, 30 minutes). After sterilization, the fermenter was aerated (3 L / min) and cooled to 37°C.

[0119] 2-1-2. Fermentation in a 5L fermenter

[0120] After the fermentation tank was prepared, the fermentation tank was aerated (3 L / min) and the culture medium was stirred (100 rpm) for 20 minutes at 37° C. Simultaneously, the dissolved oxygen electrode was calibrated (100%).

[0121] Only when the dissolved oxygen level is less than 5% can Bifidobacterium breve BCRC 911145 be introduced into the culture medium of the fermentation tank for fermentation. Before introducing the strain, it is necessary to confirm that the fermentation tank is not contaminated.

[0122] The strain implantation and fermentation conditions are as follows:

[0123] (1) The absorbance value (OD) of the suspension of the strain to be implanted in the fermentation tank at 600 nm 600 ):3.0-5.0;

[0124] (2) Implanted bacterial volume: 5% (v / v);

[0125] (3) Dissolved oxygen content: <1% (maintained by a small amount of nitrogen);

[0126] (4) Stirring rate: 25 rpm;

[0127] (5) Temperature: 37 ± 1°C;

[0128] (6) pH: 6.0 ± 0.1;

[0129] (7) Cultivation time: about 6-8 hours (cultivated until the absorbance value of the culture medium at 600 nm (OD 600 ) is 3.0-4.0).

[0130] After fermentation is complete, samples are taken for testing and microbial morphology and strain activity are confirmed.

[0131] Afterwards, the fermentation tank was cooled (the temperature was set to 4°C, and the actual temperature was less than 15°C).

[0132] 2.2 Obtaining Cultures

[0133] 2-2-1. Obtaining live cultured Bifidobacterium breve BCRC 911145

[0134] After the fermentation in the above-mentioned 2-1-2.5 L fermenter is completed, centrifugation is performed to remove the culture medium and obtain live bacteria.

[0135] The centrifugation conditions are as follows:

[0136] 10000rpm, 10 minutes.

[0137] After centrifugation, the live bacteria were freeze-dried.

[0138] Thereafter, the freeze-dried live bacteria are pulverized to form live bacteria powder.

[0139] 2-2-2. Obtaining the culture supernatant of cultured Bifidobacterium breve BCRC 911145

[0140] After the fermentation in the 2-1-2.5 L fermentation tank is completed, the fermentation tank is sterilized (steam sterilization for 30 minutes).

[0141] After cooling, the culture was centrifuged to remove the bacterial cells and obtain the culture supernatant.

[0142] The centrifugation conditions are as follows:

[0143] 10000rpm, 10 minutes.

[0144] 2-2-3. Obtaining a suspension of cultured dead Bifidobacterium breve BCRC 911145

[0145] After the fermentation in the 2-1-2.5 L fermentation tank is completed, the fermentation tank is sterilized (steam sterilization for 30 minutes).

[0146] After cooling, centrifuge to remove the culture medium and obtain dead bacteria.

[0147] The centrifugation conditions are as follows:

[0148] 5000rpm, 10 minutes.

[0149] After centrifugation, water equal in volume to the culture before centrifugation was added to the dead bacteria to form a dead bacteria suspension.

[0150] 3. Detection of glucagon-like peptide-1 (GLP-1) secreted by cells in vitro

[0151] 3-1. Materials

[0152] (1) Cell line: NCI-H716 cells (ATCC, CCL-251);

[0153] (2) Culture medium:

[0154] RPMI 1640 medium (Gibco);

[0155] 10% (v / v) fetal bovine serum (Gibco);

[0156] 2 mM L-Glutamine (Gibco);

[0157] 1% (v / v) penicillin-streptomycin (Gibco);

[0158] (3) Trypan blue dead cell stain (Lonza; Cat. 17-942E);

[0159] (4) PBS (Gibco);

[0160] (5) ELISA reader (BioTek);

[0161] (6)GLP-1 ELISA KIT (CEA804Mi).

[0162] 3-2. Methods

[0163] (1) Cells were plated at 1×10 5 The cells were seeded at a density of 10 cells / well in a 6-well cell culture plate (culture volume was 2 mL) and cultured in a 37°C, 5% CO2 incubator for 24 hours;

[0164] (2) Two days before the test, 1×10 5 Cells were seeded into 24-well plates coated with Matrix gel, and the culture medium was replaced with DMEM complete medium, and the cells were cultured for 24 hours;

[0165] (3) One day before the assay, the cell culture medium was replaced with DMEM w / o glucose medium, and forskolin (final concentration in the medium was 1 μM) and 3-isobutyl-1-methylxanthine (IBMX) (final concentration in the medium was 1 μM) were added and co-cultured for 4 hours;

[0166] (4) The supernatant in each well of the culture plate was removed, and PBS containing 1 mM CaCl2 (300 μL / well) was added to each well. Different test samples, 1 μM forskolin, and 1 μM 3-isobutyl-1-methylxanthine were also added, and the cells were cultured for 24 hours.

[0167] (5) After 24 hours of culture, the supernatant was aspirated from each well and the GLP-1 content in the supernatant was detected using a GLP-1 ELISA KIT (CEA804Mi) to confirm the amount of GLP-1 secreted by the cells into the culture medium;

[0168] (6) Statistical analysis was performed using Excel with one-tailed Student's t-test (T-TEST) (*: p < 0.05, **: p < 0.01, ***: p < 0.001 compared to Mock).

[0169] 4. Detection of PYY gene expression in cells in vitro

[0170] 4-1. Materials and Instruments

[0171] (1) Cell line: NCI-H716 cells (ATCC, CCL-251);

[0172] (2) Culture medium:

[0173] 89% (v / v) RPMI 1640 medium (Gibco);

[0174] 10% (v / v) fetal bovine serum (Gibco);

[0175] 2 mM L-Glutamine (Gibco);

[0176] 1% (v / v) penicillin-streptomycin (Gibco);

[0177] (3)RNA Extraction Kit (Geneaid);

[0178] (4) III reverse transcriptase (Invitrogen);

[0179] (5) Primer set: a primer set targeting the PYY gene and a primer set targeting the GAPDH gene as an internal control;

[0180] (6)KAPA CYBR FAST qPCR Kits (2x) (KAPA Biosystems);

[0181] (7) StepOnePlus real-time polymerase chain reaction instrument (ABI).

[0182] 4-2. Methods

[0183] 4-2-1. Cell culture and pretreatment

[0184] (1) Cells were plated at 1×10 5 The cells were seeded at a density of 10 cells / well in a 6-well cell culture plate (culture volume was 2 mL) and cultured in a 37°C, 5% CO2 incubator for 24 hours;

[0185] (2) Afterwards, the cells were treated with different test samples for 24 hours and 48 hours;

[0186] (3) Collect cells for gene expression analysis.

[0187] 4-2-2. Gene expression analysis

[0188] (1) Extract cellular RNA using an RNA Extraction Kit (Geneaid);

[0189] (2) III Reverse Transcriptase (Invitrogen) to reverse transcribe the above RNA (2000 ng) into cDNA;

[0190] (3) Using KAPA SYBR FAST qPCR Kits (2x) (KAPA Biosystems) with a StepOnePlus real-time polymerase chain reaction instrument, real-time polymerase chain reaction was performed on the cDNA to measure the expression level of the target gene;

[0191] (4) Analyze the melting curve during real-time polymerase chain reaction;

[0192] (5) Use 2 -ΔΔ C T The relative quantification of gene expression was determined by the threshold cycle (C) of GAPDH reference gene. T The relative fold change was calculated according to the formulas (I) to (III) below using the internal control and the mock group as the control:

[0193] (6) The standard deviation of the relative expression of genes was calculated using STDEV in Excel;

[0194] (7) Statistical analysis was performed using Excel with one-tailed Student's t-test (T-TEST) (*: p < 0.05, **: p < 0.01, ***: p < 0.001).

[0195] 5. Detection of the expression of fat-related genes (CEBPA, UCP, ATGL, PLIN1, PPARG2 and LIPE (HSL)) in cells in vitro

[0196] 5.1 Materials and Instruments

[0197] (1) Cell line: mouse bone marrow stromal cells (OP9) (BCRC; Cat. 60566);

[0198] (2) Culture medium:

[0199] Minimum essential medium α (MEMα) (Gibco; Cat. 12000-022, or equivalent);

[0200] 2% (v / v) fetal bovine serum (Gibco; Cat. 10437-028);

[0201] 1% (v / v) antibiotic (Gibco; Cat. 15240-062, or equivalent);

[0202] (3) 10X DPBS (Gibco; Cat. 14200-075, or equivalent);

[0203] (4) Trypan blue dead cell stain (Lonza; Cat. 17-942E, or equivalent);

[0204] (5) RB Buffer (Geneaid; Cat. RP050, or equivalent);

[0205] (6) 10X trypsin solution (SIGMA; Cat. 59427C, or equivalent);

[0206] (7)RNA Extraction Kit (Geneaid);

[0207] (8) III reverse transcriptase (Invitrogen);

[0208] (9) Primer set: primer sets for each gene to be tested (CEBPA, UCP, ATGL, PLIN1, PPARG2, and LIPE (HSL)) and a primer set for the GAPDH gene as an internal control;

[0209] (10)KAPA CYBR FAST qPCR Kits (2x) (KAPA Biosystems);

[0210] (11) StepOnePlus real-time polymerase chain reaction instrument (ABI).

[0211] 5-2. Method

[0212] 5-2-1. Preparation of operating reagents

[0213] (1) 1X DPBS: Dilute 10X DPBS 10-fold with sterilized ddH2O;

[0214] (2) 1X trypsin solution: Dilute 10X trypsin solution 10-fold with 1X DPBS.

[0215] 5-2-2. Cell culture and pretreatment

[0216] (1) Cells were plated at 1×10 5 The cells were seeded at a density of 10 cells / well in a 6-well cell culture plate (culture volume was 2 mL) and cultured in a 37°C, 5% CO2 incubator for 24 hours.

[0217] (2) Afterwards, the cells were divided into a mock group and an experimental group (high-dose group and low-dose group). After adding different test samples, the cells were placed in a 37°C, 5% CO2 incubator for 6 hours, 24 hours, and 48 hours;

[0218] (3) After the treatment is completed, remove the culture medium;

[0219] (4) Wash the cells with 1X DPBS (1 mL / well) and then remove the 1X DPBS;

[0220] (5) Repeat step (4);

[0221] (6) Add RB Buffer (600 μL / well) to lyse the cells to obtain cell lysate;

[0222] (7) Collect the cell lysate in a 1.5 mL microcentrifuge tube and store in a -80°C refrigerator.

[0223] 5-2-3. Gene expression analysis

[0224] (1) Extract cellular RNA using an RNA Extraction Kit (Geneaid);

[0225] (2) III Reverse Transcriptase (Invitrogen) to reverse transcribe the above RNA (2000 ng) into cDNA;

[0226] (3) Using KAPA SYBR FAST qPCR Kits (2x) (KAPA Biosystems) with a StepOnePlus real-time polymerase chain reaction instrument, real-time polymerase chain reaction was performed on the cDNA to measure the expression level of the target gene;

[0227] (4) Analyze the melting curve during real-time polymerase chain reaction.

[0228] (5) Use 2 -ΔΔ C T The relative quantification of gene expression was determined by the threshold cycle (C) of GAPDH reference gene. T The relative fold change was calculated according to the formulas shown in the previous method (I) to formula (III).

[0229] 6. Detection of fat accumulation in cells in vitro

[0230] OP9 mouse embryonic stem cells were used as a cell platform to confirm whether the test samples in the experimental group had the effect of reducing oil droplet formation.

[0231] 6-1. Materials

[0232] (1) Cell line: OP9 mouse embryonic stem cell (ATCC; Cat. CRL-2749);

[0233] (2) Culture medium:

[0234] 79% (v / v) minimum essential medium alpha (MEMα) (Gibco; Cat. 12561072);

[0235] 20% (v / v) fetal bovine serum (Gibco; Cat. 10437-028);

[0236] 1% (v / v) penicillin-streptomycin (Gibco; Cat. 15140122);

[0237] (3) 10X DPBS (Gibco; Cat. 14200-075, or equivalent);

[0238] (4) Trypan blue dead cell stain (Lonza; Cat. 17-942E, or equivalent);

[0239] (5) 10X trypsin solution (Gibco; Cat. 15400-054);

[0240] (6) Oil red O (Sigma; Cat. O0625-25G);

[0241] (7) Isopropanol (Jingming; Cat. 184130010);

[0242] (8) Formaldehyde (Jingming; Cat. 119690010).

[0243] 6-2 Method

[0244] 6-2-1. Preparation of operating reagents

[0245] (1) 1X DPBS: Dilute 10X DPBS 10-fold with sterilized ddH2O;

[0246] (2) 1X trypsin solution: 10X trypsin solution was diluted 10 times with 1X DPBS;

[0247] (3) 10% formaldehyde: dilute formaldehyde with sterilized ddH2O;

[0248] (4) 50% and 60% isopropyl alcohol: dilute isopropyl alcohol with sterilized ddH2O;

[0249] (5) Oil Red O stock solution: Prepare Oil Red O with 100% isopropanol to a final concentration of 3 mg / mL.

[0250] (6) Oil Red O working solution: Mix the Oil Red O stock solution and ddH2O in a ratio of 3:2 to form a mixed solution. Centrifuge the mixed solution at 1000 x g for 10 minutes and remove the supernatant into a new 15 mL centrifuge tube for later use.

[0251] 6-2-2. Cell culture and pretreatment

[0252] (1) Cells were plated at 8x10 4 The cells were seeded at a density of 10 cells / well in a 24-well cell culture plate (culture volume was 500 μL / well) and cultured in a CO2 incubator for 7 days, with the culture medium replaced every 3 days.

[0253] (2) After 7 days, the formation of oil droplets in the cells was observed under a microscope to confirm cell differentiation;

[0254] (3) The culture medium in the culture plate was replaced with the culture medium for the experimental group (containing the test sample) and the culture medium for the control group (Mock), and cultured in a CO2 incubator for 7-10 days, with the culture medium replaced every 3 days.

[0255] 6-2-3. Determination of oil droplet content

[0256] (1) After the culture is complete, remove the liquid from the plate and rinse the cells twice with 1X DPBS;

[0257] (2) Add 10% methanol (1 mL / well) to the cells and fix the cells at room temperature for 30 minutes;

[0258] (3) Remove the liquid in the well plate and rinse the cells twice with 1X DPBS;

[0259] (4) Remove the liquid from the plate, add 60% isopropanol (1 mL / well), and let it stand at room temperature for 1 minute;

[0260] (5) Remove the liquid from the plate, add Oil Red O working solution (1 mL / well), and stain in the dark at room temperature for 1 hour;

[0261] (6) Remove the liquid from the plate and add 60% isopropanol (1 mL / well) to destain for 5 seconds;

[0262] (7) Remove the liquid from the well plate and add ddH2O (1 mL / well);

[0263] (8) Observe and take photos using a microscope;

[0264] (9) Remove the liquid from the plate, add 100% isopropyl alcohol, and shake the plate on an orbital shaker for 10 minutes to precipitate the dye;

[0265] (10) Add the liquid in the well plate to a 96-well plate (100 μL / well) and measure its absorbance at a wavelength of 510 nm (OD510) using a spectrophotometer;

[0266] (11) Statistical analysis was performed using Excel with one-tailed Student's t-test (T-TEST) (*: p < 0.05, **: p < 0.01, ***: p < 0.001).

[0267] 7. Detection of lipolysis in cells in vitro

[0268] Using OP9 mouse embryonic stem cells as a cell platform, the effect of stimulation with the test sample in the experimental group on the amount of glycerol released into the culture medium during OP9 adipogenic differentiation was determined, thereby determining whether the test sample has a lipolysis effect.

[0269] 7-1. Materials

[0270] (1) Cell line: OP9 mouse embryonic stem cells (ATCC; Cat. CRL-2749);

[0271] (2) Culture medium:

[0272] 79% (v / v) minimum essential medium alpha (MEMα) (Gibco; Cat. 12561072);

[0273] 20% (v / v) fetal bovine serum (Gibco; Cat. 10437-028);

[0274] 1% (v / v) penicillin-streptomycin (Gibco; Cat. 15140122);

[0275] (3) 10X DPBS (Gibco; Cat. 14200-075, or equivalent);

[0276] (4) Trypan blue dead cell stain (Lonza; Cat. 17-942E, or equivalent);

[0277] (5) 10X trypsin solution (Gibco; Cat. 15400-054);

[0278] (6) Glycerol cell-based assay kit (Cayman; Cat. 10011725):

[0279] Glycerol standard solution (Cayman; Cat. 10009952);

[0280] Free glycerol assay reagent (Cayman; Cat. 10009953);

[0281] Chloroquine positive control (25 mM) (Cayman; Cat. 10012670).

[0282] 7-2. Methods

[0283] 7-2-1. Preparation of operating reagents

[0284] (1) 1X DPBS: Dilute 10X DPBS 10-fold with sterilized ddH2O;

[0285] (2) 1X trypsin solution: Dilute 10X trypsin solution 10-fold with 1X DPBS.

[0286] (3) Free glycerol assay reagent: Open the labeled vial (Cat. 10009953) in the kit and add 5 mL of ddH2O to dissolve the contents. This will prepare the free glycerol assay reagent. One vial is sufficient for 48 samples (half a 96-well plate). The reagent can be stored at 4°C for 2 weeks.

[0287] (4) Glycerol standard solution: The concentration of the glycerol stock solution (Cat. 10009952) in the vial with this label included in the kit is 125 μg / mL. Serially dilute this stock solution. Number eight clean 1.5 mL microcentrifuge tubes, 1 through 8. Add 200 μL of the glycerol stock solution to the first tube. Add 100 μL of 1X DPBS to each of the remaining seven tubes. Next, transfer 100 μL from tube 1 to tube 2, mix thoroughly, and then transfer 100 μL from tube 2 to tube 3. Repeat this sequence until the 7th tube is reached, obtaining glycerol solutions with concentrations of 125 μg / mL (tube 1), 62.5 μg / mL (tube 2), 31.25 μg / mL (tube 3), 15.625 μg / mL (tube 4), 7.8125 μg / mL (tube 5), 3.90625 μg / mL (tube 6), 1.953125 μg / mL (tube 7), and 0 μg / mL (tube 8).

[0288] 7-2-2. Cell culture and pretreatment

[0289] (1) Cells were plated at 8x10 4The cells were seeded at a density of 100 cells / well in a 24-well cell culture plate (the culture medium volume was 500 μL / well) and cultured in a CO2 incubator for 7 days, with the culture medium changed every 3 days.

[0290] (2) After 7 days, the formation of oil droplets in the cells was observed under a microscope to confirm cell differentiation;

[0291] (3) The culture medium was replaced with the experimental group (test sample) and control group (Mock) culture medium, and cultured in a CO2 incubator for 7-10 days, with the culture medium changed every 3 days.

[0292] 7-2-3. Determination of glycerol content released into culture medium

[0293] (1) After the culture is completed, collect the cell culture medium of the last culture;

[0294] (2) Add 25 μL of cell culture medium, glycerol standard solution of various concentrations, and chloroquine positive control to a 96-well plate;

[0295] (3) Add free glycerol assay reagent (100 μL / well) and react at room temperature for 15 minutes;

[0296] (4) Measure the absorbance of each well at a wavelength of 540 nm (OD540) using a spectrophotometer;

[0297] (5) Statistical analysis was performed using Excel with one-tailed Student's t-test (T-TEST) (*: p < 0.05, **: p < 0.01, ***: p < 0.001).

[0298] 8. Detection of lipid-related genes (CETP, SCARB1, ABCA1, LDLR) in vitro cells

[0299] 8.1 Materials and Instruments

[0300] (1) Cell line: human hepatocellular carcinoma cell line HepG2 (ATCC; Cat. HB-8065);

[0301] (2) Culture medium:

[0302] Dulbecco's modified Eagle's medium (DMEM) (Gibco; Cat. 11965-092);

[0303] 10% (v / v) fetal bovine serum (Gibco; Cat. 10437-028, or equivalent);

[0304] 1% (v / v) penicillin-streptomycin (Gibco; Cat. 15140122);

[0305] (3) 10X DPBS (Gibco; Cat. 14200-075, or equivalent);

[0306] (4) Trypan blue dead cell stain (Lonza; Cat. 17-942E, or equivalent);

[0307] (5) RB Buffer (Geneaid; Cat. RP050, or equivalent);

[0308] (6) 10X trypsin solution (SIGMA; Cat. 59427C, or equivalent);

[0309] (7)RNA Extraction Kit (Geneaid);

[0310] (8) III reverse transcriptase (Invitrogen);

[0311] (9) Primer set: primer set for each gene to be tested (CETP, SCARB1, ABCA1, and LDLR) and primer set for the GAPDH gene as an internal control;

[0312] (10)KAPA CYBR FAST qPCR Kits (2x) (KAPA Biosystems);

[0313] (11) StepOnePlus real-time polymerase chain reaction instrument (ABI).

[0314] 8-2. Methods

[0315] 8-2-1. Cell culture and pretreatment

[0316] (1) Cells were plated at 1×10 6 Cells / well were seeded in 6-well plates and cultured overnight (n=3);

[0317] (2) Treat cells with different test samples for 6, 24, and 48 hours;

[0318] (3) After removing the supernatant, wash the cells once with 1X PBS;

[0319] (4) After adding 600 μL of RB Buffer to lyse the cells, collect the cell lysate in a centrifuge tube and store it in a -80°C refrigerator.

[0320] 8-2-2. Gene expression analysis

[0321] (1) Extract cellular RNA using an RNA Extraction Kit (Geneaid);

[0322] (2) III Reverse Transcriptase (Invitrogen) to reverse transcribe the above RNA (2000 ng) into cDNA;

[0323] (3) Using KAPA SYBR FAST qPCR Kits (2x) (KAPA Biosystems) with a StepOnePlus real-time polymerase chain reaction instrument, real-time polymerase chain reaction was performed on the cDNA to measure the expression level of the target gene;

[0324] (4) Analyze the melting curve during real-time polymerase chain reaction;

[0325] (5) Use 2 -ΔΔ C T Methods: Relative quantification of gene expression was determined. The threshold cycle (CT value) of the GAPDH reference gene was used as an internal control and the mock group, and the relative fold change was calculated according to the formula shown in the previous method (I) to formula (III).

[0326] 9. Detection of Glut4 expression in cells in vitro

[0327] 9.1 Materials and Instruments

[0328] (1) Cell line: human hepatocellular carcinoma cell line HepG2 (ATCC; Cat. HB-8065);

[0329] (2) Culture medium:

[0330] Dulbecco's modified Eagle's medium (DMEM) (Gibco; Cat. 12100046);

[0331] 10% fetal bovine serum (Gibco; Cat. 10437-028, or equivalent);

[0332] 1% antibiotic (Gibco; Cat. 15240-062, or equivalent);

[0333] Sodium bicarbonate (Sigma; Cat. S5761-500G, or equivalent);

[0334] Sodium hydroxide (NaOH) (Sigma; Cat. 221465-500G, or equivalent);

[0335] Hydrogen chloride (HCl) (Sigma; Cat. 7647-01-0, or equivalent);

[0336] (3) 10X DPBS (Gibco; Cat. 14200-075, or equivalent);

[0337] (4) Fetal bovine serum (Gibco; Cat. 10437-028, or equivalent);

[0338] (6) 10X trypsin solution (Gibco; Cat. 15400054, or equivalent);

[0339] (7) Alexa Fluor 488 goat anti-mouse IgG antibody (Invitrogen; Cat. A-11001, or equivalent);

[0340] (8) GLUT4 antibody (Invitrogen; Cat. MA5-17176, or products with equivalent functions);

[0341] (9) Insulin (Sigma; Cat. I9278-5ML, or products with equivalent functions);

[0342] (10) Trypan blue dead cell stain (Lonza; Cat. 17-942E, or equivalent);

[0343] (11) Flow cytometer (BD, Accuri C6 Plus, or equivalent).

[0344] 9-2-1. Preparation of operating reagents

[0345] (1) 1X DPBS: Dilute 10X DPBS 10-fold with sterilized ddH2O;

[0346] (2) 1X trypsin solution: dilute 10X trypsin solution 10 times with pure water;

[0347] (3) 2% fetal bovine serum solution: Mix 1 mL of fetal bovine serum with 49 mL of 1X DPBS in a 50 mL centrifuge tube.

[0348] 9-2-2. Cell culture and pretreatment

[0349] (1) Cells were plated at 1×10 5 The cells were seeded at a density of 10 cells / well in a 6-well cell culture plate (culture volume was 2 mL) and cultured in a 37°C, 5% CO2 incubator for 24 hours.

[0350] (2) Afterwards, the cells were divided into Mock group and experimental group (high-dose group and low-dose group);

[0351] (3) Optional: Remove the original culture medium and add serum-free culture medium containing 1 μM insulin to the cells. Incubate at 37°C in a 5% CO2 incubator for 72 hours. Then remove the culture medium.

[0352] (4) Adding culture medium containing different concentrations of the test sample to the cells, and then culturing in a 37°C, 5% CO2 incubator for 24 hours;

[0353] (5) Optional: Add 0.5 μM insulin to the cells and culture in a 37°C, 5% CO2 incubator for 24 hours;

[0354] (6) After the treatment is completed, remove the culture medium;

[0355] (7) Wash the cells with 1X DPBS (1 mL / well) and then remove the 1X DPBS;

[0356] (8) Repeat step (7);

[0357] (9) Add 1X trypsin solution (200 μL / well) and incubate the cells in a 37°C, 5% CO2 incubator in the dark for 5 minutes. Tap the culture plate to confirm that the cells are detached.

[0358] (10) Add culture medium (800 μL / well) to terminate the action of trypsin;

[0359] (11) Transfer the cell suspension in each well to a separate microcentrifuge tube and centrifuge at 400 × g for 5 minutes to remove the supernatant;

[0360] (12) Add 200 μL of 1X DPBS to each tube to wash the cells, then centrifuge at 400 × g for 5 minutes and remove the supernatant;

[0361] (13) Add 200 μL of 2% fetal bovine serum solution to each tube and mix well to fix the cells. Incubate at room temperature in the dark for 30-60 minutes.

[0362] (14) Centrifuge each tube at 400 × g for 5 minutes to remove the supernatant;

[0363] (15) Add 200 μL of 1X DPBS and 1 μL of GLUT4 antibody (1:200) to each tube and mix well. Incubate at room temperature in the dark for 30 minutes.

[0364] (16) Centrifuge each tube at 400 × g for 5 minutes to remove the supernatant;

[0365] (17) Add 200 μL of 1X DPBS to each tube to wash the cells and centrifuge at 400 × g for 5 minutes to remove the supernatant;

[0366] (18) Repeat step (17);

[0367] (19) Add 200 μL of 1X DPBS and 1 μL of Alexa Fluor 488 goat anti-mouse IgG secondary antibody (1:200) to each tube and mix well. Incubate at room temperature for 10 minutes in the dark.

[0368] (20) Centrifuge each tube at 400 × g for 5 minutes to remove the supernatant;

[0369] (21) Add 200 μL of 1X DPBS to each tube to wash the cells and centrifuge at 400 × g for 5 minutes to remove the supernatant;

[0370] (22) Repeat step (21);

[0371] (23) Add 200 μL of 1X DPBS to each tube to resuspend the cells;

[0372] (24) Analyze the fluorescence intensity of fluorescein isothiocyanate (FITC) by flow cytometery;

[0373] (25) Statistical analysis was performed using Excel with one-tailed Student's t-test (T-TEST) (*: p < 0.05, **: p < 0.01, ***: p < 0.001).

[0374] 10. Preparation of Soybean Extract

[0375] The soybean extract was produced on behalf of FUJI OIL CO., LTD., Japan.

[0376] The main processes are as follows in order:

[0377] (1) Soy protein isolate;

[0378] (2) Enzyme hydrolysis;

[0379] (3) separation;

[0380] (4) Sterilization;

[0381] (5) Filtering;

[0382] (6) spray drying; and

[0383] (7) Obtain the product.

[0384] 11. Preparation of Apple Extract

[0385] The main production process of apple extract is as follows:

[0386] (1) Coarsely chop the apples (coarse chopping aperture: 12 mm);

[0387] (2) The roughly chopped apples were extracted with RO water to obtain a crude extract. The extraction conditions were as follows:

[0388] The weight ratio of coarsely chopped apples to RO water is 1:6;

[0389] Extraction temperature: 85±5℃;

[0390] Extraction time: 60 minutes;

[0391] Reflux pump 3.0±0.5kg / cm 2 ;

[0392] (3) Filter the crude extract (pore size: 400 mesh) and take a sample for testing after filtration (Brix: 8.0 ± 0.5 (20°C));

[0393] (4) Concentrating the filtrate and taking samples to check whether the concentrate meets the predetermined specifications (Brix sugar content: 8.0±0.5; CA: 0.3±0.2 (20°C); pH: 2.7±1.0 (20°C));

[0394] (5) After reaching the specifications, the concentrated liquid can be used as the apple extract used in the present disclosure.

[0395] B. Component Analysis

[0396] 1. Analysis of short-chain fatty acids (SCFA) in the culture supernatant of cultured Bifidobacterium breve BCRC 911145

[0397] Short-chain fatty acids are known to stimulate the secretion of glucagon-like peptide-1 (GLP-1). Therefore, the short-chain fatty acids contained in the culture supernatant of cultured Bifidobacterium breve BCRC 911145 were analyzed to determine its potential to stimulate glucagon-like peptide-1 (GLP-1) secretion.

[0398] The results are shown in Table 2.

[0399] Table 2 Short-chain fatty acids in the culture supernatant of cultured Bifidobacterium breve BCRC 911145

[0400] As shown in Table 2, the culture supernatant of cultured Bifidobacterium breve BCRC 911145 contains at least short-chain fatty acids such as formic acid, acetic acid, propionic acid, butyric acid, isobutyric acid, and 3-methylbutyric acid, and therefore should have the ability to stimulate the secretion of glucagon-like peptide-1.

[0401] C. In vitro experiments

[0402] 1. Confirmation of the efficacy of culture supernatant and dead bacterial suspension in promoting GLP-1 secretion and reducing appetite

[0403] Glucagon-like peptide-1 (GLP-1) is the main intestinal secretin in the human body. It can promote insulin secretion and inhibit glucagon secretion, which has the effects of regulating blood sugar, reducing appetite, and controlling weight. The PYY gene is related to appetite suppression.

[0404] The effects of culture supernatant and killed cell suspension of Bifidobacterium breve BCRC 911145 on glucagon-like peptide-1 (GLP-1) secretion and PYY gene expression were analyzed to evaluate the efficacy of Bifidobacterium breve BCRC 911145 in appetite control, weight control, and blood sugar regulation.

[0405] The effects of culture supernatant and killed cell suspension of Bifidobacterium breve BCRC 911145 (tested in NCI-H716 cells) on GLP-1 secretion and PYY gene expression were determined using the methods described in "3. Detection of GLP-1 Secretion in Vitro" and "4. Detection of PYY Gene Expression in Vitro" in "A. Materials and Methods" above.

[0406] FIG1A and Table 3 show the relative amount of glucagon-like peptide-1 (GLP-1) secreted into the culture medium by cells treated with the culture supernatant of Bifidobacterium breve BCRC 911145 (the final concentration of the culture supernatant of Bifidobacterium breve BCRC 911145 in the culture medium of the cells was 0.25%).

[0407] FIG1B and Table 4 show the relative expression of PYY gene in cells treated with culture supernatant of Bifidobacterium breve BCRC 911145 (cells were cultured for 48 hours, the final concentration of culture supernatant of Bifidobacterium breve BCRC 911145 in the cell culture medium was 0.5%).

[0408] FIG2A and Table 5 show the relative amount of glucagon-like peptide-1 (GLP-1) secreted into the culture medium by cells treated with a suspension of killed cells of Bifidobacterium breve BCRC 911145 (the final concentration of the suspension of killed cells of Bifidobacterium breve BCRC 911145 in the culture medium was 0.125%).

[0409] FIG2B and Table 6 show the relative expression of the PYY gene in cells treated with a suspension of killed cells of Bifidobacterium breve BCRC 911145 (cells were cultured for 24 hours, and the final concentration of the suspension of killed cells of Bifidobacterium breve BCRC 911145 in the cell culture medium was 0.125%).

[0410] Table 3. Relative amount of glucagon-like peptide-1 secreted into the culture medium by cells treated with the culture supernatant of Bifidobacterium breve BCRC 911145 (the final concentration of the culture supernatant of Bifidobacterium breve BCRC 911145 in the culture medium of the cells was 0.25%)

[0411] Compared with the Mock group, ***: p < 0.001.

[0412] Table 4. Relative expression of the PYY gene in cells treated with the culture supernatant of Bifidobacterium breve BCRC 911145 (cell culture for 48 hours, the final concentration of the culture supernatant of Bifidobacterium breve BCRC 911145 in the culture medium was 0.5%)

[0413] Table 5. Relative amount of glucagon-like peptide-1 secreted into the culture medium by cells treated with a suspension of killed cells of Bifidobacterium breve BCRC 911145 (the final concentration of the suspension of killed cells of Bifidobacterium breve BCRC 911145 in the culture medium was 0.125%)

[0414] Compared with the Mock group, **: p < 0.05.

[0415] Table 6. Relative expression of the PYY gene in cells treated with a suspension of killed cells of Bifidobacterium breve BCRC 911145 (cells were cultured for 24 hours, the final concentration of the suspension of killed cells of Bifidobacterium breve BCRC 911145 in the culture medium was 0.125%)

[0416] These results indicate that both the culture supernatant and the dead cell suspension of Bifidobacterium breve BCRC 911145 can effectively promote cell secretion of glucagon-like peptide-1 (GLP-1) and effectively increase the expression of the PYY gene in cells.

[0417] Specifically, the culture supernatant of Bifidobacterium breve BCRC 911145 can promote the secretion of glucagon-like peptide-1 (GLP-1), increasing the secretion amount by 51.8%. It can also effectively enhance the expression of the appetite-suppressing gene PYY by as much as 79.8%.

[0418] Similarly, a suspension of killed Bifidobacterium breve BCRC 911145 also boosted the secretion of glucagon-like peptide-1 (GLP-1) by 20.5%, while also effectively increasing the expression of the appetite-suppressing gene PYY by 48.4%.

[0419] In combination with the above, both the culture supernatant and the killed bacteria suspension of Bifidobacterium breve BCRC 911145 can be used for applications related to appetite control, weight control and / or blood sugar regulation.

[0420] 2. Confirmation of the efficacy of culture supernatant in inhibiting adipocyte maturation and reducing fat accumulation

[0421] The effects of culture supernatant of Bifidobacterium breve BCRC 911145 on C / EBPA gene expression and fat accumulation were determined using the methods described in "5. Detection of Fat-Related Gene Expression in Cells (C / EBPA, UCP, ATGL, PLIN1, PPARG2, LIPE (HSL))" and "6. Detection of Fat Accumulation in Cells in Vitro" in "A. Materials and Methods" above.

[0422] The C / EBPA gene is a key regulatory gene for energy homeostasis and lipid storage in the body. Its low expression will terminate the differentiation of fat droplet cells.

[0423] The effects of the culture supernatant of Bifidobacterium breve BCRC 911145 on C / EBPA gene expression and fat accumulation were analyzed to evaluate the ability of Bifidobacterium breve BCRC 911145 to inhibit adipocyte maturation and reduce fat accumulation.

[0424] The results are shown in Tables 7 and 8 and Figures 3A and 3B.

[0425] Table 7 and FIG3A show the relative expression of C / EBPA genes in cells treated with culture supernatant of Bifidobacterium breve BCRC 911145 (cell culture for 6 hours, final concentration of culture supernatant of Bifidobacterium breve BCRC 911145 in the cell culture medium was 0.25%).

[0426] Table 8 and FIG. 3B show the oil red (lipid) content in cells treated with the culture supernatant of Bifidobacterium breve BCRC 911145.

[0427] Table 7. Relative expression of C / EBPA genes in cells treated with culture supernatant of Bifidobacterium breve BCRC 911145 (cell culture for 6 hours, final concentration of culture supernatant of Bifidobacterium breve BCRC 911145 in the culture medium was 0.25%)

[0428] Table 8. Oil red (lipid) content in cells treated with culture supernatant of Bifidobacterium breve BCRC 911145 (the final concentration of culture supernatant of Bifidobacterium breve BCRC 911145 in the culture medium of the cells was 0.25%)

[0429] Based on the above results, the culture supernatant of Bifidobacterium breve BCRC 911145 can effectively inhibit C / EBPA gene expression, that is, inhibit adipocyte maturation, with an inhibition rate as high as 83.4%, and can successfully reduce fat accumulation by 21.0%.

[0430] In view of the above, the culture supernatant of Bifidobacterium breve BCRC 911145 can be used in fat reduction and other related applications.

[0431] 3. Confirmation of the efficacy of culture supernatant and dead bacteria suspension in promoting fat burning and / or fat decomposition

[0432] According to the methods described in "5. Detection of the Expression of Fat-Related Genes (CEBPA, UCP, ATGL, PLIN1, PPARG2, LIPE (HSL)) in Cells in Vitro" and "7. Detection of Lipolysis in Cells in Vitro" in "A. Materials and Methods" above, the effects of the culture supernatant of Bifidobacterium breve BCRC 911145 on the expression of UCP1 and UCP2 genes and lipolysis were determined.

[0433] Increased expression of the UCP1 and UCP2 genes helps convert fat cells and promotes fat burning.

[0434] The potential of Bifidobacterium breve BCRC 911145 in promoting fat burning and lipolysis was evaluated by analyzing the effects of culture supernatant and killed cell suspension of Bifidobacterium breve BCRC 911145 on UCP1 and UCP2 gene expression, as well as the effects of culture supernatant of Bifidobacterium breve BCRC 911145 on lipolysis.

[0435] The results are shown in Tables 9, 10, and 11 and Figures 4A, 4B, and 5.

[0436] Table 9 and FIG4A show the relative expression of UCP1 and UCP2 genes in cells treated with culture supernatant of Bifidobacterium breve BCRC 911145 (cells were cultured for 24 hours, and the final concentration of culture supernatant of Bifidobacterium breve BCRC 911145 in the cell culture medium was 0.125%).

[0437] Table 10 and FIG4B show the glycerol content in the culture medium of cells treated with the culture supernatant of Bifidobacterium breve BCRC 911145 (the final concentration of the culture supernatant of Bifidobacterium breve BCRC 911145 in the culture medium of the cells was 0.25%).

[0438] Table 11 and Figure 5 show the relative expression of UCP1 and UCP2 genes in cells treated with a suspension of killed cells of Bifidobacterium breve BCRC 911145 (cells were cultured for 24 hours, and the final concentration of the suspension of killed cells of Bifidobacterium breve BCRC 911145 in the cell culture medium was 0.25%).

[0439] Table 9. Relative expression of UCP1 and UCP2 genes in cells treated with culture supernatant of Bifidobacterium breve BCRC 911145 (cell culture for 24 hours, final concentration of culture supernatant of Bifidobacterium breve BCRC 911145 in the culture medium was 0.125%)

[0440] Table 10. Glycerol content in the culture medium of cells treated with the culture supernatant of Bifidobacterium breve BCRC 911145 (the final concentration of the culture supernatant of Bifidobacterium breve BCRC 911145 in the culture medium of the cells was 0.25%)

[0441] Compared with the Mock group, **: p < 0.05.

[0442] Table 11. Relative expression of UCP1 and UCP2 genes in cells treated with a suspension of killed cells of Bifidobacterium breve BCRC 911145 (cells were cultured for 24 hours, the final concentration of the suspension of killed cells of Bifidobacterium breve BCRC 911145 in the culture medium was 0.25%)

[0443] Based on the above results, it can be seen that both the culture supernatant and the killed bacteria suspension of Bifidobacterium breve BCRC 911145 can effectively promote fat burning and have the effect of reducing fat.

[0444] Specifically, the culture supernatant of Bifidobacterium breve BCRC 911145 can effectively increase the expression of fat-burning-related genes UCP1 and UCP2, with the increase rate reaching up to approximately 4.35 times, and at the same time can increase fat decomposition efficiency by approximately 20.6%.

[0445] Similarly, the dead bacteria suspension of Bifidobacterium breve BCRC 911145 can also effectively increase the expression of fat burning-related genes UCP1 and UCP2, with the increase rate reaching up to about 1.3 times.

[0446] In summary, both the culture supernatant and the killed bacteria suspension of Bifidobacterium breve BCRC 911145 can be used in fat reduction and other related applications.

[0447] 4. Confirmation of the efficacy of culture supernatant and dead bacteria suspension in fat metabolism

[0448] According to the method described in "5. Detection of the expression of fat-related genes (CEBPA, UCP, ATGL, PLIN1, PPARG2, LIPE (HSL)) in cells in vitro" in "A. Materials and Methods" above, the effect of the culture supernatant of Bifidobacterium breve BCRC 911145 on the expression of fat-related genes was confirmed.

[0449] ATGL is involved in the first step of fat metabolism in adipocytes, hydrolyzing triglycerides. The PLIN1 gene regulates fat metabolism within adipocytes; its low expression promotes fat metabolism. PPARG2 regulates the β-oxidation of fatty acids; its low expression promotes fat metabolism. LIPE (HSL) hydrolyzes triglycerides into free fatty acids, responsible for converting cholesterol esters into free cholesterol. CEBPA, on the other hand, terminates adipocyte differentiation and is a key regulatory gene for energy homeostasis and lipid storage.

[0450] The effects of culture supernatant and killed cell suspension of Bifidobacterium breve BCRC 911145 on the expression of the above genes were analyzed to evaluate the efficacy of Bifidobacterium breve BCRC 911145 in promoting fat metabolism.

[0451] The results are shown in Tables 12 and 13 and Figures 6A to 6D and Figures 7A to 7D.

[0452] Table 12 and Figures 6A to 6D show the relative expression of ATGL, PLIN1, PPARG2, and LIPE (HSL) genes in cells treated with the culture supernatant of Bifidobacterium breve BCRC 911145.

[0453] Table 13 and Figures 7A to 7D show the relative expression of ATGL, PLIN1, PPARG2, and CEBPA genes in cells treated with a suspension of killed cells of Bifidobacterium breve BCRC 911145.

[0454] Table 12. Relative expression of ATGL, PLIN1, PPARG2, and LIPE (HSL) genes in cells treated with culture supernatant of Bifidobacterium breve BCRC 911145

[0455] Table 13. Relative expression of ATGL, PLIN1, PPARG2, and CEBPA genes in cells treated with culture supernatant of Bifidobacterium breve BCRC 911145

[0456] The above results indicate that both the culture supernatant and the dead bacteria suspension of Bifidobacterium breve BCRC 911145 can effectively promote fat metabolism and have the effect of reducing fat.

[0457] Specifically, the culture supernatant of Bifidobacterium breve BCRC 911145 can effectively promote fat metabolism, with the increase rate reaching up to approximately 3.39 times.

[0458] Similarly, a suspension of killed Bifidobacterium breve BCRC 911145 can also effectively promote fat metabolism, with an average increase rate of approximately 10%.

[0459] In summary, both the culture supernatant and the killed bacteria suspension of Bifidobacterium breve BCRC 911145 can be effectively used in fat reduction and other related applications.

[0460] 5. Confirmation of the efficacy of culture supernatant and dead bacterial suspension in regulating blood lipids and promoting high-density lipoprotein cholesterol (HDL-C)

[0461] According to the method described in "8. Detection of the expression of blood lipid-related genes (CETP, SCARB1, ABCA1, LDLR) in cells in vitro" in "A. Materials and Methods" above, the effects of the culture supernatant and dead cell suspension of Bifidobacterium breve BCRC 911145 on the expression of blood lipid-related genes were determined.

[0462] The CETP, SCARB1, and ABCA1 genes are associated with the synthesis of high-density lipoprotein cholesterol (HDL-C), while the LDLR gene is associated with the metabolism of low-density lipoprotein cholesterol (LDL-C).

[0463] The effects of culture supernatant and killed cell suspension of Bifidobacterium breve BCRC 911145 on the expression of the above genes were analyzed to evaluate the efficacy of Bifidobacterium breve BCRC 911145 in regulating blood lipids.

[0464] The results are shown in Tables 14 and 15 and Figures 8A to 8D and 9 .

[0465] Table 14 and Figures 8A to 8D show the relative expressions of the CETP gene, SCARB1 gene, ABCA1 gene, and LDLR gene in cells treated with the culture supernatant of Bifidobacterium breve BCRC 911145.

[0466] Table 15 and FIG9 show the relative expression of CETP gene, SCARB1 gene and ABCA1 gene in cells treated with a suspension of killed cells of Bifidobacterium breve BCRC 911145.

[0467] Table 14. Relative expression of CETP, SCARB1, ABCA1, and LDLR genes in cells treated with culture supernatant of Bifidobacterium breve BCRC 911145

[0468] Table 15. Relative expression of CETP, SCARB1, and ABCA1 genes in cells treated with a suspension of killed bacteria of Bifidobacterium breve BCRC 911145

[0469] The above results indicate that both the culture supernatant and the dead bacteria suspension of Bifidobacterium breve BCRC 911145 can effectively regulate blood lipids and promote the synthesis of high-density lipoprotein cholesterol.

[0470] Specifically, the culture supernatant of Bifidobacterium breve BCRC 911145 can effectively promote the synthesis of high-density lipoprotein cholesterol, with the increase rate reaching up to approximately 9.2 times, and can also promote the metabolism of low-density lipoprotein cholesterol.

[0471] Similarly, the killed bacteria suspension of Bifidobacterium breve BCRC 911145 can also effectively promote fat metabolism and the synthesis of high-density lipoprotein cholesterol, with the highest increase rate reaching approximately 89.7%.

[0472] 6. Confirmation of the blood sugar regulation efficacy of culture supernatant and dead bacteria suspension

[0473] According to the method described in "9. Detection of Glut4 Expression in Cells in Vitro" in "A. Materials and Methods" above, the effects of the culture supernatant and the dead cell suspension of Bifidobacterium breve BCRC 911145 on the expression of blood glucose-related genes were determined.

[0474] It is currently known that glucose transporter type 4 (GLUT4) is a member of the integral membrane glucose transporter (GLUT) family. It is expressed in muscle, fat, and liver tissues. It absorbs glucose and brings glucose into cells for use, thereby balancing blood sugar.

[0475] The effects of culture supernatant and killed cell suspension of Bifidobacterium breve BCRC 911145 on the expression of glucose transporter type 4 (GLUT4) were analyzed to evaluate the efficacy of Bifidobacterium breve BCRC 911145 in regulating blood glucose.

[0476] The results are shown in Tables 16 and 17 and Figures 10 and 11.

[0477] Table 16 and FIG10 show the relative expression of glucose transporter type 4 (GLUT4) in cells treated with the culture supernatant of Bifidobacterium breve BCRC 911145.

[0478] Table 17 and FIG. 11 show the relative expression of glucose transporter type 4 (GLUT4) in cells treated with a suspension of killed cells of Bifidobacterium breve BCRC 911145.

[0479] Table 16. Expression of glucose transporter type 4 (GLUT4) in cells treated with culture supernatant of Bifidobacterium breve BCRC 911145

[0480] Compared with the Mock group, **:p<0.01.

[0481] Table 17. Relative expression of glucose transporter type 4 (GLUT4) in cells treated with a suspension of killed bacteria of Bifidobacterium breve BCRC 911145

[0482] Compared with the Mock group, ***: p < 0.001.

[0483] The above results indicate that both the culture supernatant and the killed bacteria suspension of Bifidobacterium breve BCRC 911145 can effectively regulate blood sugar.

[0484] Specifically, the culture supernatant of Bifidobacterium breve BCRC 911145 can increase the expression of glucose transporter type 4 (GLUT4) by approximately 20.4%.

[0485] Similarly, a suspension of killed Bifidobacterium breve BCRC 911145 increased the expression of glucose transporter type 4 (GLUT4) by approximately 116%.

[0486] D. Human trials

[0487] D-1. Efficacy test of live bacteria powder

[0488] Objective: To examine the weight loss effect of Bifidobacterium breve BCRC 911145.

[0489] Experimental content:

[0490] 1. Dosage form and dosage: Bifidobacterium breve BCRC 911145 live bacteria capsules (100 mg live bacteria powder / capsule, equivalent to 1×10 9 CFU / capsule), 1 capsule daily after dinner.

[0491] 2. Experimental Design

[0492] Take one live bacteria capsule daily for 4 consecutive weeks, and perform tests before and after taking it for 4 weeks.

[0493] 3. Subject conditions:

[0494] Healthy adults over 20 years old, with body fat >25% for men and >30% for women

[0495] 4. Number of subjects: 9

[0496] 5.Test items:

[0497] (1) Questionnaire-appetite status;

[0498] (2) Blood biochemical indicators (total cholesterol, HDL, LDL, LDL / HDL and fasting blood glucose) (tested by Liren Medical Laboratory)

[0499] The results are shown in Figures 12, 13, 14A and 14B and Table 18.

[0500] FIG12 shows the results of the questionnaire survey on the appetite of the subjects.

[0501] As shown in Figure 12, continuous use of Bifidobacterium breve BCRC 911145 live bacteria capsules for four weeks significantly reduced cravings by 25.9% and hunger by 20.4%. Furthermore, questionnaire survey results also revealed that 62.5% of participants reported improvements in cravings and 87.5% reported improvements in hunger.

[0502] Table 18. Blood biochemical indicators of subjects

[0503] According to Figure 13 and Table 18, continuous use of Bifidobacterium breve BCRC 911145 live bacteria capsules for 4 weeks can reduce total cholesterol by 4.2%, and the improvement rate reaches 77.8%.

[0504] 14A to 14C and Table 18 show that continuous use of the live bacteria capsules of Bifidobacterium breve BCRC 911145 for four weeks significantly increased high-density lipoprotein cholesterol (HDL-C) by 3.9% and decreased low-density lipoprotein cholesterol (LDL-C) by 8.2%. The ratio of LDL-C to HDL-C (LDL / HDL) was also reduced by approximately 9.3%. The percentages of patients whose cholesterol levels improved in these three indicators were 88.9%, 77.8%, and 77.8%, respectively.

[0505] In addition, according to Figure 15 and Table 18, continuous use of Bifidobacterium breve BCRC 911145 live bacteria capsules for 4 weeks can significantly reduce fasting blood sugar by 9.1%, and the improvement rate of the number of people reached 90.0%.

[0506] From the above, we can see that the live bacteria Bifidobacterium breve BCRC 911145 do have the effects of reducing appetite, lowering total cholesterol and low-density lipoprotein cholesterol (LDL-C), increasing high-density lipoprotein cholesterol (HDL-C), and lowering fasting blood sugar.

[0507] D-2. Efficacy test of live bacterial composition

[0508] D-2-1. Efficacy test of a composition containing live bacteria and soybean extract

[0509] Objective: To examine the weight loss effect of a composition containing live bacteria of Bifidobacterium breve BCRC 911145 and soybean extract.

[0510] Experimental content:

[0511] 1. Dosage form: powder pack

[0512] 2. Dosage: 4000mg / pack of a composition containing live bacteria of Bifidobacterium breve BCRC 911145 and soybean extract (containing 150mg of live bacteria powder of Bifidobacterium breve BCRC 911145 (equivalent to 2×10 8 CFU / g) with 150mg of soy extract and additives)

[0513] 3. Experimental design: Subjects took one pack of the drug one hour before lunch daily for 60 consecutive days.

[0514] 4. Number of participants: 30

[0515] 5. Subject inclusion criteria: Male or female aged 20-40 years old

[0516] 6.Test items:

[0517] Weight and body fat

[0518] The results are shown in Table 19 and Figures 16A and 16B.

[0519] Table 19. Changes in weight and body fat percentage of subjects

[0520] According to Table 19 and Figures 16A and 16B , the composition containing live bacteria of Bifidobacterium breve BCRC 911145 and soybean extract can indeed reduce body weight and body fat percentage, and thus has a weight loss effect.

[0521] D-2-2. Efficacy test of a composition containing live bacteria, soybean extract, and apple extract

[0522] D-2-2-1. Blood sugar regulation efficacy test of a composition containing live bacteria, soybean extract, and apple extract

[0523] Method 1

[0524] Objective: To examine the blood sugar regulating effect of a composition containing live bacteria of Bifidobacterium breve BCRC 911145, soy extract, and apple extract.

[0525] Experimental content:

[0526] 1. Dosage form: powder pack

[0527] 2. Dosage: 800mg / pack of a composition containing live bacteria of Bifidobacterium breve BCRC 911145, soybean extract and apple extract (containing 150mg of live bacteria powder of Bifidobacterium breve BCRC 911145 (equivalent to 5×10 9 CFU / g), soy extract 150mg, apple extract 150mg and additives)

[0528] 3. Number of subjects: 5

[0529] 4. Subject inclusion criteria: Male or female aged 20-40 years

[0530] 5. Experimental Design

[0531] Self-controlled experiment: Blood samples were collected from the same group of subjects at the same time intervals without taking the composition of the present disclosure (control group) and after taking the composition of the present disclosure (group taking the composition of the present disclosure).

[0532] The subjects took one packet of the aforementioned composition on day 1 of the experiment and did not take the composition on day 7. On day 1 of the experiment, blood was collected from the subjects while fasting. The subjects were then asked to take one packet of the aforementioned composition, and blood was collected 15, 45, 60, 120, and 240 minutes after taking the composition. After a 7-day washout, blood was collected again while fasting (on day 9 of the experiment), and then 15, 45, 60, 120, and 240 minutes after taking the composition.

[0533] 6.Test items:

[0534] (1) Changes in serum GLP-1 concentration;

[0535] (2) Changes in blood glucose concentration

[0536] The results are shown in Figures 17 and 18.

[0537] Figure 17 shows that 60 minutes after taking the aforementioned composition, the serum GLP-1 concentration in the subjects increased significantly by 17.6%, indicating that the composition effectively promotes GLP-1 secretion in the body. In contrast, when the composition was not taken, the serum GLP-1 concentration in the subjects fluctuated significantly, with no trend of steady increase.

[0538] FIG18 shows that, compared with not taking the aforementioned composition, the blood sugar levels of the subjects after taking the aforementioned composition were more stable and without fluctuations, indicating that taking the aforementioned composition can effectively help regulate blood sugar and achieve the effect of controlling appetite by stabilizing blood sugar levels.

[0539] From the above, it can be seen that the aforementioned composition does have the effect of regulating blood sugar and can thereby control appetite.

[0540] Method 2

[0541] Objective: To examine the blood sugar regulating effect of a composition containing live bacteria of Bifidobacterium breve BCRC 911145, soy extract, and apple extract.

[0542] Experimental content:

[0543] 1. Dosage form: powder pack

[0544] 2. Dosage: 800mg / pack of a composition containing live bacteria of Bifidobacterium breve BCRC 911145, soybean extract and apple extract (containing 150mg of live bacteria powder of Bifidobacterium breve BCRC 911145 (equivalent to 5×10 9 CFU / g), soy extract 150mg, apple extract 150mg and additives)

[0545] 3. Number of subjects: 4

[0546] 4. Subject inclusion criteria: Male or female aged 20-40 years

[0547] 5. Experimental Design

[0548] Self-controlled experiment: Blood samples were collected from the same group of subjects at the same time intervals without taking the composition of the present disclosure (control group) and after taking the composition of the present disclosure (group taking the composition of the present disclosure).

[0549] On the first day of the experiment, fasting blood samples were collected from the subjects. The subjects were then asked to consume breakfast (a corn omelet) within 15 minutes. Blood samples were collected 2 and 4 hours after breakfast. Three days later (day 5 of the experiment), fasting blood samples were collected. The subjects were then asked to consume one packet of the aforementioned composition and the same breakfast within 15 minutes. Blood samples were collected 2 and 4 hours after breakfast. After the subjects consumed the composition for three consecutive days (days 5 to 7 of the experiment), fasting blood samples were collected again (day 8 of the experiment).

[0550] 6.Test items:

[0551] (1) Changes in serum glucagon-like peptide-1 (GLP-1) concentration;

[0552] (2) changes in blood glucose concentration and insulin concentration;

[0553] (3) fasting blood glucose concentration;

[0554] (4) Appetite questionnaire survey.

[0555] The results are shown in FIG. 19 , FIG. 20A and FIG. 20B , FIG. 21 , and FIG. 22A to FIG. 22C .

[0556] Figure 19 shows that serum GLP-1 levels in the subjects remained steadily elevated 2 and 4 hours after taking the aforementioned composition. This indicates that the composition effectively promotes GLP-1 secretion in the body, helping to regulate blood sugar and appetite. In contrast, serum GLP-1 levels in the control group did not increase.

[0557] As shown in Figures 20A and 20B , the subjects' postprandial blood glucose levels increased significantly 4 hours after breakfast. In contrast, the increase in postprandial blood glucose levels in the subjects who took the aforementioned composition 4 hours later was significantly lower than that in the control group, indicating that the aforementioned composition can effectively help stabilize postprandial blood glucose levels.

[0558] As shown in FIG21 , after taking the aforementioned composition for 3 consecutive days, the subjects' fasting blood glucose levels improved, which means that the aforementioned composition can effectively help stabilize fasting blood glucose.

[0559] As shown in Figures 22A to 22C , after taking the aforementioned composition, the subjects reported lower hunger and appetite levels four hours after breakfast compared to the control group. Furthermore, the subjects were less likely to feel hungry and less likely to crave food before lunch.

[0560] From the above, it can be seen that the above composition does have the effects of regulating blood sugar and reducing appetite.

[0561] Although the present disclosure has been disclosed above with reference to preferred embodiments, this is not intended to limit the present disclosure. Anyone skilled in the art may make slight changes and modifications without departing from the spirit and scope of the present disclosure. Therefore, the scope of protection of the present disclosure shall be determined by the scope of the appended patent applications.

[0562] Deposit of biological materials

[0563] Domestic Storage

[0564] 1. Bifidobacterium breve TCI761

[0565] Bioresource Conservation and Research Center, Food Industry Development Institute, Taiwan, China

[0566] August 8, 2022

[0567] BCRC 911145

[0568] Overseas Storage

[0569] 1. Bifidobacterium breve TCI761

[0570] German Collection of Microorganisms and Cell Cultures GmbH (DSMZ)

[0571] September 7, 2022

[0572] DSM 34383

Claims

1. A novel Bifidobacterium breve, which is deposited in the Bioresource Conservation and Research Center of the Food Industry Development Institute and has a deposit number of BCRC 911145.

2. Use of the culture according to claim 1 in preparing a composition for reducing appetite and / or promoting the secretion of glucagon-like peptide-1 (GLP-1).

3. Use of the novel Bifidobacterium breve or a culture thereof as claimed in claim 1 in preparing a composition for inhibiting adipocyte maturation.

4. Use of the novel Bifidobacterium breve or a culture thereof as claimed in claim 1 in preparing a composition for reducing fat accumulation and / or promoting fat metabolism.

5. Use of the novel Bifidobacterium breve or a culture thereof as claimed in claim 1 in the preparation of a composition for reducing body weight.

6. Use of the novel Bifidobacterium breve or a culture thereof as claimed in claim 1 in the preparation of a composition for regulating blood lipids.

7. The method of claim 6, wherein the blood lipids are regulated include: Lowers total cholesterol; Promote the synthesis of high-density lipoprotein cholesterol (HDL-C); and / or Promotes low-density lipoprotein cholesterol cholesterol, LDL-C) metabolism.

8. Use of the novel Bifidobacterium breve or a culture thereof as claimed in claim 1 in preparing a composition for regulating blood sugar.

9. The use according to any one of claims 2, 3, 4, 5, 6 and 8, wherein the culture comprises the cultured novel Bifidobacterium breve.

10. The use according to any one of claims 2, 3, 4, 5, 6 and 8, wherein the cultured novel Bifidobacterium breve is active or viable.

11. The use according to any one of claims 2, 3, 4, 5, 6 and 8, wherein the cultured novel Bifidobacterium breve is free of its own physiological activity or is dead.

12. The use according to any one of claims 2, 3, 4, 5, 6 and 8, wherein the culture comprises a culture supernatant of the novel Bifidobacterium breve.

13. A probiotic composition, include: The novel Bifidobacterium breve or a culture thereof as claimed in claim 1; as well as 1. Prebiotics.

14. The probiotic composition of claim 13, wherein the weight ratio of the novel Bifidobacterium breve or its culture to the prebiotic is 1-6:1-12.

15. The probiotic composition of claim 13, wherein the culture comprises the cultured novel Bifidobacterium breve. 16 . The probiotic composition of claim 15 , wherein the cultured novel Bifidobacterium breve is active or viable.

17. The probiotic composition of claim 15, wherein the cultured novel Bifidobacterium breve is free of its own physiological activity or is dead.

18. The probiotic composition of claim 13, wherein the probiotic culture comprises a culture supernatant of the novel Bifidobacterium breve.

19. The probiotic composition of claim 13, wherein the prebiotic comprises at least one of the following ingredients: Soybean extract, oat extract, barley extract, apple extract, banana extract, tomato extract, chicory extract, asparagus extract, beet extract, garlic extract, onion extract, seaweed extract, pectin extract and tea extract.

20. The probiotic composition of claim 19, wherein the prebiotic comprises soybean extract.

21. The probiotic composition of claim 19, wherein the probiotic comprises soybean extract and apple extract.

22. The probiotic composition of claim 13, wherein the probiotic composition is a food composition.

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