Synbiotic composition, metabolite and application of synbiotic composition and metabolite to prevention and treatment of obesity-related diseases

Abstract

The invention provides a synbiotic composition which comprises litchi polyphenol, bifidobacterium longum and methionine. The invention also provides a synbiotics composition for preventing and treating obesity-related diseases. The invention also provides a metabolite which is used for preventing and treating obesity-related diseases.

Description

Synbiotic compositions, metabolites, and uses thereof in preventing and treating obesity-related diseases Technical Field

[0001] The present invention relates to a composition, metabolites and uses thereof, and in particular to a synbiotic composition, metabolites and uses thereof for preventing and treating obesity-related diseases. Background Art

[0002] The global prevalence of obesity has nearly tripled since 1975, primarily due to unhealthy eating habits. The growing prevalence of obesity has become a major global public health issue, as it is associated with increased susceptibility to multiple chronic diseases.

[0003] Dietary patterns and their corresponding gut microbiome profiles have been linked to a variety of health conditions. For example, a Western diet, typically high in fat and animal protein, increases the abundance of trimethylamine (TMA)-producing bacteria in the gut, which in turn increases the risk of cardiovascular disease and other health conditions.

[0004] Therefore, there is a need to improve the existing technology in order to provide a composition for preventing and treating obesity-related diseases.

[0005] Summary of the Invention

[0006] One embodiment of the present invention provides a synbiotic composition comprising litchi polyphenols, Bifidobacterium longum and methionine, wherein the Bifidobacterium longum comprises Bifidobacterium longum strains BCRC910812, BCRC12585, BCRC14602, BCRC11847, DSMZ20104, BCRC14607, BCRC11844, BCRC11846, BCRC14601, BCRC14606, BCRC14604, BCRC12584, or a combination thereof.

[0007] In some embodiments, the litchi polyphenols include procyanidin, epicatechin (EC), or a combination thereof.

[0008] In some embodiments, based on 100% by weight of the total weight of the synbiotic composition, the weight percentage of litchi polyphenols is 1% to 30%, the weight percentage of Bifidobacterium longum is 10% to 50%, and the weight percentage of methionine is 0.5% to 20%.

[0009] In some embodiments, the Bifidobacterium longum is a live bacterium.

[0010] In some embodiments, the content of Bifidobacterium longum is 1×10 9 CFU / g to 1×10 11CFU / g.

[0011] Another embodiment of the present invention provides a use of a synbiotic for preparing a composition for preventing and treating obesity-related diseases, the synbiotic comprising litchi polyphenols, Bifidobacterium longum, and methionine, wherein the Bifidobacterium longum comprises Bifidobacterium longum strains BCRC, BCRC12585, BCRC14602, BCRC11847, DSMZ20104, BCRC14607, BCRC11844, BCRC11846, BCRC14601, BCRC14606, BCRC14604, BCRC12584, or a combination thereof.

[0012] In some embodiments, the litchi polyphenols comprise procyanidins, epicatechins, or a combination thereof.

[0013] In some embodiments, based on 100% by weight of the total weight of the synbiotic composition, the weight percentage of litchi polyphenols is 1% to 30%, the weight percentage of Bifidobacterium longum is 10% to 50%, and the weight percentage of methionine is 0.5% to 20%.

[0014] In some embodiments, the Bifidobacterium longum is a live bacterium.

[0015] In some embodiments, the obesity is diet-induced obesity.

[0016] In some embodiments, the obesity-related disease is selected from the group consisting of type 2 diabetes, hyperglycemia, glucose intolerance, dyslipidemia, insulin resistance, hyperinsulinemia, fatty liver, cardiovascular disease, stroke, cancer, and combinations thereof.

[0017] In some embodiments, the composition is a food composition or a pharmaceutical composition.

[0018] In some embodiments, the pharmaceutical composition is in a dosage form for oral administration or topical administration.

[0019] Another embodiment of the present invention provides a use of a metabolite for preparing a composition for inhibiting fat accumulation, wherein the metabolite comprises 5'-methylthioadenosine, valine, pyroglutamate, glutamate, methionine, adenosine, 3-adenosine monophosphate, pyroglutamylvaline, or a combination thereof.

[0020] Another embodiment of the present invention provides a use of a metabolite for preparing a composition for preventing and treating obesity-related diseases, wherein the metabolite comprises 5'-methylthioadenosine, pyroglutamate, valine, glutamate, methionine, adenosine, 3-adenosine monophosphate, pyroglutamylvaline, or a combination thereof. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] The various aspects of the present invention will be most readily understood when the following detailed description is read in conjunction with the accompanying drawings. It should be noted that, in accordance with standard industry practice, various features may not be drawn to scale. Indeed, the dimensions of various features may be arbitrarily increased or decreased for clarity of discussion. To make the above and other objects, features, advantages, and embodiments of the present invention more readily apparent, the accompanying drawings are described as follows:

[0022] Figures 1A to 1Q illustrate that litchi polyphenols according to one embodiment of the present invention can reduce obesity caused by a high-fat diet (HFD), improve gut microbiome imbalance, and enrich Bifidobacterium longum, thereby exerting anti-obesity activity. Figures 1A-1K: Effects of litchi polyphenols on host metabolism and gut microbial composition. Figure 1A shows weight change. Figure 1B shows weight gain during the experiment. Figure 1C shows visceral fat weight. Figure 1D shows plasma triglyceride (TG). Figure 1E shows fasting blood glucose. Figure 1F shows the area under the curve (AUC) from an oral glucose tolerance test (OGTT). Figure 1G shows the AUC from an insulin tolerance test (ITT). Figure 1H shows gut microbiome composition, represented by principal coordinate analysis (PCoA) of Bray-Curtis distances. Figure 1I shows the observed operational taxonomic unit (OUT). Figure 1J shows the abundance of Bifidobacterium genus in feces. Figure 1K shows the abundance of Bifidobacterium longum in feces. Figure 1L shows the effects of B. longum metabolites on lipid accumulation in HepG2 cells. Figures 1M-1Q show the effects of B. longum on host metabolism. Figure 1M shows changes in body weight. Figure 1N shows weight gain during the experiment. Figure 1O shows fasting blood glucose. Figure 1P shows the AUC from the OGTT. Figure 1Q shows the AUC from the ITT. Data are means and standard deviations. Statistical analysis was performed using one-way analysis of variance and Tukey's range test (*, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001). In Figures 1A-1K, N = 13, 13, and 10 mice per group, with 3 fecal samples per group. In Figures 1M-1Q, N = 5, 4, and 5 mice per group. Abbreviations: AUC, area under the curve; B. longum: Bifidobacterium longum; HFD, high-fat diet; NCD, normal diet; OGTT, oral glucose tolerance test; Phe, litchi polyphenols; UA, uric acid.

[0023] Figures 2A to 2L illustrate the identification of bioactive metabolites of Bifidobacterium longum according to various embodiments of the present invention. Figure 2A shows a workflow for identifying bioactive metabolites of Bifidobacterium longum. Figure 2B shows the effects of crude extracts and fractionation of Bifidobacterium longum on lipid accumulation in HepG2 cells (n=3). Figure 2C shows the effects of 5'-methylthioadenosine (MTA) on lipid accumulation in HepG2 cells (n=3). Figure 2D shows correlation analysis between obesity-related phenotypes and the abundance of bioactive metabolites in mouse feces (n=9). The colors and numbers in each column represent Spearman's r. Figures 2E to 2F show the abundance of methionine (Figure 2E) and MTA (Figure 2F) in mouse feces (n=4-5). Figures 2G-2I show that Bifidobacterium longum can produce 13C2H3-MTA from 13C2H3-S-adenosyl-L-methionine. Figure 2G shows the experimental design: Bifidobacterium longum was incubated with isotope-labeled methionine (-13C2H3) at gradient concentrations, and bacterial metabolites were extracted for detection of isotope-labeled MTA. Figures 2H-2I: Tandem mass verification (Figure 2H) and extracted ion chromatogram (Figure 2I) of isotope-labeled S-adenosyl-L-methionine (m / z 403.1657). Figures 2J-2K: Tandem mass verification (Figure 2J) and extracted ion chromatogram (Figure 2K) of isotope-labeled MTA (m / z 302.1173) produced by Bifidobacterium longum. Figure 2L: Abundance of MTA in feces of mice administered methionine (n = 4). Data are means and standard deviations. Statistical analysis was performed using one-way analysis of variance and Tukey's range test (*, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001). Abbreviations: AUC, area under the curve; B. longum: Bifidobacterium longum; HFD, high-fat diet; NCD, normal diet; OGTT, oral glucose tolerance test.

[0024] Figures 3A to 3K show that MTA according to other embodiments of the present invention improves obesity and metabolic disorders. Figure 3A shows weight change. Figure 3B shows weight gain during the experiment. Figure 3C shows visceral fat weight. Figure 3D shows subcutaneous fat weight. Figure 3E shows fasting blood glucose. Figure 3F shows the plasma glucose curve measured during the oral glucose tolerance test (OGTT). Figure 3G shows the AUC derived from the oral glucose tolerance test (OGTT). Figure 3H shows the plasma glucose curve measured during the insulin tolerance test. Figure 3I shows the AUC derived from the insulin tolerance test. Figure 3J shows the liver weight. Figure 3K shows representative histological features of H&E-stained liver tissue. Data are means and standard deviations. Statistical analysis was performed using one-way analysis of variance and Tukey's range test (*, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001). N = 5, 4, and 5 mice per group. Abbreviations: AUC, area under the curve; HFD, high-fat diet; MTA, 5'-methylthioadenosine; NCD, normal diet; OGTT, oral glucose tolerance test.

[0025] Figures 4A to 4E show the effects of litchi polyphenols, Bifidobacterium longum, and methionine, either individually or in combination, on inhibiting fat accumulation according to other embodiments of the present invention. Figure 4A shows the change in body weight, p < 0.0001, paired t-test; Figure 4B shows the change in body fat mass, p < 0.0001, paired t-test; Figure 4C shows the change in body fat percentage, p < 0.0001, paired t-test; Figure 4D shows the change in visceral fat level, p < 0.0001, paired t-test; and Figure 4E shows the change in waist circumference, p = 0.002, paired t-test.

[0026] Figures 5A and 5B show the fat accumulation inhibitory effects of metabolites according to other embodiments of the present invention. Figure 5A shows the fat accumulation inhibitory effect of MTA, *: p < 0.05, t-test; Figure 5B shows the fat accumulation inhibitory effect of pyroglutamate, *: p < 0.05, **: p < 0.01, t-test. DETAILED DESCRIPTION

[0027] To provide a more detailed and complete description of the present invention, the following provides illustrative descriptions of embodiments and specific examples of the present invention. However, these descriptions are not intended to be the only forms of implementing or using the embodiments of the present invention. The embodiments disclosed below may be combined or substituted with one another where beneficial, and other embodiments may be added to one embodiment without further description or explanation. In the following description, many specific details are detailed to enable the reader to fully understand the following embodiments. However, the embodiments of the present invention may be practiced without these specific details.

[0028] In this document, unless the context specifically limits the use of the articles, "a," "an," and "the" may refer to one or more. It will be further understood that the use of "comprise," "include," "have," and similar words herein specify the features, regions, integers, steps, operations, elements, and / or components described herein, but do not exclude the presence of one or more other features, regions, integers, steps, operations, elements, components, and / or groups thereof.

[0029] Furthermore, when a number or a range of numbers is described by "about," "approximately," and the like, the terms are intended to encompass numbers that are within a reasonable range taking into account variations that inherently occur during manufacturing as understood by those skilled in the art. For example, based on known manufacturing tolerances associated with manufacturing features having the characteristics associated with the number, a number or range of numbers encompasses a reasonable range that includes the described number, such as within + / -10% of the described number.

[0030] In this article, the term "synbiotic" refers to a nutritional food or medicine that combines prebiotics and probiotics, and may also contain precursors of postbiotics to promote human health.

[0031] As used herein, the term "prebiotic" refers to substances that can be decomposed and utilized by probiotics and promote the growth of probiotics, thereby producing beneficial effects on health.

[0032] As used herein, the term "postbiotic" refers to a product secreted by a non-living microorganism or its fragments that provides a physiological benefit to the host.

[0033] In some embodiments of the present invention, the synbiotic composition is administered to a subject orally or parenterally. In some embodiments of the present invention, the synbiotic composition is formulated into an oral dosage form selected from the group consisting of a solution, a suspension, an emulsion, a powder, a lozenge, a pill, a syrup, a buccal lozenge, a tablet, a chewing gum, and a capsule for administration to a subject.

[0034] In some embodiments, pharmaceutically acceptable carriers include, but are not limited to, water, alcohols, glycols, preservatives, antioxidants, solvents, emulsifiers, suspending agents, decomposers, binding agents, excipients, stabilizing agents, chelating agents, diluents, gelling agents, preservatives, lubricants, absorption enhancers, active agents, humectants, odor absorbers, fragrances, pH adjusting agents, occlusive agents, emollients, thickeners, solubilizing agents, penetration enhancers, enhancers), anti-irritants, colorants, propellants, surfactants, and other similar or suitable carriers for the present invention.

[0035] In some embodiments of the present invention, the synbiotic composition can be a food composition. For example, it can be added to an edible material as a food additive to prepare a food product for human or animal consumption. Food compositions include, but are not limited to, general foods, health foods, beverages, nutritional supplements, dairy products, or feed. In the case of oral dosage forms, the synbiotic composition can optionally include pharmaceutically and food-acceptable carriers, excipients, and / or additives. In other examples, the dosage form of the composite probiotic composition can include, but is not limited to, powders, tablets, granules, suppositories, microcapsules, ampoules, liquid sprays, or plugs.

[0036] In some embodiments of the present invention, the synbiotic composition includes litchi polyphenols, Bifidobacterium longum, and methionine.

[0037] In some embodiments, the total weight of the synbiotic composition is 100%, and the weight percentage of litchi polyphenols is 1% to 30%, the weight percentage of Bifidobacterium longum is 10% to 50%, and the weight percentage of methionine is 0.5% to 20%. The weight percentage of litchi polyphenols is 1% to 30%, such as 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, or any value between any two of these values. The weight percentage of Bifidobacterium longum is 10% to 50%, such as 15%, 20%, 25%, 30%, 35%, 40%, 45%, or any value between any two of these values. The weight percentage of methionine is 0.5% to 20%, for example, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 2%, 4%, 6%, 8%, 10%, 12%, 14%, 16%, 18%, or any value in between any two of these values.

[0038] In some embodiments, the content of Bifidobacterium longum is 1×10 9 CFU / g to 1×10 11 CFU / g, for example 2×10 9 CFU / g, 3×10 9 CFU / g, 4×10 9 CFU / g, 5×10 9 CFU / g, 6×10 9 CFU / g, 7×10 9 CFU / g, 8×10 9 CFU / g, 9×10 9 CFU / g, 1×10 10 CFU / g, 2×10 10 CFU / g, 3×10 10 CFU / g, 4×10 10 CFU / g, 5×10 10 CFU / g, 6×10 10 CFU / g, 7×10 10 CFU / g, 8×10 10 CFU / g, 9×10 10 CFU / g, or any value between any two of these values.

[0039] In some embodiments of the present invention, a use of a metabolite for preparing a composition for inhibiting fat accumulation is provided, wherein the metabolite comprises 5'-methylthioadenosine, valine, pyroglutamate, glutamate, methionine, adenosine, 3-adenosine monophosphate, pyroglutamylvaline, or a combination thereof.

[0040] In some embodiments of the present invention, a metabolite is provided for use in preparing a composition for preventing and treating obesity-related diseases, wherein the metabolite comprises 5'-methylthioadenosine, valine, pyroglutamate, glutamine, methionine, adenosine, 3-adenosine monophosphate, pyroglutamylvaline, or a combination thereof.

[0041] Several examples and experimental examples are listed below to further illustrate the synbiotic composition of the present invention. However, these examples are for illustrative purposes only and are not intended to limit the present invention. The scope of protection of the present invention shall be determined by the scope defined in the appended claims.

[0042] Example

[0043] Example 1 Litchi polyphenols can alleviate HFD-induced obesity and improve intestinal microbial imbalance

[0044] Litchi fruits (Litchi chinensis Sonn.) were obtained from a local market in Taipei, Taiwan. After manually removing the peel and seeds, the pulp was frozen in liquid nitrogen and dehydrated using a freeze dryer. Finally, the pulp was ground into a fine powder. Dried lychee pulp powder (300 g) was extracted with 70% methanol in water (2.4 L) using an ultrasonic generator (30 W, 60 kHz) for 30 minutes to obtain an extract. After filtering the extract through filter paper, the same extraction steps were used to further extract the residual compounds twice to obtain a filtrate. A rotary evaporator (45° C.) was used to remove all methanol from the filtrate. The polysaccharides in the filtrate were removed via an Oasis HLB column (20 cc / 1 g, Waters, Milford, MA, USA). The remaining compounds were centrifuged to obtain a crude extract. The Global Natural Products Social (GNPS) analyzed litchi phenolic compounds in the crude extract. This is a molecular networking method based on the principle that molecules with similar chemical structures exhibit similar MS / MS fragmentation patterns. The crude extract contains litchi polyphenols, including proanthocyanidins (proanthocyanidin B2), epicatechin ((-)-epicatechin, EC), quercetin rhamnosyl-rutinoside, kaempferol rhamnosyl-rutinoside, isorhamnetin rhamnosyl-rutinoside, rutin, kaempferol rutinoside, and isorhamnetin rutinoside.

[0045] We found that litchi polyphenols alleviated high-fat diet-induced obesity, reduced hyperlipidemia, and improved glucose tolerance and insulin sensitivity (Figures 1A-1G). Administration of litchi polyphenols enriched the diversity of the microbiota and improved HFD-induced microbiota dysbiosis (Figures 1H, 1I). In addition, litchi polyphenols increased the abundance of Bifidobacterium, with a slight increase in Bifidobacterium longum (Figures 1J, 1K).

[0046] Example 2 Bifidobacterium longum has anti-obesity activity

[0047] Next, we sought to investigate the regulatory capacity of Bifidobacterium metabolites on host metabolism. To this end, we purchased all commercial Bifidobacterium strains from Taiwan (11 strains: BCRC12585, BCRC14602, BCRC11847, DSMZ20104, BCRC14607, BCRC11844, BCRC11846, BCRC14601, BCRC14606, BCRC14604, BCRC12584, BCRC is the Bioresource Conservation and Research Center of the Food Industry Development Institute, Taiwan, and DSMZ is the German Collection of Microorganisms and Cell Cultures GmbH), and then colonized them into C57BL / 6 germ-free mice. Germ-free mice were orally administered 1×10 9 Colony-forming units (CFUs) of Bifidobacterium spp. (11 strains were mixed evenly) were added to 0.2 mL of sterile PBS solution. Germ-free mice were fed with 0.2 mL of sterile PBS via tube as a control group. All mice were fed a high-fat diet (OpenSource Diets TM To investigate whether Bifidobacterium metabolites could reduce obesity, mouse feces (100 mg) were extracted with 1 mL of 70% methanol in water containing cholic acid-d4 (2 ppm) as an internal standard. After homogenization using an ultrasonic generator (30 W, 60 kHz) for 30 minutes, the extract was centrifuged at 12,000 rpm for 10 minutes at 4°C. The supernatant, representing the metabolites, was collected and dried.

[0048] Lipid accumulation assay was performed. In brief, HepG2 cells grown at 70% were cultured in RPMI 1640 culture medium containing 120 μg / mL uric acid for 24 hours to increase lipid accumulation. After washing with PBS buffer, the cells were maintained for another 24 hours in new RPMI 1640 culture medium containing metabolites (5.0, 1.0, 0.5, 0.1 and 0.05 μg / mL, and the dried metabolites were dissolved back in RPMI 1640 culture medium) and 120 μg / mL uric acid. Cells maintained in pure RPMI 1640 culture medium were used as controls. The results showed that the metabolites extracted from the feces of mice fed with Bifidobacterium spp. significantly reduced lipid accumulation in a dose-dependent manner (not shown in the figure) by testing the lipid accumulation activity of HepG2 cells. At the same time, the metabolites of each strain of Bifidobacterium spp. also exerted anti-obesity activity (Figure 1L is Bifidobacterium longum BCRC12585 and the other strains are not shown). These findings suggest that the anti-obesity effects of polyphenols may be attributed to the abundance of Bifidobacterium. As we sought to identify specific chemicals produced by probiotic microorganisms that have therapeutic potential and can be easily scaled up in industry, we selected Bifidobacterium longum subspecies longum BCRC12585 (hereafter referred to as B. longum), a commercial strain isolated from humans that is easily scaled up, for further exploration of its bioactive metabolites in a mouse model.

[0049] Next, 10 8 CFU of Bifidobacterium longum (BCRC12585) were administered to C57BL / 6 mice once daily until one week before sacrifice. The results showed that the mice showed weight loss and reduced weight gain (Figure 1M, 1N), while the visceral and subcutaneous fat of mice fed with Bifidobacterium longum was slightly reduced (Figure not shown). In addition, mice treated with Bifidobacterium longum showed a significant decrease in fasting blood glucose (Figure 1O, 1P, 1Q) and a slight improvement in glucose and insulin tolerance (Figure not shown).

[0050] Example 3 Identification of bioactive metabolites produced by Bifidobacterium longum

[0051] To further identify the bioactive metabolites produced by Bifidobacterium longum, we applied a fractionation method, followed by liquid chromatography-mass spectrometry (LC-MS) combined with an in vitro screening strategy (Figure 2A). In short, the extraction method of Bifidobacterium longum BCRC12585 metabolites (i.e., Bifidobacterium longum metabolites, or Bifidobacterium longum crude extract) was the same as in Example 2, and then 25 fractions from Bifidobacterium longum metabolites were prepared by pre-HPLC column (pre-HPLC column), and then the anti-lipid accumulation activity of HepG2 cells was tested separately. It is worth noting that fractions A to H with short retention times strongly inhibited lipid accumulation (Figure 2B), indicating that the active compounds are mostly small molecules with high polarity. In order to identify candidate metabolites from each active component, liquid chromatography combined with high-resolution mass spectrometry (LC-MS / MS) was used for further analysis. Fourteen compounds, including nine amino acids (proline, valine, glutamine, glutamic acid (GA), arginine, pyroglutamine (PyroGA), methionine, isoleucine, and tryptophan), two peptides (cyclo-(His-Pro) and pyroglutamyl-valine (Pyro-glu-val), and adenosine and its two derivatives (3-adenosine monophosphate (3-AMPP) and 5'-methylthioadenosine (MTA)), were identified and further validated using authentic standards (not shown). Among these identified metabolites, eight (MTA, valine, pyroglutamine, glutamic acid, methionine, adenosine, 3-AMPP, and pyroglutamyl-valine) significantly inhibited lipid accumulation in HepG2 cells (MTA, Figure 2C; the others are not shown). Notably, MTA abundance was negatively correlated with most mouse obesity biomarkers (Figure 2D). Overall, we integrated in vitro, in vivo, and metabolomics data to identify potential bioactive metabolites produced by B. longum.

[0052] Interestingly, both MTA and its precursor, methionine, were identified in the active fraction of B. longum and exhibited lipid-lowering effects in HepG2 cells (MTA shown in Figure 2C; other figures not shown). We next investigated whether B. longum could produce MTA from methionine. The experiment involved administering a high-HFD for 17 weeks, followed by gavage of MTA at a dose of 100 mg / kg body weight daily (100 mg / kg / day) starting in week 8. Fecal methionine, a precursor for MTA synthesis, decreased in B. longum-treated mice, while MTA increased (Figures 2E and 2F). Next, we investigated whether B. longum could convert methionine into MTA using in vitro isotope labeling experiments coupled with mass spectrometry. Briefly, B. longum was cultured on plates coated with 1 mL of isotope-labeled methionine (Iso-met, -13C2H3) at 0 mM, 1 mM, and 10 mM concentrations (Figure 2G). The results showed that 13C2H3-MTA (m / z 302.1173 (M+4)) and its intermediate 13C2H3-S-adenosyl-L-methionine (m / z 403.1657 (M+4)) were detected in a dose-dependent manner (Figures 2H–2K), indicating that Bifidobacterium longum can utilize methionine to synthesize MTA. Furthermore, administration of methionine increased the amount of MTA in mouse feces (Figure 2L). Furthermore, we found that other Bifidobacterium species used in this study, including Bifidobacterium breve, Bifidobacterium adolescentis, Bifidobacterium infantis, Bifidobacterium longum, Bifidum bifidum, and Bifidobacterium animalis, can also utilize methionine to synthesize MTA (not shown). Here, we discovered that MTA, a bioactive metabolite with anti-obesity potential, can be produced by Bifidobacterium through the conversion of dietary amino acids.

[0053] Example 4 MTA is a methionine-derived metabolite produced by Bifidobacterium longum that can improve obesity and metabolic disorders

[0054] As a biosynthetic precursor for several key chemical compounds, MTA plays a role in energy metabolism, including regulation of the immune system, gene expression, and the synthesis of important biomolecules such as DNA and proteins. Therefore, we next investigated the effects of MTA on host metabolism by treating C57BL / 6 mice fed a high-flown diet (HFD) with or without MTA. The experiment involved administering a high-flown HFD for 17 weeks, followed by daily MTA gavage at 100 mg per kilogram of body weight (100 mg / kg / day) starting in week 8. MTA administration significantly reduced metabolic parameters in mice, including body weight, weight gain during the study period, and visceral and subcutaneous fat mass (Figures 3A to 3E). Similar reductions were observed in glucose tolerance and insulin sensitivity (Figures 3F to 3I). Furthermore, liver weight and liver H&E staining revealed that MTA attenuated hepatic steatosis in obese mice (Figures 3J and 3K). These results suggest that MTA can effectively regulate lipid metabolism and insulin sensitivity, thereby reducing obesity and hepatic steatosis.

[0055] Example 5 MTA exerts multiple regulatory effects on liver energy metabolism

[0056] To more fully understand the potential of MTA as an anti-obesity molecule, liver RNA sequencing was performed on mice treated with MTA and Bifidobacterium longum to identify its potential mechanisms of obesity prevention. Gene ontology (GO) enrichment analysis of the top 30 differentially expressed molecular functions revealed that MTA significantly modulated genes involved in fatty acid metabolism (Ltb4r1, Insig2, Elovl3, and Trib3), neurotransmitter signaling (Hcn3 and Adrb2), growth hormone signaling (Enho and Arntl), leptin signaling, and bile acid metabolism (not shown), while B. longum primarily regulated genes involved in fatty acid metabolism, bile acid metabolism, and immune response (not shown). We then conducted an in-depth analysis of the regulatory genes involved in these biological functions. Notably, expression of insulin-induced gene 2 (Insig2), which influences hepatic cholesterol metabolism, lipogenesis, and glucose homeostasis, was decreased in MTA-treated mice. MTA treatment also reduced fatty acid elongase 3 (Elovl3), an enzyme responsible for the elongation of very-long-chain fatty acids. These findings suggest that MTA's inhibition of Insig2 and Elovl3 may contribute to fatty acid remodeling in the liver and contribute to weight regulation. Furthermore, the β-2-adrenergic receptor gene (Adrb2) and the leptin receptor gene (Lepr), which promote metabolic signaling related to energy expenditure via epinephrine and leptin, respectively, were upregulated in MTA-treated mice. Furthermore, increased expression of adropin (encoded by the energy homeostasis-related gene Enho) in MTA-treated mice may improve glucose metabolism by enhancing glucose utilization. Similarly, overexpression of inhibin β-A, encoded by the Inhba gene, improved glucose metabolism, stimulated mitochondrial energy metabolism, and increased energy expenditure. These results suggest that MTA may improve energy metabolism by modulating the signaling of these metabolic hormones and proteins. In summary, these results suggest that the effects of MTA in preventing obesity and insulin resistance may be due to multiple energy metabolism-regulating activities, including fatty acid biosynthesis, glucose utilization, and energy metabolism, which may be regulated by signaling of hormones and proteins with metabolic regulatory effects.

[0057] Bile acids are considered to be important signaling molecules that regulate fatty acid, cholesterol, energy, and glucose homeostasis. Therefore, enhancing bile acid synthesis may be a strategy to avoid diet-induced obesity. In addition, increased expression of Cyp7a1 (encoding a limiting enzyme in the typical bile acid synthesis pathway) can prevent obesity, insulin resistance, and atherosclerosis caused by a high-fat diet. Given that the expression of Cyp7a1 was increased in MTA-treated mice (not shown), liver bile acid analysis was performed to determine whether the regulated expression of Cyp7a1 contributes to bile acid composition. The results showed that mice administered MTA showed a trend of increased primary bile acids in the liver. In summary, these results indicate that MTA can increase the expression level of Cyp7a1 and regulate its downstream bile acid synthesis and fatty acid metabolism.

[0058] Example 6 Effect of the combination of litchi polyphenols, Bifidobacterium longum and methionine on inhibiting fat accumulation

[0059] The synbiotic composition comprises litchi polyphenols, Bifidobacterium longum and methionine. The weight percentage of the synbiotic composition is 100%, and the litchi polyphenols in the synbiotic composition are purchased from commercial (The main active ingredients include proanthocyanidins (proanthocyanidins A1, A2, B1, B2), catechin, epicatechin (EC), epicatechin gallate (ECG), epigallocatechin gallate (EGCG), epicatechin-(4β→8, 2β→O7) ericatechin-(4β→8)-ericatechin (epicatechin-(4β→8, 2β→O→7) eicatechin-(4β→8)-eicatechin, A2-EC), and green tea polyphenols, with a content of 10 wt%; Bifidobacterium longum was obtained from B. longum subsp. infantis BLI-02 strain (deposited at the Bioresource Conservation and Research Center, Food Industry Development Research Institute, Taiwan, China BCRC910812 (this strain is freely transferable) or China General Microbial Culture Collection Center CGMCC) No.15212), with a content of 30 wt%, of which the effective living bacteria include 1×10 10 CFU / g (for example, the total weight of the synbiotic composition is 0.5 g); the methionine content is 2 wt %. The synbiotic composition is prepared in the form of capsules, and the above weight percentages do not include the weight of the capsules.

[0060] 27 volunteers met the following screening criteria, including 8 males and 19 females, with ages ranging from 18 to 65 years old. The embodiment of this example is to provide the subjects with a synbiotic composition for five consecutive weeks, with a dosage of two capsules twice a day. The nutritionist will give the subjects a calorie and diet control that is consistent with their weight based on the calorie intake recommended in the body composition analysis results to control variables. The subjects were measured by body fat machine ( Model: InBody 570, an instrument that uses bioelectric impedance (BIA) to analyze body composition to measure weight, body fat mass, body fat percentage, and visceral fat level.

[0061] The results are shown in Figures 4A to 4E. Figure 4A shows that compared to pre-use weight, the subjects' weight decreased significantly after taking the synbiotic composition for five weeks, with an average weight loss of 1.39 kg for the 27 subjects. Figure 4B shows that compared to pre-use body fat weight, the subjects' body fat weight decreased significantly after taking the synbiotic composition for five weeks, with an average body fat loss of 1.27 kg for the 27 subjects. Figure 4C shows that compared to pre-use body fat percentage, the subjects' body fat percentage decreased significantly after taking the synbiotic composition for five weeks, with an average body fat loss of 1.21% for the 27 subjects. Figure 4D shows that compared to pre-use visceral fat level, the subjects' visceral fat level decreased significantly after taking the synbiotic composition for five weeks, with an average visceral fat level loss of 0.7 grade for the 27 subjects. Figure 4E shows that compared to pre-use waist circumference, the subjects' waist circumference decreased significantly after taking the synbiotic composition for five weeks, with an average waist circumference loss of 2.57 cm for the 27 subjects.

[0062] In some embodiments, the lychee polyphenols in the synbiotic composition are obtained from Example 1, and the Bifidobacterium longum is obtained from BCRC12585, BCRC14602, BCRC11847, DSMZ20104, BCRC14607, BCRC11844, BCRC11846, BCRC14601, BCRC14606, BCRC14604, BCRC12584, or a combination thereof, which can also achieve similar effects of reducing body weight, reducing body fat weight, reducing body fat percentage, lowering visceral fat levels, and reducing waist circumference.

[0063] Example 7 Effect of MTA and pyroglutamic acid in inhibiting fat accumulation

[0064] 3T3-L1 cells were seeded in 12-well plates at a cell density of 3 × 10 33T3-L1 cells were cultured in DMEM supplemented with 10% bovine calf serum (CS), 100 U / mL penicillin, and 100 μg / mL streptomycin for 3 to 4 days. The medium was changed every 2 days until the cell density reached approximately 70%, at which point differentiation was initiated. The medium was then changed to DMEM supplemented with 0.5 mM 3-isobutyl-1-methylxanthine (IBMX), 1 μM dexamethasone, 10 μg / mL insulin, 10% fetal bovine serum (FBS), 100 U / mL penicillin, and 100 μg / mL streptomycin for 2 days. Following insulin induction, 3T3-L1 cells were induced to accumulate adipocytes by switching to DMEM supplemented with 10 μg / mL insulin, 10% FBS, 100 U / mL penicillin, and 100 μg / mL streptomycin. At the same time as induction, different compounds (MTA, pyroglutamate) were treated for 2 days.

[0065] The results, as shown in Figure 5A, show that both 1 μg / mL and 5 μg / mL MTA significantly inhibited triglyceride (TG) accumulation in 3T3-L1 adipocytes. Figure 5B shows that 10 μg / mL MTA significantly inhibited TG accumulation in 3T3-L1 adipocytes, and administration of 2.5 μg / mL or 5 μg / mL MTA also showed an inhibitory trend.

[0066] In some embodiments of the present invention, various methods reveal the molecular mechanism by which polyphenols can prevent obesity by enriching bifidobacteria and MTA, a bioactive metabolite of bifidobacteria with anti-obesity activity. Furthermore, polyphenols, bifidobacteria, and methionine (a precursor of MTA) can be combined as a symbiotic product, each with anti-diabetic properties. MTA, a metabolite of Bifidobacterium longum, and pyroglutamate have the effect of inhibiting fat accumulation.

[0067] Although the present invention has been disclosed above in terms of embodiments, this is not intended to limit the present invention. Anyone skilled in the art may make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention shall be determined by the scope defined by the appended claims.

Claims

1. A synbiotic composition comprising:

1. Lychee polyphenols; a Bifidobacterium longum, wherein the Bifidobacterium longum comprises the Bifidobacterium longum strain BCRC910812, BCRC12585, BCRC14602, BCRC11847, DSMZ20104, BCRC14607, BCRC11844, BCRC11846, BCRC14601, BCRC14606, BCRC14604, BCRC12584, or a combination thereof; and Methionine.

2. The synbiotic composition according to claim 1, wherein the litchi polyphenols comprise proanthocyanidins, epicatechins, or a combination thereof.

3. The synbiotic composition according to claim 1, wherein the total weight of the synbiotic composition is 100% by weight, the weight percentage of the litchi polyphenol is 1% to 30%, the weight percentage of the Bifidobacterium longum is 10% to 50%, and the weight percentage of the methionine is 0.5% to 20%. The synbiotic composition according to claim 1 , wherein the Bifidobacterium longum is a live bacterium.

5. The synbiotic composition according to claim 4, wherein the content of Bifidobacterium longum is 1×10 9 CFU / g to 1×10 11 CFU / g.

6. A use of a synbiotic for preparing a composition for preventing and treating obesity-related diseases, the synbiotic comprising litchi polyphenols, Bifidobacterium longum, and methionine, wherein the Bifidobacterium longum comprises Bifidobacterium longum strains BCRC, BCRC12585, BCRC14602, BCRC11847, DSMZ20104, BCRC14607, BCRC11844, BCRC11846, BCRC14601, BCRC14606, BCRC14604, BCRC12584, or a combination thereof.

7. The use according to claim 6, wherein the litchi polyphenols comprise proanthocyanidins, epicatechin, or a combination thereof.

8. The use according to claim 6, wherein the total weight of the synbiotic composition is 100% by weight, the weight percentage of the litchi polyphenol is 1% to 30%, the weight percentage of the Bifidobacterium longum is 10% to 50%, and the weight percentage of the methionine is 0.5% to 20%.

9. The use according to claim 6, wherein Bifidobacterium longum is a live bacterium.

10. The use according to claim 6, wherein the obesity is diet-induced obesity.

11. The use according to claim 6, wherein the obesity-related disease is selected from the group consisting of type II diabetes, hyperglycemia, glucose intolerance, dyslipidemia, insulin resistance, hyperinsulinemia, fatty liver, cardiovascular disease, stroke, cancer and a combination thereof.

12. The use according to claim 6, wherein the composition is a food composition or a pharmaceutical composition.

13. The use according to claim 12, wherein the pharmaceutical composition is in a dosage form for oral administration or topical administration.

14. Use of a metabolite for preparing a composition for preventing and treating obesity-related diseases, wherein the metabolite comprises 5'-methylthioadenosine, pyroglutamic acid, valine, glutamic acid, methionine, adenosine, 3-adenosine monophosphate, pyroglutamylvaline, or a combination thereof.

15. The use according to claim 14, wherein the obesity is diet-induced obesity.

16. The use according to claim 14, wherein the obesity-related disease is selected from the group consisting of type II diabetes, hyperglycemia, glucose intolerance, dyslipidemia, insulin resistance, hyperinsulinemia, fatty liver, cardiovascular disease, stroke, cancer and a combination thereof.

17. The use according to claim 14, wherein the composition is a food composition or a pharmaceutical composition.

18. The use according to claim 17, wherein the pharmaceutical composition is in a dosage form for oral administration or topical administration.

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