TWI725464B - The reduced fat probiotic strain, composition thereof and use thereof - Google Patents

Abstract

The present invention provides a reduced fat probiotic strain, composition thereof and use thereof. The probiotic strain is Lactobacillus plantarum TCI378 (BCRC910760), which itself and its metabolites can inhibit the accumulation of fat in fat cells, and can effectively reduce the amount of fat in cells so as to achieve the goal of reducing fat and obesity. Wherein the Lactobacillus plantarum TCI378 metabolites of the present invention contains an effective component of Pyroglutamyl- tryptophan, 1-Methyl-1,2,3,4-tetrahydro-β-carboline-3-carboxylic acid, and/or 3-Phenyllactic acid.

Description

Fat-reducing probiotic strain and its composition and use

The present invention relates to a fat-reduced probiotic strain and its composition and use, in particular, a Lactobacillus plantarum TCI378 or its metabolite is used to prepare a composition that inhibits fat accumulation in adipocytes and reduces the content of fat in cells Uses, wherein the metabolite of Lactobacillus plantarum TCI378 contains tryptophanyl-tryptophan (Pyroglutamyl-tryptophan), 1-methyl-1,2,3,4-tetrahydro-β-carboline-3-carboxylate The effective component of acid (1-Methyl-1,2,3,4-tetrahydro-β-carboline-3-carboxylic acid) and/or 3-Phenyllactic acid.

The World Health Organization (WHO) uses "infectious disease" to describe the rapidly spreading obesity and calls it "Global Obesity" (Globesity). According to statistics from the World Health Organization, in the United States, among the top 20 countries in the world, more than 80% of adult men and 70% of adult women are overweight (BMI over 25), and over 50% of men and women in South Korea are overweight and obese. With changes in eating habits and improved quality of life, the prevalence of obesity in Taiwan has been increasing year by year. According to the "2013-2014 National Nutrition and Health Status Change Survey" published by the National Health Administration of the Ministry of Health and Welfare, the prevalence rate of adult overweight or obesity is 43%. , And the male to female ratios were 48.9% and 38.3%. In other words, one in two men in Taiwan is overweight or obese, and one in two to three women is overweight or obese.

Therefore, obesity is one of the most important diseases in modern society. Obesity is mainly caused by excessive fat intake. Obesity not only causes appearance defects, but also forms psychological and social barriers, and further affects working ability. It is also easy to develop many physical symptoms, including edema and cardiac obesity. Large, fatty liver, gallstones, musculoskeletal pain, gynecological breast or uterine tumors, hyperuricemia (gout), hyperlipidemia, angina, diabetes, high blood pressure, stroke, etc. Among the top ten causes of death in the Chinese people, eight causes of death, including malignant tumors, heart disease, cerebrovascular disease, diabetes, chronic lower respiratory disease, hypertension, chronic liver disease and cirrhosis, and chronic kidney disease, are all related to obesity, so maintain health. Weight and normal body fat have become goals that modern people must strive for.

However, the most effective way to treat obesity is surgery. In addition, legal drugs (currently only Roche fresh), exercise, calorie control, and low-calorie meal replacements have also been proven effective. However, in addition to surgical treatment, most patients use other methods to lose weight. Most of the patients lose their alertness and regain weight after the weight loss treatment is over. Such a phenomenon of losing weight (yo-yo effect) affects the body. It hurts more.

In summary, in response to the obesity faced by modern people due to changes in living and eating habits and the overall health problems caused by obesity, and based on the improvement of modern people’s living standards and the improvement of the concept of health care, we have developed an effective method that can effectively reduce body fat content. The composition of the ingredients is really necessary.

For this reason, one object of the present invention is to provide a metabolite of Lactobacillus, comprising a compound selected from the group consisting of: tryptophanyl-tryptophan (Pyroglutamyl-tryptophan), 1-methyl -1,2,3,4-tetrahydro-β-carboline-3-carboxylic acid (1-Methyl-1,2,3,4-tetrahydro-β-carboline-3-carboxylic acid), 3-benzene Lactic acid (3-Phenyllactic acid), or any combination thereof.

Another object of the present invention is to provide a use of the aforementioned Lactobacillus metabolite for preparing a fat-reducing composition.

Another object of the present invention is to provide a pharmaceutical composition for the preparation of a fat-reducing medicine, wherein the pharmaceutical composition comprises a compound selected from the group consisting of: tryptophan-based pyrobran Amide, 1-methyl-1,2,3,4-tetrahydro-β-carboline-3-carboxylic acid, 3-phenyllactic acid, or any combination thereof, and a pharmaceutically acceptable carrier.

In an embodiment of the present invention, the Lactobacillus strain is a Lactobacillus plantarum (Lactobacillus plantarum), and its deposit number is BCRC910760; and the metabolite of the Lactobacillus is the secretion of the Lactobacillus, including culturing the Lactobacillus The culture solution; the metabolite of the Lactobacillus contains an extract obtained by extracting the metabolites of the Lactobacillus with methanol, and the metabolites of the Lactobacillus contain a compound selected from the group consisting of ：Tryptophanyl pyroglutamine, 1-methyl-1,2,3,4-tetrahydro-β-carboline-3-carboxylic acid, 3-phenyllactic acid, or any combination thereof; wherein the milk The concentration of metabolites of bacilli is at least 1 ppm.

In another embodiment of the present invention, the tryptophanyl pyroglutamine, 1-methyl-1,2,3,4-tetrahydro-β-carboline-3-carboxylic acid, or 3-benzene Lactic acid is obtained by the separation and purification of the metabolites of Lactobacillus as described above; and the tryptophanyl pyroglutamine, 1-methyl-1,2,3,4-tetrahydro-β-carboline- The concentration of 3-carboxylic acid or 3-phenyllactic acid is at least 10 μg/mL.

In another embodiment of the present invention, the fat reduction system inhibits the accumulation of fat in adipocytes to reduce the fat content in adipocytes.

The Lactobacillus plantarum TCI378 or its metabolites of the present invention effectively inhibit the accumulation of fat in adipocytes and reduce the fat content in the cells; wherein the methanol extract of the metabolites of Lactobacillus plantarum TCI378 contains tryptophan-based pyrogluten The effective components of amine, 1-methyl-1,2,3,4-tetrahydro-β-carboline-3-carboxylic acid, and 3-phenyllactic acid can also effectively inhibit the accumulation of fat in fat cells, and Reduce the fat content in the cells. Therefore, the Lactobacillus plantarum TCI378, its metabolites, and tryptophanyl pyroglutamine, 1-methyl-1,2,3,4-tetrahydro-β-carboline purified from its metabolites of the present invention -3-carboxylic acid and 3-phenyllactic acid can be used to prepare a fat-reducing composition. The composition is a medicine or a food and can be administered to a body by oral administration or the like.

The following will further illustrate the implementation of the present invention in conjunction with the drawings. The following examples are used to illustrate the present invention and are not intended to limit the scope of the present invention. Anyone who is familiar with the art will not depart from the spirit and spirit of the present invention. Within the scope, some changes and modifications can be made. Therefore, the scope of protection of the present invention shall be subject to the scope of the attached patent application.

Figure 1 is a hydrogen nuclear magnetic resonance spectrum of purified TCI378-1 from a metabolite of Lactobacillus plantarum TCI378 in an embodiment of the present invention.

Fig. 2 is a mass spectrum of purified TCI378-1 from a metabolite of Lactobacillus plantarum TCI378 in an embodiment of the present invention.

Fig. 3 is a hydrogen NMR spectrum of purified TCI378-2 from a metabolite of Lactobacillus plantarum TCI378 in an embodiment of the present invention.

Fig. 4 is a mass spectrum of purified TCI378-2 from a metabolite of Lactobacillus plantarum TCI378 in an embodiment of the present invention.

Figure 5 is a hydrogen nuclear magnetic resonance spectrum of purified TCI378-3 from a metabolite of Lactobacillus plantarum TCI378 in an embodiment of the present invention.

Fig. 6 is a mass spectrum of purified TCI378-3 from a metabolite of Lactobacillus plantarum TCI378 in an embodiment of the present invention.

Fig. 7 is a bar graph showing the inhibition of fat accumulation of purified TCI378-1, TCI378-2, and TCI378-3 from a metabolite of Lactobacillus plantarum TCI378 according to an embodiment of the present invention. *p value<0.05.

The numerical values used herein are approximate values, and all experimental data are expressed in the range of 20%, preferably in the range of 10%, and most preferably in the range of 5%.

Use Excel software for statistical analysis. Data are expressed as mean±standard deviation (SD), and the differences between groups are analyzed by student's t-test.

definition

The Lactobacillus plantarum of the present invention is a probiotic strain capable of reducing fat. The present invention is a novel Lactobacillus plantarum, named TCI378 in this article. It was deposited in the Food Industry Development Institute on January 13, 2006, with the deposit number BCRC910760. This strain has been patented in the Republic of China Patent Announcement No. TWI645854B. , The present invention has confirmed through in vitro experiments that the Lactobacillus plantarum TCI378, its metabolites, and the compounds purified from the metabolites tryptophanyl-tryptophan (Pyroglutamyl-tryptophan), 1-methyl-1, 2,3,4-tetrahydro-β-carboline-3-carboxylic acid (1-Methyl-1,2,3,4-tetrahydro-β-carboline-3-carboxylic acid), and 3-phenyllactic acid (3 -Phenyllactic acid) can inhibit the accumulation of fat in fat cells. It is shown that the Lactobacillus plantarum TCI378 and its metabolites of the present invention can be used to prepare a fat-reducing composition, and the composition is a medicine or a food, which can be administered to a body by oral administration or the like.

A probiotic strain (probiotic or probiotic bacteria) is a microorganism whose bacteria, mixed strains, extracts or metabolites have a positive effect on the host itself, and are usually derived from living bacteria in the human body that are beneficial to intestinal health. It can refer to certain microorganisms that are supplemented by external sources and may be beneficial to the body, wherein the metabolites of the probiotic strain are the secretions of the probiotic strain, and include the culture medium for cultivating the bacteria.

According to the present invention, the three compounds purified from the metabolites of Lactobacillus plantarum TCI378 of the present invention by column chromatography and thin layer chromatography (TLC) are respectively colored Pyroglutamyl-tryptophan, 1-methyl-1,2,3,4-tetrahydro-β-carboline-3-carboxylic acid (1-Methyl-1,2,3,4 -tetrahydro-β-carboline-3-carboxylic acid), and 3-Phenyllactic acid (3-Phenyllactic acid), and will be named TCI378-1, TCI378-2, and TCI378-3 respectively herein.

According to the present invention, the operating procedures and parameter conditions related to bacterial culture fall within the scope of professionalism and routine technology of those who are familiar with the technology.

As used herein, the term "metabolite" means the substance secreted into the bacterial culture solution after being metabolized by the bacteria when the bacteria are cultured, and includes the culture solution in which the bacteria are cultured.

According to the present invention, the medicine can be manufactured into a dosage form suitable for parenterally or topically by using techniques well known to those skilled in the art. This includes, but is not limited to: injections (injection) [e.g., sterile aqueous solution or dispersion], sterile powder, external preparation, and the like.

According to the present invention, the medicine may further include a pharmaceutically acceptable carrier which is widely used in medicine manufacturing technology. For example, the pharmaceutically acceptable carrier may include one or more reagents selected from the group consisting of solvent, buffer, emulsifier, suspending agent, decomposer ), disintegrating agent, dispersing agent, binding agent, excipient, stabilizing agent, chelating agent, diluent , Gelling agent, preservative, wetting agent, lubricant, absorption delaying agent, liposome and the like. The selection and quantity of these reagents fall within the scope of professionalism and routine techniques of those who are familiar with this technology.

According to the present invention, the pharmaceutically acceptable carrier includes a solvent selected from the group consisting of water, normal saline (normal saline), phosphate buffered saline (PBS), Aqueous solution containing alcohol and combinations thereof.

According to the present invention, the drug can be administered by a parenteral route selected from the group consisting of: subcutaneous injection, intraepidermal injection, intradermal injection Injection (intradermal injection) and intralesional injection (intralesional injection).

According to the present invention, the medicine can be manufactured into an external preparation suitable for topical application to the skin using techniques well known to those skilled in the art. This includes, but Not limited to: emulsion, gel, ointment, cream, patch, liniment, powder, aerosol, spray ( spray), lotion, serum, paste, foam, drop, suspension, salve and bandage.

According to the present invention, the external preparation is prepared by mixing the medicine of the present invention with a base well known to those skilled in the art.

According to the present invention, the substrate may contain one or more additives selected from the following: water, alcohols, glycols, hydrocarbons (such as petroleum jelly) and White petrolatum (white petrolatum,)], wax (such as paraffin and yellow wax), preserving agents, antioxidants, surfactants, absorption enhancers (absorption enhancers), stabilizers (stabilizing agents), gelling agent (gelling agents) [such as Carbopol ^® 974P (carbopol ^® 974P), microcrystalline cellulose (microcrystalline cellulose) and carboxymethyl cellulose (carboxymethylcellulose)] , Active agents, humectants, odor absorbers, fragrances, pH adjusting agents, chelating agents, emulsifiers, occlusion Occlusive agents, emollients, thickeners, solubilizing agents, penetration enhancers, anti-irritants, colorants, and propellants (Propellants) and so on. The selection and quantity of these additives fall within the scope of professionalism and routine technology of those who are familiar with this technology.

According to the present invention, a food product can be used as a food additive, which can be added during the preparation of raw materials by a conventional method, or added during the production process of the food, and formulated with any edible material for supply Food products consumed by humans and non-human animals.

According to the present invention, the types of food products include, but are not limited to: beverages, fermented foods, bakery products, health foods, and dietary supplements.

Chemical analysis materials

The separation of the compounds uses methanol (Methanol) and acetonitrile (Acetonitrile) as solvents. The chemical structure of the compound was analyzed using deuterated methanol d ₄ (deuteration 99.5%) as the solvent, and these materials were purchased from Merck, Taiwan.

Chemical analysis equipment

The separation of compounds utilizes column chromatography and thin layer chromatography (TLC). The medium pressure liquid chromatography (MPLC) system is CombiFlash ^® Rf ⁺ (Teledyne ISCO, Lincoln, NE); the column system is selected from Sephadex LH-20 (Pharmacia, Piscataway, NJ, USA), Diaion HP-20 (Mitsubishi Chemical Co., Japan), Silica gel 0.040-0.023mm and LiChroprep® RP-18 (0.040-0.023mm) (Merck, EMD Millopore Co., Germany). High Performance Liquid Chromatography (HPLC) system is Agilent 1200 series: degassing device is Agilent vacuum gas storage device 1322A; extraction solvent delivery system is Agilent quaternary pump G1311A; variable wavelength detector system ( Multiple Wavelength Detector, MWD) Agilent G1314B; Diode Array Detector (DAD) is Agilent 1260 Infinity DAD VL G1315D, with detection wavelengths of 210nm, 280nm, 320nm, 365nm (Agilent Germany). The column is Luna® 5μm C18(2)100Å (250 x 10mm, Phenomenex, USA). Thin layer chromatography sheet is an _{aluminum sheet of Silica gel 60 F 254} (0.25mm; Merck, EMD Millopore Co., Germany) or RP-18 F ₂₅₄ -S (0.25mm; Merck, EMD Millopore Co., Germany)

The chemical structure of the compound was analyzed by mass spectrometry (MS) and nuclear magnetic resonance spectrometry (NMR). Mass spectrometer (Mass Spectrometer, MS) is a series of tandem mass spectrometry-two-dimensional ion trap tandem Fourier transform mass spectrometry and ESI-MS/MS: measured by Bruker amaZon SL system, the unit is m/z. Nuclear magnetic resonance spectrometer (Nuclear Magnetic Resonance Spectrometer, NMR), 1D and 2D spectroscopy uses Ascend 400MHz (Bruker Co., Germany), and δ represents the chemical shift (Chemical shift), and the unit is ppm. Among them, Rotary vacuum evaporator is a rotary concentrator, used to remove solvents, the model and source are Laborota 4000, Heidolph instruments GmbH & Co. KG Germany.

According to the present invention, the operating procedures and parameter conditions related to the chemical separation and chemical structure analysis of the mixture fall within the professional quality and routine technology of those who are familiar with the technology.

The present invention provides a Lactobacillus plantarum TCI378, its metabolites or Pyroglutamyl-tryptophan (Pyroglutamyl-tryptophan), 1-methyl-1,2,3,4- Tetrahydro-β-carboline-3-carboxylic acid (1-Methyl-1,2,3,4-tetrahydro-β-carboline-3-carboxylic acid), or 3-Phenyllactic acid (3-Phenyllactic acid) is used For the preparation of fat-reducing composition, the metabolite of Lactobacillus plantarum of the present invention is obtained by taking the culture solution of Lactobacillus plantarum TCI378, which contains the active ingredients tryptophanyl pyroglutamine, 1-methyl-1,2, 3,4-Tetrahydro-β-carboline-3-carboxylic acid, and 3-phenyllactic acid, the Lactobacillus plantarum TCI378, its metabolites, and tryptophanyl pyroglutamine purified from its metabolites, 1 -Methyl-1,2,3,4-tetrahydro-β-carboline-3-carboxylic acid and 3-phenyllactic acid can be used to inhibit fat accumulation in adipocytes.

At the same time, the composition for reducing fat of the present invention can also contain an effective amount of Lactobacillus plantarum TCI378, its metabolites, tryptophanyl pyroglutamine, 1-methyl-1, 2,3,4-tetrahydro-β-carboline-3-carboxylic acid, or 3-phenyllactic acid, and a pharmaceutically acceptable carrier, the composition is a medicine or a food.

The detailed preparation method of the Lactobacillus plantarum TCI378 metabolite of the present invention will be described in detail below, and the active substances tryptophanyl pyroglutamine and 1-methyl-1 are isolated from the metabolites of Lactobacillus plantarum TCI378 of the present invention. Detailed methods of 2,3,4-tetrahydro-β-carboline-3-carboxylic acid and 3-phenyllactic acid, and the efficacy test of these active ingredients in inhibiting fat accumulation in fat cells. To prove the vegetable milk of the present invention Bacillus TCI378, its metabolites, and tryptophanyl pyroglutamine, 1-methyl-1,2,3,4-tetrahydro-β-carboline-3-carboxylic acid purified from its metabolites 3-Phenyllactic acid has the effect of inhibiting the accumulation of fat in fat cells, and can be used to prepare fat-reducing compositions.

Example 1 Preparation method of Lactobacillus plantarum TCI378 metabolite of the present invention

In the embodiment of the present invention, the cryopreserved strain of Lactobacillus plantarum TCI378 is cultured once to activate it, and then cultivated in MRS (de Man, Rogosa and Sharpe, BD Difco ^{In TM} Lactobacilli MRS Broth) medium, it is preferable to add 0.1 mL of activated bacteria to 10 mL of MRS. After culturing at 37°C for 18 hours, the culture broth is centrifuged at 5000 rpm for 10 minutes, and the supernatant is collected. It is the metabolite of Lactobacillus plantarum TCI378 of the present invention.

Example 2 Analysis of the active ingredients in the metabolites of Lactobacillus plantarum TCI378 of the present invention

One embodiment of the present invention is to analyze the active ingredients in the metabolites of Lactobacillus plantarum TCI378 of the present invention. In the process of separation and purification of functional compounds, the selection of layering, sub-layering, and sub-layering is based on Bioassay guided fractionation (results not shown). First, 10 liters of the metabolites of Lactobacillus plantarum TCI378 of the present invention are removed most of the water with a vacuum cyclone concentrator to obtain 2 liters of concentrated solution, and then the concentrated solution is columned with the macroporous resin Diaion HP-20 Chromatography (70cm x 7cm), where the extraction gradient is 100% water, 20% methanol aqueous solution, 40% methanol aqueous solution, 60% methanol aqueous solution, and 100% methanol, and a total of 5 layers are obtained. Subsequent RP-C18 fast column chromatography was performed from layer 3, with a linear gradient from 20% methanol to 100% methanol, and then 8 sub-layers were obtained by thin layer chromatography. Take the sub-layer 7 and perform column chromatography with Sephadex LH-20 gel, and then obtain 8 sub-layers by thin layer chromatography, and the sub-layer 7 is purified by RP-HPLC (methanol/water=2/ 3) to obtain compounds TCI-378-1 and TCI-378-2; and to obtain compound TCI-378-3 by RP-HPLC purification (methanol/water=2/3) from sub-layer 5.

TCI378-1 was subjected to hydrogen-nuclear magnetic resonance (Figure 1), carbon 13-nuclear magnetic resonance (results not shown), HSQC resonance (results not shown), HMBC resonance (results not shown), COSY resonance (results not shown) and electrospray After analyzing its chemical structure by ionization mass spectrometry (negative ion mode) (Figure 2), it was confirmed that it was a tryptophan-based pyroglutamyl-tryptophan (Pyroglutamyl-tryptophan), which is the first known compound in nature.

The chemical structure of TCI378-2 was analyzed by hydrogen nuclear magnetic resonance (Figure 3) and electrospray ionization mass spectrometry (negative ion mode) (Figure 4), and it was confirmed to be 1-methyl-1,2,3,4-tetrahydro -β-carboline-3-carboxylic acid (1-Methyl-1,2,3,4-tetrahydro-β-carboline-3-carboxylic acid) is a known compound.

TCI378-3 was subjected to hydrogen-nuclear magnetic resonance (Figure 5), carbon 13-nuclear magnetic resonance (results not shown), HSQC resonance (results not shown), HMBC resonance (results not shown), COSY resonance (results not shown) and electrical After spraying ionization mass spectrometry (negative ion mode) (Figure 6) to analyze its chemical structure, it was confirmed that it is 3-Phenyllactic acid as a known compound.

The chemical structures of compounds TCI378-1, TCI378-2, and TCI378-3 were determined by the analysis of mass spectrometer and nuclear magnetic resonance spectrometer, and their names and structural formulas are shown in Table 1 below.

Example 3 The effect of TCI378-1, TCI378-2, and TCI378-3 in inhibiting fat accumulation among the metabolites of Lactobacillus plantarum TCI378 of the present invention

In this example, mouse bone marrow stromal cells OP9 cells were used to test the efficacy of TCI378-1, TCI378-2, and TCI378-3 in the metabolites of Lactobacillus plantarum TCI378 of the present invention in inhibiting fat accumulation. The mouse bone marrow stromal cell line was purchased from the American Type Culture Collection (USA), under the number CRL-2749 ^™ . The cells were cultured in pre-adipocyte expansion medium (Pre-adipocyte Expansion Medium) before differentiation, which contained 90% MEMAM (Minimum Essential Medium Alpha Medium, purchased from Gibco, USA, FBS: Cat#10437-028) Cell culture medium, 20% fetal bovine serum (Fetal Bovine Serum, purchased from Gibco, USA), and 0.1% penicillin/streptomycin (Penicillin-streptomycin, purchased from Gibco, USA); and use differentiation medium ( Differentiation Medium) to differentiate mouse bone marrow stromal cells, which contains 90% MEMAM cell culture medium, 20% fetal bovine serum, and 0.1% penicillin/streptomycin. The lipids in the cells are stained with Oil Red O reagent (purchased from Sigma, USA, No. O0625), in which a 3mg/mL oil red O stock solution is prepared with 100% isopropanol, and the stock solution is prepared with ddH2O to 60% The role of the solution.

In order to confirm that TCI378-1, TCI378-2, and TCI378-3 in the metabolites of Lactobacillus plantarum TCI378 of the present invention have the effect of inhibiting fat accumulation, firstly, mouse bone marrow stromal cells were differentiated into adipocytes, and ⁸ ×10 4 mice Bone marrow stromal cells were cultured in a 24-well culture dish containing 0.5μL of the above-mentioned pre-adipocyte expansion medium, cultured at 37°C for 7 days, and the above-mentioned differentiation medium was replaced every 3 days. After 7 days, the above-mentioned differentiation medium was replaced with a microscope. Observe the formation of lipid droplets to ensure that the cells are fully differentiated, and then divide the cells into the following four groups: (1) control group containing only cell culture fluid, (2) experimental group added with 10μg/mL TCI378-1, (3) Add 10μg/mL TCI378-2 to the experimental group, and (4) Add 10μg/mL TCI378-3 to the experimental group, and incubate at 37°C for 7-10 days, and also replace the fresh differentiation medium every 3 days .

Next, oil red O was used to stain the lipids in the cells to evaluate whether the active ingredients in the metabolites of Lactobacillus plantarum TCI378 of the present invention can indeed reduce fat accumulation. First, the medium was gently taken out and buffered with 1 mL of phosphate Wash the cells twice with the solution (Phosphate buffered saline, PBS), then add 1 mL of 10% formaldehyde (purchased from Echo chemical, Taiwan, Cat.TG1794-4-0000-72NI) and react at room temperature for 30 minutes to fix the cells. Then remove the formaldehyde and gently wash the cells twice with 1 mL of PBS, then add 1 mL of 60% isopropanol (purchased from Echo chemical, Taiwan, PH-3101) to each well of the cells and react for 1 minute, then remove After isopropanol, add 1mL of oil red O solution, react at room temperature for 1 hour, then remove the oil red O solution and quickly decolorize with 1mL of 60% isopropanol for 5 seconds, then use a microscope to take pictures and Quantify. Then, add 100% isopropanol to the stained cells, and place on a shaker to react for 10 minutes to dissolve the dye, then take 100μL to a 96-well culture dish, and use a measuring ELISA reader to read the OD _{510nm of each group.} Value to quantify oil red O. Then use Excel software to perform student t-test to determine whether there is a statistically significant difference between the two sample groups (*p value<0.05; **p value<0.01; ***p value<0.001).

The results of testing the inhibition of fat accumulation by purified TCI378-1, TCI378-2, and TCI378-3 in the metabolites of Lactobacillus plantarum TCI378 of the present invention are shown in FIG. 7. After treatment with TCI378-1 of the present invention, compared with the control group, cell fat accumulation was significantly reduced by about 11.5%; after treatment with TCI378-2 of the present invention, compared with the control group, cell fat was significantly reduced by about 12.4% After the accumulation is treated with TCI378-3 of the present invention, compared with the control group, the cell fat accumulation is significantly reduced by about 21.5%. This result shows that the metabolites of Lactobacillus plantarum TCI378 of the present invention are TCI378-1, TCI378-2, and TCI378-3 can effectively inhibit the accumulation of fat in fat cells and reduce the fat content in cells.

In summary, the Lactobacillus plantarum TCI378 or its metabolites of the present invention effectively inhibit the accumulation of fat in adipocytes and reduce the fat content in the cells; wherein the methanol extract of the metabolites of Lactobacillus plantarum TCI378 contains tryptamine Acid-based pyroglutamine, 1-methyl-1,2,3,4-tetrahydro-β-carb The effective components of morpholin-3-carboxylic acid and 3-phenyllactic acid can also effectively inhibit the accumulation of fat in fat cells and reduce the content of fat in cells. Therefore, the Lactobacillus plantarum TCI378, its metabolites, and tryptophanyl pyroglutamine, 1-methyl-1,2,3,4-tetrahydro-β-carboline purified from its metabolites of the present invention The -3-carboxylic acid and the 3-phenyllactic acid can be used to prepare a fat-reducing composition. The composition is a medicine or a food, and can be administered to a body by oral administration or the like.

Claims (10)

A metabolite of Lactobacillus containing tryptophan and a compound selected from the group consisting of: 1-methyl-1,2,3,4- Tetrahydro-β-carboline-3-carboxylic acid (1-Methyl-1,2,3,4-tetrahydro-β-carboline-3-carboxylic acid), 3-Phenyllactic acid, or Any composition. For example, the metabolite of Lactobacillus in the first item of the scope of patent application, wherein the Lactobacillus is a Lactobacillus plantarum (Lactobacillus plantarum), and its deposit number is BCRC910760. A use of the metabolite of Lactobacillus described in item 1 of the scope of patent application for preparing a fat-reducing composition. The use as described in item 3 of the scope of patent application, wherein the concentration of the metabolite of the lactobacillus is at least 1 ppm. The use described in item 3 of the scope of patent application, wherein the metabolite of the lactobacillus is the secretion of the lactobacillus, including the culture medium for cultivating the lactobacillus. The use described in item 3 of the scope of patent application, wherein the metabolite of the Lactobacillus contains an extract obtained by extracting the metabolite of the Lactobacillus with methanol. The use described in item 3 of the scope of patent application, wherein the metabolites of the Lactobacillus include tryptophanyl pyroglutamine, 1-methyl-1,2,3,4-tetrahydro-β-carboline- 3-carboxylic acid and 3-phenyllactic acid. The use of a medical composition for preparing a fat-reducing medicine, wherein the medical composition comprises tryptophanyl pyroglutamine and a compound selected from the group consisting of: 1-methyl- 1,2,3,4-tetrahydro-β-carboline-3-carboxylic acid, 3-phenyllactic acid, or any combination thereof, and a pharmaceutically acceptable carrier. The use as described in item 8 of the scope of patent application, wherein the tryptophanyl pyroglutamine, 1-methyl-1,2,3,4-tetrahydro-β-carboline-3-carboxylic acid, or The concentration of 3-phenyllactic acid is at least 10μg/mL. The use described in item 3 or item 8 of the scope of patent application, wherein the fat reduction system inhibits the accumulation of fat in adipocytes to reduce the fat content in adipocytes.

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