TW202026418A - The improving respiratory health probiotic strain, composition thereof and use thereof - Google Patents

Abstract

The present invention provides an improving respiratory health probiotic strain, composition thereof and use thereof. The probiotic strain is a new Lactobacillus helveticus (BCRC910846), which itself and its metabolites can inhibit the bacterial growth, improve the repair ability of lung epithelial cells, and promote the phagocytic ability of macrophages to effectively reduce the chance of infection in the respiratory tract, improve the repair ability of the respiratory tract, and promote the elimination of PM2.5 in the human body and remove foreign matter from the respiratory tract. Wherein the Lactobacillus helveticus metabolites of the present invention contains an effective component of 1,2,3,4-tetrahydro-β-carboline-3-carboxylic acid.

Description

Probiotic strain for improving respiratory health and its composition and use

The present invention relates to a probiotic strain for improving the health of the respiratory tract and its composition and use, in particular to a kind of Lactobacillus helveticus and its metabolites used in the preparation of inhibiting bacterial growth, improving the repair ability of lung epithelial cells, and promoting the phagocytic ability of macrophages The use of the composition, wherein the metabolite of Lactobacillus helveticus contains a 1,2,3,4-tetrahydro-β-carboline-3-carboxylic acid (1,2,3,4-tetrahydro-β-carboline -3-carboxylic acid).

The development of industrial activities and the heavy demand for energy in modern life, but the process of obtaining energy has caused serious air pollution. Harvard University’s research on air pollution problems in Asia and Southeast Asia pointed out that in addition to direct PM2.5 emissions from coal-fired power plants, the emissions of sulfide (SO ₂ ), nitrogen oxides (NOX), soot and dust will indirectly promote PM2. .5 Substances formed. PM2.5 particles are very small and respirable particles, which can easily break through the human body’s nasal villi and tracheal ciliary mucus and other respiratory defense mechanisms, enter the bronchi and alveoli, and cause serious health effects, including respiratory diseases and chronic bronchi. Inflammation, lung cancer and cardiovascular diseases are more likely to cause premature birth of newborns, affect the development of the cognitive system, and induce the incidence of chronic diseases for pregnant women. Severe cases will lead to death.

However, at present, only passive methods can be used to slow down the inhalation of PM2.5 to prevent or slow the harm caused by PM2.5 to the human body. Among them, PM2.5 in the atmospheric environment can be reduced by reducing outdoor activity time and Wear personal protective equipment such as masks to reduce the risk of exposure, but PM2.5 will infiltrate the indoor environment through ventilation and air conditioning systems, doors and windows, and gaps in buildings, increasing indoor air pollution and exposure risks. And many studies have found that in most cases, the indoor environment will cause the PM2.5 concentration due to cooking fumes, dust from cleaning, and biological sources including viruses and bacteria in droplets, mold spores and dust feces. It is much higher than the one from outside. Therefore, for environmental issues, in addition to working together to improve energy usage habits and the development of clean energy, while the problem has not yet been alleviated, you must protect your own respiratory tract to prevent air pollution from harming the human body. In addition, in addition to man-made pollution, there are biological factors that threaten the human body in the air, such as infections of the respiratory tract caused by pathogens such as bacteria or viruses.

Based on the above, in order to effectively reduce the damage of PM2.5 to the human lungs, develop a system that can effectively promote the phagocytosis of macrophages to increase the body's ability to eliminate PM2.5, and at the same time can effectively inhibit the growth of lung bacteria, And a composition that enhances the repairing ability of lung epithelial cells is really necessary.

For this reason, one objective of the present invention is to provide a probiotic strain for improving respiratory tract health, wherein the probiotic strain is a Lactobacillus helveticus , and its deposit number is BCRC910846.

Another object of the present invention is to provide a use of the aforementioned probiotic strain for preparing a composition for improving respiratory health.

Another object of the present invention is to provide a use of the metabolite of the probiotic strain as described above for preparing a composition for improving respiratory health.

Another object of the present invention is to provide a use of 1,2,3,4-tetrahydro-β-carboline-3-carboxylic acid for preparing a composition for improving respiratory tract health, wherein the 1,2,3, 4-tetrahydro-β-carboline-3-carboxylic acid has the following chemical formula (I):

Another object of the present invention is to provide a composition for improving the health of the respiratory tract, comprising a probiotic strain selected from the first described in the scope of the patent application, a metabolite of the probiotic strain, and 1,2,3,4-tetra Any one of hydrogen-β-carboline-3-carboxylic acid or any combination thereof.

In an embodiment of the present invention, the concentration of the probiotic strain is at least 1×10 ⁸ cfu/mL.

In another embodiment of the present invention, the concentration of the metabolite of the probiotic is at least 1 ppm, and the metabolite of the probiotic strain is the secretion of the probiotic strain, including the culture medium for cultivating the probiotic.

In another embodiment of the present invention, the metabolite of the probiotic strain contains 1,2,3,4-tetrahydro-β-carboline-3-carboxylic acid (1,2,3,4-tetrahydro-β -carboline-3-carboxylic acid), and the 1,2,3,4-tetrahydro-β-carboline-3-carboxylic acid has the following chemical formula (I):

In another embodiment of the present invention, the concentration of the 1,2,3,4-tetrahydro-β-carboline-3-carboxylic acid is at least 1 ppm.

In another embodiment of the present invention, the improvement of respiratory tract health inhibits bacterial growth, enhances the repair ability of lung cells and/or enhances the phagocytic ability of macrophages.

The Lactobacillus helveticus and its metabolites of the present invention can effectively inhibit the growth of bacteria, effectively enhance the repair ability of lung epithelial cells, and effectively promote the phagocytic ability of macrophages, thereby reducing the chance of respiratory infection, improving the repair ability of the respiratory tract, and Promote the elimination of PM2.5 from the human body and remove foreign bodies in the respiratory tract. Among them, the metabolite of Lactobacillus helveticus of the present invention has 1,2,3,4-tetrahydro-β-carboline-3-carboxylic acid (1,2,3,4-tetrahydro-β-carboline-3-carboxylic acid). The effective ingredients of acid) have been verified by efficacy to effectively enhance the repair ability of lung epithelial cells. Therefore, the Lactobacillus helveticus, its metabolites, and the 1,2,3,4-tetrahydro-β-carboline-3-carboxylic acid purified from its metabolites of the present invention can be used to prepare compositions for improving respiratory health Purpose: The composition is a medicine or a food, which can be administered to a body by oral administration.

The following will further illustrate the embodiments 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 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 protection scope of the present invention shall be subject to those defined by the attached patent scope.

Figure 1 is a photograph showing the inhibition of bacterial growth by a metabolite of Lactobacillus helveticus in an embodiment of the present invention.

Fig. 2A is a photograph of a metabolite of Lactobacillus helveticus promoting the repair of lung epithelial cells in an embodiment of the present invention.

Fig. 2B is a bar graph of a metabolite of Lactobacillus helveticus promoting the repair of lung epithelial cells in an embodiment of the present invention. **p value<0.01.

Fig. 3 is a bar graph and photograph of a metabolite of Lactobacillus helveticus promoting the phagocytosis of PM2.5 by macrophages in an embodiment of the present invention.

Fig. 4 is a bar graph of the ethyl acetate layer extract, the n-butanol layer extract, and the water layer extract of the metabolites of Lactobacillus helveticus in one embodiment of the present invention promoting the repair of lung epithelial cells. **p value<0.01; ***p value<0.001.

Fig. 5 is a hydrogen nuclear magnetic resonance spectrum of purified TCI-TCI357-01 from a metabolite of Lactobacillus helveticus in an embodiment of the present invention.

Fig. 6 is a carbon nuclear magnetic resonance spectrum chart of purified TCI-TCI357-01 from a metabolite of Lactobacillus helveticus in an embodiment of the present invention.

Figure 7 is a two-dimensional NMR COSY spectrum of purified TCI-TCI357-01 from a metabolite of Lactobacillus helveticus in an embodiment of the present invention.

Fig. 8 is a two-dimensional nuclear magnetic resonance HSQC spectrum diagram of purified TCI-TCI357-01 from a metabolite of Lactobacillus helveticus in an embodiment of the present invention.

Figure 9 is a two-dimensional nuclear magnetic resonance HMBC spectrum of purified TCI-TCI357-01 from a metabolite of Lactobacillus helveticus in an embodiment of the present invention.

Figure 10 is a mass spectrum of TCI-TCI357-01 purified from a metabolite of Lactobacillus helveticus in an embodiment of the present invention.

Fig. 11 is a mass spectrum of TCI-TCI357-02 purified from a metabolite of Lactobacillus helveticus in an embodiment of the present invention.

Fig. 12 is a hydrogen nuclear magnetic resonance spectrum of purified TCI-TCI357-02 from a metabolite of Lactobacillus helveticus in an embodiment of the present invention.

Fig. 13 is a carbon nuclear magnetic resonance spectrum of purified TCI-TCI357-02 from a metabolite of Lactobacillus helveticus in an embodiment of the present invention.

Figure 14 is a two-dimensional NMR COSY spectrum of purified TCI-TCI357-02 from a metabolite of Lactobacillus helveticus in an embodiment of the present invention.

Fig. 15 is a two-dimensional NMR spectrum of the TCI-TCI357-02 purified from the metabolite of Lactobacillus helveticus in an embodiment of the present invention.

Figure 16 is a two-dimensional nuclear magnetic resonance HMBC spectrum of purified TCI-TCI357-02 from a metabolite of Lactobacillus helveticus in an embodiment of the present invention.

Figure 17 is an HPLC fingerprint of the metabolites TCI-TCI357-01 and TCI-TCI357-02 of Lactobacillus helveticus in an embodiment of the present invention.

Fig. 18 is a bar graph of the active ingredients TCI-TCI357-01 and TCI-TCI357-02 in the metabolites of Lactobacillus helveticus in an embodiment of the present invention promoting the repair of lung epithelial cells. **p value<0.01.

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. The data are expressed as mean ± standard deviation (SD), and the differences between groups are analyzed by student's t- test.

definition

The Lactobacillus helveticus of the present invention is a probiotic bacteria that can improve the health of the respiratory tract. The present invention is a novel Lactobacillus helveticus, deposited in the Food Industry Development Research Institute on August 15, 1987, with the deposit number BCRC910846. The present invention has been verified through in vitro experiments that the Lactobacillus helveticus and its metabolites can inhibit bacterial growth. , In order to reduce the chance of infection of the respiratory tract; cell experiments confirmed that the Lactobacillus helveticus and its metabolites of the present invention can enhance the repair ability of lung epithelial cells, promote the phagocytic ability of macrophages, so as to enhance the repair ability of the respiratory tract, and promote the elimination of people The effect of PM2.5 in the body and removal of foreign bodies in the respiratory tract. It is shown that the Lactobacillus helveticus and its metabolites of the present invention can be used to prepare a composition for improving respiratory tract health, 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, including the culture medium for cultivating the bacteria.

According to the present invention, two compounds are purified from the metabolites of Lactobacillus helveticus of the present invention by column chromatography and thin layer chromatography (TLC), 1, 2, 3,4-tetrahydro-β-carboline-3-carboxylic acid (1,2,3,4-tetrahydro-β-carboline-3-carboxylic acid) and Flazin will be named TCI-TCI357- 01 and TCI-TCI357-02.

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 a 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 solvents, buffers (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 agents, liposomes 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 technique.

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 by using techniques well known to those skilled in the art. This includes, but is not limited to: emulsion, coagulation Gel, ointment, cream, patch, liniment, powder, aerosol, spray, lotion, milk 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, Occlusive agents, emollients, thickeners, solubilizing agents, penetration enhancers, anti-irritants, colorants, and Propellants, etc. The selection and quantity of these additives fall within the scope of professionalism and routine techniques of those who are familiar with this technology.

According to the present invention, a food product can be used as a food additive, which is added during the preparation of raw materials by a conventional method, or added during the preparation of food, and is 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

Hexane (n -hexane), ethyl acetate (ethyl acetate), acetone (acetone), methanol (methanol), ethyl alcohol (ethanol), n-butanol (n -butanol), acetonitrile (acetonitrile), chloroform - d ₁ ( deuteration degree 99.5%), methanol - d ₆ (deuterated degree 99.5%), heavy water (deuterium oxide, deuterated degree of greater than 99.8%), and dimethyl sulfoxide _{- d 6 (dimethyl sulfoxide- d 6} , deuterated Degree>99.9%) purchased from Merck, Taiwan.

Chemical analysis equipment

The separation of compounds utilizes column chromatography (Column chromatography) and Thin layer chromatography (Thin layer chromatography, TLC). The Medium pressure liquid chromatography (MPLC) system is CombiFlash ^® Rf ⁺ (Teledyne ISCO); the column is selected from Sephadex LH-20 (Amersham Biosciences), Diaion HP-20 (Mitsubishi Chemical), Merck Kieselgel 60 (40-63μm, Art. 9385), and Merck LiChroprep ^® RP-18 (40-63um, Art. 0250). High Performance Liquid Chromatography (HPLC) system equipped with Hitachi L-2310 series pumps, Hitachi L-2420 UV-VIS detector (detection wavelength from 200nm to 380nm), and D-2000 Elite data processing Software; the column is selected from the analytical grade Discovery ^® HS C18 (250×4.6mm, 5μm; SUPELCO) and Mightysil RP-18 GP 250 (250×4.6mm, 5μm; Kanto Chemical), and the semi-preparative Discovery ^® HS C18 (250×10.0mm, 5μm; SUPELCO) and preparative Discovery ^® HS C18 (250×21.0mm, 5μm; SUPELCO). Chromatography system is equipped with UV lamp UVP UVGL-25 (wavelength is 254nm and 365nm). Thin layer chromatography film is coated with silicone 60 F254 (0.25mm; Merck) or RP-18 F254S (0.25mm; Merck) aluminum film.

The chemical structure of the compound was analyzed by mass spectrometry (MS) and nuclear magnetic resonance spectrometry (NMR). Specifically, two-dimensional ion trap tandem Fourier transform mass spectrometry (Bruker amaZon SL system) and electrospray ionization tandem mass spectrometry (ESI-MS/MS, Thermo Scientific Orbitrap Elite system) were used; and 400MHz Varian 400 FT-NMR was used to obtain one Two-dimensional and two-dimensional NMR spectroscopy, with δ representing the chemical shift (Chemical shift), where the unit is ppm; Tetramethylsilane (TMS) is used as the internal standard; the coupling constant (J) is in Hz and s Form peak (Singlet), d means doublet (Doublet), d means Triplet, q means Quarret, p means quintet, m means Multiplet, and brs means broad peak.

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 helveticus , its metabolite or 1,2,3,4-tetrahydro-β-carboline-3-carboxylic acid purified from its metabolite for preparing a combination for improving respiratory health The use of the substance, the metabolite of Lactobacillus helveticus of the present invention is obtained from the culture broth of Lactobacillus helveticus, which contains the active ingredient 1,2,3,4-tetrahydro-β-carboline-3-carboxylic acid (1, 2,3,4-tetrahydro-β-carboline-3-carboxylic acid), the Lactobacillus helveticus, its metabolites, and 1,2,3,4-tetrahydro-β-carboline- 3-carboxylic acid can be used to inhibit bacterial growth, enhance the repair ability of lung epithelial cells, and promote the phagocytic ability of macrophages.

At the same time, the composition for improving respiratory health of the present invention may also contain an effective amount of Lactobacillus helveticus of the present invention, its metabolites, or 1,2,3,4-tetrahydro-β purified from its metabolites -Carboline-3-carboxylic acid, and a pharmaceutically acceptable carrier, the composition is a medicine or a food.

Hereinafter, the detailed preparation method of the Lactobacillus helveticus metabolites of the present invention will be described in detail, and the efficacy test of Lactobacillus helveticus in inhibiting bacterial growth, the efficacy test in repairing lung epithelial cells, the efficacy test in enhancing the phagocytic ability of macrophages, Detailed method for separating active substance 1,2,3,4-tetrahydro-β-carboline-3-carboxylic acid from the metabolite of Lactobacillus helveticus of the present invention, verification of 1,2,3,4-tetrahydro -β-carboline-3-carboxylic acid is the metabolite of Lactobacillus helveticus of the present invention, and it has been verified that 1,2,3,4-tetrahydro-β-carboline-3-carboxylic acid can repair lung epithelial cells Efficacy test. To prove that Lactobacillus helveticus, its metabolites, and the 1,2,3,4-tetrahydro-β-carboline-3-carboxylic acid purified from its metabolites of the present invention have the ability to inhibit bacterial growth and enhance lung epithelial cells Repair ability, and the ability to promote the phagocytosis of macrophages, and can be used to prepare compositions for improving respiratory health.

Example 1 Preparation method of Lactobacillus helveticus metabolites of the present invention

In the embodiment of the present invention, the frozen strain preservation tube of Lactobacillus helveticus of the present invention is cultured once for activation, and then cultured in MRS (de Man, Rogosa and Sharpe) with one percent of colonized bacteria. , BD Difco ^TM Lactobacilli MRS Broth) medium, preferably add 0.1mL of activated bacteria to 10mL of MRS, culture at 37°C for 16-18 hours, centrifuge the culture broth at 5000rpm for 10-20 minutes , The supernatant collected is the metabolite of Lactobacillus helveticus of the present invention.

Example 2 The effect of the metabolite of Lactobacillus helveticus of the present invention in inhibiting bacterial growth

An embodiment of the present invention In order to test the effect of the Lactobacillus helveticus metabolites of the present invention in inhibiting the growth of bacteria, the inhibition zone method was used to test its antibacterial effect. Among them, the inhibition zone method is also known as the diffusion method. The diffusion in agar gum inhibits the growth of the surrounding bacteria to form a transparent circle, that is, a bacteriostatic circle. The method of determining the antibacterial effect of the test substance according to the size of the bacteriostatic circle is often used for the isolation and screening of antibiotic-producing bacteria . First, the Lactobacillus helveticus of the present invention was cultured in MRS medium, and after culturing at 37°C for 16 hours, the bacterial solution was centrifuged at 5000 rpm for 10 minutes, and the supernatant was taken as a test sample. Then, 1x10 ⁸ cfu/mL Escherichia coli ( E.coli ) was cultured in 5mL LB (Lysogeny broth) medium, and after acting at 37°C for 8 hours, 100 μL of this medium was applied to LB agar (LA After digging and preparing the hole, take 50 μL of the metabolite of Lactobacillus helveticus of the present invention in the LA hole that has been coated with E.coil , and culture it at 37°C for 24 hours to observe whether a zone of inhibition is produced.

The experimental results of the Lactobacillus helveticus metabolites of the present invention inhibiting bacterial growth are shown in Figure 1. An obvious inhibition zone is formed around the LA cavity containing the Lactobacillus helveticus 7 metabolite of the present invention. This result shows that the Lactobacillus helveticus of the present invention can produce substances that inhibit the growth of bacteria and can effectively inhibit the growth of bacteria to reduce respiratory tract Probability of infection.

Example 3 The effect of Lactobacillus helveticus and its metabolites in repairing lung epithelial cells of the present invention

In one embodiment of the present invention, human bronchial epithelial cells BEAS-2b (purchased from the American Type Culture Collection, number CRL-9609) were tested by cell scratch damage repair experiments to test the Lactobacillus helveticus and its metabolites in the present invention. The effect of repairing lung epithelial cells. The cell line was cultured in Bronchial epithelial cell basal Medium (BEBM, purchased from Lonza, Switzerland), in which the cell scratch damage repair experiment system is to observe the cell refilling a scratch created artificially to know the healing ability of the cell . First, culture 1.5x10 ⁵ BEAS-2b cells in each well of a 24-well culture plate, incubate at 37°C for 16-18 hours to spread the cells on the bottom of the plate well, and then use a 200μL micropipette Scratch a line on the monolayer of cells to create a scar, remove the culture medium and wash with Phosphate Buffered Saline (PBS), then divide the cells into the following two groups (n=3) and mix without Serum-free the above-mentioned culture medium, and the final volume is 1mL: (1) MRS, and (2) 1% Lactobacillus helveticus or its metabolites of the present invention, and add only serum-free culture medium As the blank control group. Then observe with a microscope and take a photo to record. The size of the scar at this time point is used as the reference point. After 16 hours of exposure in a 37°C, 5% CO ₂ incubator, remove the culture medium and add the same volume of After incubating the serum-containing culture medium at 37°C for 24 hours, observe the scars with a microscope and record them with the same method. Finally, analyze the images of each group with Image J software, and then use Excel software to perform Student t-test to determine the coefficient of variation and Whether there are statistically significant differences.

The experimental results of Lactobacillus helveticus and its metabolites in promoting lung epithelial cell repair are shown in Figure 2A and Figure 2B, in which the blank control group is taken as 100%. After the action of Lactobacillus helveticus or its metabolites of the present invention, the scratch damage of lung epithelial cells can be repaired by 218.2%, which is significantly higher than that of the blank control group and the MRS group. Among them, the MRS group only has 115.7% of the scratch damage repair . This result shows that the Lactobacillus helveticus and its metabolites of the present invention can effectively enhance the repair ability of lung epithelial cells and can enhance the repair ability of the respiratory tract.

Example 4 The effect of Lactobacillus helveticus and its metabolites of the present invention in enhancing the phagocytic ability of macrophages

One embodiment of the present invention differentiates human monocyte THP-1 (purchased from the American Type Culture Collection, number TIB202) into macrophages, and observes that the macrophages phagocytize fireflies of the same size as PM2.5 The ability of light particles to test the effect of the Lactobacillus helveticus and its metabolites of the present invention in promoting the elimination of fine suspended particulate PM2.5 from the human body. Among them, there are macrophages in the human lungs, which can participate in the phagocytosis and removal of foreign dust particles in the lungs Or pathogens. The THP-1 cells were cultured in RPMI-1640 cell culture medium (purchased from Gibco, USA), which contained 10% Fetal Bovine Serum, 0.05 mM 2-mercaptoethanol, 100 units /mL of penicillin and 100μg/mL of streptomycin. First, culture 1.5x10 ⁵ THP-1 cells in each well of a 6-well culture plate, and add 500 nM of phorbol-12-tetradecane-13-acetate ( Phorbol 12-myristate 13-acetate, PMA, purchased from Sigma, No. P8139), and incubate at 37°C in an incubator containing 5% CO ₂ for 48 hours, then replace it with fresh cell culture medium and monitor it Continue to culture for 48 hours to differentiate THP-1 cells into macrophages, and then divide the cells into the following two groups: (1) the experimental group with 1% Lactobacillus helveticus or its metabolites of the present invention, and (2) only Add the blank control group of cell culture medium, react for 24 hours, add 0.5% fluorescent particles (Fluorescent particles, yellow high intensity, 1% w/w, 1.7-2.2μm, purchased from Spherotech, USA, code FH- 2052-2) Act for 4 hours, then remove the culture medium and wash the cells with 1xPBS for three times, observe with a microscope and take photos for recording, and use a flow cytometer to pass FL1 photomultiplier (emission wavelength: 515-545nm) detection Fluorescent signal from macrophages, in which the fluorescent signal of cell culture medium (air culture medium) is used as the background reading. Then use Excel software to perform Student t-test to determine whether the coefficient of variation is statistically significant.

The experimental results of the Lactobacillus helveticus and its metabolites of the present invention in promoting the phagocytosis of fine suspended particulate PM2.5 by macrophages are shown in FIG. 3. After the action of Lactobacillus helveticus or its metabolites of the present invention, it can be clearly observed that the number of fluorescent particles phagocytosed by macrophages is more than that of the control group, and the fluorescent signal is also 3.5% higher than that of the control group. This result shows that the Lactobacillus helveticus and its metabolites of the present invention can effectively promote the phagocytic ability of macrophages, promote the elimination of PM2.5 from the human body, and help the removal of foreign bodies in the respiratory tract.

Example 5 Analysis of active ingredients in the metabolites of Lactobacillus helveticus of the present invention

An embodiment of the present invention is to analyze the active ingredients in the metabolites of Lactobacillus helveticus of the present invention. First, take 5L of the Lactobacillus helveticus metabolite of the present invention, mix it with ethyl acetate (Ethyl acetate) and water in a ratio of 1:1, divide it into 3 times for liquid phase partition extraction, and divide the ethyl acetate layer for 3 times. After mixing the extracts, they were concentrated and dried under reduced pressure to obtain 8.28 g of ethyl acetate layer extract. The remaining aqueous layer extracts were continued to be extracted 3 times in a liquid phase partition extraction method with an equal ratio of n-butanol and water at a ratio of 1:1. The combined extracts were concentrated and dried under reduced pressure to obtain a total of 18.20 g of n-butanol layer extracts, and A total of 255.0 g of the aqueous layer extract. Then, the ethyl acetate layer extract, the n-butanol layer extract, and the water layer extract were preliminarily tested for their efficacy in repairing lung epithelial cell damage.

Then, human bronchial epithelial cells BEAS-2b (purchased from the American Type Culture Collection, number CRL-9609) were used for cell scratch damage repair experiments to analyze the efficacy of the above three extracts in repairing lung epithelial cells. The cell line was cultured in Bronchial epithelial cell basal Medium (BEBM, purchased from Lonza, Switzerland). First, culture 1.5x10 ⁵ BEAS-2b cells in each well of a 24-well culture plate, incubate at 37°C for 16-18 hours to spread the cells on the bottom of the plate well, and then use a 200μL micropipette Scratch a line on the monolayer of cells to create a scar, remove the culture medium and wash with Phosphate Buffered Saline (PBS), divide the cells into the following four groups (n=3) and mix them without Serum (Serum-free) in the above-mentioned culture medium, and the final volume is 1mL: (1) 1% of the Lactobacillus helveticus metabolite stock solution of the present invention, (2) 1% ethyl acetate layer extract, (3) 1 % N-butanol layer extract, and (4) 1% water layer extract, and only cells containing serum-free culture medium were used as the blank control group. Then observe with a microscope and take a photo to record. The size of the scar at this time point is used as the reference point. After 16 hours of exposure in a 37°C, 5% CO ₂ incubator, remove the culture medium and add the same volume of After incubating the serum-containing culture medium at 37°C for 24 hours, observe the scars with a microscope and record them with the same method. Finally, analyze the images of each group with Image J software, and then use Excel software to perform Student t-test to determine the coefficient of variation and Whether there are statistically significant differences.

The results of the ethyl acetate layer extract, the n-butanol layer extract, and the water layer extract of the metabolites of Lactobacillus helveticus of the present invention are shown in FIG. 4. Among them, the metabolite stock solution of Lactobacillus helveticus was significantly higher than the blank control group by 179.2% times, indicating that the metabolites of Lactobacillus helveticus of the present invention indeed have a good effect of repairing lung epithelial cells, and the ethyl acetate layer extract is more The blank control group was 22% higher, the n-butanol layer extract was 116.7%, and the water layer extract was 20.3%. These results show that the metabolite stock solution of Lactobacillus helveticus, the ethyl acetate layer extract, the n-butanol layer extract, and the water layer extract of the present invention have a good effect of repairing lung epithelial cells, especially the n-butanol layer The extract showed the best effect of repairing lung epithelial cells. Therefore, components that improve respiratory health are further separated from the n-butanol extract.

Then, the active ingredients in the n-butanol extract were tracked according to the Bioassay guided fractionation method. First, a total of 18.2g of n-butanol layer extracts were used Diaion HP-20 (purchased from Mitsubishi Chemical, Japan) as the chromatographic material, water and methanol mixed in a ratio of 3:2 were used as the eluent, after column chromatography A total of 3 divided layers (F1-F3) were separated. Then the second divided layer (F2) mixed with dichloromethane and methanol in a ratio of 9:1 was used as the eluent, and after column chromatography through a silica gel column, a total of 7 divided layers (F2- 1-F2-7). Then the subdivision layer F2-7 was mixed with dichloromethane and methanol at a ratio of 4:1 as the eluent, and after column chromatography on a silica gel column, a total of 20.0 mg of TCI-TCI357-01 was separated. In addition, the third divided layer (F3) was extracted with dichloromethane and methanol as the eluent, and then subjected to column chromatography through a silica gel column. A total of 7 divided layers (F3-1- F3-7), F3-5 was purified by High Performance Liquid Chromatography (HPLC), where water and methanol mixed in a ratio of 3:7 were used as the eluent, and a total of 5.2 mg was obtained. TCI-TCI357-02.

TCI-TCI357-01 is a white powder compound. Its NMR hydrogen spectrum is shown in Figure 5. The analysis shows that this compound has a set of ABCD system aromatic ring proton absorption signal δ H 7.40(1H,d,J= 8.0Hz), δ H 7.30 (1H, d, J=8.0 Hz), δ H 7.03 (1H, t, J=8.0 Hz), δ H 6.95 (1H, t, J=8.0 Hz), and different atoms The connected CH ₂ absorbs the signal δ H 4.15 (2H, d, J=15.6 Hz). The NMR carbon spectrum is shown in Figure 6, and the analysis shows that this compound has 12 carbon signals, including one carbonyl absorption signal. In order to confirm the structure of this compound, NMR analysis was performed. The two-dimensional spectrum COSY is shown in Figure 7, HSQC is shown in Figure 8, and HMBC is shown in Figure 9, and the mass spectrum is shown in Figure 10. These results confirm that this compound is 1,2,3,4-tetrahydro-β-carboline-3-carboxylic acid (1,2,3,4-tetrahydro-β-carboline-3-carboxylic acid). The structural formula is shown in Table 1 below and named TCI-TCI357-01.

TCI-TCI357-02 is a yellow powder compound. Its mass spectrum is shown in Figure 11. A molecular ion peak is found to be 331[M+Na] ⁺ , and the molecular weight of this compound is estimated to be 308. In addition, its NMR hydrogen spectrum is shown in Figure 12. From the analysis, it can be seen that this compound has a set of ABCD aromatic ring proton absorption signals δ H 8.40 (1H, d, J=8.0 Hz), δ H 7.78 (1H, d) , J=8.0Hz), δ H 7.62 (1H, t, J=8.0 Hz), and δ H 7.32 (1H, t, J=8.0 Hz), a group of aromatic ring proton coupling signals δ H 7.39 (1H, d, J=3.2Hz), δ H 6.59 (1H, d, J=3.2Hz), a set of aromatic ring single-peak proton absorption signals δ H 8.81 (1H, s), and a set of single atoms connected to different atoms The peak proton CH ₂ absorption signal δ H 4.65 (1H, s). The NMR carbon spectrum is shown in Figure 13. The analysis shows that the compound has a total of 17 carbons, including a carbonyl absorption signal. In order to confirm the structure of this compound, NMR analysis was performed. The two-dimensional spectrogram COSY is shown in Figure 14, HSQC is shown in Figure 15, and HMBC is shown in Figure 16. From these results, the structural formula of this compound is connected. After comparison with the literature, it was confirmed that this compound is Flazin, and its structure is shown in Table 1 below, and it is named TCI-TCI357-02.

The chemical structures of the compounds TCI-TCI357-01 and TCI-TCI357-02 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 6 TCI-TCI357-01 and TCI-TCI357-02 are metabolites of Lactobacillus helveticus of the present invention

An example of the present invention is to verify that the compounds TCI-TCI357-01 and TCI-TCI357-02 are metabolites produced by Lactobacillus helveticus of the present invention, and compare the components in the blank of the bacterial culture medium and the culture broth of Lactobacillus helveticus of the present invention. First, take 20 μL of the 50 mg/mL Lactobacillus helveticus supernatant of the present invention of the n-butanol layer extract and 20 μL of the 50 mg/mL bacterial culture medium blank solution of the n-butanol layer extract for HPLC fingerprint analysis, wherein Use Octadecylsilyl (Octadecylsilyl, Mightysil RP-18 GP 250) Kanto Chemical Company column (inner diameter (ID) is 10mm, length (L) is 250mm, particle size is 5μm, flow rate is 1.0mL/min) , And the mobile phase composition is a mixed solution of methanol (Methanol) and water, the extraction conditions are shown in Table 2 below, and a signal with a wavelength of 280nm is detected.

The HPLC fingerprints of the n-butanol layer extract of the metabolites of Lactobacillus helveticus and the n-butanol layer extract of the blank medium of the present invention are shown in Figure 17, where it can be seen that there is no TCI-TCI357-01 and the blank medium. The TCI-TCI357-02 compound exists, and these two compounds only appear in the culture broth inoculated with Lactobacillus helveticus of the present invention. This result proves that these two compounds are indeed metabolites produced by Lactobacillus helveticus of the present invention. Metabolites produced after Lactobacillus helveticus.

Example 7 The effect of the active ingredients in the metabolites of Lactobacillus helveticus of the present invention in repairing lung epithelial cells

One embodiment of the present invention is to re-verify that TCI-TCI357-01 and TCI-TCI357-02 isolated from the metabolites of Lactobacillus helveticus of the present invention have effective components for repairing lung epithelial cells, using human bronchial epithelial cells BEAS -2b Perform cell scratch damage repair experiment. First, culture 1.5x10 ⁵ BEAS-2b cells in each well of a 24-well culture plate, incubate at 37°C for 16-18 hours to spread the cells on the bottom of the plate well, and then use a 200μL micropipette Scratch a line on the monolayer of cells to create a scar, remove the culture medium and wash with Phosphate Buffered Saline (PBS), then divide the cells into the following two groups (n=3) and mix without Serum-free the above-mentioned culture medium, and the final volume is 1mL: (1) 1% TCI-TCI357-01 and (2) 1% TCI-TCI357-02, and add only serum-free The cells in the culture medium served as the blank control group. Then observe with a microscope and take a photo to record. The size of the scar at this time point is used as the reference point. After 16 hours of exposure in a 37°C, 5% CO ₂ incubator, remove the culture medium and add the same volume of After incubating the serum-containing culture medium at 37°C for 24 hours, observe the scars with a microscope and record them with the same method. Finally, analyze the images of each group with Image J software, and then use Excel software to perform Student t-test to determine the coefficient of variation and Whether there are statistically significant differences.

The experimental results of the compounds TCI-TCI357-01 and TCI-TCI357-02 isolated from the metabolites of Lactobacillus helveticus of the present invention in promoting the repair ability of lung epithelial cells are shown in FIG. 18. After the compound TCI-TCI357-01 is used, compared with the blank control group, the scratch damage repair rate of lung epithelial cells can be significantly increased by 38%. After the compound TCI-TCI357-02 is used, compared with the control group. There is no significant difference in the scratch damage repair of lung cells. This result shows that the compound with lung protection effect in the metabolite of Lactobacillus helveticus of the present invention is TCI-TCI357-01 (1,2,3,4-tetrahydro-β-carboline-3-carboxylic acid) in the n-butanol layer .

In summary, the Lactobacillus helveticus and its metabolites of the present invention can effectively inhibit bacterial growth, effectively enhance the repair ability of lung epithelial cells, and effectively promote the phagocytic ability of macrophages, thereby reducing the chance of respiratory tract infection and improving the respiratory tract It has the ability to repair and promote the elimination of PM2.5 from the body, and can remove foreign bodies in the respiratory tract. Among them, the metabolite of Lactobacillus helveticus of the present invention has 1,2,3,4-tetrahydro-β-carboline-3-carboxylic acid (1,2,3,4-tetrahydro-β-carboline-3-carboxylic acid). The effective ingredients of acid) have been verified by efficacy to effectively enhance the repair ability of lung epithelial cells. Therefore, the Lactobacillus helveticus, its metabolites, and the 1,2,3,4-tetrahydro-β-carboline-3-carboxylic acid purified from its metabolites of the present invention can be used to prepare compositions for improving respiratory health Purpose: The composition is a medicine or a food, which can be administered to a body by oral administration.

【Biological Material Deposit】

Food Industry Development Research Institute (Taiwan); August 15, Republic of 107; No. BCRC910846.

Claims (12)

A probiotic strain for improving respiratory health, wherein the probiotic strain is a Lactobacillus helveticus ( Lactobacillus helveticus ), and its deposit number is BCRC910846. A use of the probiotic strain described in item 1 of the scope of patent application for preparing a composition for improving respiratory health. The use as described in item 2 of the scope of patent application, wherein the concentration of the probiotic strain is at least 1x10 ⁸ cfu/mL. A use of the metabolite of the probiotic strain as described in item 1 of the scope of patent application for preparing a composition for improving respiratory health. The use as described in item 4 of the scope of patent application, wherein the concentration of the metabolites of the probiotic bacteria is at least 1 ppm. The use described in item 4 of the scope of patent application, wherein the metabolite of the probiotic strain is the secretion of the probiotic strain, including the culture medium for cultivating the probiotic strain. The use described in item 4 of the scope of patent application, wherein the metabolite of the probiotic strain contains 1,2,3,4-tetrahydro-β-carboline-3-carboxylic acid (1,2,3,4- tetrahydro-β-carboline-3-carboxylic acid), and the 1,2,3,4-tetrahydro-β-carboline-3-carboxylic acid has the following chemical formula (I):

A use of 1,2,3,4-tetrahydro-β-carboline-3-carboxylic acid for preparing a composition for improving respiratory tract health, wherein the 1,2,3,4-tetrahydro-β-carbohydrate The morpholine-3-carboxylic acid has the following chemical formula (I):

The use described in item 9 of the scope of patent application, wherein the concentration of the 1,2,3,4-tetrahydro-β-carboline-3-carboxylic acid is at least 1 ppm. The use described in any one of item 2, item 4, or item 8 of the scope of patent application, wherein the improvement of respiratory tract health inhibits bacterial growth, enhances the repair ability of lung cells and/or enhances the phagocytic ability of macrophages . A composition for improving the health of the respiratory tract, comprising a probiotic strain, a metabolite of the probiotic strain, and 1,2,3,4-tetrahydro-β-carboline-3 as described in item 1 of the scope of patent application -Any one of carboxylic acids or any combination thereof. The composition according to claim 11, wherein the improvement of respiratory tract health inhibits bacterial growth, enhances the repair ability of lung cells and/or enhances the phagocytic ability of macrophages.

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