CN112618578B

CN112618578B - Use of leuconostoc mesenteroides TCI007 or metabolite thereof for improving allergic conditions

Info

Publication number: CN112618578B
Application number: CN202011019107.2A
Authority: CN
Inventors: 林咏翔; 邱渝凯
Original assignee: TCI Co Ltd
Current assignee: TCI Co Ltd
Priority date: 2019-09-24
Filing date: 2020-09-24
Publication date: 2022-03-15
Anticipated expiration: 2040-09-24
Also published as: CN112625936B; CN112625936A; TW202113065A; TW202113066A; TWI815042B; CN112618578A; TWI764295B

Abstract

Use of Leuconostoc mesenteroides TCI007 or metabolites thereof for improving allergic conditions, wherein Leuconostoc mesenteroides TCI007(Leuconostoc mesenteroides TCI007) is deposited at the institute for food industry development of the treasury legal, institute for food industry (deposit No. BCRC910981) and at the german collection of microorganisms and collections (DSMZ) (deposit No. DSM 33502).

Description

Use of leuconostoc mesenteroides TCI007 or metabolite thereof for improving allergic conditions

Technical Field

The invention relates to Leuconostoc mesenteroides TCI007(Leuconostoc mesenteroides TCI007) or a metabolite thereof, in particular to application of Leuconostoc mesenteroides TCI007 or the metabolite thereof to improvement of allergic conditions.

Background

Probiotics is generally understood to mean edible microorganisms that are beneficial to the host (or recipient, such as an animal or human) after ingestion. Its nomenclature derives from the Greek language "for life", also known as "primary health-care bacterial species"; and, the probiotics mainly refer to lactic acid bacteria and part of yeast.

In general, "Lactic Acid Bacteria (Lactic Acid Bacteria)" refers to Bacteria capable of producing large amounts of Lactic Acid by fermentation using carbohydrates, and is a relatively bulky and complicated bacterial group. Among the strains of lactic acid bacteria, some strains of lactic acid bacteria have been studied to find that they are beneficial for the health of the recipient, and thus such strains are considered as probiotics. In other words, the probiotic bacteria are mostly lactic acid bacteria, but only a few strains of lactic acid bacteria that have been studied to prove beneficial to the health of the recipient can be referred to as probiotics.

For example, common species of lactic acid bacteria that can be used as probiotics include Enterococcus (Enterococcus), Lactobacillus (Lactobacillus), Bifidobacterium (Bifidobacterium), Bacillus (Bacillus), and the like, and species of yeast that can be used as probiotics include Saccharomyces (Saccharomyces).

Lactobacillus is a group of gram-positive bacilli that are fermentative, facultative anaerobic, and non-spore-forming. Lactobacillus is so named because it can ferment carbohydrates into lactic acid, and can be used to produce various fermented foods such as liquid yogurt, solid cheese, etc.

Disclosure of Invention

An object of the present invention is to provide a use of Leuconostoc mesenteroides TCI007 and/or metabolites thereof in the preparation of a composition for improving allergic conditions, wherein Leuconostoc mesenteroides TCI007(Leuconostoc mesenteroides TCI007) is deposited at institute for food industry development of the financial group legal and is deposited with number BCRC 910981.

In some embodiments, the Leuconostoc mesenteroides TCI007 strain comprises the sequence shown as SEQ ID NO: 1.

In some embodiments, the leuconostoc mesenteroides TCI007 strain has the ability to produce polysaccharides.

In some embodiments, leuconostoc mesenteroides TCI007 and/or metabolites thereof are used to reduce histamine levels in tissues.

In some embodiments, leuconostoc mesenteroides TCI007 and/or metabolites thereof are used to improve gut flora.

In some embodiments, the effective dose of Leuconostoc mesenteroides TCI007 is 5X10⁹ cells。

In some embodiments, leuconostoc mesenteroides TCI007 improves the intestinal phase to bifidobacteria (bifidobacteria) increase.

In some embodiments, leuconostoc mesenteroides TCI007 improves enterobacterial phase to Haemophilus depravens (Haemophilus).

Another object of the present invention is to provide a use of Leuconostoc mesenteroides TCI007 and/or metabolites thereof in preparing immunomodulatory compositions, wherein Leuconostoc mesenteroides TCI007(Leuconostoc mesenteroides TCI007) is deposited at institute for development of food industry, and deposited under the number BCRC 910981.

In some embodiments, leuconostoc mesenteroides TCI007 and/or its metabolites have the function of promoting neutrophilic phagocytosis.

The detailed technical disclosure and some embodiments of the present invention will be described in the following for the purpose of explaining the features of the present invention to those skilled in the art.

Drawings

FIG. 1 is a graph showing the results of histamine content measurement in cell experiments;

FIG. 2 is a graph showing the relative expression of IL-8 gene in cell experiments (marked differences compared with blank group, p < 0.01; # shows marked differences compared with comparative group, p < 0.05);

FIG. 3 is a graph of the results of the ability of neutrophilic sphere phagocytosis in cell experiments (. lambda.indicates a significant difference compared to the blank group, p < 0.01;. lambda.indicates a significant difference compared to the comparative group, p < 0.05);

fig. 4 is a graph of histamine measurements at week 0, week 4, and week 8 in human experiments (marked differences from week 0, p < 0.01);

FIG. 5 is a graph showing the results of measurement of the ratio of Bifidobacterium (richness of species) at

weeks

0, 4 and 8 in human experiments;

FIG. 6 is a graph showing the results of the measurement of the proportion of Haemophilus proportion (abundance of species) at

weeks

0, 4 and 8 in human experiments.

Preservation of biological materials

Leuconostoc mesenteroides TCI007, filed 2019 on 27.03.month to the institute of food industry development and research, Taiwan treasury people, China, with the address: taiwan new bamboo city, preservation number: BCRC 910981;

leuconostoc mesenteroides TCI007, filed on 17.4.2020 of German Collection of microorganisms DSMZ (Deutsche Sammlung von Mikroorganismen und Zellkulturen), on the address: the German Branrex city Neuhen street 7B has a preservation number: DSM 33502.

Detailed Description

To enable one of ordinary skill in the art to understand the features of the present invention, the following descriptions and claims refer to terms and words used in the specification and claims for general description and definition. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

The term "ameliorating an allergic condition" as used herein refers to alleviating symptoms associated with allergy, and in particular, reducing one or more indicators of allergic reactions, such as, but not limited to, reducing histamine, improving intestinal flora, reducing runny nose, nasal congestion. In a preferred embodiment, it refers to the reduction of histamine in serum, the increase of bifidobacteria in the gastrointestinal tract and the reduction of haemophilus in the gastrointestinal tract.

The term "intestinal bacteria phase" as used herein refers to the distribution of microorganisms in the intestine of an individual, including the type and proportion of microorganisms. The term "improving the intestinal microbial phase" as used herein refers to changing the type and species of microorganisms in the intestine of an individual, in particular the type and proportion of probiotics in the intestine, in order to direct the physiological state of reduced allergic reactions.

The term "composition" as used herein refers to a food, a cosmetic or a pharmaceutical, in particular a food.

The term "phagocytosis" as used herein refers to a special form of endocytosis, in which solid particles in the surrounding environment, such as bacteria, etc., are engulfed into the interior of the cell in the form of vesicles, and in a preferred embodiment, refers to non-specific phagocytosis induced by neutrophiles (neutrophiles) when a foreign body invades the body.

The present inventors selected a novel strain from human breast milk, and identified it as Lactobacillus gasseri (herein after named Leuconostoc mesenteroides TCI007) by 16S ribosomal ribonucleic acid (16S rRNA) sequence analysis and as Leuconostoc mesenteroides TCI007 (hereinafter called Leuconostoc mesenteroides TCI007), which was deposited at the institute for food industry development of the finance and law (deposited under the accession number BCRC910981) and the German Collection of microorganisms (DSMZ) (deposited under the number DSM 33502). The Leuconostoc mesenteroides TCI007 has a 16S ribosomal ribonucleic acid (16S rRNA) fragment shown in SEQ ID NO. 1.

The inventor finds that leuconostoc mesenteroides TCI007 and/or metabolites thereof have the effect of improving allergic conditions. Therefore, the invention provides a use of leuconostoc mesenteroides TCI007 and/or metabolites thereof in preparing a composition for improving allergic conditions, wherein the composition for improving allergic conditions can be prepared from the following components: reducing histamine content, improving gut flora, or a combination thereof.

The inventor researches and discovers that leuconostoc mesenteroides TCI007 and/or metabolites thereof have the effect of immunoregulation. Therefore, the invention provides the use of leuconostoc mesenteroides TCI007 and/or metabolites thereof for preparing a composition for improving immune regulation, wherein the improvement of allergic conditions refers to the improvement of neutrophilic phagocytosis.

According to the present invention, the metabolite of Leuconostoc mesenteroides TCI007 can be produced by culturing in an environment suitable for the growth of Leuconostoc mesenteroides TCI 007. For example: leuconostoc mesenteroides TCI007 is cultured in a suitable culture solution, and then solid substances such as bacterial cells in the culture solution are removed as required to obtain a metabolite-containing liquid. For example, leuconostoc mesenteroides TCI007 is cultured in a suitable culture medium, and then the cells are sedimented at the bottom by centrifugation or the like, and the supernatant is directly aspirated to obtain a metabolite-containing solution.

Any suitable culture medium can be selected for culturing the leuconostoc mesenteroides TCI007 to provide the desired metabolites, as long as the culture medium can provide nutrients (such as yeast extract, protein and glucose) and conditions (such as pH value) required for the growth and metabolism of the leuconostoc mesenteroides TCI 007. In addition, the culturing time of the leuconostoc mesenteroides TCI007 is not particularly limited as long as it is sufficient for the leuconostoc mesenteroides TCI007 to complete at least one metabolic cycle. For example, in one embodiment of the invention, Leuconostoc mesenteroides TCI007 is cultured in MRS broth for 18 hours to allow the strain to metabolize and produce metabolites.

In the present invention, a culture solution containing leuconostoc mesenteroides TCI007 and metabolites, which undergoes the metabolic cycle of leuconostoc mesenteroides TCI007, or a metabolite-containing liquid from which solids such as leuconostoc mesenteroides TCI007 have been removed, may be used as it is. Any convenient procedure may be employed to remove the solids so long as the desired benefits of the metabolites produced after culturing are not adversely affected. Generally, physical means are employed to remove solids, including, for example: centrifuging, filtering with filter membrane, and settling and decanting. If necessary, the physical operations described above may be repeated or combined to remove as much as possible solid matter such as cells in the culture.

The composition provided by the invention can be a food composition, and the food composition can be a health food, a functional food, a nutritional supplement or a special nutritional food, and can be prepared into products such as dairy products, meat processed products, breads, wheaten foods, biscuits, buccal tablets, capsules, juices, teas, sports drinks, nutritional drinks and the like, but not limited thereto.

The health food, functional food, nutritional supplement, and special nutritional food provided according to the present invention can be taken at different frequencies such as once a day, multiple times a day, or once a few days, depending on the age, weight, and health condition of the individual to whom it is administered. The content of leuconostoc mesenteroides TCI007 and/or metabolites thereof in the health food, functional food, nutritional supplement and special nutritional food provided according to the present invention can also be adjusted to the needs of a specific population, for example, to the amount that should be taken daily.

For the health food, functional food, nutritional supplement and/or special nutritional food provided by the present invention, the recommended dosage, the standard and condition for use of a specific group (for example, hyperlipidemia patients, pregnant women, etc.), or the recommended items for co-administration with other foods or medicines may be marked on the outer package thereof, so that the user can take the food by himself without the guidance of a doctor, pharmacist or related medical practitioner without safety worry. In the food composition according to the present invention, the states of the Leuconostoc mesenteroides TCI007 and/or the metabolites thereof are as described above.

The food composition or feed composition provided according to the present invention may further contain, as necessary, a suitable amount of additives such as a flavoring agent, a coloring agent, and the like, which can improve the mouth-feel and visual-feel of the pharmaceutical composition, food composition, or feed composition upon administration, and a buffer, a preservative, an antibacterial agent, an antifungal agent, and the like, which can improve the stability and storability of the pharmaceutical composition, food composition, or feed composition.

The following examples further illustrate the invention. The examples are provided for illustration only and are not intended to limit the scope of the present invention. The scope of the invention is indicated in the claims.

Examples

[ preparation examples ]

A: screening and identification of leuconostoc mesenteroides TCI007

(A-1) screening

Crushing the whole fruit of natural organic fig (producing area: taiwan) into viscous juice, and mixing with MRS culture solution (MRS broth) in a volume ratio of 1:100 (volume of fig is 1) and anaerobic culture (oxygen concentration is 10-1%, preferably below 5%) at 37 deg.C for 18 hr. Thereafter, the aforementioned culture solution was spread on MRS solid medium (MRS agar) and placed at 37 ℃ for anaerobic culture to generate different colonies. Next, to ensure the uniqueness of the strain, a colony was picked using a sterile inoculating loop, streaked on a solid medium, and cultured in an anaerobic incubator at 37 ℃ until a single colony (single colony) appeared.

(A-2) identification

A strain of the single colony isolated in (A-1) was selected and subjected to genome analysis (genetic analysis), and it was confirmed that the strain had a 16S ribosomal ribonucleic acid (16S rRNA) fragment shown in SEQ ID NO: 1. Using the NCBI (national Center for Biotechnology information) online database for alignment, the sequence of the gene shown in SEQ ID NO:1 was aligned with the 16SrDNA sequence of other Leuconostoc mesenteroides, respectively, and it was found that the 16S rDNA sequence of the isolated strain was similar to the 16SrDNA sequence of other Leuconostoc mesenteroides as shown in Table 1.

Therefore, this isolated strain was named Leuconostoc mesenteroides TCI007 (hereinafter referred to as TCI 007). The Leuconostoc mesenteroides TCI007 was deposited at the institute for food industry development under accession number BCRC910981 and at the German Collection of microorganisms and cell cultures (DSMZ) under accession number DSM 33502.

TABLE 1

(A-3) preservation

The single leuconostoc mesenteroides strain obtained in (a-1) was cultured in MRS medium using liquid culture to provide a bacterial solution, 25% glycerol was added to the bacterial solution, and the resulting mixture was transferred to a cryopreservation tube and stored at-80 ℃.

B. Activation and sample preparation of leuconostoc mesenteroides TCI007

(B-1) activation

The cryopreservation tube in which the Leuconostoc mesenteroides TCI007 strain was stored was thawed to 1% of the inoculum size (about 1X 10)⁴CFU/mL) TCI007 strain was inoculated in MRS broth and placed at 37 ℃ for anaerobic culture for 18 hours to provide leuconostoc mesenteroides TCI007 bacterial solution for use in subsequent experiments.

(B-2) sample preparation

Taking the leuconostoc mesenteroides TCI007 bacterial liquid provided by (B-1), and planting the leuconostoc mesenteroides TCI007 bacterial liquid in a bacterial quantity of 1% (about 1x 10)⁴CFU/mL) were inoculated in MRS broth and incubated anaerobically at 37 ℃ for 18 hours. Then, the cultured bacterial liquid was centrifuged at 5000rpm for 5 minutes, and the obtained supernatant was filtered with a 0.2 μm filter membrane to obtain a filtrate, i.e., a TCI007 sample (containing a metabolite of leuconostoc mesenteroides TCI 007).

[ Experimental example ]

Example 1: polysaccharide content of Leuconostoc mesenteroides TCI007

Leuconostoc mesenteroides TCI007 was activated by the procedure described for activation in preparation example (B-1) above. Subsequently, the activated leuconostoc mesenteroides TCI007 bacterial liquid was inoculated in an amount of 1% (about 1 × 10)⁴CFU/mL) was inoculated in 20mL of liquid Lactobacillus MRS medium (BD Difco)^TMLactobacillus MRS Broth), then anaerobically cultured at 37 ℃, and sampling procedures were performed at 12 hours and 24 hours of culture, respectively, the sampling procedures including: taking 1ml of bacterial liquid, centrifuging (5000 rpm) for 20 minutes, and taking supernatant (containing no thallus, namely, metabolite of leuconostoc mesenteroides TCI007 or supernatant of TCI007), wherein the supernatant is the test sample for detecting polysaccharide content in the embodiment.

The polysaccharide content was determined as follows:

the relative content of total polysaccharide is expressed in terms of D-Glucose (D-Glucose) equivalent. Therefore, a calibration curve (Standard curve) was prepared using D-Glucose (D-Glucose) as a Standard.

First, 10mg of D-Glucose (D-Glucose) (available from J.T. Baker, stock No. 1916-01) was precisely weighed and placed in a 10mL volumetric flask with redistilled water (ddH)₂O) was quantified to 10mL and prepared as a 1mg/mL D-glucose solution.

Next, the above-mentioned standard was serially diluted to give a D-glucose solution of 0,20,50,100,150, 200. mu.g/mL in double distilled water. The standard solution may be prepared in the manner of table 2 below:

table 2: preparation method of standard solution of D-glucose solution

Thereafter, 100. mu.L each of the standard solutions was placed in a glass test tube, and 500. mu.L each of a 5% strength Phenol solution (Phenol) (available from Merck, stock No.: 1.00206.0250) was added thereto.

Then 2.5mL of a concentrated sulfuric acid solution (H) having a concentration of 95.5% and a specific gravity of 1.84 was slowly added₂SO₄) (from Showa, feed number: 1970-5250). After ensuring bubble-free via vortexing (vortex), it was allowed to stand for 20 minutes. Finally, 200. mu.L of the sample was placed in a 96-well plate, and the absorbance at 490nm was measured and a standard curve was drawn.

The supernatant of TCI007 obtained by 12 hours and 24 hours of the above-mentioned culture was diluted 20-fold to 1200ml with water, and a volume of 100. mu.L was taken in a glass tube, and each set of samples was repeated three times. Thereafter, 500. mu.L of a 5% strength Phenol solution (Phenol) (from Merck, stock No.: 1.00206.0250) were added.

Then 2.5mL of a concentrated sulfuric acid solution (H) having a concentration of 95.5% and a specific gravity of 1.84 was slowly added₂SO₄) (from Showa, feed number: 1970-5250). After ensuring bubble-free via vortexing (vortex), it was allowed to stand for 20 minutes. Finally, 200. mu.L of the sample was placed in a 96-well plate, the absorbance was measured at 490nm, and the concentration was calculated by interpolation and multiplied by the dilution factor to obtain the original supernatant concentration of TCI 007.

Table 3: TCI007 polysaccharide assay

As shown in table 3, as the culture time increases, the polysaccharide content in the culture solution of leuconostoc mesenteroides TCI007 is increased, and it can be known that leuconostoc mesenteroides TCI007 can secrete or be metabolized to increase the polysaccharide content in the culture solution, for example, the polysaccharide content in the supernatant (containing no thallus and being a metabolite of leuconostoc mesenteroides TCI007) obtained after the culture solution of leuconostoc mesenteroides TCI007 cultured for 12 hours is centrifuged is 11.15 g/L; the polysaccharide content in the supernatant (containing no thallus and being the metabolite of the leuconostoc mesenteroides TCI007) obtained by centrifuging the culture solution of the leuconostoc mesenteroides TCI007 cultured for 24 hours is 32.8 g/L.

Example 2: cell experiment for leuconostoc mesenteroides TCI007 to regulate allergy-related factors

(2-1) experiment of ability of TCI007 to inhibit histamine

Histamine (histamine) is an organic nitrogen-containing cyclic compound that acts as a chemical conductor in the body, affecting many cellular responses including allergic, inflammatory reactions, and acting as a mediator center of itch and regulating the physiological functions of the intestinal tract. Therefore, when an allergic reaction occurs, reduction of histamine is also an important means for improving allergic symptoms.

In this example, the ability of TCI007 samples to modulate histamine content was tested using the cellular pattern of DNP-HAS to induce histamine of basic granulocytes.

A test materials

1. Cell lines: rat basophilic leukemia cell RBL-2H3(Rat basophilic leukemia cells, available from ATCC under the accession number CRT-2256).

2. Culture solution: minimum Essential Medium (MEM) (Gibco; Cat.11095080) was added 15% fetal bovine serum FBS (Gibco; Cat.10437-028) and 1% penicillin-streptomycin (Gibco; Cat.15140122).

3. Histamine assay reagent kit (purchased from USCN, model CEA927Ge) containing the following solutions:

3-1: standard liquid (Standard)

3-2: standard diluent (Standard diluent)

3-3: detection reagent A (detection reagent A)

3-4: detection reagent B (detection reagent B)

3-5: analytical diluent A (assay diluent A)

3-6: analytical diluent B (assay diluent B)

3-7: TMB liquid (TMB substrate)

3-8: stop solution (Stop solution)

3-9: cleaning fluid (Wash buffer)

4. anti-DNP-IgE (purchased from Sigma, model D8406).

DNP-HAS solution (dinitrophenyl-human serum album) (purchased from Biosearch, model D-5059).

Preparation before experiment

1.1 times DPBS: diluting 10 times of DPBS by sterilized dd water;

2.1-fold trypsin: 10-fold dilution of 10-fold trypsin with 1X DPBS;

3. DNP-HAS solution: prepared to a final concentration of 1. mu.g/ml with serum-free MEM;

4.1 times washing solution: 20ml of 30-fold wash was added to 580ml of ddH₂O, mixing uniformly for later use;

5. detection reagent a operating solution: detection reagent a was diluted 100-fold with assay diluent a.

6. Detection reagent B procedure solution: detection reagent B was diluted 100-fold with assay diluent B.

7. Standard sequence dilution: redissolving with 2ml standard diluent, standing for at least 10 minutes to fully dissolve to the final concentration of 100ng/ml, preparing 5 new 1.5ml microcentrifuge tubes, respectively adding 600. mu.l of standard diluent, then adding 300. mu.l of standard solution into the first tube, mixing uniformly, taking 300. mu.l from the first tube to the second tube, diluting to the 4 th tube in sequence, and finally obtaining the serial dilutions with the concentrations of 100ng/ml, 33.33ng/ml, 11.11ng/ml, 3.70ng/ml and 1.23 ng/ml.

The following brief description of the histamine determination procedure, and the detailed procedures can be performed with reference to the instructions attached to the histamine determination reagent.

First, RBL-2H3 cells were plated at 1.5X10⁵The cells/well are seeded into 24-well cell culture plates at a density of 500. mu.l/well in CO₂The culture was carried out in an incubator for 18 hours. After the cells were attached, the culture medium was changed to experimental and control medium, and the medium components and additional additive components are shown in table 4 below.

TABLE 4

Next, anti-DNP-IgE was added at a final concentration of 0.2. mu.g/ml, and the reaction was carried out for 30 minutes. The liquid in the well plate was removed, and 500. mu.l of DNP-HAS solution was added and reacted at 37 ℃ for 30 minutes. And collecting the supernatant culture solution in the hole plate for later use after the reaction is finished. Subsequently, 50. mu.l/well of the supernatant culture (without cells) of the serial dilutions (at the above-mentioned concentrations), the blank, the control and the experimental groups were added to a pre-coated ready-to-use 96-well plate (included in the histamine detection reagent kit), 50. mu.l/well of the detection reagent A working solution was immediately added thereto, and the reaction was carried out at 37 ℃ for 1 hour after shaking. Pouring out the liquid in the hole plate, adding 350 mul of cleaning solution with the volume being 1 time of the hole, and standing for 1-2 minutes. Inverted on a paper towel to try to remove all liquid from the wells.

Next, 100. mu.l/well of the working solution of the detection reagent B was added, and the reaction was carried out at 37 ℃ for 30 minutes. Adding 90. mu.l/well of TMB solution, and reacting at 37 ℃ for 10-20 minutes (which needs to be protected from light, and the action can not exceed 30 minutes). Add 50. mu.l/well of stop solution and tap the well plate to mix well (change color).

Finally, the measured light intensity was measured by a spectrophotometer (Thermo Fisher Scientific)

) The O.D.450 value was measured and compared with the absorbance value of the sequence dilution to determine the histamine concentration of the samples (each group of cell culture fluid) and statistical analysis was performed using student t-test in Excel software.

Through comparison and conversion of light absorption values, the histamine content measured by a blank group is 0.025ng/ml, the histamine content measured by a control group is 0.041ng/ml, and the histamine content measured by an experimental group is 0.011 ng/ml; in detail, histamine was not inhibited after DNP-HAS induction of histamine from basic granulocytes followed by 24 hours of treatment with 0.125% by volume (compared to medium) MRS medium, whereas histamine was significantly inhibited after DNP-HAS induction of histamine from basic granulocytes followed by 24 hours of treatment with 0.125% by volume (compared to medium) TCI007 supernatant.

Please refer to fig. 1. When the histamine content (0.025ng/ml) of the blank group was regarded as 1 (i.e., 100%), the histamine content (0.011ng/ml) of the control group was 1.6238 (i.e., 162.38%), and the histamine content of the experimental group relative to the blank group was 0.433 (i.e., 43.30%), representing that the histamine content of the cells was reduced to 0.43 times that of the blank group after the cells were treated with the supernatant of TCI 007.

Therefore, when basophils are treated by the TCI007 sample, the histamine content is inhibited, which means that the TCI007 sample can effectively inhibit the histamine content and can inhibit the allergic condition.

(2-2) experiment of TCI007 Regulation of immune-related Gene

This example uses RNA extraction kit, reverse transcriptase, KAPA

The FAST qPCR reagent set is combined with a quantitative PCR instrument to determine the change of immune related genes in cells of the human monocyte cell strain after being treated by the TCI007 sample.

For example, the immune-related gene is IL-8 gene, IL-8 is a multifunctional factor capable of specifically chemotactic neutrophils to enter inflammatory tissues and regulate immunity, and the IL-8 gene is taken as a target for immune-related gene analysis.

Materials and instruments

1. Cell line (hereinafter referred to as cell): human monocyte cell line THP-1(ATCC company (Switzerland), TIB 202).

2. Culture medium: RPMI 1640 medium (Gibco RPMI medium 1640; Thermo Fisher Scientific) supplemented with 10% Fetal Bovine Serum (FBS), 0.05mM 2-mercaptoethanol (2-mercaptoethanol), 100 units/mL penicillin, and 100. mu.g/mL streptomycin.

RNA extraction reagent set (from Geneaid corporation, Taiwan, type:. FC24015-G)

4. Reverse transcriptase (A)

III Reverse Transcriptase) (Invitrogen corporation, USA, No. 18080-

5. The target gene primer was measured, which contained the IL-8 gene, and additionally included an internal control group (GAPDH gene).

6.KAPA

FAST qPCR reagent set (purchased from Sigma, USA, No. 38220000000)

7.ABI StepOnePlus^TMReal-time PCR System (ABI StepOnePlus)^TMReal-Time PCR system (Thermo Fisher Scientific, USA)).

TCI007 sample: the TCI007 sample used in this experiment was obtained through the (B-2) sample preparation procedure as in the preparation example above.

First, human monocyte cells (simply referred to as cells in this example) were cultured at 1X10 cells per well⁵The individual cells were cultured in a six-well plate containing 2mL of the above-mentioned culture solution and cultured at 37 ℃ for 16 hours, and then the cells were divided into the following three groups: control, experimental and blank groups.

Table 5: test conditions

In detail, the experimental group was prepared by culturing human monocyte cell strain in 2ml of a 0.125% (v/v) concentration medium of TCI007 sample prepared as described in example (B-2) above for 16 hours; in the control group, 2ml of a 0.125% (v/v) MRS culture medium was cultured in human monocyte cell lines for 16 hours.

The blank group was used as experimental blank group by culturing human monocyte cells with 2ml of culture medium alone without adding other additives for 16 hours.

The above experimental and blank groups were performed in quadruplicates per group.

Treated cells (i.e., control, experimental) and nullThe white group of cells were disrupted with cell lysates to form three groups of cell solutions. Next, RNA in the three cell solutions was extracted using an RNA extraction reagent kit (purchased from Geneaid, Taiwan, Lot No. FC24015-G). Then, 1000 nanograms (ng) of extracted RNA was taken as template for each group, and the RNA was permeated

III reverse transcriptase (from Invitrogene, USA, No. 18080-051) reverse transcribes the extracted RNA into the corresponding cDNA. Then using ABI StepOneplus^TMReal-time PCR System (ABI StepOnePlus)^TMReal-Time PCR system (Thermo Fisher Scientific Co., U.S.), KAPA SYBR FAST (available from Sigma, U.S. Pat. No. 38220000000) and primers of Table 6 (SEQ ID NO:2 to SEQ ID NO:5) quantitative Real-Time reverse transcription polymerase chain reaction (quantitative Real-Time reverse transcription polymerase reaction) was performed on the cDNAs of the two groups to observe the expression of IL-8 gene in the human monocytes of the three groups. The quantitative real-time reverse transcription polymerase chain reaction apparatus was set to react at 95 ℃ for 20 seconds, then at 95 ℃ for 3 seconds, at 60 ℃ for 30 seconds, and repeated for 40 cycles, and gene quantification was performed using the 2- Δ Ct method. Here, the quantitative real-time reverse transcription polymerase chain reaction using cDNA can indirectly quantify the mRNA expression level of the IL-8 gene, and further estimate the expression level of the protein encoded by the IL-8 gene.

TABLE 6

Primer name	Sequence numbering	Sequence of	Length of
				IL-8-F	SEQ ID NO:2	TTTTGCCAAGGAGTGCTAAAGA	22
IL-8-R	SEQ ID NO:3	AACCCTCTGCACCCAGTTTTC	21
				GAPDH-F	SEQ ID NO:4	CTGGGCTACACTGAGCACC	19
GAPDH-R	SEQ ID NO:5	AAGTGGTCGTTGAGGGCAATG	21

It should be noted that the relative gene expression of each gene shown in the figures mentioned below is presented in relative magnification, wherein the standard deviation is calculated using the STDEV formula of Excel software, and whether there is a statistically significant difference is analyzed in the Excel software by single Student t test (Student t-test). In the drawings, the term "indicates a p value of less than 0.05, the term" indicates a p value of less than 0.01, and the term "indicates a p value of less than 0.001. As more "x", the more significant the statistical difference.

Please refer to fig. 2. When the expression level of IL-8 gene in the blank group was regarded as 1 (i.e., 100%), the expression level of IL-8 gene in the control group relative to the blank group was 1.02 (i.e., 102%), and the expression level of IL-8 gene in the experimental group relative to the blank group was 3.05 (i.e., 305%), which means that the expression level of IL-8 gene was increased to 3.05 times that in the blank group after the cells were treated with the TCI007 sample.

It is known that when the cells are treated by the TCI007 sample, the expression level of the genes related to immunity in the cells is increased, which indicates that the TCI007 sample has the function of regulating immunity.

(2-3) test for increasing activity of TCI007 on neutrophilic globus

The most abundant leukocyte in vivo is the neutrophilic globulin, which can rapidly aggregate when the foreign body invades, phagocytose the foreign body or pathogen through nonspecific phagocytosis, and kill the pathogen through specific ferment and peroxide in the cell, playing an important role in the human immune system.

In the experiment, after a sample and green fluorescent particles are added into a culture solution together for culturing for four hours, the ability of the centromere phagocytosis is judged by measuring the fluorescence intensity of the fluorescent particles in cells.

Experimental Material

1. Cell lines: neutrophilic sphere neutrophile (isolated from human blood)

2. Culture solution: serum-free Hematopoietic Cell Medium (X-VIVOTM 15 Serum-free Hematopoietic Cell Medium) (X-VIVOTM 15) (from Lonza; model 04-418Q).

SPHERO Fluorescent Yellow Particles (Fluorescent Yellow Particles) (available from spheriotech; model Cat. FH-2052-2)

Experimental procedure

First, the neutrophilic sphere is separated from the blood of the human body at 10 deg.C⁶cells/well were seeded into 6-well cell culture plates and cultured in six-well plates containing 2mL of the above medium, and the cells were then divided into the following three groups: blank group, control group and experimental group;

TABLE 7

The cells were then harvested and washed 1 times with 1-fold PBS after cell harvest, the supernatant removed (to remove non-phagocytosed fluorogenic yellow particles), the cells resuspended in 1 ml/tube of 1-fold PBS, the fluorescence signal measured by flow cytometry (from Beckman) and statistically analyzed using student t-test in Excel software for 4 hours.

Please refer to fig. 3. When the fluorescence signal expression amount of the blank group is regarded as 1 (namely 100%), the fluorescence signal expression amount of the control group relative to the blank group is 1.05 (namely 105%), the fluorescence signal expression amount of the experimental group relative to the blank group is 1.13 (namely 113%), the fluorescence signal is the fluorescence generated by the fluorescent yellow particles phagocytized by the centromere, and the stronger the fluorescence signal is, the more the fluorescent yellow particles phagocytized by the centromere are, the stronger the phagocytosis is; therefore, representative cells treated with TCI007 sample had increased phagocytosis of the neutrophils, 1.13-fold that of the blank.

Example 3: human experiment for adjusting allergy by leuconostoc mesenteroides TCI007

(3-1) test of ability of TCI007 to inhibit histamine in human tissues

To further confirm the effect of TCI007 on amine content in human tissues. Each capsule contains 5X10⁹cells (i.e. 5X 10)⁹cells/cap) were provided to 6 subjects (persistent allergic rhinitis population), one per day before sleep, for 8 weeks. And blood test of the subject was carried out before the administration of the TCI007 capsule (considered as week 0), 4 weeks after the administration of the TCI007 capsule (considered as week 4), and 8 weeks after the administration of the TCI007 capsule (considered as week 8), wherein the test items included the histamine content in the blood of the subject. Wherein, the content of the somatic histamine in the blood of the subject can be detected by a hydroxylamine histamine Kit (trademark: Elablcience, detection Kit name Hydroxyproline (Hydrop) Colorimetric Assay Kit) according to the attached instruction of the detection Kit or executed by adopting external medical inspection; preferably, the subject blood of this embodimentThe somatic histamine content in (c) was performed by the institute of the same assignee (taiwan).

Referring to FIG. 4, the mean blood histamine content of 6 subjects was 0.94ug/ml (FIG. 4 indicates week 0) before taking the TCI007 capsule, the mean blood histamine content of 6 subjects was reduced to 0.41ug/ml (FIG. 4 indicates week 4) after taking the TCI007 capsule for 4 weeks, and the mean blood histamine content of 6 subjects was reduced to 0.21ug/ml (FIG. 4 indicates week 8) after taking the TCI007 capsule for 8 weeks. In other words, the mean blood histamine content of 6 subjects decreased by 0.53ug/ml, equivalent to a 57% decrease, compared to week 0 after 4 weeks of administration of the TCI007 capsule; the mean blood histamine content of 6 subjects was reduced by 0.73ug/ml, equivalent to a 78% reduction, compared to week 0, after 8 weeks of administration of TCI007 capsule.

In other words, when the subject takes 5X10 daily⁹cells (i.e. 5X 10)⁹cells/cap), the histamine content in blood decreased by 57% after 4 weeks of continuous administration; after 8 weeks of continuous administration, the histamine content in the blood decreased by 78%.

Therefore, it is known that TCI007 is effective in suppressing histamine levels and suppressing allergic conditions.

(3-2) examination result of questionnaire for improving allergic symptoms by TCI007

First, the above 6 subjects were subjected to the examination of allergic conditions at week 0 (before the administration of the viable cell capsule of leuconostoc mesenteroides TCI007), week 4 (after the administration of the viable cell capsule of leuconostoc mesenteroides TCI007) and week 8, and the skin conditions in the examination were examined in the examination paper in the following manner and scored as shown in table 8. Wherein, each score of the first part represents the severity of each symptom, 0 is no trouble, 1 is mild, 2 is moderate, 3 is slightly severe, 4 is severe, and 5 is very severe.

TABLE 8

A first part: symptoms/scores	0	1	2	3	4	5
							1. Severe nasal discharge or nasal obstruction
2. The allergic condition is severe
							3. Frequent sneezing

Among them, the severe allergic condition refers to the evaluation of the degree of the subject's own overall allergic condition (e.g., allergic eczema such as skin itching, or allergic rhinitis such as rhizine or nasal rubbing).

Item 1 questionnaire results are shown in table 9 below

TABLE 9

Wherein, the average score is obtained by averaging the scores corresponding to the severity filled by all the subjects, and the decrease of the average score represents the reduction of the condition of the item.

From the above, it can be seen from table 9 that after taking the viable capsules of leuconostoc mesenteroides TCI007 for 4 weeks and 8 weeks, the subjects were improved in terms of item 1, severe rhinorrhea or nasal obstruction, item 2, severe allergy, and item 3, frequent sneezing (results not shown).

Example 4: human experiment of leuconostoc mesenteroides TCI007 regulating mycophase

To further confirm the effect of leuconostoc mesenteroides TCI007 on the human intestinal phase. Before (as the 0 th week) and after (as the 8 th week) the subject takes the TCI007 capsule, the intestinal bacteria phase detection of the subject is carried out after (as the 4 th week) and after (as the 8 th week) the TCI007 capsule, wherein the detection method comprises the steps of collecting a stool sample of the subject, extracting DNA from the stool, carrying out quantitative real-time polymerase chain reaction (qRT-PCR) on 16S ribosomal gene (16S ribosomal gene), and detecting the bacteria number ratio of common intestinal bacteria by using a primer sequence of known bacteria; in the detection method, after DNA extraction of feces, Sequencing of the 16S Amplicon (16S amplification Sequencing) of the specimen may be performed in a Next Generation Sequencing (NGS) manner, in which the V3-V4 region of the 16S DNA is amplified and the subsequent Sequencing analysis is performed. In this example, the Sequencing was performed by next generation Sequencing, and the Sequencing of the 16S Amplicon (16S amplification Sequencing) was performed by Torus Biotech, Inc., and species annotation and abundance analysis were performed by means of Paired-End Sequencing (Paired-End) through Reads splicing filtration and OTU (operational Taxonomic units) clustering to reveal the sample species composition and the ratio of microorganisms in the sample.

Referring to fig. 5, administration of the TCI007 capsule for 4 weeks resulted in an increase of about 155% in bifidobacteria (Bifidobacterium) compared to administration prior to (week 0), and administration of the TCI007 capsule for 8 weeks resulted in an increase of about 117% in bifidobacteria.

Referring to fig. 6, administration of TCI007 capsules for 8 weeks resulted in a reduction of Haemophilus (haempohilus) of about 52% compared to administration prior to (week 0).

The literature proves that the bifidobacterium is a common probiotic in the intestinal tract and has the effects of regulating immunity and resisting allergy. Haemophilus belongs to the normal adult intestinal flora phase, rarely causes diseases, and is proved by literature that when allergy occurs, the Haemophilus stimulates basophilus to generate more histamine so as to make allergic reaction more serious. See, for example: a uplink from laboratory and Clinical data,2010 British Society for Immunology, Clinical and Experimental Immunology,160: 295-304, endogenous from Haemophilus influenzae enginehances IgE-treated and non-immunological histamine release, allergy.1990 Jan; 45(1) 10-7, which is incorporated herein by reference in its entirety.

Through sequencing detection of a fecal sample of the subject, the capability of TCI007 on the regulation of the bacterial phase of the human body is analyzed, and the results show that after the TCI007 capsule is taken, bifidobacteria in the gastrointestinal tract of the subject are obviously increased, which means that the TCI007 has the effects of regulating immunity and resisting allergy; after taking the TCI007 capsule, the haemophilus in the gastrointestinal tract of the subject is obviously reduced, which means that TCI007 has the effects of reducing histamine and reducing anaphylactic reaction.

In conclusion, it can be seen from example (2-1) that Leuconostoc mesenteroides TCI007 can reduce histamine content in the cell model. As is clear from example (2-2), Leuconostoc mesenteroides TCI007 has the ability to modulate immune-related genes, particularly the IL-8 gene. As shown in examples (2-3), Leuconostoc mesenteroides TCI007 was found to promote phagocytosis of neutrophiles, indicating that TCI007 could promote activity of neutrophiles. As is clear from example (3-1), in the human experiment, the amine content in the human tissue was reduced by administering the cell body of Leuconostoc mesenteroides TCI 007. As is clear from example (3-3), it is found that the administration of the cell of Leuconostoc mesenteroides TCI007 can improve the allergic conditions such as nasal discharge and nasal obstruction. As shown in example 4, after taking the leuconostoc mesenteroides TCI007, the bacterial phase in the gastrointestinal tract of a human body can be adjusted, and particularly, bifidobacteria can be increased, haemophilus can be reduced, so that the histamine can be reduced, and the anaphylactic reaction can be reduced.

The present invention is capable of other embodiments, and various changes and modifications may be made by one skilled in the art without departing from the spirit and scope of the invention as defined in the appended claims.

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Claims

1. Use of Leuconostoc mesenteroides TCI007 and/or metabolites thereof for the preparation of a composition for ameliorating allergic conditions, wherein Leuconostoc mesenteroides TCI007(Leuconostoc mesenteroides TCI007) is deposited at the german collection of microorganisms and cell z mz (DSMZ) under the deposit number DSM 33502.

2. The use of claim 1, wherein the Leuconostoc mesenteroides TCI007 strain comprises the sequence shown in SEQ ID NO. 1.

3. The use of claim 1, wherein the Leuconostoc mesenteroides TCI007 strain has the ability to produce polysaccharides.

4. The use of claim 1, wherein the Leuconostoc mesenteroides TCI007 and/or metabolites thereof is used to reduce histamine levels.

5. The use of claim 1, wherein the leuconostoc mesenteroides TCI007 and/or metabolites thereof is used to improve intestinal phase.

6. The use of claim 4 or 5, wherein the effective dose of Leuconostoc mesenteroides TCI007 is 5x10⁹cells。

7. The use of claim 5, wherein the gut-improving bacterial phase is increased bifidobacteria (Bifidobacterium).

8. The use of claim 5, wherein the gut-improving bacterial phase is Haemophilus norphilus (Haemophilus).

9. Use of Leuconostoc mesenteroides TCI007 and/or metabolites thereof for the preparation of an immunomodulatory composition, wherein Leuconostoc mesenteroides TCI007(Leuconostoc mesenteroides TCI007) is deposited at the german collection of microorganisms and cell z mz (DSMZ) under the accession number DSM 33502.

10. The use of claim 9, wherein the leuconostoc mesenteroides TCI007 and/or metabolites thereof has the function of promoting phagocytosis by neutrophilic globulism (phagocytosis).