TWI754302B - Application of Bifidobacterium lactis TCI604 and its metabolites - Google Patents

Abstract

A kind of Bifidobacterium lactis ( Bifidobacterium lactis ) TCI604 and the application of the Bifidobacterium lactis TCI604 and its metabolites, wherein the Bifidobacterium lactis TCI604 is deposited in the Food Industry Research Institute of a consortium corporation (the deposit number is BCRC910887), and the German national bacteria The Bifidobacterium lactis TCI604 and its metabolites have the effect of regulating the expression of CRP , IL-1β , IL8 , IL6 , IL10 , IL17A , TNF-α , CYP7A1 , and APC genes, so It can be used to reduce the incidence of colorectal polyps, reduce the incidence of colorectal cancer, reduce the incidence of peptic ulcer, reduce the incidence of gastroesophageal reflux, help digestion, improve gastrointestinal function, regulate immunity, and regulate blood lipids.

Description

Application of Bifidobacterium lactis TCI604 and its metabolites

The present invention relates to a novel Bifidobacterium lactis ( Bifidobacterium lactis , also known as Reiterella bifidii) TCI604 (deposited in the Food Industry Research Institute of a consortium corporation, the deposit number is BCRC910887; and deposited in the German National Culture Collection Center , the accession number is DSM33303) and the application of its metabolites, especially the application of the aforementioned Bifidobacterium lactis TCI604 and its metabolites in at least one of the following: reducing the incidence of colorectal polyps, reducing the incidence of colorectal cancer, reducing the gastrointestinal tract Ulcer incidence, reduce the incidence of gastroesophageal reflux, help digestion, improve gastrointestinal function, regulate immunity, regulate blood lipids, regulate CRP gene expression, regulate IL-1β gene expression, regulate IL8 gene expression, regulate IL6 gene expression, and regulate IL10 gene expression , regulate the expression of IL17A gene, regulate the expression of TNF-α gene, regulate the expression of CYP7A1 gene , and regulate the expression of APC gene.

Modern people generally have problems such as abnormal work and rest, life pressure, and lack of exercise. Coupled with the long-term intake of excessive fat and insufficient dietary fiber intake, the incidence of colorectal polyps is increasing year by year. Colorectal polyps can be mainly divided into hyperplastic polyps, inflammatory polyps, and adenomatous polyps. Among them, adenomatous polyps have the highest probability of canceration. Most colorectal cancers are derived from adenomatous polyps.

Poor eating habits may cause symptoms such as indigestion, flatulence, poor bowel movements, immune disorders, and hyperlipidemia. For example, a high-fat diet can also easily lead to gastric acid secretion disorders, so that the mucosa of the stomach, duodenum and other tissues are eroded by gastric acid, resulting in damage to the walls of the digestive tract that penetrate deep into the tissue, and ultimately form a digestive tract ulcer. Severe peptic ulcer may even have complications such as gastrointestinal bleeding and perforation. Gastroesophageal reflux is another common digestive tract disease. The causes are also related to abnormal diet and gastric acid secretion disorders. When the patient's gastric acid retrogrades to the esophagus, it may also cause chest pain, indigestion, dysphagia, and chronic cough. , throat foreign body sensation, asthma and other symptoms.

Maintaining regular exercise and consuming more fruits and vegetables can help digestion, improve gastrointestinal function, and promote fat metabolism, but for modern people who are busy and often eat out, other more convenient solutions are still needed. When necessary, drugs (such as antacids, hypolipidemic drugs) are used clinically to relieve the discomfort of patients, but only temporary effects can be achieved; if treatment is performed by surgery, patients still have to face disease recurrence, and risks of complications.

The inventors of the present case screened out a Bifidobacterium lactis TCI604 from breast milk. The strain and its metabolites have the functions of regulating the expression of CRP gene, regulating the expression of IL-1β gene, regulating the expression of IL8 gene, regulating the expression of IL6 gene, regulating the expression of IL10 gene, regulating IL17A gene expression, regulating TNF-α gene expression, regulating CYP7A1 gene expression , and regulating the effect of APC gene expression, so it can be used to reduce the incidence of colorectal polyps, reduce the incidence of colorectal cancer, reduce the incidence of peptic ulcer, reduce gastroesophageal reflux Incidence, help digestion, improve gastrointestinal function, regulate immunity, and regulate blood lipids, which meet the needs of modern people.

One object of the present invention is to provide a kind of Bifidobacterium lactis TCI604, which is deposited in the Food Industry Research Institute of a consortium (the deposit number is BCRC910887) and the German National Culture Collection Center (the deposit number is DSM33303).

Another object of the present invention is to provide a use of the above-mentioned Bifidobacterium lactis TCI604 and/or its metabolites for preparing a composition, wherein the composition is used for at least one of the following: reducing the incidence of colorectal polyps, Reduce the incidence of colorectal cancer, reduce the incidence of peptic ulcer, and reduce the incidence of gastroesophageal reflux. Preferably, the composition is a pharmaceutical composition, and the peptic ulcer is at least one of duodenal ulcer and gastric ulcer.

Another object of the present invention is to provide a use of the above-mentioned Bifidobacterium lactis TCI604 and/or its metabolites for at least one of assisting digestion, improving gastrointestinal function, regulating immunity, and regulating blood lipids. Preferably, the Bifidobacterium lactis TCI604 and/or the metabolites are used in the form of food compositions. More preferably, the food composition is a health food, health food, functional food, nutritional supplement, or special nutritional food.

Another object of the present invention is to provide a composition comprising the above-mentioned Bifidobacterium lactis TCI604 and/or its metabolites. Preferably, the composition is a pharmaceutical composition or a food composition. More preferably, the food composition is a health food, health food, functional food, nutritional supplement, or special nutritional food.

Yet another object of the present invention is to provide a use of the above-mentioned Bifidobacterium lactis TCI604 and/or its metabolites for preparing a composition, wherein the composition is used for at least one of the following: regulating CRP gene expression, Regulates IL-1β gene expression, IL8 gene expression, IL6 gene expression, IL10 gene expression, IL17A gene expression, TNF-α gene expression, CYP7A1 gene expression , and APC gene expression. Preferably, the pharmaceutical composition is used for at least one of the following: reducing CRP gene expression, increasing CYP7A1 gene expression, and increasing APC gene expression. Preferably, the composition is a pharmaceutical composition.

Some specific embodiments according to the present invention will be described below; however, without departing from the spirit of the present invention, the present invention can still be practiced in many different forms, and the protection scope of the present invention should not be construed as being limited to what is specifically stated in the specification. or as defined by the scope of the patent application attached hereto.

Unless otherwise specified in the context, the terms "a", "the" and similar terms used in this specification (especially in the scope of the patent application described later) shall be construed to include both singular and plural forms; the so-called "metabolites" are not limited to The bacterial liquid obtained by bacterial culture (that is, the bacterial-containing culture liquid) also includes a solid-liquid separation operation (for example: centrifugation, filtration) or a separation and purification operation (for example: column chromatography, reduction pressure concentration) to obtain products such as supernatant, stratified liquid or concentrated liquid; the so-called "individual" refers to human or non-human mammals (for example: dogs, cats).

Numerical ranges (eg, 5 to 100) used herein should be understood to also include all rational numbers in the range and any rational numbers in the range. Therefore, the numerical ranges used in this specification are inclusive of All possible combinations of values between the lowest and highest values recited. In addition, when "about" is used herein before a numerical value, it means that the numerical value can be increased or decreased by an amount that is recognized by one of ordinary skill in the art as a normal and reasonable amount, for example, when used herein before a numerical value When "about", it substantially means that the difference from the stated value is within 10%, preferably within 5%.

Studies have shown that the expression of genes such as tumor necrosis factor-α (Tumor Necrosis Factor Alpha, TNF -α ) and C-Reactive Protein (C-Reactive Protein, CRP ) is related to the occurrence of colorectal polyps, all belonging to inflammatory genes, TNF - The increased expression of α gene or CRP gene represents an imbalance in the regulation of inflammation in the body, and repeated inflammation of the large intestine is likely to lead to the formation of colorectal polyps. For the foregoing see, eg, Serum CRP and IL-6, genetic variants and risk of colorectal adenoma in a multiethnic population. Cancer Causes Control (2010) 21:1131-1138, and Defensin expression in chronic pouchitis in patients with ulcerative colitis or familial adenomatous polyposis coli. World J Gastroenterol 2006 February 21; 12(7):1056-1062, which is incorporated herein by reference in its entirety. Therefore, if the expression levels of the aforementioned genes can be reduced, the effect of reducing the incidence of colorectal polyps can be achieved.

In addition, TNF -α gene, as well as interleukin-1β (interleukin-1β, IL- 1β ) , interleukin 8 ( IL8 ) , interleukin 6 ( IL6 ) , interleukin 10 ( IL10 ) , The expression changes of genes such as interleukin 17A ( IL17A ) are closely related to the immune regulation in the organism. Generally speaking, if an active ingredient can increase the gene expression of IL-1β , IL8 , IL6 , IL10 , and/or TNF-α in normal cells, it means that the active ingredient has the effect of enhancing immunity; however, If an active ingredient can reduce the gene expression of IL-1β , IL6 , IL8 , and/or IL17A in cells in an inflamed state, it means that the active ingredient has anti-inflammatory properties, reduces the incidence of peptic ulcers, and reduces gastroesophageal tract The effect of the incidence of backflow. The foregoing can be found, for example, in: The Effects of Genetic Polymorphisms of IL-6, IL-8, and IL-10 on Helicobacter pylori-induced Gastroduodenal Diseases in Korea. J Clin Gastroenterol . Volume 43, Number 5, May-June 2009, Interleukin- 1 b and -10 polymorphisms influence erosive reflux esophagitis and gastritis in Taiwanese patients. Journal of Gastroenterology and Hepatology 25 (2010) 1443-1451, Association of Genetic Polymorphisms in IL17A and IL17F with Gastro-Duodenal Diseases. J Gastrointestin Liver Dis Actions . 2012 Sep;21(3):243-9, and Immunomodulating effect of antimicrobial agents on cytokine production by human polymorphonuclear neutrophils. International Journal of Antimicrobial Agents Volume 23, Issue 2, February 2004, Pages 150-154, the full text of which is incorporated Here for reference. Therefore, if the expression of the aforementioned genes can be regulated, the effects of regulating immunity, reducing the incidence of peptic ulcer and reducing the incidence of gastroesophageal reflux can be achieved.

In addition, it is known that the CYP7A1 gene is involved in the metabolism of cholesterol, and a decrease in the expression level of the CYP7A1 gene increases the amount of cholesterol in the living body. In addition, patients with hypercholesterolemia are at high risk of developing colorectal polyps and/or colorectal cancer. For the foregoing see, for example: The Risk of Colorectal Adenomatous Polyp in Relation to Serum Total Cholesterol Levels in Japanese Men Classified by Age Group. Sangyo Eiseigaku Zasshi. 2000 May;42(3):97-101, and Human cholesterol 7α-hydroxylase (CYP7A1 ) deficiency has a hypercholesterolemic phenotype. J Clin Invest. 2002 Jul 1; 110(1): 109-117, the full text of which is incorporated herein by reference. Therefore, if the expression of the CYP7A1 gene can be increased, the effect of reducing the incidence of colorectal polyps and the incidence of colorectal cancer can be achieved.

It is known that the APC gene is a tumor suppressor-related gene, and when its expression level decreases, it represents an increased risk of colorectal cancer. In addition, the deletion of the APC gene can cause familial colorectal polyposis, and patients with colorectal polyposis will have thousands of polyps in the intestine, which is a high-risk group for colorectal cancer. The foregoing can be found, for example, in: A nonsense mutation in exon 8 of the APC gene (Arg283Ter) auses clinically variable FAP in a Malaysian Chinese family. Cancer Sci . August 2003, vol. 94, no. 8, 725-728, in its entirety and included here for reference. Therefore, if the expression of APC gene can be increased, the effect of reducing the incidence of colorectal polyps and the incidence of colorectal cancer can be achieved.

The inventors of this case screened out a Bifidobacterium lactis TCI604 from breast milk, and identified it as Bifidobacterium lactis (Chinese name is Bifidobacterium lactis, also known as Bifidobacterium ray) according to the 16S ribosomal ribonucleic acid (16S rRNA) sequence analysis. tertella), and named Bifidobacterium lactis TCI604 (hereinafter referred to as Bifidobacterium lactis TCI604), which is deposited in the Food Industry Research Institute of the Consortium (Deposit No. BCRC910887), and the German National Culture Collection Center (Deposit No. for DSM33303). The Bifidobacterium lactis TCI604 line has a 16S rRNA fragment shown in SEQ ID NO: 1.

The inventors of the present application found that Bifidobacterium lactis TCI604 has protease activity, excellent gastric acid resistance and bile salt resistance, and can grow smoothly in the intestinal tract. IL-1β gene expression, IL8 gene expression, IL6 gene expression, IL10 gene expression, IL17A gene expression, TNF-α gene expression, CYP7A1 gene expression , and APC gene expression. It can reduce the incidence of colorectal polyps, reduce the incidence of colorectal cancer, reduce the incidence of peptic ulcer, reduce the incidence of gastroesophageal reflux, help digestion, improve gastrointestinal function, regulate immunity, and regulate blood lipids.

Therefore, the present invention relates to the application of strains (ie, Bifidobacterium lactis TCI604) screened from breast milk and their metabolites, the aforementioned applications include: a method using Bifidobacterium lactis TCI604 and/or its metabolites in the preparation of a Use of a composition (especially a pharmaceutical composition) for reducing the incidence of colorectal polyps, reducing the incidence of colorectal cancer, reducing the incidence of peptic ulcers, and/or reducing the incidence of gastroesophageal reflux; a use of Bifidobacterium lactis TCI604 and/or Use of its metabolites for at least one of helping digestion, improving gastrointestinal function, regulating immunity, and regulating blood lipids; a composition (especially a pharmaceutical composition or a food composition) comprising Bifidobacterium lactis TCI604 and/or its metabolites and a kind of using Bifidobacterium lactis TCI604 and/or its metabolites to prepare a method for regulating CRP gene expression, regulating IL-1β gene expression, regulating IL8 gene expression, regulating IL6 gene expression, regulating IL10 gene expression, regulating IL17A gene expression Use of a composition (especially a pharmaceutical composition) that expresses, modulates the expression of the TNF-α gene, modulates the expression of the CYP7A1 gene , and/or modulates the expression of the APC gene.

The metabolites of Bifidobacterium lactis TCI604 used according to the present invention may be those produced by culturing in an environment suitable for the growth of Bifidobacterium lactis TCI604. For example, firstly culture Bifidobacterium lactis TCI604 with an appropriate culture medium, and then remove solids such as bacterial cells in the culture medium as necessary to obtain a liquid containing metabolites.

Any suitable medium can be used for the cultivation of Bifidobacterium lactis TCI604 to provide the desired metabolites, as long as the medium can provide the nutrients required for the growth and metabolism of Bifidobacterium lactis TCI604 (such as yeast extract, protein and glucose) and conditions (such as pH, sodium content). In addition, the cultivation time of Bifidobacterium lactis TCI604 is not particularly limited, as long as it is sufficient for Bifidobacterium lactis TCI604 to complete at least one metabolic cycle. For example, in a specific embodiment of the present invention, Bifidobacterium lactis TCI604 is cultured in MRS (de Man, Rogosa and Sharpe medium) medium for 18 hours to allow the strain to metabolize and produce metabolites.

In the use of the metabolites of Bifidobacterium lactis TCI604 according to the present invention, the culture solution containing Bifidobacterium lactis TCI604 and the metabolites that have undergone the metabolic cycle of Bifidobacterium lactis TCI604 can be directly used, or the culture solution that has undergone the metabolic cycle of Bifidobacterium lactis TCI604 and metabolites can be used. Liquid containing metabolites of solids such as Fidobacteria TCI604. Any suitable procedure can be used to remove solids as long as the desired benefit of the metabolites produced after incubation is not adversely affected. Generally speaking, physical means are used to remove solids, and the physical means include, for example, operations such as centrifugation, membrane filtration, precipitation decantation, and the like. If necessary, the aforementioned physical operations can be repeated or combined to remove as much as possible solids such as bacterial cells in the culture solution.

The pharmaceutical composition provided by the present invention can be delivered through an oral drug delivery system, and can be delivered through systems such as liposomes, microcapsules, and nanoparticles. delivery, in order to achieve the effects of improving bioavailability, controlling drug release rate, precise drug delivery to lesions, and reducing drug side effects.

The pharmaceutical composition provided according to the present invention can be in any suitable form without special limitation, and is in a corresponding appropriate dosage form depending on the intended use; for example, but not limited thereto, the pharmaceutical composition The substance can be administered to an individual in need thereof by oral administration. Depending on the form of use and purpose, a pharmaceutically acceptable carrier can be selected to provide the pharmaceutical composition, wherein the carrier is well known to those skilled in the pharmaceutical technology, including excipients, diluents, adjuvants, and stabilizers , Absorption accelerator, disintegrating agent, solubilizer, emulsifier, antioxidant, binder, binder, tackifier, dispersant, suspending agent, lubricant, hygroscopic agent, etc.

Taking the oral dosage form as an example, any suitable method can be used to provide the pharmaceutical composition in a dosage form suitable for oral administration, wherein, the liquid dosage form suitable for oral administration includes syrup, oral liquid, suspension, elixirs, etc., suitable for oral administration. Solid dosage forms for oral administration include powders, granules, buccal lozenges, sugar-coated lozenges, enteric-coated lozenges, chewable lozenges, foam lozenges, film-coated lozenges, capsules, and long-acting sustained-release lozenges. Any pharmaceutically acceptable carrier that does not adversely affect the desired benefits of the active ingredient (ie, Bifidobacterium lactis TCI604 and/or its metabolites) may be included in the pharmaceutical composition provided according to the present invention. By way of example, but not limitation, examples of pharmaceutically acceptable carriers for the aforementioned liquid dosage forms include: water, saline, dextrose, glycerol, ethanol or the like, oils (such as olive oil, castor oil, cottonseed oil, peanut oil, corn oil, and germ oil), glycerin, polyethylene glycol, and combinations of the foregoing; examples of pharmaceutically acceptable carriers for the foregoing solid dosage forms include: cellulose, starch, kaolin clay (kaolinite), bentonite (bentonite), sodium citrate, gelatin, agar, carboxymethyl cellulose, acacia, algin, glyceryl monostearate, calcium stearate, and combination of the foregoing.

The food composition provided according to the present invention can be a beverage, a solid food, or a semi-solid food, and can be provided in the form of health food, health food, functional food, nutritional supplement or special nutritional food. For example, but not limited to this, the food composition can be dairy products, processed meat products, bread, pasta, biscuits, ice products, lozenges, capsules, juices, teas, sparkling water, Alcoholic beverages, sports beverages, nutritional beverages, infant weaning food and other products. Preferably, the food composition is provided in the form of health food or health food.

In addition, depending on the usage form and requirements, any suitable food additives may be included in the food composition provided according to the present invention. Examples include, but are not limited to, preservatives, bactericides, antioxidants, bleaching agents, color retention agents, bulking agents, nutritional additives, coloring agents, flavoring agents (eg, sweeteners), thickening agents, binding agents , Food industry chemicals, emulsifiers, and quality improvement, brewing and food manufacturing agents.

The recommended usage amount, the usage standards and conditions of specific groups (for example: pregnant women, children, etc.), Or the recommendations for co-administration with other foods or medicines, so that users can take it by themselves without safety concerns without the guidance of doctors, pharmacists or relevant deacons.

If necessary, the pharmaceutical composition or food composition provided according to the present invention may further contain additives in appropriate amounts, such as toners, colorants, etc., which can improve the feeling of the composition during use, and can Buffers, preservatives, preservatives, antibacterial agents, antifungal agents, etc. to improve the stability and storage properties of the composition.

The pharmaceutical composition or food composition provided according to the present invention may additionally contain one or more other active ingredients (such as antacids, hypolipidemic drugs, dietary fiber) as needed to further enhance the efficacy of the composition or increase the formulation. The flexibility and formulation of the other active ingredients are sufficient as long as the benefits of the active ingredients of the present invention (ie, Bifidobacterium lactis TCI604 and/or its metabolites) are not adversely affected.

The pharmaceutical composition or food composition provided according to the present invention contains, based on the total weight of the composition, at least about 0.0001, 0.0002, 0.0003, 0.0004, 0.0005, 0.001, 0.0015, 0.002, 0.0025, 0.003, 0.0035, 0.004 , 0.0045, 0.005, 0.0055, 0.006, 0.0065, 0.007, 0.0075, 0.008, 0.009, 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6 , 0.8, 0.9, 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 100 wt% activity ingredients (ie, Bifidobacterium lactis TCI604 and/or its metabolites), and useful ranges may be selected from any two of the foregoing values, for example: about 0.0001% to about 90% by weight, about 0.001% to about 25% by weight % by weight, from about 0.01% by weight to about 10% by weight, from about 0.01% by weight to about 5% by weight, from about 0.05% by weight to about 1% by weight, and from about 0.05% by weight to about 0.5% by weight.

The pharmaceutical composition or food composition provided by the present invention can be administered at different frequencies such as once a day, multiple times a day, or once a few days, depending on the individual's needs, age, weight, and health conditions and The purpose of administration varies. The content of Bifidobacterium lactis TCI604 and/or its metabolites in the pharmaceutical composition or food composition provided according to the present invention can also be adjusted according to practical application requirements, for example, adjusted to the amount that should be taken daily or for external use. In general, when used in the form of a food composition, the recommended dosage is 5×10 ⁸ to 5× ^{10 10} cfu of Bifidobacterium lactis TCI604 per day, preferably 5× ^{10 9} cfu of Bifidobacterium lactis TCI604 per day.

The invention is further illustrated by the following examples. The embodiments are provided for illustration only, but not for limiting the protection scope of the present invention. The protection scope of the present invention is shown in the appended patent application scope.

Example

[ Preparation Examples ]

A. Screening and identification of Bifidobacterium lactis TCI604

A-1. Screening

The present inventors sampled from breast milk (obtained from voluntary donors), spread the collected samples on MRS medium (BD ^™ Difco ^™ Lactobacilli MRS Broth) and cultured at 37°C for 3 days. Thereafter, a single colony was picked using a sterile inoculation loop for subsequent 16S ribosomal ribonucleic acid (16S rRNA) species identification.

A-2. Identification

The strain of the single colony isolated from A-1 was taken, and phylogenetic analysis was performed to confirm that the strain had the 16S rRNA fragment shown in SEQ ID NO: 1. Using the NCBI (National Center for Biotechnology Information) online database for comparison, it was confirmed that the SEQ ID NO: 1 line and the 16S rRNA fragment of Bifidobacterium lactis strain have about 98% similarity, so they are related The target strain was identified as Bifidobacterium lactis and named as Bifidobacterium lactis TCI604.

A-3. Save

The monogenic strain obtained by A-1 was cultivated in MRS medium (BD ^TM Difco ^TM Lactobacilli MRS Broth) by liquid culture to provide bacterial liquid, then 25% glycerol was added to the bacterial liquid, and the obtained The mixture was transferred to a cryopreservation tube and stored at -80°C.

B. Preparation of metabolites of Bifidobacterium lactis TCI604

The cryopreservation tube of A-3 containing Bifidobacterium lactis TCI604 strain was thawed, and the TCI604 strain was inoculated into MRS medium (BD ^™ Difco ^™ Lactobacilli MRS Broth) and cultured at 37°C to activate the strain. Next, the once activated TCI604 strain was inoculated into MRS medium (BD ^™ Difco ^™ Lactobacilli MRS Broth) at 1% of the planting amount, and placed at 37° C. for 18 hours to obtain Bifidobacterium lactis TCI604 bacteria liquid. Thereafter, the bacterial solution was centrifuged (5,000 rpm, 20 minutes), and the supernatant was collected for subsequent experiments.

Example 1 : Bifidobacterium lactis TCI604 protease activity assay

Thaw the cryopreservation tube containing Bifidobacterium lactis TCI604 strain from [Preparation Example A-3], inoculate the TCI604 strain into MRS medium (BD™ Difco™ Lactobacilli MRS Broth), and cultivate at 37°C, to activate the strain. Next, take a piece of Skim milk agar and dig a hole in it. Then, 50 microliters of the once activated TCI604 strain was injected into the holes on the aforementioned agar and incubated at 37°C for 24 hours. Finally, observe whether there is a transparent ring on the cultured agar (if there is a transparent ring, it means that the cultured strain has protease activity), and take pictures to record. The results are shown in Figure 1.

It can be seen from FIG. 1 that Bifidobacterium lactis TCI604 of the present invention can form a transparent ring on skim milk agar. The aforementioned results show that the Bifidobacterium TCI604 of the present invention does have protease activity, so it can be used to help digestion and improve gastrointestinal function.

Example 2 : Bifidobacterium lactis TCI604 Gastric acid and bile salt tolerance

Thaw the cryopreservation tube containing Bifidobacterium lactis TCI604 strain from [Preparation Example A-3], inoculate the TCI604 strain into MRS medium (BD™ Difco™ Lactobacilli MRS Broth), and cultivate at 37°C, to activate the strain. The activated TCI604 strain was then rinsed twice with 1x Phosphate-buffered saline (PBS), and then reconstituted with 0.3 ml of PBS (total bacterial count was about 1x10 ¹⁰ ). Divide the lysate after the aforementioned treatment into three groups (each group contains 0.1 ml of the lysate), and add each group of lysates to the following solutions: 1. Control group: 99 ml of 0.2M KCl solution (pH 7) ). 2. Gastric acid group: 99 ml of 0.2M KCl solution (pH1.2). 3. Bile salt group: 99 ml of 0.2M KCl solution (containing 0.3% bile salt).

Next, the solutions provided by the above groups were subjected to the following treatments: reacted at 50 rpm for 3 hours, then, the reacted solutions were spread on MRS medium, and incubated at 37° C. for 72 hours. Finally, the number of viable cells of TCI604 was counted. The results are shown in Figure 2.

As can be seen from Figure 2, the viable counts of both the "gastric acid group" and the "bile salt group" were comparable to the "control group". The foregoing results show that the bifidobacteria TCI604 of the present invention has excellent gastric acid resistance and bile salt resistance, and is not easily disturbed by the environment in the digestive tract when administered to a living body, and can reach the intestinal tract smoothly.

Example 3 : Bifidobacterium lactis TCI604 intestinal colonization effect of

( 3-1 ) The human colon adenocarcinoma cell line Caco-2 was cultured in a triangular dish (T75 Flask) for 120 hours. Next, the culture medium was removed, the cells were rinsed twice with 1x PBS, and then 1 ml of 1% trypsin (Trypsin/EDTA) was added to suspend the cells. The aforementioned cell suspension was injected into a 24-well dish (4×10 ⁵ cells/well) and incubated overnight at 37°C, 5% CO ₂ . Afterwards, after rinsing the cells twice with 1x PBS, 900 microliters of fresh antibiotic-free culture medium was added to each well.

( 3-2 ) Thaw the cryopreservation tube containing the Bifidobacterium lactis TCI604 strain from [Preparation Example A-3], inoculate the TCI604 strain into MRS medium (BD™ Difco™ Lactobacilli MRS Broth), and store it at 37°C. C to activate the strain. The once-activated TCI604 strain was then rinsed twice with 1x PBS and reconstituted with 1x PBS (total bacterial count approximately 1x10 ¹⁰ ). Take 1 ml of the lysate and centrifuge at 8000×g for 10 minutes. The strains were rinsed twice with 1xPBS and reconstituted with 1 ml of fresh antibiotic-free medium. Take 100 μl of the lysate (about 5×10 ⁷ cfu/ml), add it to each well of the 24-well plate provided in 3-1., and incubate it in an incubator at 37°C, 5% CO ₂ . Incubate for 2 hours. After removing the culture medium, each well was rinsed twice with 1×PBS to wash away the TCI604 that was not adsorbed on the Caco-2 cells.

Next, lysis buffer (Triton X-100 Lysis Buffer) was added to each well of the 24-well plate for cell lysis. The cell lysate was taken and spread on MRS medium, and cultured at 37°C for 72 hours, and the number of viable bacteria of lactic acid bacteria was counted. Finally, carry out the calculation of the colonization rate with the following formula, and obtain the colonization rate=41 of Bifidobacterium lactis TCI604 of the present invention: Colonization rate = number of viable bacteria of lactic acid bacteria/total number of cells

It can be seen from the above colonization rate that the Bifidobacterium lactis TCI604 of the present invention has an excellent intestinal colonization effect. The aforementioned results show that the Bifidobacterium TCI604 of the present invention can grow smoothly in the intestinal tract after being administered to the organism, and provide desired efficacy.

Example 4 : Bifidobacterium lactis TCI604 metabolites in adjust IL-1β , IL8 , IL6 , IL10 , TNF-α , CRP , CYP7A1 ,and APC The effect of gene expression

As described above, it is known that if an active ingredient can increase the gene expression levels of IL-1β , IL8 , IL6 , IL10 , and/or TNF-α in normal cells, it means that the active ingredient has the effect of enhancing immunity . In addition, CYP7A1 is related to cholesterol metabolism, and the expression of CRP and APC genes is related to the occurrence of colorectal polyps and colorectal cancer. In order to know whether Bifidobacterium lactis TCI604 of the present invention has the ability to regulate immunity, regulate blood lipids, reduce the incidence of colorectal polyps, and reduce the incidence of colorectal cancer, the following experiments were carried out.

In this example, the materials and equipment used are as follows: 1. Cell line: peripheral blood mononuclear cell (PBMC), purchased from ATCC, serial number: PCS-800-011. 2. Medium: X-VIVO ^™ 10 medium, purchased from Lonza. 3. RNA Extraction Kit: purchased from Geneaid. 4. Reverse transcriptase (SuperScript® III Reverse Transcriptase): purchased from Invitrogen. 5. KAPA CYBR FAST qPCR Kits (2x): purchased from KAPA Biosystems. 6. Step One Plus: Purchased from ABI. 7. Introduction: the following table Gene Introduction primer sequence Primer length IL- 1β IL-1β-F AGCTACGAATCTCCGACCAC 20 IL-1β-R CGTTATCCCATGTGTCGAAGAA twenty two IL-8 IL-8-F TTTTGCCAAGGAGTGCTAAAGA twenty two IL-8-R AACCCTCTGCACCCAGTTTTC twenty one IL-6 IL-6-F ACTCACCTCTTCAGAACGAATTG twenty three IL-6-R CCATCTTTGGAAGGTTCAGGTTG twenty three IL-10 IL-10-F TCAAGGCGCATGTGAACTCC 20 IL-10-R GATGTCAAAACTCACTCATGGCT twenty two IL-17A IL-17A-F AGATTACTACAACCGATCCACCT twenty three IL-17A-R GGGGACAGAGTTCATGTGGTA twenty one TNF -α TNF-α-F GAGGCCAAGCCCTGGTATG 19 TNF-α-R CGGGCCGATTGATCTCAGC 19 CRP CRP-F CCAGACAGACATGTCGAGGAA twenty one CRP-R CCGTGTAGAAGTGGAGGCAC 20 CYP7A1 CYP7A1-F TTAGGAGAAGGCAAACGGGTG twenty one CYP7A1-R TTCCGTGGCACAACACCTTA 20 APC APC-F AAGAGGAGGAGGCAGGTCCAA twenty one APC-R CATCTTCAGTGCCCTCAACTTGC twenty two

PBMC cells were cultured in X-VIVO ^™ 10 medium for 24 hours. Next, the cells were divided into two groups and subjected to the following treatments: 1. Control group: cells were cultured in X-VIVO ^™ 10 medium for 6 hours. 2. Experimental group: cells were cultured in X-VIVO ^™ 10 medium containing 2% of the supernatant obtained from [Preparation Example] B for 6 hours.

The above cells were collected, RNA extraction was performed with an RNA extraction kit, and the RNA was reverse transcribed into cDNA with reverse transcriptase. Next, use ABI Step One Plus instrument and KAPA SYBR FAST qPCR kit to perform qPCR (quantitative polymerase chain reaction) on the provided cDNA to detect IL-1β , IL8 , IL6 , IL10 , TNF-α , Gene expression levels of CRP , CYP7A1 , and APC . Finally, the relative expression of genes in the experimental group was calculated using the control group as the benchmark (ie, the gene expression in the control group was set to be 1-fold). The data were statistically analyzed using Excel software, and the Student's t-test was used to test whether the statistical significance was reached. The results are shown in FIGS. 3 and 4 .

It can be seen from Figure 3 and Figure 4 that, compared with the "control group", the cells of the "experimental group" have higher expression levels of IL-1β , IL8 , IL6 , IL10 , TNF-α , CYP7A1 , and APC genes. In addition, compared with the "control group", the cells in the "experimental group" had lower CRP gene expression levels. The foregoing results show that the Bifidobacterium lactis TCI604 and its metabolites of the present invention indeed have the effects of regulating immunity, regulating blood lipids, reducing the incidence of colorectal polyps, and reducing the incidence of colorectal cancer.

Example 5 : Human experimentation

( 5-1 ) sample collection

Volunteer subjects (8 people in total, all of whom were gastrointestinal discomfort with long-term peptic ulcer and/or gastroesophageal reflux) were recruited, and the intestinal conditions of the subjects were evaluated. Before the start of the experiment, each subject was asked to fill in the intestinal status questionnaire (ie, the 0th week intestinal status questionnaire, the content of the questionnaire is shown in Table 1 below), and the blood of each subject was collected (ie, the 0th week blood) for subsequent experiments. Next, the subjects were asked to take one capsule containing 5× ^{10 9} cfu of Bifidobacterium lactis TCI604 of the present invention every day for four weeks (that is, each subject took the capsule 28 times), after that, each subject was asked again The subjects filled out the intestinal status questionnaire (ie, the 4th week intestinal status questionnaire, the content of the questionnaire was the same as the 0th week intestinal status questionnaire), and collected the blood of each subject (ie, the 4th week blood) for subsequent experiments. .

Table 1

(5-2) Assessment of the severity of intestinal conditions

Analysis of the 5-1 Week 0 Gut Status Questionnaire and the 4th Week Gut Status Questionnaire for heartburn (gastroesophageal reflux), bloating (feeling of fullness), belching (burping), flatulence (feeling of gas), and Five gastrointestinal discomfort conditions, including diarrhea, were calculated for each subject's severity score (0-6 points from least severe to most severe). The results are shown in FIG. 5 .

As can be seen from Figure 5, compared with week 0, at week 4, the subjects' fire heartburn (gastroesophageal reflux), abdominal distension (feeling of fullness), belching (burping), flatulence (feeling of gas), and Diarrhea severity scores were significantly reduced. The foregoing results show that the Bifidobacterium lactis TCI604 and its metabolites of the present invention indeed have the effects of reducing the incidence of gastroesophageal reflux, helping digestion, and improving gastrointestinal function.

(5-3) Expression evaluation of colorectal polyps and peptic ulcer-related genes

As described above, if the expression levels of TNF-α and CRP genes can be reduced, the effect of reducing the incidence of colorectal polyps can be achieved. In addition, it is known that if an active ingredient can reduce the gene expression of IL-1β , IL6 , IL8 , and/or IL17A in cells in an inflammatory state, it means that the active ingredient has an anti-inflammatory effect. Herein, the subjects recruited in this example are all gastrointestinal discomfort patients with chronic peptic ulcer and/or gastroesophageal reflux (that is, the cells of these subjects are in an inflamed state for a long time). To find out whether Bifidobacterium lactis TCI604 of the present invention has the ability to reduce the incidence of peptic ulcer, reduce the incidence of gastroesophageal reflux, and reduce the incidence of colorectal polyps, the following experiments were carried out.

The 0th and 4th week blood of each subject provided in 5-1. was taken, and peripheral blood mononuclear cells were separated therefrom with Ficoll-Paque Plus separating medium. Next, by the analysis method of Example 4, the expression levels of TNF-α , CRP , IL-1β , IL6 , IL8 , IL17A and other genes in peripheral blood mononuclear cells of each group of subjects were detected. Based on the results of the 0th week (ie, the gene expression level of the 0th week was set to be 1-fold), the gene relative expression level of the 4th week was calculated. The results are shown in FIGS. 6 and 7 .

As can be seen from Figure 6 and Figure 7, compared with the 0th week, the expression levels of TNF-α , CRP , IL-1β , IL6 , IL8 , and IL17A genes in peripheral blood mononuclear cells in the 4th week were significantly decreased. The foregoing results show that the Bifidobacterium lactis TCI604 of the present invention and its metabolites indeed have the effects of reducing the incidence of peptic ulcer, reducing the incidence of gastroesophageal reflux, and reducing the incidence of colorectal polyps.

The above results show that the Bifidobacterium lactis TCI604 of the present invention has protease activity, excellent gastric acid resistance and bile salt resistance, and can grow smoothly in the intestinal tract, and Bifidobacterium lactis TCI604 and its metabolites have the ability to regulate the expression of CRP genes, The effects of regulating IL-1β gene expression, regulating IL8 gene expression, regulating IL6 gene expression, regulating IL10 gene expression, regulating IL17A gene expression, regulating TNF-α gene expression, regulating CYP7A1 gene expression, and regulating the expression of APC gene can be used for Reduce the incidence of colorectal polyps, reduce the incidence of colorectal cancer, reduce the incidence of peptic ulcer, reduce the incidence of gastroesophageal reflux, help digestion, improve gastrointestinal function, regulate immunity, and regulate blood lipids.

【Deposit of Biological Materials】

TW Republic of China Food Industry Development Research Institute 2019/4/15 BCRC 910887

DE Germany German National Culture Collection DSMZ (Deutsche Sammlung von Mikroorganismen und Zellkulturen) 2019/9/19 DSM33303

Figure 1 is a photograph taken with a camera, showing the transparent ring produced by culturing Bifidobacterium lactis TCI604 of the present invention on skim milk agar;

Figure 2 shows the number of viable bacteria in the "control group", "gastric acid group", and "bile salt group", in which Bifidobacterium lactis TCI604 in the "control group" was cultured in a solution of pH 7, and the "gastric acid group" had Bifidobacterium lactis TCI604 was cultured in a solution with pH 1.2, and Bifidobacterium lactis TCI604 in the "bile salt group" was cultured in a solution containing 0.3% bile salts;

Figure 3 shows the expression levels of IL-1β , IL8 , IL6 , IL10 , and TNF-α genes in peripheral blood mononuclear cells (PBMC) of the "control group" and "experimental group", where " The peripheral blood mononuclear cells of the "control group" were cultured in a medium containing no metabolites of Bifidobacterium lactis TCI604, while the peripheral blood mononuclear cells of the "experimental group" were cultured in a medium containing metabolites of Bifidobacterium lactis TCI604 ( ***line indicates p-value < 0.001 compared to the control group);

Figure 4 shows the expression levels of CRP , CYP7A1 , and APC genes in peripheral blood mononuclear cells of the "control group" and "experimental group", wherein the peripheral blood mononuclear cells of the "control group" were cultured in milk-free cells The medium containing the metabolites of Bifidobacterium TCI604, the peripheral blood mononuclear cells of the "experimental group" were cultured in the medium containing the metabolites of Bifidobacterium lactis TCI604 (* means p value < 0.05 compared to the control group; ** *Denotes p-value < 0.001 compared to the control group);

Figure 5 shows the severity scores of gastrointestinal distress conditions for subjects at week 0 (ie, before taking Bifidobacterium lactis TCI604) and week 4 (ie, after taking Bifidobacterium lactis TCI604 for 4 consecutive weeks). (score 0-6 from least severe to most severe) (* means p-value < 0.05 compared to week 0);

Figure 6 shows the subjects' peripheral blood mononuclear cells at week 0 (ie, before taking Bifidobacterium lactis TCI604) and week 4 (ie, after taking Bifidobacterium lactis TCI604 for 4 consecutive weeks). The expression levels of TNF-α and CRP genes (*** means p value < 0.001 compared with the control group);

Figure 7 shows the subjects' peripheral blood mononuclear cells at week 0 (ie, before taking Bifidobacterium lactis TCI604) and week 4 (ie, after taking Bifidobacterium lactis TCI604 for 4 consecutive weeks). IL-1β , IL6 , IL8 , and IL17A gene expression levels (* means p-value <0.05 compared to the control group; *** means p-value <0.001 compared to the control group).

Claims (10)

A Bifidobacterium lactis TCI604 is deposited in the Food Industry Research Institute of a consortium (the deposit number is BCRC910887) and the German National Culture Collection Center (the deposit number is DSM33303). A use of Bifidobacterium lactis TCI604 and/or its metabolites as claimed in item 1 for preparing a composition, wherein the composition is used for at least one of the following: reducing the incidence of colorectal polyps and reducing the incidence of colorectal cancer , reduce the incidence of peptic ulcer, and reduce the incidence of gastroesophageal reflux. A use of Bifidobacterium lactis TCI604 and/or its metabolites as claimed in item 1 in the preparation of a composition, wherein the composition is used for at least one of the following: aiding digestion, improving gastrointestinal function, regulating immunity, and Regulate blood lipids. The use of claim 3, wherein the composition is a food composition. The use according to claim 4, wherein the food composition is a health food, health food, functional food, nutritional supplement, or special nutritional food. A composition comprising Bifidobacterium lactis TCI604 and/or its metabolites as claimed in item 1. The composition of claim 6, which is a pharmaceutical composition or a food composition. The composition of claim 7, wherein the food composition is a health food, health food, functional food, nutritional supplement, or special nutritional food. A method of using Bifidobacterium lactis TCI604 as claimed in item 1 and/or its metabolites in the preparation of a composition, wherein the composition is used for at least one of the following: regulating CRP gene expression, regulating IL-1β gene expression , regulate the expression of IL8 gene, regulate the expression of IL6 gene, regulate the expression of IL10 gene, regulate the expression of IL17A gene, regulate the expression of TNF-α gene, regulate the expression of CYP7A1 gene, and regulate the expression of APC gene. The use of claim 9, wherein the composition is used for at least one of the following: reducing CRP gene expression, increasing CYP7A1 gene expression, and increasing APC gene expression.

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