WO2019227414A1 - Composition and uses thereof - Google Patents

Abstract

Provided is a composition and the uses thereof, the composition comprising Lactobacillus gasseri and/or a metabolite thereof, Lactobacillus acidophilus and/or a metabolite thereof and pseudo small chain Bifidobacteria and/or a metabolite thereof. Said composition can treat inflammation including ulcerative colitis and related diseases thereof.

Description

Composition and application thereof Technical field

The present application relates to the field of microbial preparations, and in particular, to a microorganism or a microorganism-related composition and application thereof.

Background technique

Inflammatory bowel disease (IBD) is a chronic inflammatory bowel disease of unknown etiology, which is easy to repeat and seriously affects the quality of life of patients. Modern medicine believes that the factors that cause inflammatory bowel disease (IBD) include heredity, diet, infection, autoimmunity, psychological factors, and the environment. Inflammatory bowel diseases include ulcerative enteritis (UC) and Crohn's disease (CD), which are all inflammation-related diseases.

Ulcerative colitis (UC) is an important type of inflammatory bowel disease (IBD). The cause of the disease is unknown. The main lesion is in the submucosa of the colonic mucosa, which is a chronic disease. Intestinal diseases. Based on the current research, the main causes of UC are genetic susceptibility to the host, intestinal flora, and immune response of the intestinal mucosa. The clinical and pathological manifestations are persistent abdominal pain, diarrhea, and mucus and bloody stools, and the condition is recurrent. The number of patients showed a clear upward trend.

At present, the clinical medications for UC mainly include salicylic acid drugs, adrenal glucocorticoid drugs and immunosuppressants. These three types of drugs can alleviate UC to a certain extent, but they also have shortcomings. Salicylic acid drugs can better inhibit prostaglandin synthesis and scavenge oxygen free radicals in order to alleviate the inflammatory response, but they can only be relieved in a short period of time and cannot be cured. Salicylic acid western medicines commonly used in the clinical treatment of UC are mainly Sulfasalazine (SASP), mainly for patients with mild, moderate and chronic UC. In addition, salicylic acid drugs also have many side effects, such as triggering gastrointestinal reactions, headaches, increased reticulocytes, decreased sperm, and rashes, liver toxicity, leukopenia, and anemia caused by allergic reactions. These drugs also have Antibacterial effect, easy to cause flora disorder and enhanced drug resistance. Adrenal glucocorticoids are the first choice for patients with severe or explosive UC. Typical drugs such as betamethasone; however, adrenal glucocorticoids can cause side effects such as metabolic disorders and water retention. They can only be used as emergency medication. Cannot be taken for a long time. Immunosuppressive agents, such as cyclosporine, can suppress UC by inhibiting the production of IL-2 in T cells, which affects the progress of the immune response; however, immunosuppressive therapy is highly drug-dependent, has a long treatment cycle, and is easy Causes nephrotoxicity and secondary infections, and can only be used as a means of adjuvant therapy. Therefore, there is currently no safe and effective treatment for inflammation and related diseases, especially ulcerative enteritis.

Summary of the Invention

The purpose of this application is to provide a composition and its use.

This application uses the following technical solutions:

One aspect of the present application discloses a composition comprising Lactobacillus gasseri and / or a metabolite thereof, Lactobacillus acidophilus and / or a metabolite thereof, and Bifidobacterium pseudosmallii and / or metabolite.

It should be noted that the key to this application is that the study found that the combined use of Lactobacillus gasseri, Lactobacillus acidophilus, and Bifidobacterium pseudoparvum can prevent and treat inflammation and inflammation-related diseases, and can effectively prevent and treat Treatment of ulcerative enteritis; research shows that its preventive or therapeutic preventive effect is mainly composed of two aspects. On the one hand, Lactobacillus gasseri, Lactobacillus acidophilus and Bifidobacterium pseudosmall chain improve the intestinal microecology in vivo. Forms an ecological protective barrier composed of beneficial bacteria, thereby preventing and treating inflammation or inflammation-related diseases; on the other hand, the metabolites of Lactobacillus gasseri, Lactobacillus acidophilus, and Bifidobacterium pseudosmall chain are used as Probiotic materials play a role in the prevention and treatment of inflammation or inflammation-related diseases. Therefore, the composition of the present application is mainly used for preventing or treating inflammation or inflammation-related diseases, especially for preventing or treating ulcerative enteritis or related diseases.

It should also be noted that in an implementation manner of the present application, a composition consisting of Lactobacillus gasseri, Lactobacillus acidophilus and Bifidobacterium pseudosmall chain is used to improve the intestinal microecology of the body through three bacteria To form an ecological protective barrier composed of beneficial bacteria, and to prevent and treat ulcerative enteritis. It can be understood that this micro-ecological improvement not only has prevention and treatment effects on ulcerative enteritis, but also has effects on other micro-ecological-related diseases, such as common enteritis or gastritis; therefore, the composition of the present application can be used for Prevent or treat inflammation or inflammation-related diseases.

Preferably, Lactobacillus gasseri is Lactobacillus gasseri TF08-1 with accession number GDMCC 60092, Lactobacillus acidophilus is Lactobacillus acidophilus AM13-1 with accession number GDMCC 60091, and Bifidobacterium pseudosmall chain is GDMCC 60089 Pseudo-small Bifidobacterium TM12-14.

It should be noted that the key to this application is that the research found that the combined use of Lactobacillus gasseri, Lactobacillus acidophilus and Bifidobacterium pseudosmall chain can prevent and treat inflammation and inflammation-related diseases, and Lactobacillus gasseri TF08- 1. Lactobacillus acidophilus AM13-1 and Bifidobacterium pseudominis TM12-14. These three strains are the three strains found to have a good combination effect during the research process of this application. Therefore, three strains were tested separately. Preserved. It can be understood that on the one hand, under the inventive idea of the present application, other strains of Lactobacillus garnerii, Lactobacillus acidophilus, and Bifidobacterium pseudoparvum can also be used to achieve or even exceed the effects of the three strains of the present application. ; On the other hand, in the case of lower requirements for the prevention or treatment of inflammation or inflammation-related diseases, other strains of Lactobacillus garnerii, Lactobacillus acidophilus, and Bifidobacterium pseudoparvum can also be used.

Preferably, the composition of the present application further contains other probiotics and / or prebiotics.

It should be noted that the key to the composition of the present application is that the combined use of Lactobacillus gasseri, Lactobacillus acidophilus and Bifidobacterium pseudoparvum can prevent and treat inflammation and inflammation-related diseases. It can be understood that In the case of affecting the combined effect of Lactobacillus gasseri, Lactobacillus acidophilus and Bifidobacterium pseudominis, other probiotics or prebiotics may be added, so that the composition of the present application has more functions, or The original efficacy is strengthened. These probiotics or prebiotics can use the probiotics or prebiotics reported in the existing research, and are not specifically limited here.

Preferably, in one implementation of the present application, the prebiotics in the composition of the present application are selected from the group consisting of fructooligosaccharide (FOS), galactooligosaccharide (GOS), xylooligosaccharide (XOS) and lactulose (LACT), soy oligosaccharide (SOS), inulin and oligosaccharide.

Preferably, the composition of the present application further contains a substance that helps maintain the viability of at least one of Lactobacillus gasseri, Lactobacillus acidophilus, and Bifidobacterium pseudoparvum.

It can be understood that, in order to maintain the vigor of Lactobacillus garneri, Lactobacillus acidophilus, and Bifidobacterium pseudofimbriae in the composition, and to ensure its efficacy, various substances to maintain the viability of the strain can also be added to the composition. These vital substances It may be an active substance that has been reported in existing research, and is not specifically limited here.

Preferably, in an implementation manner of the present application, a substance that is helpful for maintaining the viability of at least one of Lactobacillus gasseri, Lactobacillus acidophilus, and Bifidobacterium pseudosmall chain is selected from cysteine, At least one of glutathione, butylhydroxyanisole, dibutylmethyltoluene, tocopherol, bamboo leaf antioxidants, D-isoascorbic acid or its sodium salt, sodium ascorbate, calcium ascorbate, phospholipids, vitamin C and vitamin E One.

Preferably, the composition of the present application further comprises a pharmaceutically or food-acceptable carrier or excipient.

It should be noted that the composition of the present application has the effect of preventing and treating inflammation and inflammation-related diseases. In one implementation manner of the present application, the composition of the present application is mainly used to achieve the prevention and treatment effect. Therefore, a pharmaceutically or food-acceptable carrier or excipient may also be included in the composition for ease of use.

Preferably, a pharmaceutically or food-acceptable carrier or excipient is selected from glucose, lactose, sucrose, starch, mannitol, dextrin, fatty acid glyceride, polyethylene glycol, hydroxyethyl starch, ethylene glycol, poly At least one of oxyethylene sorbitan fatty acid ester, amino acid, gelatin, albumin, water, and physiological saline.

The other side of the present application discloses the application of the composition of the present application in the preparation of food, health products, food additives or medicines for treating or preventing inflammation or inflammation-related diseases.

It can be understood that the composition of the present application has the effects of preventing and treating inflammation and inflammation-related diseases. In order to facilitate the use, the composition of the present application can be made into various foods, health products, food additives or drugs.

Preferably, the composition of the present application can be used in particular for preparing food, health products, food additives or drugs for treating or preventing ulcerative enteritis or related diseases.

Another aspect of the present application discloses the use of the composition of the present application in the preparation of a food, a health product, a food additive, or a medicine for controlling weight loss in a mammal.

Among them, the weight loss of mammals, especially the weight loss of mammals due to inflammation.

Preferably, the inflammation is ulcerative enteritis, that is, controlling weight loss caused by ulcerative enteritis in a mammal.

Another aspect of the present application discloses the use of the composition of the present application in the preparation of a food, a health product, a food additive, or a medicine for reducing the disease activity index of a mammal.

Another aspect of the present application discloses the application of the composition of the present application in the preparation of foods, health products, food additives, or medicines for improving intestinal lesions in mammals.

It should be noted that the composition of the present application can prevent and treat inflammation and inflammation-related diseases, and the key lies in being able to control the weight loss caused by inflammation and inflammation-related diseases and reduce the disease activity index caused by inflammation and inflammation-related diseases And improve mammalian intestinal pathology; therefore, the composition of the present application can also be used alone to prepare foods, health products, and food additives that control mammalian weight loss, reduce mammalian disease activity index, or improve mammalian intestinal pathology. Or medicine.

Another aspect of the present application discloses a method for treating or preventing inflammation or inflammation-related diseases using the composition of the present application.

Another aspect of the present application discloses a method for controlling weight loss in a mammal using the composition of the present application.

Another aspect of the present application discloses a method for reducing the disease activity index of a mammal using the composition of the present application.

Another aspect of the present application discloses a method for improving intestinal pathological changes in mammals by using the composition of the present application.

It should be noted that, in the above various methods, the composition of the present application is mainly used to treat or prevent inflammation or inflammation-related diseases, control the weight loss of mammals, reduce the mammalian disease activity index, or improve the mammalian intestine. The effect of pathological changes.

Another aspect of the present application discloses a food, which contains the composition of the present application.

Preferably, the food is a lactic acid drink or a soy milk drink. The food in the present application is an edible article existing in any form in a broad sense, and is not limited to a lactic acid drink or a soy milk drink. For example, the food may be a fermented food or an animal feed.

It should be noted that, since the food of the present application contains the composition of the present application, it also has the effects of treating or preventing inflammation or inflammation-related diseases, controlling weight loss of mammals, reducing mammalian disease activity index, and improving mammalian intestinal diseases. . It can be understood that the key to the food of the present application is that it contains the composition of the present application. As for the specific form of the food, such as solid, liquid, etc., it can be determined according to different food products or use requirements, and is not specifically limited here. In an implementation manner of the present application, the composition of the present application is mainly made into a common lactic acid drink or soy milk drink for easy drinking; of course, it can also be made into solid foods such as milk slices and cheese bars, etc. Be specific.

It should also be noted that the amount of active bacteria or ingestion of Lactobacillus gasseri, Lactobacillus acidophilus, and Bifidobacterium pseudofimbriae in the food of the present application is not specifically limited. In practical applications, it can be flexibly selected according to the actual situation . Taking Lactobacillus gasseri TF08-1, Lactobacillus acidophilus AM13-1, and Bifidobacterium pseudosmall chain TM12-14 as examples, the study of this application shows that daily intake of Lactobacillus gasseri TF08-1, acidophilus milk The concentration of Bacillus AM13-1 and Bifidobacterium pseudomonas TM12-14 are both 10 ⁹ cfu / mL, 0.2mL of the composition, which has a good therapeutic effect on ulcerative enteritis. The active bacteria dose can be used as food and health products , The reference amount of food additives or medicines or the reference intake.

Another aspect of the present application discloses a health product, which contains the composition of the present application.

It should be noted that, since the health product of the present application contains the composition of the present application, it also has the advantages of treating or preventing inflammation or inflammation-related diseases, controlling weight loss of mammals, reducing mammalian disease activity index, and improving mammalian intestinal diseases. effect.

Another aspect of the present application discloses a food additive containing the composition of the present application.

It should be noted that the composition of the present application can be used in combination with common food materials. For example, cereals and potatoes, cereals include rice, noodles, miscellaneous grains, potatoes including potatoes, sweet potatoes, etc .; animal foods, including meat, poultry, fish, milk, eggs, etc .; beans and their products, including soybeans and other dried Beans; vegetables and fruits, including fresh beans, rhizomes, leafy vegetables, eggplants, etc .; pure thermal energy foods, including animal and vegetable oils, starch, edible sugar, and alcohol; therefore, the composition of the present application can be used alone as a food additive Or the modulator is added to various foods and eaten directly to play a therapeutic or preventive effect on inflammation and related diseases.

Another aspect of the present application discloses a medicine containing the composition of the present application.

Preferably, the medicine is a tablet, granule, powder, enteric solvent, solution or suspension.

It should be noted that the medicine of the present application has the composition of the present application, so it has the effect of treating or preventing inflammation and related diseases; the medicine of the present application may be the composition of the present application alone, or it may be combined with other inflammations. The drugs can be used in combination as long as they do not affect the activity of each other. It can be understood that, as long as the medicine of the present application does not affect the activity of each strain in the composition, the medicine can adopt various existing dosage forms. The medicines in the present application may further include auxiliary materials commonly used in medicines or dosage forms, such as stabilizers, wetting agents, emulsifiers, adhesives, isotonic agents, and the like.

The medicine of the present application can be administered in any form of oral solution, tablet, injection, orally disintegrating tablet, lyophilized powder preparation or enteric solvent type. An enteric solvent type, such as a capsule or an enteric tablet, is preferred so that the active ingredient of the medicine, that is, the microorganism can pass through the stomach smoothly without being destroyed by gastric acid. More preferably, the medicine of the present application can be made into enteric tablets for oral use.

The enteric solvent type in the present application refers to a pharmaceutical dosage form that does not disintegrate in gastric juice and can be disintegrated and absorbed in intestinal fluid. The enteric solvent type includes capsules and enteric tablets. Among them, capsules are formed by powdered medicines enclosed in capsule shells that are allowed for conventional medicines; enteric-coated tablets are formed by wrapping an enteric coating on the outside of ordinary tablet medicines. The "enteric coating" referred to in the present application is referred to as "enteric coating", and includes all the coatings allowed for conventional drugs. These coatings are not degraded by gastric acid, but can be fully decomposed in the small intestine and quickly release the drug of the present application. For example, the casing of the present application can be maintained at 36-38 ° C. for more than 2 hours in a synthetic gastric acid such as a HCl solution of pH = 1, and preferably decomposed within 1.0 hour in a synthetic intestinal juice such as a buffer solution of pH = 7.0.

Preferably, in the enteric-coated tablets of the present application, the thickness of the casing is 5-100 μm, and the ideal thickness is 20-80 μm. The casing components are selected from conventional materials which are publicly known.

The active bacteria content or the dosage of each strain of the probiotic composition in the medicine of the present application is not particularly limited, and in practical applications, it can be flexibly selected according to the health status of the subject to be administered. However, the study of this application shows that the daily intake of Lactobacillus garnerii TF08-1, Lactobacillus acidophilus AM13-1, and Bifidobacterium pseudominis TM12-14 are all 10 ⁹ cfu / mL of probiotic combination 0.2mL, which has a good therapeutic effect on ulcerative enteritis. This amount can be used as a reference for the content of active bacteria in the drug or the dosage.

The beneficial effects of this application are:

The composition of the present application, through the combined use of Lactobacillus gasseri, Lactobacillus acidophilus, and Bifidobacterium pseudoparvum, can have good treatment and prevention effects on inflammation, especially ulcerative enteritis, and related diseases. For the treatment and prevention of inflammation and related diseases, it provides a new safe, effective, small toxic and side effect composition, and is not easy to produce resistance.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a curve of body weight changes of mice in the Control group, the model group, the VSL ^# 3 group, and the probiotic composition treatment group in the examples of the present application;

FIG. 2 is a change curve of the DAI index of mice in the Control group, the model group, the VSL ^# 3 group, and the probiotic composition treatment group in the examples of the present application.

The Lactobacillus gasseri TF08-1 of the present application was deposited at the Guangdong Provincial Center for Microbial Strains on October 13, 2016. The address of the deposit unit is the fifth floor of the Provincial Institute of Microbiology, 100 Xianlie Middle Road, Guangzhou, Guangdong Province, China. The number is GDMCC 60092.

Lactobacillus acidophilus AM13-1 was deposited in the Guangdong Provincial Center for Microbial Strains on October 13, 2016. The address of the deposit unit is the fifth floor of the Provincial Institute of Microbiology, 100 Xianlie Middle Road, Guangzhou, Guangdong Province, China, and the deposit number is GDMCC. 60091.

Pseudomonas aeruginosa TM12-14 was deposited in the Guangdong Provincial Center for Microbial Strains on October 13, 2016. The address of the deposit unit is the fifth floor of the Provincial Institute of Microbiology, 100 Xianlie Middle Road, Guangzhou, Guangdong, China. The number is GDMCC 60089.

Detailed ways

With the in-depth study of intestinal microecology, it is found that the pathogenesis of ulcerative enteritis is closely related to the composition of intestinal microorganisms. The imbalance of intestinal bacteria is closely related to the intestinal inflammatory response. The excessive proliferation of harmful bacteria can trigger an inflammatory response. , And then induce the onset of ulcerative enteritis. There are a large number of beneficial bacteria in the healthy intestine, and these beneficial bacteria constitute the first biological barrier of the intestine.

Based on the above research and knowledge, a new composition is developed and proposed in the present application. The composition includes Lactobacillus gasseri and / or its metabolites, Lactobacillus acidophilus and / or its metabolites, and a pseudo-small chain. Bifidobacterium and / or its metabolites. The composition of the present application not only has the function of treating and preventing ulcerative enteritis, but also has effects on other microecological-related diseases, such as common enteritis or gastritis; therefore, the composition of the present application can be used to prevent or treat inflammation Or inflammation-related diseases.

In an implementation manner of the present application, Lactobacillus gasseri TF08-1 with a deposit number of GDMCC 60092, Lactobacillus acidophilus AM13-1 with a deposit number of GDMCC 60091, and Bifidobacterium pseudoparvum with a deposit number of GDMCC 60089 The composition consisting of TM12-14 has a better therapeutic effect on ulcerative enteritis than the VSL ^# 3 composite probiotic agent produced by the American company Alfasigma. It can be used to prepare foods, health products and food additives for the treatment or prevention of inflammation and related diseases. Or medicine.

The following further describes this application in detail through specific embodiments. The following examples only further illustrate the present application, and should not be construed as limiting the present application.

Example one

In this example, a mouse model of ulcerative enteritis induced by sodium dextran sulfate (DSS) with a molecular weight of 36,000-50000 is used as the research object. Lactobacillus gasseri TF08-1 and Lactobacillus acidophilus were studied. The therapeutic effect of AM13-1 and Bifidobacterium pseudocatenulatum TM12-14 probiotic composition on ulcerative enteritis. details as follows:

I. Materials and methods

1. Strain culture and identification

1.1 Lactobacillus gasseri TF08-1

In this example, Lactobacillus gasseri TF08-1 was isolated using PYG medium, and the isolation condition was 37 ° C anaerobic condition. TF08-1 was cultured in PYG medium for 2 days. The colonies were white, low raised, nearly round, and wavy. The diameter of the colonies was about 1-2 mm. The microscopic morphology of the bacteria was rod-shaped. Gram-positive and non-producing. Spores and flagella. The strains were deposited in the Guangdong Provincial Center for Microbial Strains, and the accession number was GDMCC 60092.

The specific isolation and identification steps of Lactobacillus gasseri TF08-1 are as follows:

1.1.1 Sample Collection

The isolated sample was from a fecal sample of a 16-year-old healthy female volunteer living in Shenzhen, Guangdong. The diet and physical conditions of the volunteer were recorded in detail.

1.1.2 Isolation and culture of strains

The isolation medium was prepared in advance. The medium was selected from PYG medium and purchased from Huankai Microbial Technology Co., Ltd. The specific ingredients were: peptone 5g, tryptic casein 5g, yeast powder 10g, beef paste 5g, glucose 5g, K ₂ HPO ₄ 2g, Tween 80 1mL, Cysteine-HCl · H ₂ O 0.5g, sodium sulfide 0.25g, heme 5mg, vitamin K ₁ 1μL, inorganic salt solution 40mL, resazurin 1mg, distilled water 950mL, pH 6.8 ～ 7.0, 115 Sterilize at ℃ for 25min. The solid medium was added with 1.5% agar and poured in an anaerobic operation box. Each 1 L of the inorganic salt solution contains 0.25 g of CaCl ₂ · 2H ₂ O, 0.5 g of MgSO ₄ · 7H ₂ O, 1 g of K ₂ HPO _4, 1 g of KH ₂ PO ₄ , 10 g of NaHCO ₃ , and 2 g of NaCl.

The collected fresh stool samples were transferred to an anaerobic box, 0.2 g of stool was suspended in 1 mL of sterile phosphate buffered saline (abbreviated PBS), thoroughly mixed, and then subjected to gradient dilution, and 100 μL of the diluted solution was plate coated, 37 ° C For 3-4 days of anaerobic culture, the anaerobic gas composition is N ₂ : CO ₂ : H ₂ = 90: 5: 5. When the colonies grow on the plate, select a single colony to separate and purify, obtain a pure culture strain, and then perform identification and functional verification.

16S rDNA identification of 1.1.3 strains

The isolated strain was identified by 16S rDNA to determine the species classification information of the strain. The obtained isolated strain was cultured in liquid PYG medium for 24 hours, and 1 mL of the bacterial solution was centrifuged at 10,000 r / min for 5 min. The bacterial cells were collected, the genomic DNA of the strain was extracted, and 16S rDNA was amplified using the genomic DNA as a template. 16S rDNA was used. Universal primers.

The 16S rDNA PCR amplification system is: 10 × PCR buffer 3 μL, dNTP 2.5 μL, upstream primer 27F 0.5 μL, downstream primer 1492R 0.5 μL, Taq enzyme 0.3 μL, template 1 μL, ddH ₂ O 18.2 μL.

The 16S rDNA amplification conditions are: pre-denaturation at 95 ° C for 4 min, and then enters 30 cycles: denaturation at 95 ° C for 30s, annealing at 57 ° C for 40s, and extension at 72 ° C for 1min to 30s.

The PCR amplified product of 16S rDNA was purified, sequenced at 3730 to obtain the 16S rDNA sequence of the strain, and then compared with the NCBI database.

The upstream and downstream primers of the 16S rDNA universal primer in this experiment are the sequences shown in SEQ ID NO.1 and SEQ ID NO.2, respectively. The 16SrDNA sequence of the isolated strain TF08-1 showed the sequence shown in SEQ ID NO.3. The NCBI blast comparison showed that the TF08-1 strain isolated in this example had the highest homology with Lactobacillus gasseri, with a similarity of 99.9%. Therefore, TF08-1 was judged as Lactobacillus gasseri and was named Lactobacillus gasseri TF08-1 And preserve it.

SEQ ID NO. 1: 5’-AGAGTTTGATCATGGCTCAG-3 ’

SEQ ID NO. 2: 5’-TAGGGTTACCTTGTTACGACTT-3 ’

1.2 Lactobacillus acidophilus AM13-1

Lactobacillus acidophilus AM13-1 was isolated using PYG medium under 37 ° C anaerobic conditions. The colonies of AM13-1 cultured in PYG medium for 2 days were white, raised, thick, opaque, round, with neat edges, about 2-3 mm in diameter. The microscopic morphology of the cells was rod-shaped and Gram-positive. No spores and flagella. The strain was deposited in the Guangdong Provincial Center for Microbial Strains, and the accession number was GDMCC 60091.

The specific isolation and identification steps of Lactobacillus acidophilus AM13-1 are as follows:

1.2.1 Isolation culture

The isolated sample was from a fecal sample from a healthy male in Shenzhen. The isolation process of Lactobacillus acidophilus AM13-1 was as follows:

(1) Transfer the sample to an anaerobic box, take about 0.2g of the sample and suspend it in 1mL of sterile PBS, mix thoroughly, and then perform gradient dilution;

(2) Take 100 μL of the diluted solution on a PYG medium plate, and then coat it. After the coating is uniform, place it in a 37 ° C anaerobic environment for cultivation. The anaerobic gas composition is: nitrogen: hydrogen: carbon dioxide = 90: 5. : 5; PYG medium is the same as "Isolation and culture of 1.1.2 strain";

(3) Cultivate for 4 days. After colonies grow on the plate, select single colonies for streaking and separation, and anaerobic culture at 37 ° C.

(4) Glycerin preservation and vacuum freeze-drying preservation of the separated single bacteria.

1.2.2 16S rDNA identification of AM13-1

Genomic DNA was extracted, and 16SrDNA was amplified using DNA as a template. The universal primers for 16SrDNA were used. The amplification conditions were predenatured at 95 ° C for 4min, and then entered into 30 cycles: denaturation at 95 ° C for 30s, annealing at 57 ° C for 40s, and 72 ° C extension 1min 30s. The amplified PCR product was purified and sequenced at 3730 to obtain the full 16S rDNA sequence of AM13-1. The 16S rDNA sequence of AF13-1 was compared in the NCBI database.

The 16S rDNA universal primer and PCR amplification system in this test are the same as "16. rDNA identification of 1.1.3 strain". The 16SrDNA sequence of the isolated strain AM13-1 showed the sequence shown in SEQ ID NO.4. NCBI blast comparison results showed that the AM13-1 strain isolated in this example had the highest homology with Lactobacillus acidophilus, with a similarity of 100%. Therefore, AM13-1 was judged as Lactobacillus acidophilus and named Lactobacillus acidophilus AM13-1 And preserve it.

1.3 Bifidobacterium pseudotm TM12-14

Pseudo-small chain Bifidobacterium TM12-14 was isolated using PYG medium under 37 ° C anaerobic conditions. The colonies of TM12-14 cultured in PYG medium for 2 days were white, raised, round, and the edges were neat. The diameter of the colonies was about 1-2 mm. The microscopic morphology of the bacteria showed a divergent rod shape. The Gram stain was positive and no spore And flagella are produced. The strain was deposited in the Guangdong Provincial Center for Microbial Strains, and the accession number was GDMCC 60089.

The specific isolation and identification steps of Bifidobacterium pseudomonas TM12-14 are as follows:

1.3.1 Sample Collection

The isolated sample was from the feces of a 14-year-old healthy male. The feces were collected into a sterile sample tube and brought back to the laboratory for sorting within 1 hour.

1.3.2 Isolation and purification of Bifidobacterium pseudominis

The collected fresh samples were immediately transferred to the anaerobic operation box, 0.2g of the sample was taken in 1mL of sterile PBS, thoroughly shaken and mixed, and then gradient dilution coating was performed. The medium was plated with PYG medium and anaerobic at 37 ° C. During culturing, the anaerobic gas composition was N ₂ : CO ₂ : H ₂ = 90: 5: 5. After culturing for 3 days, single colonies were picked and purified by streaking to obtain a pure culture of each single strain. Among them, the PYG medium is the same as "Isolation culture of the 1.1.2 strain".

1.3.3 Preservation of bacteria

The obtained pure culture strain was cultured to a concentration of about 10 ⁹ cfu / mL, and 400 μL of the bacterial solution was added to 400 μL of 40% glycerol to make the glycerol concentration reach 20%, and then stored at -80 ° C. at ultra-low temperature.

1.3.4 16S rDNA identification

The obtained isolated strain was cultured in liquid PYG medium for 24 hours, and 1 mL of the bacterial solution was centrifuged at 10,000 r / min for 5 minutes. The bacterial cells were collected and genomic DNA was extracted. Using genomic DNA as a template, 16S rDNA universal primers were used for PCR amplification. The PCR amplification primers, systems and conditions were the same as "16. rDNA identification of strain 1.1.3".

The 16S rDNA amplification products obtained were subjected to electrophoretic detection, purification, and 3730 sequencing to obtain the 16S rDNA sequences of the strains, and then the comparison of the NCBI database was performed.

The sequencing results showed that the 16SrDNA of strain TM12-14 was 1400bp in length and the sequence is shown in SEQ ID NO.5. NCBI blast comparison results showed that strain TM12-14 has the highest homology with Bifidobacterium pseudocatenulatum. It was identified as Bifidobacterium pseudocatenulatum and named as Bifidobacterium TM12-14 and stored. .

2. Mouse model

The mouse model selected in this example is: DSS (dextran sodium sulfate Dextran Sulfate, Na, molecular weight 36000-50000) induced ulcerative enteritis mouse model.

Specifically, 48 mouse strains of C57bl / 6 mice purchased from Hubei Medical Laboratory Animal Center were used. All mice were 8 weeks old and weighed 20g ± 2g. They were reared in a SPF-grade mouse room environment. Forty-eight mice were randomly divided into 4 groups, and 12 mice in each group were followed up.

DSS modeling: mice were continued to drink 0.15% DSS for seven days to obtain a mouse model of ulcerative enteritis.

3. Test method

Forty-eight mice were randomly divided into four groups of 12 each, and the four groups were the normal group (ie, the control group), the model group, the probiotic composition treatment group, and the VSL ^# 3 treatment group. The specific treatment methods of each group are as follows :

Normal group: they were fed with ordinary feed, and each mouse was fed with 0.2 mL of PBS buffer.

Model group: fed with the same feed, and subjected to DSS modeling: DSS was added to the drinking water of the mice, the DSS was added at a final concentration of 0.15%, and fed for seven days. Each mouse was fed with 0.2 mL of PBS buffer daily. liquid.

Probiotic composition treatment group: fed with the same feed, 3 days before DSS modeling, each mouse was administrated with 0.2 mL of probiotic composition liquid daily, and then DSS modeling: DSS modeling It was added to the drinking water of mice, and the DSS was added at a final concentration of 0.15%. After being fed for seven days, each mouse was fed with 0.2 mL of the probiotic composition bacterial solution daily.

VSL ^# 3 treatment group: fed with the same feed, 3 days before DSS modeling, each mouse was given 0.2 mL of VSL ^# 3 bacteria solution daily, and then DSS modeling was performed: DSS was added to Mice drank water, the DSS was added at a final concentration of 0.15%, and they were fed for seven days. Each mouse was fed with 0.2 mL of VSL ^# 3 bacteria solution daily.

The bacterial solution of the probiotic composition is prepared by the following method:

Lactobacillus gasseri TF08-1, Lactobacillus acidophilus AM13-1 and Bifidobacterium pseudofimbriae TM12-14 were cultured for 24 hours, the cells were collected by centrifugation, suspended with PBS, and the bacteria concentration was adjusted to 10 ⁹ cfu / mL The three strains are mixed according to 1: 1: 1 to prepare a probiotic composition liquid.

VSL ^# 3 bacteria solution is prepared by the following method:

VSL ^# 3 was purchased from the American company Alfasigma. It is a strain containing Lactobacillus casei, Lactobacillus plantarum, Lactobacillus acidophilus, Lactobacillus germani subsp. Thermophilus, Bifidobacterium longum, Bifidobacterium breve, and infant. Compound probiotics of 8 beneficial bacteria such as Bifidobacterium. Similarly, VSL ^# 3 was suspended in PBS and adjusted to a concentration of 10 ⁹ cfu / mL to obtain a VSL ^# 3 bacterial solution.

The DSS model was used to record the body weight, diet and drinking water of the mice, and observe the fecal characteristics and occult blood of the mice. The disease activity of the mice was calculated on the 1st, 3rd, 5th, and 7th days, respectively. Index (abbreviation DAI), DAI scoring standards are shown in Table 1. Mice were sacrificed at the end of the experiment. All mice were bled, neck removed, colon removed, photographed, weighed, and colon length measured. Colon tissue was stored in a -80 ° C refrigerator and paraformaldehyde. Among them, the recording time of the normal group was the same as that of DSS modeling.

Table 1 DAI index score table

In Table 1, "stool characteristics", "normal" stools refer to shaped stools, "loose" stools refer to pasty, semi-formed stools that do not adhere to the anus, and "thin stools" refer to thin stools that can adhere to the anus. Like water. In "fecal occult blood / weak eye blood stool", "normal" means that the blood in the stool of the mouse is negative; "naked eye blood stool" means that red or brown blood in the stool can be directly observed by the naked eye; "positive blood" means insignificant blood in the stool, Tetramethylbenzidine was tested positive for blood in the stool. The DAI index is equal to the sum of the three points of body weight, "fecal traits" and "fecal occult blood / weak eye bloody stool".

Results and analysis

Weight change

The weights of the mice on the 1st, 3rd, 5th, and 7th days were counted, and the average weights of the mice in each group are shown in Table 2 and FIG. 1.

Table 2 Average weight of each mouse

Group Day 1 (g) Day 3 (g) Day 5 (g) Day 7 (g) Control 22.32 ± 0.45 23.73 ± 0.64 24.98 ± 0.96 25.52 ± 1.26 Model group 22.41 ± 0.52 21.65 ± 0.71 * 20.02 ± 1.26 * 18.21 ± 1.57 ** Probiotics 22.38 ± 0.61 21.97 ± 0.85 21.47 ± 1.35 21.32 ± 1.49 ^▲ VSL ^# 3 22.54 ± 0.71 21.87 ± 0.92 21.32 ± 1.48 20.01 ± 1.66 ^▲

In Table 2, "Control" refers to the normal group, "Probiotics" is the probiotic composition treatment group, and "VSL ^# 3" is the VSL ^# 3 treatment group. "*" Refers to the significant difference in body weight of the model group relative to the normal group of mice P <0.05, "**" refers to the significant difference in weight of the model group to the normal group of mice P <0.01, "▲" refers to The "probiotics" and "VSL ^# 3" groups of mice were significantly different from the model group at a level of P <0.05.

The results in Table 2 and Figure 1 show that the weight of mice in the normal group is slowly increasing. The DSS-induced mice, that is, the model group, the "probiotic" group, and the "VSL ^# 3" group, all of the three groups have the same weight. Continued decline. Compared with the control group, the weight loss of the model group began to be significant on the 3rd day (ie * P <0.05), and on the 7th day, the difference between the two was more significant (ie ** P <0.01). And the intervention of probiotic composition and VSL ^# 3 can slow down the weight loss of UC mice. On the seventh day, the control of the weight loss of the two groups of "probiotics" and "VSL ^# 3" was compared with the model group. Significant (ie ▲ P <0.05). It shows that the probiotic composition and VSL ^# 3 can control the weight loss caused by UC. In addition, the weight of mice in the probiotic composition group (ie, "probiotics") on the seventh day was slightly higher than that of VSL ^# 3, indicating that the effect of the probiotic composition in controlling weight loss of UC mice was slightly better than that of VSL ^# 3.

2. Changes in DAI

DSA-induced ulcerative enteritis in mice caused changes in DAI index due to changes in body weight, stool characteristics, and blood in the stool. The DAI index of mice on day 1, 3, 5, and 7 was as follows: Table 3 and Figure 2. In Table 3, the DAI of each group of mice is taken as the average value of each group of mice.

Table 3 DAI values of mice

Group Day 1 Day 3 Day 5 Day 7 Control 1.1 ± 0.5 1.1 ± 0.7 1.2 ± 0.8 1.3 ± 0.8 Model group 1.1 ± 0.5 3.6 ± 1.1 * 7.2 ± 1.6 ** 9.4 ± 2.0 ** Probiotics 1.2 ± 0.4 3.3 ± 1.2 6.0 ± 1.5 ^▲ 6.5 ± 1.8 ^▲ VSL ^# 3 1.1 ± 0.4 3.4 ± 1.3 6.6 ± 1.6 7.8 ± 1.9 ^▲

In Table 3, "Control" refers to the normal group, "Probiotics" is the probiotic composition treatment group, and "VSL ^# 3" is the VSL ^# 3 treatment group. "*" Refers to the significant difference in the DAI index of the model group relative to the normal group of mice P <0.05, "**" refers to the significant difference in the DAI index of the model group to the normal group mice P <0.01, "▲" It means that the "probiotics" and "VSL ^# 3" groups of mice have a significant difference with respect to the model group at a level of P <0.05.

The data in Table 3 and Figure 2 show that the DAI of the normal group of mice is basically the same, and with the induction of DSS, the DAI of the model group, the probiotic group and the VSL ^# 3 group of mice gradually increases. The DAI of the model group mice began to be significant (ie * P <0.05), and the DAI of the model group mice reached the highest level on the seventh day (ie, relative to the control group ** P <0.01). The intervention of probiotics can control the increase of DAI. The DAI values of mice in the probiotic group on the 5th and 7th days were significantly controlled relative to the model group (ie, P <0.05), and, on the 7th day The DAI of the mice in the probiotic intervention group was slightly lower than VSL # 3, indicating that the effect of the probiotic composition of this example in controlling the increase of DAI in UC mice was better than VSL # 3.

3. Changes in colon length

The colon tissue of UC model mice will change, mainly due to the shortening of colon tissue due to the occurrence of ulcers and inflammation. After the treatment, the colon length of the mouse measured by anatomy is shown in Table 4.

Table 4 Mouse colon length

Group Colon length (cm) Control 8.38 ± 0.49 Model group 5.02 ± 0.87 ** VSL ^# 3 6.30 ± 0.67 ^▲ Probiotics 6.63 ± 0.71 ^▲

In Table 4, "Control" refers to the normal group, "Probiotics" is the probiotic composition treatment group, and "VSL ^# 3" is the VSL ^# 3 treatment group. "**" refers to the significant difference in colon length between the model group and the normal group. P <0.01, "▲" refers to the "probiotic" and "VSL ^# 3" groups of mice relative to the model group's colon length. Significant difference level P <0.05.

The results in Table 4 show that, after 7 days of DSS induction in mice, the shortening of colon tissue in the model group was more serious, which was very significant compared with the control group (** P <0.01). And the intervention of probiotic composition and VSL ^# 3 can significantly control the shortening of mouse colon, which is significantly controlled relative to the model group (* P <0.05). According to the data in the table, it can be found that the colon length of the mice in the probiotic composition group is longer than that of the VSL ^# 3 group mice, which can explain that the probiotic composition has a stronger ability to control colon shortening in UC mice than VSL ^# 3.

The results in Tables 2 to 4 and Figures 1 and 2 show that the probiotic composition of this example has a therapeutic and preventive effect on ulcerative enteritis, and its therapeutic effect is slightly better than existing VSL ^# 3 products.

Example two

In this example, the composition having the effect of treating and preventing ulcerative enteritis in Example 1 is made into a common food, as follows:

Milk, vitamin C and white sugar and other excipients are mixed with the cultured Lactobacillus gasseri TF08-1, Lactobacillus acidophilus AM13-1, and Bifidobacterium pseudominis TM12-14 according to the formula in Table 5 to prepare ulcerative Food for enteritis treatment and prevention.

Table 5 Food formula containing probiotic composition

raw material Mass percentage (%) Lactobacillus gasseri TF08-1 0.15 Lactobacillus acidophilus AM13-1 0.15 Bifidobacterium pseudocatenulatumTM12-14 0.15 milk 90.0 White sugar 9.0 Vitamin C 0.55

Mix the milk and sugar according to the formula ratio in Table 5, stir until completely mixed, preheat, homogenize at 20Mpa pressure, sterilize at about 90 ° C for 5-10 minutes, cool to 40-43 ° C, mix in the protective agent (ie, vitamin C), and Inoculate 1-100 × 10 ⁶ cfu / g Lactobacillus gasseri TF08-1, Lactobacillus acidophilus AM13-1, and Bifidobacterium pseudocatenulatum TM12-14 as three mixed probiotic foods combination.

The cow's milk product of this example was added to the feed of mice modeled by DSS, and fed and tested according to the probiotic composition treatment group of Example 1. The difference was that only the cow's milk of this example was added to the feed. The product does not additionally administer the probiotic composition bacterial liquid. The test results show that the milk product of this example can also control the weight loss of UC mice, reduce the disease activity index (DAI) of mice, improve the intestinal lesions, and has ulcerative enteritis treatment and prevention effects.

Example three

In this example, the probiotic composition confirmed in Example 1 to have ulcerative enteritis treatment and prevention effects is made into a medicament for treating ulcerative enteritis, and the formula is shown in Table 6:

Table 6 Pharmaceutical formula containing probiotic composition

raw material Mass percentage (%) Lactobacillus gasseri TF08-1 0.5% Lactobacillus acidophilus AM13-1 0.5% Bifidobacterium pseudocatenulatumTM12-14 0.5% lactose 2.0% yeast 2.0% Peptone 1.0% pure water 93% Vitamin C 0.5%

Mix lactose, yeast powder, and peptone with purified water according to the ratio in Table 6. Preheat to 60-65 ° C, homogenize at 20Mpa pressure, sterilize at about 90 ° C for 20-30 minutes, cool to 36-38 ° C, and mix in a protective agent. (Ie, vitamin C), access 1-50 × 10 ⁶ cfu / mL Lactobacillus gasseri TF08-1, Lactobacillus acidophilus AM13-1, and Bifidobacterium pseudocatenulatum TM12-14 The live bacteria are fermented to a pH value of 6.0 at 36-38 ° C, centrifuged, and freeze-dried to a moisture content of less than 3% to prepare a freeze-dried product of combined probiotics. Weigh 0.5 grams of freeze-dried product and mix it with an equal amount of maltodextrin and fill it in a capsule, which is made into Lactobacillus gasseri TF08-1, Lactobacillus acidophilus AM13-1, and pseudo-small chain double Capsule pharmaceutical composition of Bifidobacterium pseudocatenulatum TM12-14 three strains combined with probiotics.

The capsule pharmaceutical composition of this example was used to replace the bacterial solution of the probiotic composition in the treatment group of the probiotic composition of Example 1, and the capsule pharmaceutical composition of this example was orally administered in the same manner as in Example 1. One capsule was tested in the same way as in Example 1. The results show that the capsule pharmaceutical composition of this example can also control the weight loss of UC mice, reduce the disease activity index (DAI) of mice, improve the intestinal lesions, and has ulcerative enteritis treatment and prevention effects.

Example 4

Preparation method of medicine for treating ulcerative enteritis (UC)

1. Preparation of bacterial solution: 1 × 10 ⁹ cfu / mL Lactobacillus gasseri TF08-1, Lactobacillus acidophilus AM13-1 and Bifidobacterium pseudocatenulatum TM12-14 were subjected to anaerobic treatment, respectively. For culture, PYG medium was used as the anaerobic medium. After anaerobic fermentation at 37 ° C for 2-3 days, the mixture was mixed in equal proportions to a final concentration of about 1 × 10 ⁹ cfu / mL.

2. Preparation of growth factors: The skimmed milk and casein are mixed, centrifuged, and ultra-filtered to obtain a crude extract of milk growth factors, which contains vitamins, purines, and pyrimidines.

3. Preparation of pharmaceutical dosage forms: 5 volumes of growth factor and 1 volume of protective agent (ie, vitamin C) are added to 100 volumes of fermented bacterial liquid mixture, thoroughly stirred, and then starch auxiliary materials such as maltodextrin are added to prepare Pharmaceutical dosage form.

The pharmaceutical dosage form can be further made into pharmaceuticals such as slurry, powder or granule.

The pharmaceutical dosage form of this example was directly added to the DSS modeled mouse feed, and fed and tested according to the probiotic composition treatment group of Example 1. The difference is that this example only added this The medicinal dosage form of the example does not additionally administer the bacterial solution of the probiotic composition. The test results show that the drug product of this example can also control the weight loss of UC mice, reduce the disease activity index DAI of the mice, improve the intestinal lesions, and have ulcerative enteritis treatment and prevention effects.

The above examples demonstrate that the combined use of Lactobacillus gasseri, Lactobacillus acidophilus, and Bifidobacterium pseudoparvum can treat and prevent ulcerative enteritis; and the composition of the three bacteria can be made into various foods or drugs Use; Of course, it can be understood that since the composition of the three bacteria can be made into various foods and medicines, it can also be made into various health products or food additives.

In addition, research shows that the treatment effect of the three bacteria is largely based on the improvement of microecology, and this microecological improvement not only has therapeutic and preventive effects on ulcerative enteritis, but also other microecological-related diseases, such as general Enteritis, gastritis and the like also have effects; therefore, the composition of the present application can be used to prevent or treat inflammation or inflammation-related diseases, especially various enteritis and gastritis.

The above content is a further detailed description of the present application in combination with specific embodiments, and it cannot be considered that the specific implementation of the present application is limited to these descriptions. For those of ordinary skill in the technical field to which this application belongs, without deviating from the concept of this application, several simple deductions or replacements can be made, which should all be regarded as falling within the protection scope of this application.

Claims (25)

A composition, characterized in that the composition comprises Lactobacillus garneri and / or its metabolites, Lactobacillus acidophilus and / or its metabolites, and Bifidobacterium pseudoparvum and / or its metabolites . The composition according to claim 1, wherein the Lactobacillus gasseri is Lactobacillus gasseri TF08-1 with accession number GDMCC 60092, and the Lactobacillus acidophilus is Lactobacillus acidophilus with accession number GDMCC 60091 AM13-1, the pseudo-small-chain Bifidobacterium is pseudo-small-chain Bifidobacterium TM12-14 with the accession number GDMCC 60089. The composition according to claim 1 or 2, wherein the composition further contains other probiotics and / or prebiotics; preferably, the prebiotics are selected from the group consisting of fructooligosaccharides, galactooligosaccharides, and At least one of polyxylose, lactulose oligosaccharide, soy oligosaccharide, inulin and oligosaccharide. The composition according to claim 1 or 2, characterized in that the composition further contains the ability to help maintain the activity of at least one of Lactobacillus gasseri, Lactobacillus acidophilus, and Bifidobacterium pseudoparvum The substance. The composition according to claim 4, characterized in that the substance which helps to maintain the viability of at least one of Lactobacillus gasseri, Lactobacillus acidophilus, and Bifidobacterium pseudosmall chain is selected from the group consisting of Cystine, glutathione, butylhydroxyanisole, dibutylmethyltoluene, tocopherol, bamboo leaf antioxidants, D-isoascorbic acid or its sodium salt, sodium ascorbate, calcium ascorbate, phospholipids, vitamin C and vitamins At least one of E. The composition according to claim 1 or 2, wherein the composition further comprises a pharmaceutically or food-acceptable carrier or excipient. The composition according to claim 6, characterized in that the pharmaceutically or food-acceptable carrier or excipient is selected from the group consisting of glucose, lactose, sucrose, starch, mannitol, dextrin, fatty acid glyceride, polyethylene At least one of glycol, hydroxyethyl starch, ethylene glycol, polyoxyethylene sorbitan fatty acid ester, amino acid, gelatin, albumin, water, and physiological saline. Use of the composition according to any one of claims 1-7 in the preparation of a food, a health product, a food additive, or a medicament for treating or preventing inflammation or an inflammation-related disease. The use according to claim 8, wherein the inflammation is ulcerative enteritis. Use of the composition according to any one of claims 1 to 7 in the preparation of a food, a health product, a food additive or a medicament for controlling weight loss in a mammal. The application according to claim 10, wherein the mammal's weight loss refers to the mammal's weight loss due to inflammation; preferably, the inflammation is ulcerative enteritis. Use of the composition according to any one of claims 1 to 7 in the preparation of a food, a health product, a food additive or a medicine for reducing the disease activity index of a mammal. Use of the composition according to any one of claims 1 to 7 in the preparation of a food, a health product, a food additive, or a medicament for improving intestinal disease in mammals. A method for treating or preventing inflammation or inflammation-related diseases using the composition according to any one of claims 1-7. A method for controlling weight loss in a mammal using the composition according to any one of claims 1-7. A method for reducing the disease activity index of a mammal using the composition according to any one of claims 1-7. A method for improving intestinal lesions in mammals using the composition according to any one of claims 1-7. A food, characterized in that the food contains the composition according to any one of claims 1-7. The food according to claim 18, wherein the food is a lactic acid drink or a soy milk drink. A health-care product, characterized in that the health-care product contains the composition according to any one of claims 1-7. A food additive, characterized in that the food additive contains the composition according to any one of claims 1-7. A medicine, characterized in that the medicine contains the composition according to any one of claims 1-7. The medicine according to claim 22, wherein the medicine is a tablet, granule, powder, enteric solvent, solution or suspension. The medicine according to claim 22, wherein the medicine is an enteric solvent, and the enteric solvent is a capsule or an enteric-coated tablet; preferably, the medicine is an enteric-coated tablet. The medicine according to claim 24, wherein the thickness of the enteric coating of the enteric tablet is 5-100 μm, preferably 20-80 μm.

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