CN117866831A

CN117866831A - Lactobacillus rhamnosus and application thereof

Info

Publication number: CN117866831A
Application number: CN202410047053.2A
Authority: CN
Inventors: 吕洁; 迮晓雷; 李良
Original assignee: BY Health Co Ltd
Current assignee: BY Health Co Ltd
Priority date: 2024-01-12
Filing date: 2024-01-12
Publication date: 2024-04-12

Abstract

The present application relates to a lactobacillus rhamnosus. In particular, the present application relates to a lactobacillus rhamnosus 203-10 and compositions, cultures, food products and pharmaceutical compositions comprising the same. The application also relates to the use of lactobacillus rhamnosus 203-10 and compositions, cultures, food products and pharmaceutical compositions comprising same for the preparation of a medicament.

Description

Lactobacillus rhamnosus and application thereof

Technical Field

Background

In recent years, probiotics have received much attention to their function of protecting the intestinal barrier against damage. More and more evidence indicates that probiotics can regulate the intestinal mucosa immune system and relieve intestinal inflammation; improving intestinal microecology and recovering intestinal flora balance; the production of active metabolites has a beneficial effect on the host.

Impaired intestinal barrier or inflammatory bowel disease (inflammatory bowel diseases, IBD) is characterized by impaired intestinal epithelial barrier function and impaired intestinal immune function, which often causes recurrent abdominal pain and diarrhea, severely affecting people's quality of life, with a long-term chronic disease course, a tendency to relapse for life-long, and an increased risk of cancer at the affected parts of the intestine. Intestinal mucosal barriers include mechanical barriers, biological barriers, immune barriers, and chemical barriers. The mechanical barrier is an important component of the intestinal mucosa barrier, and is composed of Tight Junction (TJ) between intestinal mucosa epithelium, epithelial cells, mucus secreted by the epithelial cells, and bacterial membranes on the surface. The zonulin (OCLN), the claudin 1 (CLDN1), and the zonulin 1 (ZO-1) are the main proteins that constitute the tight junctions of the intestines, forming the structural basis of the intestinal barrier function on the epithelial cell-side membrane surface of the intestinal mucosa, and are important components of the intestinal barrier function. When the integrity of the intestinal epithelium is compromised, the permeability of the intestine increases and bacteria and products thereof in the intestine can penetrate the intestinal barrier into the blood, causing abnormal immune responses and activation of inflammatory cytokines, leading to inflammatory responses in the colonic mucosal tissue. The current therapeutic drugs commonly used for inflammatory bowel disease are: aminosalicylic acid preparation, glucocorticoid, immunosuppressant, biological preparation, etc. However, most of these drugs do not address the underlying problems of IBD, such as intestinal mucosa injury, impaired intestinal barrier function and unbalanced intestinal flora. In addition, long-term use of these drugs is prone to cause serious adverse reactions.

Probiotics have a higher safety profile than conventional therapeutic drugs. Therefore, the development and screening of the probiotics capable of improving the damage of intestinal barriers, relieving intestinal inflammation and promoting intestinal health have important significance and wide application prospect.

Disclosure of Invention

The inventor of the application screens a strain of lactobacillus rhamnosus with obvious strain-specific probiotic potential from 265 strains of healthy neonatal intestinal sources through a large number of experiments. The inventors of the present application confirmed through a large number of experiments that the lactobacillus rhamnosus has good tolerance to gastric acid environment and bile salts, and good adhesion to intestinal epithelial cells, and can regulate immunity, improve intestinal barrier damage and colonic inflammation, and thus completed the present invention.

Thus, in a first aspect, the present application provides a lactobacillus rhamnosus deposited with the cantonese collection of microbial strains under accession number gdmccno.61778.

In certain embodiments, the Lactobacillus rhamnosus (Lacticaseibacillus rhamnosus) has a 16S rDNA sequence that is 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, 99.95% or 100% identical to SEQ ID NO. 1.

In certain embodiments, the 16S rDNA, after amplification using the primers shown in SEQ ID NO. 2 and SEQ ID NO. 3, produces a 1442bp fragment.

In certain embodiments, the colonies of lactobacillus rhamnosus are white or somewhat transparent, and the colonies are round with smooth edges.

In certain embodiments, the lactobacillus rhamnosus is a gram-positive bacterium.

In certain embodiments, the bacterial form of the lactobacillus rhamnosus is rod-shaped or rod-shaped, and is free of spores.

In certain embodiments, the lactobacillus rhamnosus is resistant to gastric acid and/or bile salts.

In certain embodiments, the lactobacillus rhamnosus has an adhesive capacity to the intestinal tract or intestinal epithelium.

In certain embodiments, the lactobacillus rhamnosus is capable of inhibiting pathogenic bacteria.

In certain embodiments, the lactobacillus rhamnosus is capable of preventing and/or treating intestinal barrier damage.

In certain embodiments, the lactobacillus rhamnosus is capable of preventing and/or treating gut-related inflammation.

In certain embodiments, the pathogenic bacteria are selected from clostridium perfringens (Clostridium perfringens), escherichia coli (Escherichia coli), klebsiella pneumoniae (Klebsiella pneumoniae) and gardnerella (Gardnerella vaginalis).

In a second aspect, the present application provides a composition comprising the lactobacillus rhamnosus.

In certain embodiments, the lactobacillus rhamnosus may be used in combination with one or more other species of microorganisms capable of having a beneficial effect on the health of the host to which it is administered. Thus, in certain embodiments, the composition further comprises additional bacteria and/or fungi (e.g., yeast).

In certain embodiments, the additional bacteria and/or fungi are probiotics or are edible.

As used herein, the term "probiotic" is defined as any non-pathogenic bacteria that, when administered to a host in a sufficient quantity in a viable bacteria, can have a beneficial effect on the health of the host.

In certain embodiments, the additional bacteria are selected from the group consisting of lactobacillus, bifidobacterium, lactobacillus mucilaginosus, lactobacillus plantarum, lactobacillus in combination, lactobacillus guangdalina, streptococcus, lactococcus, propionibacterium, leuconostoc, pediococcus, westmann, zoococcus, staphylococcus, or any combination thereof.

In certain embodiments, the yeast is selected from the group consisting of s.parapsilosis (Brettanomyces anomalus), s.cerevisiae (Saccharomyces cerevisiae), brussels Brettanomyces (Brettanomyces bruxellensis), candida stellate (Candida stillata), schizosaccharomyces pombe (Schizosaccharomyces pombe), zygosaccharomyces bayer (Zygosaccharomyces bailii), or any combination thereof.

In certain embodiments, the yeast is kluyveromyces.

In certain embodiments, the composition further comprises additional additives.

Additional additives may be selected and adjusted as desired by those skilled in the art. In certain embodiments, the additional additive is a nutrient.

In certain embodiments, the additional additive can have a beneficial effect on the health of the host to which it is administered.

In certain embodiments, the additional additive is a nutrient selected from the group consisting of dietary fiber, prebiotic, protein, lipid, mineral, vitamin, plant extract, or any combination thereof.

In certain embodiments, the mineral is selected from the group consisting of iron, zinc, potassium, sodium, calcium, magnesium, and any combination thereof.

In certain embodiments, the vitamin is selected from the group consisting of vitamin B1, vitamin B2, vitamin B6, vitamin B12, vitamin a, vitamin C, vitamin D, vitamin E, vitamin K, and any combination thereof.

In certain embodiments, the composition is used as a starter (e.g., starter for a plant fermentation product, starter for a dairy product). In such embodiments, the lactobacillus rhamnosus in the composition participates in the fermentation process as a starter. For example, during the preparation of yogurt, lactobacillus rhamnosus is fermented as a probiotic with fresh milk to prepare yogurt.

In certain embodiments, the lactobacillus rhamnosus in the composition is at 10 ⁶ To 10 ¹² The CFU/dose amount is present (e.g. 10 ⁷ To 10 ¹⁰ CFU/dose).

In certain embodiments, the bacterium of the genus bifidobacterium is selected from the group consisting of: bifidobacterium animalis (Bifidobacterium animalis), bifidobacterium bifidum (Bifidobacterium bifidum), bifidobacterium breve (Bifidobacterium breve), bifidobacterium infantis (Bifidobacterium infantis), bifidobacterium longum (Bifidobacterium longum), bifidobacterium adolescentis (Bifidobacterium adolescentis), or any combination thereof.

In certain embodiments, the bacterium of the genus lactobacillus is selected from the group consisting of: lactobacillus paracasei (Lactobacillus paracasei), lactobacillus acidophilus (Lactobacillus acidophilus), lactobacillus brevis (Lactobacillus brevis), lactobacillus jensenii (Lactobacillus jensenii), lactobacillus inertia (Lactobacillus iners), lactobacillus casei (Lactobacillus casei), lactobacillus crispatus (Lactobacillus crispatus), lactobacillus curvatus (Lactobacillus curvatus), lactobacillus delbrueckii (Lactobacillus delbrueckii), lactobacillus fermentum (Lactobacillus fermentum), lactobacillus gasseri (Lactobacillus gasseri), lactobacillus helveticus (Lactobacillus helveticus), lactobacillus johnsonii (Lactobacillus johnsonii), lactobacillus plantarum (Lactobacillus plantarum), lactobacillus reuteri (Lactobacillus reuteri), lactobacillus rhamnosus (Lactobacillus rhamnosus), lactobacillus sake (Lactobacillus sakei), lactobacillus salivarius (Lactobacillus salivarius), or any combination thereof.

In certain embodiments, the bacteria of the genus bacillus are selected from the group consisting of: bacillus subtilis (Bacillus subtilis), bacillus coagulans (Bacillus coagulans), or any combination thereof.

In certain embodiments, the bacteria of the genus propionibacterium are selected from the group consisting of: propionibacterium scheelite (Propionibacterium shermanii), propionibacterium freudenreichii (Propionibacterium freudenreichii), propionibacterium propionicum (Propionibacterium acidipropionici), or any combination thereof.

In certain embodiments, the bacteria of the streptococcus genus are selected from: streptococcus thermophilus (Streptococcus thermophilus), streptococcus salivarius (Streptococcus salivarius), or any combination thereof.

In certain embodiments, the bacterium of the genus lactococcus is lactococcus lactis (Lactococcus lactis).

In certain embodiments, the bacterium of the enterococcus genus is selected from the group consisting of: enterococcus faecalis (Enterococcus faecalis), enterococcus faecium (Enterococcus faecium), or any combination thereof.

In a third aspect, the present application provides a food product comprising lactobacillus rhamnosus as described above or a composition as described above.

In the context of this document, the term "food" is used in a broad sense to include human foods and drinks, as well as animal foods and drinks (i.e., feeds). In certain embodiments, the food product is suitable and designed for human consumption.

It is understood that the food products of the present application may be in liquid, solid or semi-solid form, depending on the application, mode of application or mode of administration.

Thus, in certain embodiments, the food product is a solid food product (e.g., soft candy, troche, capsule, powder) a liquid food product (e.g., drink), or a semi-solid food product (e.g., jelly).

In certain embodiments, the food product is a dietary supplement, a nutritional formulation, a functional food or a beverage product.

In certain embodiments, the food product is a dairy product (e.g., yogurt, flavored fermented milk, lactobacillus beverage, cheese).

In certain embodiments, the food product is formulated for oral administration.

In certain embodiments, the food product is in the form of a pill, powder, capsule, tablet, granule, caplet, sachet, or dragee.

In certain embodiments, the food product further comprises a prebiotic. Generally, prebiotics are non-digestible in that they are not disintegrated and absorbed in the stomach or small intestine, and thus they remain intact as they pass through the stomach and small intestine to the colon. Examples of prebiotics include certain oligosaccharides such as Fructooligosaccharides (FOS), inulin, xylo-oligosaccharides (XOS), polydextrose or any mixtures thereof.

In certain embodiments, the food product may further include (but is not limited to) one or any combination of the following: probiotics (e.g. probiotic bacteria), dietary fibres, proteins (e.g. enzymes), carbohydrates, lipid substances (e.g. fats), vitamins, minerals, plant components (e.g. plant extracts), amino acids, immunomodulators, milk substitutes, or metabolites or extracts of lactobacillus rhamnosus.

In certain embodiments, the food products used in the present invention may comprise a protein source, such as an animal protein (e.g., milk, meat, or egg protein), a vegetable protein (e.g., soy, wheat, rice, or pea protein); a mixture of free amino acids; or a combination thereof.

In certain embodiments, the food products used in the present invention may comprise a source of fat, and the lipid comprising the source of fat may be any suitable fat or fat mixture. Vegetable fats such as soybean oil, palm oil, coconut oil, safflower oil, sunflower oil, corn oil, canola oil and lecithin. Animal fat, such as milk fat, may also be added if desired.

In certain embodiments, the food products useful in the present invention may comprise carbohydrates such as sucrose, lactose, glucose, fructose, corn syrup solids, maltodextrin, and mixtures thereof.

In certain embodiments, the food products used in the present invention may comprise dietary fiber. The dietary fiber may be from any suitable source including, for example, soy, pea, oat, pectin, guar gum, gum arabic, fructooligosaccharides, galactooligosaccharides, sialyllactose, and oligosaccharides derived from animal milks.

In certain embodiments, the food products useful in the present invention may also be suitable vitamins and minerals that may be included in the food product in suitable amounts.

In certain embodiments, the lactobacillus rhamnosus of the present invention may also be combined with various food acceptable adjuvants such as sweeteners or flavoring agents, color matching substances, stabilizers, glidants, bulking agents, and the like.

In certain embodiments, the lactobacillus rhamnosus is present in a concentrate form.

In certain embodiments, the lactobacillus rhamnosus is present in the food product at 10 ⁶ To 10 ¹² The CFU/dose amount is present (e.g. 10 ⁷ To 10 ¹⁰ CFU/dose).

In a fourth aspect, the present application provides a pharmaceutical composition comprising lactobacillus rhamnosus as described above or a composition as described above.

As used herein, the term "medicament" encompasses medicaments for humans as well as medicaments for animals (i.e., veterinary applications). In certain embodiments, the medicament is for use in humans.

In certain embodiments, the pharmaceutical composition comprises lactobacillus rhamnosus or a formulation of the composition.

In certain embodiments, the pharmaceutical composition comprises a pharmaceutically acceptable carrier.

In certain embodiments, the pharmaceutical composition is formulated for oral administration.

In certain embodiments, the pharmaceutical composition is in the form of a pill, powder, capsule, tablet, granule, caplet, cream, ointment, gel, lotion, foam, suppository, sachet, or dragee.

In certain embodiments, the lactobacillus rhamnosus is present in the food product at 10 ⁶ To 10 ¹² The CFU/dose amount is present (e.g. 10 ⁸ To 10 ¹⁰ CFU/dose).

In a fifth aspect, the present application provides a culture comprising lactobacillus rhamnosus as described above or a composition as described above.

In certain embodiments, the culture is a bacterial suspension of lactobacillus rhamnosus.

In certain embodiments, the culture further comprises a nutrient providing component (e.g., a solid or liquid medium, a feeder cell layer).

In certain embodiments, the nutrient providing ingredient is selected from the group consisting of a protein, a carbohydrate, a fat, a probiotic, an enzyme, a vitamin, an immunomodulator, a milk substitute, a mineral, an amino acid, or any combination thereof.

In certain embodiments, the culture further comprises a cell-free culture filtrate of lactobacillus rhamnosus.

In certain embodiments, the culture further comprises a derivative of lactobacillus rhamnosus.

In certain embodiments, the derivative is selected from a metabolite, an enzyme, a cellular structural component (e.g., a cell wall or component thereof), an extracellular polysaccharide, a bacteriocin, a compound containing an immunogenic component, or any combination thereof.

In a sixth aspect, the present application provides the use of a lactobacillus rhamnosus as described above or a composition as described above or a food product as described above or a pharmaceutical composition as described above or a culture as described above in the manufacture of a medicament capable of inhibiting a pathogenic bacterial infection or preventing and/or treating a disease and/or condition caused by a pathogenic bacterial infection.

In a further aspect, the present invention provides a method of inhibiting a pathogenic bacterial infection, or preventing and/or treating a disease and/or symptom caused by a pathogenic bacterial infection, the method comprising administering to a subject in need thereof an effective amount of lactobacillus rhamnosus or a composition as described above or a food as described above or a pharmaceutical composition as described above or a culture as described above.

In certain embodiments, the pathogenic bacteria are pathogenic bacteria.

In certain embodiments, the pathogenic bacteria are selected from the group consisting of clostridium, escherichia, klebsiella, gardnerella, neisseria, or any combination thereof.

In certain embodiments, the pathogenic bacteria are selected from clostridium perfringens (Clostridium perfringens), escherichia coli (Escherichia coli), klebsiella pneumoniae (Klebsiella pneumoniae), gardnerella (Gardnerella vaginalis), or any combination thereof.

In certain embodiments, the disease is selected from gastroenteritis, urinary tract infection, vaginitis, cervicitis, urethritis, urinary tract infection, external genital infections in men and/or women, sepsis, bacteremia, bacterial cold, meningitis, peritonitis, or any combination thereof.

In certain embodiments, the symptom is selected from chills, diarrhea, fever, cough, vomiting, or any combination thereof.

In a seventh aspect, the present application provides the use of lactobacillus rhamnosus as described above or a composition as described above or a food product as described above or a pharmaceutical composition as described above or a culture as described above for the manufacture of a medicament for inhibiting inflammation or preventing and/or treating an inflammatory disease in a subject.

In another aspect, the invention provides a method of inhibiting inflammation or alleviating inflammatory disease comprising administering to a subject in need thereof an effective amount of lactobacillus rhamnosus or a composition as described above or a food product as described above or a pharmaceutical composition as described above or a culture as described above.

In certain embodiments, the inflammatory disease is selected from diseases caused by retinal inflammation (e.g., retinitis, keratitis), diseases caused by skin inflammation (e.g., dermatitis, eczema), related diseases caused by respiratory inflammation (e.g., upper respiratory tract infection), and related diseases caused by digestive tract inflammation (e.g., inflammatory gastrointestinal disease).

In certain embodiments, the inflammatory gastrointestinal disease is selected from: inflammatory-related gastrointestinal disorders, gastrointestinal-related inflammation, intestinal barrier dysfunction and intestinal barrier damage.

In certain embodiments, the inflammatory-related gastrointestinal disorder is selected from the group consisting of non-ulcerative colitis, chronic bowel disease, crohn's disease, colonodulitis, and food-responsive diarrhea disease.

In certain embodiments, the medicament is capable of improving the barrier structure, repairing the intestinal barrier and/or improving the barrier function of the intestinal tract.

In certain embodiments, the inflammatory gastrointestinal disease is modulated by a pro-inflammatory cytokine and/or an anti-inflammatory cytokine.

In certain embodiments, the agent is capable of reducing the expression level of a proinflammatory cytokine (e.g., TNF- α, IL-1β, IL-6, IL-8, IL-12, TGF- β1, NF-kB) and/or increasing the expression level of an anti-inflammatory cytokine (e.g., IL-10).

In certain embodiments, the medicament is used alone or in combination with other antifungal agents, antiviral agents, analgesic agents, anti-inflammatory agents, healing promoting agents, or moisturizers.

In certain embodiments, the subject is a mammal.

In certain embodiments, the subject is a human.

In an eighth aspect, the invention provides the use of a lactobacillus rhamnosus as described above or a composition as described above in the preparation of a starter culture for use in the fermentation of a solid food (e.g. cheese, bread) or a liquid food (e.g. yoghurt, flavoured milk, lactic acid bacteria beverages).

Definition of terms

In the present invention, unless otherwise indicated, scientific and technical terms used herein have the meanings commonly understood by one of ordinary skill in the art. Meanwhile, in order to better understand the present invention, definitions and explanations of related terms are provided below.

As used herein, the term "repair intestinal barrier" includes, but is not limited to, restoration of the integrity of the gastrointestinal barrier. Specifically, the method comprises repairing a damaged barrier of the gastrointestinal tract and repairing gastrointestinal mucosa.

As used herein, the term "improving the barrier structure of the intestinal tract" includes, but is not limited to, strengthening the gastrointestinal barrier, repairing the integrity of the gastrointestinal barrier and tightly-coupled structures, repairing the integrity of the intestinal epithelial lining.

As used herein, the term "improving barrier function of the intestinal tract" includes, but is not limited to, improvement of gastrointestinal barrier resistance, reduction of gastrointestinal barrier permeability (e.g., reduction of translocation of pathogens from the luminal site to the mucosa, reduction of translocation of commensal bacteria from the luminal site to the mucosa, reduction of penetration of pathogens from the luminal site to the mucosa, reduction of translocation of toxic compounds from the luminal site to the mucosa, and reduction of disease susceptibility).

As used herein, the term "pharmaceutically acceptable carrier" refers to a carrier that is pharmacologically and/or physiologically compatible with the subject and active ingredient, is well known in the art (see, e.g., remington's Pharmaceutical sciences. Mediated by Gennaro AR,19th ed.Pennsylvania:Mack Publishing Company,1995), and includes, but is not limited to: pH adjusters, surfactants, adjuvants, ionic strength enhancers. For example, pH modifiers include, but are not limited to, phosphate buffers; surfactants include, but are not limited to, cationic, anionic or nonionic surfactants, such as Tween-80; ionic strength enhancers include, but are not limited to, sodium chloride.

As used herein, the term "dietary supplement" refers to an edible product that is capable of providing a benefit (e.g., a nutritional effect, a prophylactic effect, a therapeutic effect, or other benefit) to a consumer. Dietary supplements in this context encompass products such as nutraceuticals, supplements, and the like.

As used herein, the term "drug" encompasses drugs in human and veterinary medicine for use by both humans and animals, as well as drugs for incorporation into animal feed (e.g., livestock feed and/or pet food). Furthermore, the term "drug" as used herein means any substance that provides a therapeutic, prophylactic and/or beneficial effect. The term "medicament" as used herein is not necessarily limited to substances requiring a marketing license (Marketing Approval) but includes substances that may be used in cosmetics, health products, foods (including, for example, feeds and beverages), probiotic cultures and dietary supplements.

As used herein, the term "CFU (color-Forming Units)" refers to the total population of microorganisms such as bacteria, fungi, yeast, etc., in a product, typically used as a viable count calculation.

As used herein, the term "CFU/dose" means the amount of bacteria present in a composition/food/pharmaceutical composition provided to a subject daily or each time. For example, in certain embodiments, the lactobacillus rhamnosus is present in the food product at 10 ⁶ To 10 ¹² The CFU/dose amount is present (e.g. 10 ⁷ To 10 ¹⁰ CFU/dose). In such embodiments, if the lactobacillus rhamnosus is administered in a food product (e.g., in a solid beverage, yogurt), the food product provided to the subject daily or each time (e.g., solid beverage, yogurt) may contain about 10 ⁶ To 10 ¹² CFU of lactobacillus rhamnosus. Of course, alternatively, the amount of such bacteria may be divided into multiple administrations, provided that the subject receives the total amount of lactobacillus rhamnosus at any particular time (e.g. every 24 hours)Is from about 10 ⁶ To about 10 ¹² Bacteria of CFU, i.e. lactobacillus rhamnosus satisfying the above-mentioned food products or dietary supplements, are present in an amount of 10 ⁶ To 10 ¹² The CFU/dose amount is present (e.g. 10 ⁷ To 10 ¹⁰ CFU/dose).

Advantageous effects of the invention

Compared with the lactobacillus rhamnosus in the prior art, the lactobacillus rhamnosus has at least the following beneficial effects: (1) Has good tolerance to gastric acid environment and bile salt; (2) good adhesion to the intestinal tract; (3) capable of inhibiting pathogenic bacteria; (4) Can increase the expression of intestinal claudin and prevent and/or improve the damage of intestinal epithelial barrier; (5) Can remarkably improve the expression of anti-inflammatory factor IL-10 and inhibit the expression of pro-inflammatory factor, and can inhibit or improve colon inflammation and regulate immunity.

Embodiments of the present invention will be described in detail below with reference to the accompanying drawings and examples, but it will be understood by those skilled in the art that the following drawings and examples are only for illustrating the present invention and are not to be construed as limiting the scope of the present invention. Various objects and advantageous aspects of the present invention will become apparent to those skilled in the art from the following detailed description of the preferred embodiments and the accompanying drawings.

Drawings

Figure 1 shows the results of gram staining and microscopic observation of lactobacillus rhamnosus.

FIG. 2 shows the morphology of colonies of Lactobacillus rhamnosus cultured on plates.

FIG. 3 shows the effect of different treatments on the single layer epithelial barrier TEER of a colon organoid; wherein NC is a blank control group, IFN-gamma is a disease model group, L.R 203-10 is a lactobacillus rhamnosus 203-10 intervention group.

FIG. 4 shows the effect of different treatments on the permeability of the colonic organoid monolayer epithelial barrier FITC; wherein NC is a blank control group, IFN-gamma is a disease model group, L.R 203-10 is a lactobacillus rhamnosus 203-10 intervention group. And (3) injection: * Indicating significant differences compared to the blank, P <0.05; # indicates significant differences compared to the model group, P <0.05.

FIG. 5 shows the relative expression levels of intestinal claudin after treatment of different groups; wherein NC is a blank control group, IFN-gamma is a disease model group, L.R 203-10 is a lactobacillus rhamnosus 203-10 intervention group. And (3) injection: * Indicating significant differences compared to the blank, P <0.05; # indicates significant differences compared to the model group, P <0.05.

FIG. 6 shows the relative expression levels of cell inflammation-associated factors after treatment in different groups; wherein NC is a blank control group, IFN-gamma is a disease model group, L.R 203-10 is a lactobacillus rhamnosus 203-10 intervention group. And (3) injection: * Indicating significant differences compared to the blank, P <0.05; # indicates significant differences compared to the model group, P <0.05.

Sequence information

The information of the partial sequences to which the present invention relates is provided in table 1 below.

Table 1: description of the sequence

Description of preservation of biological Material

Lactobacillus rhamnosus 203-10 (Lacticaseibacillus rhamnosus 203-10) has been deposited with the guangdong province microorganism strain collection (GDMCC, guangdong Microbial Culture Collection Center) located in building 5 of institute 100, first middle road, guangzhou city, with deposit number GDMCC No.61778 and with a deposit time of 2021, month 7, and day 5.

Detailed Description

The invention will now be described with reference to the following examples, which are intended to illustrate the invention, but not to limit it.

The experiments and methods described in the examples were performed substantially in accordance with conventional methods well known in the art and described in various references unless specifically indicated. For example, for the conventional techniques of immunology, biochemistry, chemistry, molecular biology, microbiology, cell biology, genomics and recombinant DNA used in the present invention, reference may be made to Sambrook (Sambrook), friech (Fritsch) and manitis (Maniatis), molecular cloning: laboratory Manual (MOLECULAR CLONING: A LABORATORY MANUAL), edit 2 (1989); the handbook of contemporary molecular biology (CURRENT PROTOCOLS IN MOLECULAR BIOLOGY) (edited by f.m. ausubel (f.m. ausubel) et al, (1987)); series (academic publishing company) of methods in enzymology (METHODS IN ENZYMOLOGY): PCR 2: practical methods (PCR 2:A PRACTICAL APPROACH) (M.J. MaxFrson (M.J. MacPherson), B.D. Hemsl (B.D. Hames) and G.R. Taylor (G.R. Taylor) editions (1995)), and animal cell CULTURE (ANIMAL CELL CULTURE) (R.I. French Lei Xieni (R.I. Freshney) editions (1987)).

In addition, the specific conditions are not specified in the examples, and the process is carried out according to conventional conditions or conditions recommended by the manufacturer. The reagents or apparatus used were conventional products commercially available without the manufacturer's attention. Those skilled in the art will appreciate that the examples describe the invention by way of example and are not intended to limit the scope of the invention as claimed. All publications and other references mentioned herein are incorporated by reference in their entirety.

Example 1 isolation and identification of strains

The 265 strain is separated from a normal term neonatal excrement sample born by a Hospital of child-birth university Hua Xifu, and a strain of lactobacillus rhamnosus is screened from the strain, and is named as lactobacillus rhamnosus 203-10. Among these, the inclusion criteria for newborns are: residing in five urban areas of Chengdu city; the birth weight is 2500-4000g after 37-42 weeks, and the infant has no congenital abnormality or birth defect. The exclusion criteria were: the mother used antibiotics within one month before birth; parents have infectious diseases such as AIDS, tuberculosis, hepatitis B and the like; the neonate cannot collect the feces due to severe diseases such as neonatal pneumonia.

Collecting fresh feces within 1-4 months after birth of the infant is convenient for the aseptic feces collection tube. Immediately after sampling, the sample is temporarily stored at 4 ℃, and then sent to a laboratory by a sampling person at a low temperature to dilute and culture the fecal sample, if the sample cannot be immediately operated, the sample is stored at 4 ℃ anaerobically, and the sample is cultured on the same day. 0.5g of feces was weighed, 4.5ml of feces dilution (4.5 g KH2PO4, 6.0g Na2HPO4, 0.5g g L cysteine hydrochloride, 0.5g Tween-80, 1.0g agar were mixed in 1000ml distilled water, autoclaved at 121℃for 15min for use), thoroughly shaking and mixing, 10-fold serial dilution. 100. Mu.l of a fecal mixture of a suitable dilution was applied to lactobacillus selective agar (LBS selective medium 84.0g, lab-lemco powder (Oxoid) 8.0g, na.3H2O 15.0g, pure water 1000ml, acetic acid 3.7 ml) by surface coating with an L-shaped rod, and anaerobically cultured at 37℃for 48 hours. The suspected colony of lactobacillus (generally white or slightly transparent, round, smooth-edged) on the LBS plate was picked and subcultured to MRS medium (beijing land bridge) and cultured with oxygen at 37 ℃ for 48h.

Gram staining and microscopic observation were performed on energy-consuming oxygen grown strains on MRS, and the results are shown in FIG. 1. The isolated strain was initially determined to be lactobacillus based on the oxygen demand of the bacteria (facultative anaerobic) and the bacterial morphology (gram positive, diverse bacilli: long or slender rods, bent short rods, sharp corners, generally in a chain arrangement, no spores). The culture pattern on the plate is shown in FIG. 2.

After resuscitating and purifying the separated strain, the separated lactobacillus is identified as lactobacillus rhamnosus by adopting API 50CH and 16SrDNA sequencing of Mei Liai. Wherein, the amplification primers of the 16SrDNA are shown as SEQ ID NO. 2 and 3, and the sequencing result of the 16S rDNA is shown as SEQ ID NO. 1.

Further, whole genome measurement was performed on Lactobacillus rhamnosus 203-10 using PacBIO sequence 2 sequencing platform, and its genome length was 2,946,142bp, GC contained 46.61% respectively (where GC content means the ratio of guanine and cytosine among 4 bases of DNA), and the chromosome genome contained 2,873 coding genes, 54 tRNA genes, and 7 rRNA genes (including 5s rRNA, 16s rRNA and 23s rRNA).

In combination with the experimental results of PCR identification and whole genome sequencing, a brand new strain of Lactobacillus rhamnosus 203-10 is obtained and preserved in 2021 at 7 and 5 days.

EXAMPLE 2 probiotic property study of Lactobacillus rhamnosus 203-10

1. Acid-resistant and bile-salt-resistant experimental screening of Lactobacillus rhamnosus 203-10

1.1 preparation of test bacterial suspension: the lactobacillus rhamnosus 203-10 is resuscitated by MRS culture medium and passaged twice for later use. After MRS culture for 48 hours, bacteria are scraped into 2ml of excrement diluent, and the bacteria are uniformly mixed by vortex vibration, and the concentration of the bacteria is roughly regulated to 10 ⁸ -10 ⁹ CFU/ml。

1.2 preparation of simulated gastric acid and bile salt culture solution

Preparation of simulated gastric acid culture solution: preparing MRS broth culture solution, sterilizing, and regulating pH to 3.0 with 1mol/LHCL to obtain simulated gastric acid culture solution.

Preparation of simulated bile salt culture solution: preparing MRS broth culture solution, sterilizing, adding ox gall salt to a concentration of 0.1%, adjusting pH to 8.0 with 1mol/L HCL, and filtering with 0.22 μm microporous membrane for sterilization to obtain gall salt culture solution.

1.3 gastric acid and bile salt tolerance test

The strains involved in this experiment were all from a Shang Chen-fold healthy own strain library, which were isolated from normal term neonatal stool samples born at the university of Sichuan Hua Xifu birth Hospital, for specific isolation and identification methods, as described in example 1. The rhamnose strain comprises lactobacillus rhamnosus 203-10, lactobacillus rhamnosus 203-13 and lactobacillus rhamnosus 203-15.

Acid resistance test of lactobacillus rhamnosus: 0.9ml of simulated gastric acid culture solution is split into EP tubes, 0.1ml of the prepared bacterial suspension is added, the bacterial suspension is uniformly mixed by vortex, and then the bacterial suspension is placed in an anaerobic incubator at 37 ℃ for 2 hours. The tests were performed at 0h and 2h, respectively, with 3 strains set in parallel at each time point. The growth of the bacteria was observed with an unadjusted pH MRS broth (ph=7) control, excluding bacterial death due to other factors.

Lactobacillus rhamnosus bile salt tolerance test: 0.9ml of bile salt culture solution is split into EP tubes, 0.1ml of the prepared bacterial suspension is added, the bacterial suspension is uniformly mixed by vortex, and then the bacterial suspension is placed in an anaerobic incubator at 37 ℃ for culturing for 24 hours. The tests were performed at 0h and 24h, respectively, with 3 strains set in parallel at each time point. The growth of the bacteria was observed with control MRS broth without bile salts (ph=7), excluding bacterial death due to other factors.

Immediately after the culture is finished, 10 times of gradient dilution is carried out, a suitable dilution dibbling MRS plate is selected, and anaerobic culture is carried out for 24-48 hours at 37 ℃ for counting. Initial bacterial concentration was obtained as a 0 hour dilution inoculation count. The results were compared in terms of survival. The calculation formula is as follows:

survival (%) = viable bacteria concentration after culture (CFU/ml)/viable bacteria concentration after 0 hours (CFU/ml) ×100%

The experimental results are shown in Table 2.

TABLE 2 tolerance of rhamnose Strain to gastric acid and bile salts

The experimental results show that: among the above-mentioned rhamnose strains, lactobacillus rhamnosus 203-10 has the highest comprehensive tolerance in gastric acid and bile salts, and has a survival rate of 87.50% after digestion for 2 hours in simulated gastric acid culture solution with ph=3, which is 1.6 times that of the other two strains; the survival rate after 24 hours of culture in bile salt culture solution is 79.78% which is 1.8 times of that of other two strains. Therefore, the lactobacillus rhamnosus 203-10 has good tolerance to gastric acid and bile salts, and can effectively resist the extreme environment of the upstream digestive tract, so that the lactobacillus rhamnosus can smoothly reach the downstream digestive tract, such as the colon, and perform a health function. The lactobacillus rhamnosus 203-10 thus fulfils the basic requirements as a probiotic.

2. Adhesion capability of Lactobacillus rhamnosus 203-10 to intestinal epithelial organoids

2.1 isolated culture of colon organoids

Colonic organoid origin: colon biopsy samples from healthy adults were isolated from a first affiliated hospital at university of Zhejiang.

Resuscitation of colon organoids: the colonocytes were thawed in a 37℃water bath for about 2min and the colonocytes in the frozen vials were transferred with pre-moistened tips into 15ml centrifuge tubes containing pre-warmed rinse (1% BSA in DMEM/F-12). Centrifuging at 2-8deg.C for 5min with 300g, and discarding supernatant. Adding colon organoid complete medium and Matrigel in a ratio of 1:2, slowly mixing on ice, inoculating organoid suspension into a preheated 24-well plate according to 50 μl/well, placing into an incubator for solidification for 30min, adding 500 μl of colon organoid growth medium into each well for culture after Matrigel solidification, and performing three complete liquid exchange every week, and passaging every 5-10 days.

Colonic organoid passaging: mild cell dissociation reagent (GCDR) was added to the plates at 500. Mu.l/well, incubated at room temperature (15-25 ℃) for 1min, gently swirled up and down using a pre-wetted tip of GCDR, and the swirled Dome was collected in a 15ml centrifuge tube. The tube was placed on a shaker at 20rpm and incubated at room temperature (15-25 ℃) for 10min. Centrifuge at 300g at 2-8deg.C for 5min and carefully remove the supernatant. The mixture was resuspended in pre-chilled DMEM/F-12 containing 1% BSA+0.1% Y-27632 and gently blown. Further centrifuging at 2-8deg.C for 5min with 300g, and carefully removing supernatant. The organoid suspension was inoculated into a pre-heated 24-well plate at 50. Mu.l/well, placed in an incubator for 30min of clotting, and after Matrigel clotting, 500. Mu.l of colonic organoid growth medium was added to each well for culturing, three complete exchanges of fluid were performed per week, and passaging was performed every 5-10 days.

2.2 construction of a Single layer epithelial model of a colonocoid

(1) And (3) coating a culture dish: the Matrigel was diluted with D-PBS (1:49), the diluted Matrigel was added to the 24-well plate at 200ml/well, the dish was slowly tilted to bring the excess Matrigel solution to the edge of the well plate, and the excess Matrigel was aspirated.

(2) InoculatingPi Shanceng: colonocoids well grown (approximately 100-150) were harvested and GCDR was added to 24-well plates at 1ml/well and incubated for 1min at room temperature. The organoids were transferred to 15ml centrifuge tubes and gently blown multiple times until the organoids were resolved into single cells or small organoid fragments. 1ml of DMEM/F-12 was added, and after stirring, the chips were centrifuged at 200g at 2-8℃for 5min. The supernatant was discarded and resuspended in monolayer growth medium. Adding cell suspension into the well, and adding 5% CO at 37deg.C ₂ Culturing in the environment. And (3) completely changing the culture medium every 2-3 days, changing the culture medium into a differentiation culture medium after the monolayers are completely combined, and continuously culturing for 2-3 days, wherein the culture medium is used for subsequent experiments after differentiation is completed.

2.3 colonocnoid adhesion experiments

The lactobacillus rhamnosus referred to in this experiment was derived from a Shang Chen-fold healthy own strain library isolated from normal term neonatal stool samples born at the birth Hospital of Sichuan university Hua Xifu, for specific isolation and identification methods as described in example 1. The lactobacillus rhamnosus LGG is a strain commercially purchased from the ministerial company of Beijian stock.

To the washed colonochemical monolayer was added 1ml of a Lactobacillus rhamnosus suspension (1X 10) ⁹ CFU/ml) or an equivalent amount of DMEM solution, incubated at 37 ℃ for 2h. The monolayers were then washed three times with sterile PBS to remove surface unbound bacteria. 1ml of sterile PBS was added to the plates and the adherent cells were resuspended to make a bacterial suspension. The commercial strain Lactobacillus rhamnosus LGG is used as a control strain by taking a DMEM solution as a blank control, the bacterial suspension is subjected to gradient dilution by using sterile D-PBS, and is respectively coated on MRS and RCM agar culture media, 3 repeats are arranged on each gradient, the culture is carried out for 24 hours at 37 ℃, and the total bacterial count in the cell suspension is calculated according to the plate counting result.

The adhesion rate was calculated as follows:

adhesion Rate (bacteria/cell) =number of adhered lactic acid bacteria per well (bacteria)/total number of cells per well×100%

TABLE 3 results of Lactobacillus rhamnosus adhesion experiments

The results indicate that the adhesion rate of lactobacillus rhamnosus 203-10 is slightly higher than that of LGG, indicating that lactobacillus rhamnosus 203-10 has similar intestinal epithelial adhesion capacity to the commercial strain lactobacillus rhamnosus LGG, and that its adhesion is significantly higher than that of other lactobacillus rhamnosus strains of the same sample source.

Example 3 inhibition of different pathogenic bacteria by Lactobacillus rhamnosus 203-10

The present example uses a paper sheet diffusion method to evaluate the inhibition of common pathogenic bacteria by Lactobacillus rhamnosus, such as Clostridium perfringens (Clostridium perfringens, GDMCC No. 1.307), invasive E.coli (Escherichia coli, GDMCC No. 1.2987), klebsiella pneumoniae (Klebsiella pneumoniae, GDMCC No. 1.279) and Gardnerella (Gardnerella vaginalis, GDMCC No. 1.1347), infection of which may cause intestinal inflammation, genital tract inflammation, etc. Pathogenic strains according to this example were purchased from China center for type culture Collection of Industrial microorganisms.

Lactobacillus rhamnosus is inoculated in an MRS culture medium, cultured for 24 hours under anaerobic environment, bacterial fermentation supernatant is collected by centrifugation (6000 g,10 min), and is subjected to filtration treatment by a filter membrane with the diameter of 0.22 mu m, and a blank antibacterial paper sheet with the diameter of 6mm is soaked in the bacterial fermentation supernatant for 2 hours. Diluting pathogenic bacteria to 1×10 with corresponding culture medium ⁷ ～5×10 ⁷ And (3) CFU/mL, uniformly coating 0.2mL of pathogenic bacteria suspension on a corresponding flat plate, sticking a bacteriostatic paper sheet on the surface of the flat plate, setting a blank control group, culturing for 24 hours at 37 ℃, and observing and recording the size of a bacteriostatic ring. The sheet diameter was 6mm, the data were expressed as mean ± SEM, and n represents the number of sheets.

TABLE 4 inhibition of different pathogenic bacteria by Lactobacillus rhamnosus culture supernatant

The antibacterial experiment shows that the lactobacillus rhamnosus 203-10 has a good inhibition effect on clostridium perfringens and gardnerella, has a certain inhibition effect on invasive escherichia coli and klebsiella pneumoniae, and the antibacterial effect of the lactobacillus rhamnosus 203-10 is better than that of other lactobacillus rhamnosus from the same sample source.

EXAMPLE 4 protection of the intestinal barrier of the colon organoids by Lactobacillus rhamnosus 203-10

This example uses IFN-gamma induced colonocoid enteritis model to evaluate the functional effect of Lactobacillus rhamnosus 203-10 on intestinal barrier.

3.1 colonic organoid monolayer epithelial barrier transmembrane resistance (TEER) and Fluorescein Isothiocyanate (FITC) permeabilities Measurement

A colonic monolayer epithelial barrier model was constructed in a Transwell chamber (24-well, 0.22 μm) following the procedure of 2.2 in example 2, and was induced to produce inflammation and barrier damage using 200ng/mL IFN-gamma.

The constructed colon monolayer epithelial barrier model was subjected to a transmembrane resistance (TEER) assay when the colon epithelial monolayer TEER stabilized at 200-300 Ω cm ² Representing that a colonic epithelial monolayer has formed, replacing the differentiation medium for further culturing for 2-3 days, and the monolayer epithelial model after complete differentiation can be used for subsequent experiments. The TEER calculation formula is as follows:

TEER(Ω·cm ² ) = (each hole resistance value-blank hole resistance value) ×membrane area

The experiments were divided into 3 groups:

(1) Blank control (NC): TEER values were measured at three fixed locations per day for each well, and each well was repeated three times to obtain an average.

(2) IFN-gamma model group (IFN-gamma): DMEM/F-12 containing 200ng/mL IFN-. Gamma.was added and treated for 24 hours. TEER values were measured every 4h, three replicates were set for each group, three replicates were measured for each well, and the mean was taken.

(3) Lactobacillus rhamnosus 203-10 intervention group (L.R 203-10): fresh DMEM/F-12 containing 5% (v/v) of the Lactobacillus rhamnosus 203-10 fermentation supernatant was added to the lower chamber after monolayer formation for pretreatment for 6h. The upper chamber broth was replaced with fresh DMEM/F-12. After the end of the treatment the medium was removed and washed 3 times with D-PBS. TEER values were measured every 4h, three replicates were set for each group, three replicates were measured for each well, and the mean was taken.

FITC permeability test: after TEER detection, the monolayers were washed 3 times with D-PBS and equilibrated at 37℃for 30min. The cells were transferred to a new well plate and 200. Mu.L of 1mg/mL Fluorescein Isothiocyanate (FITC) solution pre-heated to 37℃was added to the upper chamber. 600. Mu.L of PBS is added into the lower chamber, the mixture is incubated for 6 hours at 37 ℃ in a dark place, 100. Mu.L of lower chamber solution is taken to set the excitation wavelength of 490nm, the fluorescence intensity is detected at the emission wavelength of 520nm, and the concentration of FITC is calculated according to standard curve.

The experimental results are shown in fig. 3 and 4. The results show that IFN-gamma induction significantly reduces the TEER value of the monolayer epithelium barrier model of the colon organoid, greatly increases the permeability of the barrier to FITC, indicating that the induction causes barrier damage to the monolayer epithelium of the colon organoid. The treated group of lactobacillus rhamnosus 203-10 fermentation supernatants significantly increased TEER values of the colonic organoid monolayer epithelial barrier model and greatly improved the barrier permeability to FITC, which suggests that lactobacillus rhamnosus 203-10 may prevent IFN- γ induced intestinal barrier damage.

3.2 expression of intestinal claudin and cell inflammation-related factor

thepacketsofthisexample3.1wereprocessedandRNAextractedforreal-timefluorescentquantitativePCR(qPCR)experimentstodeterminetherelativeexpressionofthefibronectinincludingZO-1,E-cadherin,MUC2,Occludin,Claudin-1,Claudin-3,JAM-A.

The specific operation steps are as follows:

(1) Extraction of total RNA:

(1) cleavage of colon organoids with Trizol

(2) The suspension was transferred to a fresh EP tube, 200. Mu.L of chloroform was added thereto, and the mixture was placed on a vortex mixer for shaking and mixing, and placed on ice for 10min. Thereby isolating RNA.

(3) After the end of the standing, the EP tube was transferred to a table refrigerated centrifuge pre-cooled to 4℃and centrifuged at 12000rpm at 4℃for 15min. The end of centrifugation was seen to separate the solution significantly into three layers, with the RNA layer on top.

(4) Carefully aspirate the top solution, transfer to a set of previously numbered 1.5mL RNase-freeEP tubes, add an equal volume of isopropanol solution, mix well and rest for 15min. Thereby removing moisture from the RNA.

(5) After completion of the standing, the mixture was centrifuged at 12000rpm at 4℃for 15 minutes. After centrifugation, the supernatant was discarded, and white precipitate was seen on the bottom side wall of the tube, which was RNA.

(6) 1mL of 75% ethanol was added thereto, and the mixture was left to stand for 15 minutes after being mixed uniformly. The purpose of this step is to remove residual isopropanol from the RNA.

(7) After completion of the standing, the mixture was centrifuged at 12000rpm at 4℃for 15 minutes. The supernatant was discarded after centrifugation. Putting the mixture into a centrifugal machine again for centrifugation for 1min. The residual solution was carefully removed with a pipette. The tube cap was opened and air dried for about 10min.

(8) DEPC water is added according to the size of the RNA sediment volume at the bottom of the tube, and after blowing and dissolving, an ultra-micro spectrophotometer is used for measuring the concentration and purity of RNA in each tube.

(2) Reverse transcription (cDNA Synthesis)

Taking out the RNA sample, and putting the RNA sample in a water bath at 65 ℃ for denaturation for 5min. cDNA was synthesized using the reverse transcription kit with the denatured RNA as a template. RNA was subjected to genomic DNA removal using gDNA wind Mix and treated at 42℃for 2min. Preparing a reverse transcription system, uniformly mixing the reagents, and then reacting at 50 ℃ for 10min and 85 ℃ for 5s.

(3) qPCR reaction system

Mouse colon intestinal barrier claudin-related primers were designed according to the reference, wherein qPCR reactions were performed using ChamQ Universal SYBR qPCR Master Mix purchased from the biosciences, inc. Of nanking nuozhen. The relevant reaction system is shown in Table 5, and the reaction procedure is shown in Table 6.

TABLE 5 qPCR reaction System

TABLE 6 qPCR reaction procedure

TABLE 7 primer sequences

The results of the relative expression levels of intestinal claudin in the different treatment groups are shown in FIG. 5. The results show that the pre-treatment with Lactobacillus rhamnosus 203-10 prevents or ameliorates the damage of the intestinal barrier of the colon organoid. theexpressionofthecloselyrelatedproteinsincludingZO-1,E-cadherin,MUC2,Occludin,Claudin-1,Claudin-3andJAM-Aissignificantlyimprovedcomparedwithapositivemodelgroup.

EXAMPLE 4 immune activation/modulation of colon organoid intestinal epithelial cells by Lactobacillus rhamnosus 203-10

The relative expression levels of cytokines associated with cellular inflammation in the different treatment groups, including TNF-. Alpha., IL-1β, IL-6, IL-8, IL-10, IL-12, TGF-. Beta.1, NF-kB, were determined using the same model and the same method as in example 3.

The results of the relative expression amounts of the cytokines in the different treatment groups are shown in FIG. 6. The results show that the pretreatment of lactobacillus rhamnosus 203-10 can prevent or improve intestinal inflammation. In particular, the expression of relevant inflammatory factors is remarkably reduced compared with a positive model group, such as: TNF-alpha, IL-1 beta, IL-6, IL-8, IL-12, TGF-beta 1, NF-kB, and in addition, the expression of anti-inflammatory factor IL-10 is also improved.

Although specific embodiments of the invention have been described in detail, those skilled in the art will appreciate that: many modifications and variations of details may be made to adapt to a particular situation and the invention is intended to be within the scope of the invention. The full scope of the invention is given by the appended claims together with any equivalents thereof.

Claims

1. A lactobacillus rhamnosus (Lacticaseibacillus rhamnosus) deposited with the cantonese province microorganism strain collection under accession number gdmccno.61778.

2. The lactobacillus rhamnosus of claim 1 having a 16S rDNA sequence with 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, 99.95% or 100% identity to SEQ ID No. 1;

preferably, the 16S rDNA, after amplification using the primers shown in SEQ ID NO. 2 and SEQ ID NO. 3, generates a 1442bp fragment.

3. The lactobacillus rhamnosus of claim 1 or 2 having one or more features selected from the group consisting of:

(1) The bacterial colony is white or slightly transparent, and is circular with smooth edge;

(2) Is a gram positive bacterium;

(3) The bacteria form is rod-shaped or bar-shaped, and no spores exist.

4. A lactobacillus rhamnosus according to any of claims 1-3 having one or more characteristics selected from the group consisting of:

(1) Tolerance to gastric acid and/or bile salts;

(2) Has adhesion capability to intestinal tract or intestinal epithelium;

(3) Can inhibit pathogenic bacteria;

(4) Can prevent and/or treat intestinal barrier injury;

(5) Can prevent and/or treat intestinal tract-related inflammation;

preferably, the pathogenic bacteria are selected from clostridium perfringens (Clostridium perfringens), escherichia coli (Escherichia coli), klebsiella pneumoniae (Klebsiella pneumoniae) and gardnerella (Gardnerella vaginalis).

5. A composition comprising the lactobacillus rhamnosus of any of claims 1-4;

preferably, the composition further comprises a further bacterium and/or fungus (e.g. yeast), wherein the further bacterium and/or fungus is a probiotic or is edible;

preferably, the additional bacteria are selected from the group consisting of lactobacillus, bifidobacterium, lactobacillus mucilaginosus, lactobacillus in combination, lactobacillus, streptococcus, lactococcus, propionibacterium, leuconostoc, pediococcus, wegener, zoococcus, staphylococcus, or any combination thereof;

preferably, the yeast is selected from the group consisting of Saccharomyces cerevisiae (Brettanomyces anomalus), saccharomyces cerevisiae (Saccharomyces cerevisiae), brettanomyces brucei (Brettanomyces bruxellensis), candida stellate (Candida stillata), schizosaccharomyces pombe (Schizosaccharomyces pombe), zygosaccharomyces bailii (Zygosaccharomyces bailii), or any combination thereof;

preferably, the yeast is kluyveromyces;

preferably, the composition further comprises additional additives;

Preferably, the additional additive is a nutrient selected from dietary fiber, prebiotics, proteins, lipids, minerals, vitamins, plant extracts, or any combination thereof.

6. The composition of claim 5, wherein the composition has one or more characteristics selected from the group consisting of:

(1) The bacterium of the genus lactobacillus is selected from the group consisting of: lactobacillus paracasei (Lactobacillus paracasei), lactobacillus acidophilus (Lactobacillus acidophilus), lactobacillus brevis (Lactobacillus brevis), lactobacillus jensenii (Lactobacillus jensenii), lactobacillus inertia (Lactobacillus iners), lactobacillus casei (Lactobacillus casei), lactobacillus crispatus (Lactobacillus crispatus), lactobacillus curvatus (Lactobacillus curvatus), lactobacillus delbrueckii (Lactobacillus delbrueckii), lactobacillus fermentum (Lactobacillus fermentum), lactobacillus gasseri (Lactobacillus gasseri), lactobacillus helveticus (Lactobacillus helveticus), lactobacillus johnsonii (Lactobacillus johnsonii), lactobacillus plantarum (Lactobacillus plantarum), lactobacillus reuteri (Lactobacillus reuteri), lactobacillus rhamnosus (Lactobacillus rhamnosus), lactobacillus sake (Lactobacillus sakei), lactobacillus salivarius (Lactobacillus salivarius), or any combination thereof;

(2) The bacterium of the genus bifidobacterium is selected from: bifidobacterium animalis (Bifidobacterium animalis), bifidobacterium bifidum (Bifidobacterium bifidum), bifidobacterium breve (Bifidobacterium breve), bifidobacterium infantis (bifidobacteria), bifidobacterium longum (Bifidobacterium longum), bifidobacterium adolescentis (Bifidobacterium adolescentis), or any combination thereof;

(3) The bacteria of the genus bacillus are selected from: bacillus subtilis (Bacillus subtilis), bacillus coagulans (Bacillus coagulans), or any combination thereof;

(4) The bacteria of the genus propionibacterium are selected from: propionibacterium xivians (Propionibacterium shermanii), propionibacterium freudenreichii (Propionibacterium freudenreichii), propionibacterium propionicum (Propionibacterium acidipropionici), or any combination thereof;

(5) The bacteria of the streptococcus genus are selected from: streptococcus thermophilus (Streptococcus thermophilus), streptococcus salivarius (Streptococcus salivarius), or any combination thereof;

(6) The bacterium of the genus lactococcus is lactococcus lactis (Lactococcus lactis).

7. A food product comprising the lactobacillus rhamnosus of any of claims 1-4 or the composition of claim 5 or 6;

Preferably, the food product is a solid food product (e.g., soft candy, troche, capsule, fungus powder), a liquid food product (e.g., drink), or a semi-solid food product (e.g., jelly);

preferably, the food product is a dietary supplement, a nutritional formulation, a functional food or a beverage product;

preferably, the food product is a dairy product (e.g., yogurt, flavored fermented milk, lactobacillus beverage, cheese);

preferably, the food product is formulated for oral administration;

preferably, the food product is in the form of a pill, powder, capsule, tablet, granule, film, sachet or dragee;

preferably, the food product further comprises a prebiotic;

preferably, the lactobacillus rhamnosus in the food is 10 ⁶ To 10 ¹² The CFU/dose amount is present (e.g. 10 ⁷ To 10 ¹⁰ CFU/dose).

8. A pharmaceutical composition comprising the lactobacillus rhamnosus of any of claims 1-4 or the composition of claim 5 or 6;

preferably, the pharmaceutical composition comprises a formulation of the lactobacillus rhamnosus or composition;

preferably, the pharmaceutical composition comprises a pharmaceutically acceptable carrier;

preferably, the pharmaceutical composition is formulated for oral administration;

Preferably, the pharmaceutical composition is in the form of a pill, powder, capsule, tablet, granule, caplet, cream, ointment, gel, lotion, foam, suppository, sachet or dragee;

preferably, the lactobacillus rhamnosus in the food is 10 ⁶ To 10 ¹² The CFU/dose amount is present (e.g. 10 ⁸ To 10 ¹⁰ CFU/dose).

9. A culture comprising the lactobacillus rhamnosus of any of claims 1-4 or the composition of claim 5 or 6;

preferably, the culture is a bacterial suspension of lactobacillus rhamnosus;

preferably, the culture further comprises a nutrient providing component (e.g., a solid or liquid medium, a feeder cell layer);

preferably, the nutrient providing ingredient is selected from the group consisting of proteins, carbohydrates, fats, probiotics, enzymes, vitamins, immunomodulators, milk substitutes, minerals, amino acids, or any combination thereof;

preferably, the culture further comprises a cell-free culture filtrate of lactobacillus rhamnosus;

preferably, the culture further comprises a derivative of lactobacillus rhamnosus;

preferably, the derivative is selected from the group consisting of a metabolite, an enzyme, a cell structural component (e.g., a cell wall or component thereof), an extracellular polysaccharide, a bacteriocin, a compound containing an immunogenic component, or any combination thereof.

10. Use of the lactobacillus rhamnosus of any of claims 1-4 or the composition of claim 5 or 6 or the food of claim 7 or the pharmaceutical composition of claim 8 or the culture of claim 9 in the manufacture of a medicament capable of inhibiting a pathogenic bacterial infection or preventing and/or treating a disease and/or symptom caused by a pathogenic bacterial infection;

preferably, the pathogenic bacteria are pathogenic bacteria;

preferably, the pathogenic bacteria are selected from the group consisting of clostridium, escherichia, klebsiella, gardnerella, neisseria, or any combination thereof;

preferably, the pathogenic bacteria are selected from clostridium perfringens (Clostridium perfringens), escherichia coli (Escherichia coli), klebsiella pneumoniae (Klebsiella pneumoniae), gardnerella (Gardnerella vaginalis), or any combination thereof;

preferably, the disease is selected from gastroenteritis, urinary tract infection, vaginitis, cervicitis, urethritis, urinary tract infection, external genital infection of men and/or women, septicemia, bacteremia, bacterial cold, meningitis, peritonitis, or any combination thereof;

preferably, the symptom is selected from chills, diarrhea, fever, cough, vomiting, or any combination thereof.

11. Use of the lactobacillus rhamnosus of any of claims 1-4 or the composition of claim 5 or 6 or the food of claim 7 or the pharmaceutical composition of claim 8 or the culture of claim 9 in the manufacture of a medicament for inhibiting inflammation or preventing and/or treating an inflammatory disease in a subject;

preferably, the inflammatory disease is selected from diseases caused by retinal inflammation (e.g., retinitis, keratitis), diseases caused by skin inflammation (e.g., dermatitis, eczema), related diseases caused by respiratory tract inflammation (e.g., upper respiratory tract infection), and related diseases caused by digestive tract inflammation (e.g., inflammatory gastrointestinal disease);

preferably, the inflammatory gastrointestinal disease is selected from: inflammatory-related gastrointestinal disorders, gastrointestinal-related inflammation, intestinal barrier dysfunction and intestinal barrier damage;

preferably, the inflammatory-related gastrointestinal disorder is selected from the group consisting of non-ulcerative colitis, chronic bowel disease, crohn's disease, colonosonitis, and food-responsive diarrhea disease;

preferably, the medicament is capable of improving the barrier structure of the intestine, repairing the intestinal barrier and/or improving the barrier function of the intestine;

Preferably, the inflammatory gastrointestinal disease is modulated by pro-inflammatory cytokines and/or anti-inflammatory cytokines;

preferably, the agent is capable of reducing the expression level of a proinflammatory cytokine (e.g., TNF- α, IL-1β, IL-6, IL-8, IL-12, TGF- β1, NF-kB) and/or increasing the expression level of an anti-inflammatory cytokine (e.g., IL-10);

preferably, the medicament is used alone or in combination with other antifungal agents, antiviral agents, analgesic agents, anti-inflammatory agents, healing promoting agents, or moisturizers;

preferably, the subject is a mammal;

preferably, the subject is a human.

12. Use of the lactobacillus rhamnosus of any of claims 1-4 or the composition of claim 5 or 6 in the preparation of a starter for the fermentation of solid food (e.g. cheese, bread) or liquid food (e.g. yoghurt, flavoured milk, lactic acid bacteria beverages).