Bifidobacterium longum strain or lacticaseibacillus rhamnosus strain, vesicles derived therefrom, and Anti-inflammatory and antibacterial uses thereof

Abstract

The present invention relates to a novel microorganism, a lysate thereof, a culture medium, an extract of the culture medium, vesicles, and anti-inflammatory use thereof. A novel strain and vesicles derived therefrom, according to one aspect, can be effectively used in the prevention, amelioration or treatment of inflammation-related conditions or bacterial infections.

Description

Bifidobacterium longum strain or Lacticasebacillus rhamnosus strain, its derived endoplasmic reticulum, and its anti-inflammatory and antimicrobial uses

[0001] The present invention relates to novel microorganisms, their lysates, culture media, extracts of culture media, endoplasmic reticulum, and their anti-inflammatory and / or antibacterial uses.

[0002] A microbiome refers to the microorganisms existing in a specific environment and their entire genetic information, signifying a collection of genomes, which represent the entire genetic information of a single organism. In other words, the human microbiome refers to the microorganisms inhabiting the inside and outside of the human body and their entire genetic information.

[0003] The human body lives in a symbiotic relationship with many microorganisms, and the gut, in particular, is home to the largest number of microorganisms as it provides an optimal environment for them to consume nutrients and form systematic communities. Gut microorganisms supply nutrients that cannot be produced solely by the host's own enzymes and are deeply linked to the host's metabolic and immune systems; furthermore, they are reported to be associated with the development of various diseases such as irritable bowel syndrome, obesity, atopy, depression, rheumatoid arthritis, autism spectrum disorder, and dementia.

[0004] Recently, Westernized dietary habits and the indiscriminate use of antibiotics have led to an imbalance in the gut microbiota, resulting in a deterioration of gut health. Furthermore, research on gut microbes and various diseases has highlighted their importance and is generating growing interest.

[0005] Meanwhile, endoplasmic reticulum is a nano-sized substance, approximately 20 to 200 nm in size, produced and released by cells, and it moves freely between cells. Furthermore, endoplasmic reticulum contains membrane lipids, membrane proteins, DNA, or RNA, and it is known that these genetic materials act as complexes to transmit toxic factors between cells and play roles such as regulating inflammation and immune responses. From unicellular to multicellular organisms, intercellular information exchange is an essential process of life; recently, as endoplasmic reticulum has been recognized as a mediator of intercellular information exchange, methods are being developed to utilize vesicles as drug carriers.

[0006] Accordingly, there is a need to develop substances for the improvement, prevention, or treatment of diseases using novel microorganisms derived from the human gut and endoplasmic reticulum derived therefrom.

[0007] [Prior Art Literature]

[0008] [Patent Literature]

[0009] (Patent Document 1) Korean Published Patent 10-2018-0012849

[0010] One aspect is to provide a strain of Bifidobacterium longum deposited under accession number KCTC 16468BP, a strain of Lacticaseibacillus rhamnosus deposited under accession number KCTC 16469BP, a strain of Lacticaseibacillus rhamnosus deposited under accession number KCTC 16470BP, an endoplasmic reticulum derived from said strain, a lysate of said strain, or a culture medium of said strain.

[0011] Another aspect is to provide a use for the prevention, improvement, or treatment of inflammatory diseases comprising, as an active ingredient, a Bifidobacterium longum strain or a Lacticase Bacillus rhamnosus strain, an endoplasmic reticulum derived from said strain, a lysate of said strain, a culture medium of said strain, or a mixture thereof.

[0012] Another aspect is to provide a use for the prevention, improvement, or treatment of intestinal disease and / or intestinal health comprising a Bifidobacterium longum strain or a Lacticase Bacillus rhamnosus strain, an endoplasmic reticulum derived from said strain, a lysate of said strain, a culture medium of said strain, or a mixture thereof as an active ingredient.

[0013] One aspect provides a Bifidobacterium longum strain or a Lacticase Bacillus rhamnosus strain.

[0014] The above Bifidobacterium longum strain may be the BBH110 strain deposited under accession number KCTC 16468BP.

[0015] The above Lacticase Bacillus rhamnosus strain may be the BBH111 strain deposited under accession number KCTC 16469BP.

[0016] The above Lacticase Bacillus rhamnosus strain may be the BBH112 strain deposited under accession number KCTC 16470BP.

[0017] The above Bifidobacterium longum strain may be a strain containing the 16S rRNA of Sequence No. 1.

[0018] The above Lacticase Bacillus rhamnosus strain may be a strain containing the 16S rRNA of Sequence No. 2.

[0019] The above Lacticase Bacillus rhamnosus strain may be a strain containing the 16S rRNA of Sequence No. 3.

[0020] The above strain may have anti-inflammatory and / or antibacterial activity.

[0021] The above strain can inhibit the production of nitric oxide in inflamed cells, inhibit the expression of pro-inflammatory cytokines (e.g., TNF-α, IL-6, or IL-8), or increase the expression of anti-inflammatory cytokines (e.g., IL-10).

[0022] Another aspect provides the above-mentioned Bifidobacterium longum strain, an endoplasmic reticulum derived from the strain, a lysate of the strain, or a culture medium of the strain.

[0023] Another aspect provides the above-mentioned Lacticase Bacillus rhamnosus strain, an endoplasmic reticulum derived from the strain, a lysate of the strain, or a culture medium of the strain.

[0024] In this specification, the term "vesicle" refers to a particle secreted from a cell and released into the extracellular space, and may include a number of different species such as exosomes, ectosomes, microvesicles, microparticles, and exosome-like vesicles. Extracellular vesicles may reflect the state of the originating cell (donor cell) that secretes them, exhibit various biological activities depending on which cell they were secreted from, and play an important role in intercellular interactions by transporting genetic material and proteins between cells. Furthermore, cell-derived materials containing the vesicles have the effect of causing disease or stimulating immune cells to fight against disease, and helping to break down and absorb substances that humans cannot digest through the metabolic processes of microorganisms. The above endoplasmic reticulum is a membrane-structured endoplasmic reticulum that is divided into an inside and an outside, contains plasma membrane lipids, plasma membrane proteins, nucleic acids, and cytoplasmic components, and may be smaller in size than the original cell.

[0025] In one embodiment, the endoplasmic reticulum may be isolated from cell lysates of a culture medium of Bifidobacterium longum strain or Lacticase Bacillus rhamnosus strain.

[0026] In one embodiment, the vesicle may have a diameter of 10 nm to 400 nm. For example, it may be 10 nm to 400 nm, 10 nm to 350 nm, 10 nm to 300 nm, or 10 nm to 250 nm.

[0027] In this specification, the term "culture medium" may be used interchangeably with "culture supernatant," "conditional culture medium," or "conditioning medium," and may refer to the entire medium containing said strain, said metabolites, excess nutrients, etc., obtained by culturing said strain for a certain period in a medium capable of supplying nutrients so that said strain Bifidobacterium longum or Lacticase Bacillus rhamnosus can grow and survive in a test tube. Additionally, said culture medium may refer to a culture medium from which the cells have been removed from a culture medium obtained by culturing the strain. Meanwhile, the liquid from which the cells have been removed from said culture medium is also referred to as "supernatant," and it may be obtained by leaving the culture medium undisturbed for a certain period of time to take only the liquid from the upper layer excluding the part that has settled at the bottom, by removing the cells through filtration, or by centrifuging the culture medium to remove the lower sediment and taking only the upper liquid. The above "bacterial body" refers to the strain of the present invention itself and includes the strain itself isolated and selected from a skin sample, etc., or the strain isolated from a culture medium by culturing the said strain. The said bacterial body can be obtained by centrifuging the culture medium and taking the portion that settles to the bottom layer, or by letting it sit for a certain period of time and removing the liquid from the top, as it sinks to the bottom layer of the culture medium due to gravity.

[0028] The above culture medium may include the culture medium itself, its concentrate, or freeze-dried product obtained by culturing the strain, or the culture supernatant, its concentrate, or freeze-dried product obtained by removing the strain from the culture medium.

[0029] The above culture medium may be obtained by culturing Bifidobacterium longum or Lacticase Bacillus rhamnosus in a suitable medium (e.g., R2A medium or TSA medium) at a temperature greater than 10°C or less than 40°C for a certain period of time, for example, 4 to 50 hours.

[0030] In one embodiment, the culture supernatant of the strain can be obtained by a step of removing the strain by centrifuging or filtering the strain culture medium.

[0031] In another embodiment, the concentrate may be obtained by the step of concentrating the strain culture solution itself, or the supernatant obtained after filtering the culture solution using centrifugation or a filter.

[0032] The culture medium and culture conditions for culturing the above-mentioned Bifidobacterium longum or the above-mentioned Lacticase Bacillus rhamnosus may be appropriately selected or modified and used by a person of ordinary knowledge.

[0033] In this specification, the term "lysate" may refer to a product obtained by breaking the cell walls of the strain itself by chemical or physical force.

[0034] In this specification, the term "culture medium extract" means extracted from the culture medium or its concentrate, and may include an extract, a diluted or concentrated extract, a dried product obtained by drying the extract, a modified or purified product thereof, or a fraction obtained by fractionating the same.

[0035]

[0036] Another aspect provides a composition for the prevention, improvement, or treatment of a disease comprising, as an active ingredient, a Bifidobacterium longum strain and / or a Lacticase Bacillus rhamnosus strain, an endoplasmic reticulum derived from said strain, a lysate of said strain, a culture medium of said strain, or a mixture thereof.

[0037] In this specification, the term "treat" may mean the healing of inflammation or bacterial infections, etc., in a time shorter than natural healing. The treat may include the improvement and / or alleviation of inflammation or bacterial infections. Additionally, the treat may mean the healing and / or recovery of symptoms caused by inflammation or bacterial infections.

[0038] In this specification, the term "prevention" refers to a method of partially or completely delaying or preventing the onset or recurrence of a disease, disorder, or its associated symptoms, preventing the acquisition or reacquisition of a disease or disorder, or reducing the risk of acquiring a disease or disorder. For example, the above prevention refers to any act of suppressing or delaying the occurrence of inflammation or bacterial infection by administering a composition according to the present invention.

[0039] The uses of the above strain may include the prevention, improvement, or treatment of inflammatory (inflammatory) diseases (anti-inflammatory activity), the prevention, improvement, or treatment of bacterial infections, the prevention, improvement, or treatment of intestinal diseases (antimicrobial activity), or the prevention or improvement of intestinal health.

[0040] The above inflammatory diseases may include inflammation of the digestive system (gastrointestinal tract, etc.), inflammation of the intestines, inflammation of the eyes, inflammation of the oral cavity, inflammation of the respiratory system including the lungs, inflammation of the skin, inflammation of the cardiovascular system, inflammation of the brain, inflammation of the ears, etc.

[0041] More specifically, the above-mentioned inflammatory diseases include inflammatory bowel diseases (IBD); irritable bowel syndrome; Behcet's disease; enteritis; Crohn's disease; ulcerative colitis; vasculitis; mucositis; stomatitis; peri-implantitis; periodontitis; pulpitis; gingivitis; pneumonia; dermatitis; atopic dermatitis; contact dermatitis; CREST syndrome; dermatitis herpetiformis; dermatomyositis; systemic scleroderma; erythema nodosum; Henoch-Schonlein purpura; Hidradenitis suppurativa; Lichen planus; Majeed syndrome; Schnitzler syndrome; psoriasis; eczema; acne; mouth ulcers; uveitis; pharyngitis; tonsillitis; otitis including otitis media; psoriatic arthritis; synovitis; meningitis; encephalitis; Bickerstaff's encephalitis; encephalomyelitis; spondylitis; osteomyelitis;It may include one or more selected from the group consisting of Guillain-Barre syndrome; myelitis; neuromyelitis optica; cystitis; acute inflammation at the site of infection or injury; nephritis; and glomerulonephritis.;

[0042] In addition, the improvement of the above-mentioned intestinal health may include assistance in the proliferation of beneficial bacteria and inhibition of harmful bacteria in the intestines, assistance in intestinal health by regulating immunity, or assistance in facilitating bowel movements.

[0043] Examples of the above bacterial infections may include infections caused by Gram-positive or Gram-negative bacteria. Specifically, the bacterial infection may include infections caused by bacteria belonging to the genera Clostridioides, Helicobacter, Escherichia, Salmonella, Staphylococcus, Streptococcus, Haemophilus, Klebsiella, Moraxella, Enterobacter, Proteus, Serratia, Pseudomonas, Acinetobacter, Citrobacter, Stenotrophomonas, Bacteroides, Prevotella, and Fusobacterium. More specifically, the bacterial infection may include Clostridioides difficile infection (CDI) or Clostridioides difficile-associated disease (CDAD), for example, Clostridioides difficile-associated diarrhea.

[0044] The above composition comprises, based on the total weight of the composition, 0.00001 wt% to 80 wt%, for example, 0.00001 wt% to 60 wt%, 0.00001 wt% to 40 wt%, 0.00001 wt% to 30 wt%, 0.00001 wt% to 20 wt%, 0.00001 wt% to 10 wt%, 0.00001 wt% to 5 wt%, 0.05 wt% to 60 wt%, 0.05 wt% to 40 wt%, 0.05 wt% to 30 wt%, 0.05 wt% to 20 wt%, 0.05 wt% to 10 wt%, 0.05 wt% to 5 wt%, 0.1 wt% to 60 wt%, 0.1 wt% to 40 wt%, 0.1 wt% to It may include 30% by weight, 0.1% to 20% by weight, 0.1% to 10% by weight, or 0.1% to 5% by weight of a strain, a lysate, a culture medium, or an extract of the culture medium.

[0045] The term "included as an active ingredient" means that the strain of this specification, vesicles derived from said strain, lysate, culture medium, or extract of said culture medium are added to an extent capable of producing the effects mentioned above, and includes formulation in various forms by adding various components as auxiliary ingredients for drug delivery and stabilization, etc.

[0046] In one embodiment, the composition may be a pharmaceutical composition.

[0047] The types of pharmaceutical active ingredients capable of delivering the above active ingredients into an organism may include anticancer agents, contrast agents (dye), hormone preparations, anti-hormone preparations, vitamin preparations, calcium preparations, mineral preparations, sugar preparations, organic acid preparations, protein amino acid preparations, detoxifiers, enzyme preparations, metabolic preparations, diabetes concomitant preparations, tissue revitalization agents, chlorophyll preparations, pigment preparations, tumor agents, tumor therapeutic agents, radiopharmaceuticals, tissue cell diagnostic agents, tissue cell therapeutic agents, antibiotic preparations, antiviral agents, combination antibiotic preparations, chemotherapy agents, vaccines, toxins, toxoids, antitoxins, leptospira serum, blood preparations, biological preparations, analgesics, immunogenic molecules, antihistamines, allergy preparations, non-specific immunogenic preparations, anesthetics, stimulants, psychotropic agents, small molecule compounds, nucleic acids, aptamers, antisense nucleic acids, oligonucleotides, peptides, siRNA, and microRNA, etc.

[0048] The above pharmaceutical composition may additionally comprise a pharmaceutically acceptable diluent or carrier. The diluent may be lactose, corn starch, soybean oil, microcrystalline cellulose, or mannitol, and the lubricant may be magnesium stearate, talc, or a combination thereof. The carrier may be an excipient, a disintegrant, a binder, a lubricant, or a combination thereof. The excipient may be microcrystalline cellulose, lactose, low-substituted hydroxycellulose, or a combination thereof. The disintegrant may be calcium carboxymethylcellulose, sodium starch glycolate, calcium anhydrous phosphate, or a combination thereof. The binder may be polyvinylpyrrolidone, low-substituted hydroxypropylcellulose, hydroxypropylcellulose, or a combination thereof. The lubricant may be magnesium stearate, silicon dioxide, talc, or a combination thereof.

[0049] When formulating the above pharmaceutical composition, it is prepared using commonly used diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrants, and surfactants. Preparations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, and suppositories. Propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate may be used as non-aqueous solvents and suspension solvents. Witepsol, macrogol, Tween 61, cacao oil, lyulin oil, glycerogelatin, etc. may be used as bases for suppositories.

[0050] In order to increase stability or absorption, carbohydrates such as glucose, sucrose, or dextran, antioxidants such as ascorbic acid or glutathione, chelating agents, low molecular weight proteins, or other stabilizers may be used as pharmaceutical agents in the above pharmaceutical composition.

[0051] The above pharmaceutical composition may be formulated into an oral or parenteral administration formulation. The oral administration formulation may be a granule, powder, liquid, tablet, capsule, dry syrup, or a combination thereof. The parenteral administration formulation may be an injection.

[0052] In one embodiment, the composition may be a health functional food composition.

[0053] The above-described health functional food composition may be used alone or in combination with other foods or food ingredients, or appropriately used according to conventional methods. The mixed amount of active ingredients may be appropriately determined according to the purpose of use (prevention, health, or therapeutic treatment). Generally, when manufacturing food or beverages, the composition of this specification may be added in an amount of 15 parts by weight or less relative to the raw materials. There are no special restrictions on the types of health functional foods. Among the types of health functional foods, beverage compositions may contain various flavoring agents or natural carbohydrates, etc., as additional ingredients, similar to ordinary beverages. The natural carbohydrates are monosaccharides such as glucose and fructose, disaccharides such as maltose and sucrose, polysaccharides such as dextrin and cyclodextrin, and sugar alcohols such as xylitol, sorbitol, and erythritol. As sweeteners, natural sweeteners such as taumatin and stevia extract, or synthetic sweeteners such as saccharin and aspartame may be used. The above health food composition may also contain nutritional supplements, vitamins, electrolytes, flavoring agents, coloring agents, pectic acid and its salts, alginic acid and its salts, organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohol, carbonating agents used in carbonated beverages, or combinations thereof. The above health functional food composition may also contain fruit pulp for the production of natural fruit juice, fruit juice beverages, vegetable beverages, or combinations thereof.

[0054] In one embodiment, the composition may be a cosmetic composition.

[0055] The above cosmetic composition may have, for example, a softening lotion, a nourishing lotion, a massage cream, a nourishing cream, an essence, a pack, a gel, an ampoule, or a skin-adhesive type cosmetic formulation.

[0056] The ingredients included in the above cosmetic composition may include ingredients commonly used in cosmetic compositions in addition to the above composition as active ingredients, and may include, for example, conventional auxiliary agents and carriers such as stabilizers, solubilizers, vitamins, pigments, and fragrances.

[0057] In one embodiment, the composition may be a composition for external use on the skin.

[0058] The above-mentioned topical preparation may be a cream, gel, ointment, skin emulsifier, skin suspension, transdermal patch, drug-containing bandage, lotion, or a combination thereof. The above-mentioned topical preparation may be appropriately formulated as needed with ingredients commonly used in topical preparations for the skin, such as cosmetics or pharmaceuticals, for example, aqueous ingredients, oily ingredients, powder ingredients, alcohols, moisturizers, thickeners, UV absorbers, whitening agents, preservatives, antioxidants, surfactants, fragrances, colorants, various skin nutrients, or combinations thereof. The above topical preparation may also appropriately incorporate metal chelating agents such as disodium edetate, trisodium edetate, sodium citrate, sodium polyphosphate, sodium metaphosphate, and gluconic acid; caffeine, tannin, bellapamil, licorice extract, glablidin, hot water extract of the fruit of *calin*; various herbal medicines; preparations such as tocopherol acetate, glycyrrhizic acid, tranexamic acid and its derivatives or salts; and sugars such as vitamin C, magnesium ascorbate phosphate, ascorbate glucoside, arbutin, kojic acid, glucose, fructose, and trehalose.

[0059]

[0060] Another aspect provides a method for preventing, improving, or treating the condition of an individual, comprising the step of treating or administering an effective amount of the said strain, endoplasmic reticulum derived from the said strain, lysate of the said strain, culture medium of the said strain, or composition said above to an individual in need thereof.

[0061] The condition of the above-mentioned individual may be a condition related to inflammation, a condition related to intestinal disease, or a condition related to bacterial infection.

[0062] Administration may be performed by methods known in the art. Administration may be performed directly to an individual by any means, via routes such as, for example, intravenous, intramuscular, oral, transdermal, mucosal, intranasal, intratracheal, or subcutaneous administration. The administration may be performed systemically or locally.

[0063] The above-mentioned individual may be a mammal, for example, a human, cattle, horses, pigs, dogs, sheep, goats, or cats. The above-mentioned individual may be an individual requiring an effect of improving a condition related to inflammation or a condition related to bacterial infection.

[0064] The above administration is 0.00001 mg to 1,000 mg of the composition according to one embodiment per individual per day, for example, 0.00001 mg to 500 mg, 0.00001 mg to 100 mg, 0.00001 mg to 50 mg, 0.00001 mg to 25 mg, 1 mg to 1,000 mg, 1 mg to 500 mg, 1 mg to 100 mg, 1 mg to 50 mg, 1 mg to 25 mg, 5 mg to 1,000 mg, 5 mg to 500 mg, 5 mg to 100 mg, 5 mg to 50 mg, 5 mg to 25 mg, 10 mg to 1,000 mg, 10 mg to 500 mg, 10 mg to 100 mg, 10 mg to 50 mg, or It may be administered in an amount of 10 mg to 25 mg. However, the dosage may vary depending on factors such as the formulation method, method of administration, patient's age, body weight, sex, pathological condition, food, time of administration, route of administration, excretion rate, and response sensitivity, and a person skilled in the art may appropriately adjust the dosage by considering these factors. The frequency of administration may be once a day or two or more times within the range of clinically acceptable side effects, and the administration site may be one or two or more sites. The total duration of administration may be from 1 to 30 days per treatment, administered daily or at intervals of 2 to 5 days. If necessary, the same treatment may be repeated after the appropriate time. For animals other than humans, the dosage may be the same as that for humans per kg, or an amount calculated by converting the above dosage based on, for example, the ratio of organ (e.g., heart) volume between the target animal and humans (e.g., average value).

[0065]

[0066] The present invention is capable of various modifications and may have various embodiments. Specific embodiments are illustrated in the drawings and described in detail in the detailed description below. However, this is not intended to limit the present invention to specific embodiments, and it should be understood that it includes all modifications, equivalents, and substitutions that fall within the spirit and scope of the present invention. In describing the present invention, detailed descriptions of related prior art are omitted if it is determined that such detailed descriptions may obscure the essence of the present invention.

[0067] According to a novel strain and endoplasmic reticulum derived therefrom, there is an effect that can be usefully employed in the prevention, improvement, or treatment of inflammation-related conditions, specifically intestinal inflammation-related conditions such as irritable bowel syndrome.

[0068] Figure 1a is a graph showing the change in cell viability by endoplasmic reticulum derived from the Bifidobacterium longum BBH110 strain after stimulation with LPS; N: negative control, P: untreated control, EV: endoplasmic reticulum of Example 2.

[0069] Figure 1b is a graph showing the change in cell viability by endoplasmic reticulum derived from the Lacticase-Bacillus rhamnosus BBH111 strain after stimulation with LPS; N: negative control, P: untreated control, EV: endoplasmic reticulum of Example 2.

[0070] Figure 1c is a graph showing the change in cell viability by endoplasmic reticulum derived from the Lacticase-Bacillus rhamnosus BBH112 strain after stimulation with LPS; N: negative control, P: untreated control, EV: endoplasmic reticulum of Example 2.

[0071] Figure 2a is a graph showing the change in nitric oxide production by endoplasmic reticulum derived from the Bifidobacterium longum BBH110 strain after stimulation with LPS; N: negative control, P: untreated control, EV: endoplasmic reticulum of Example 2.

[0072] Figure 2b is a graph showing the change in nitric oxide production by endoplasmic reticulum derived from the Lacticase-Bacillus rhamnosus BBH111 strain after stimulation with LPS; N: negative control, P: untreated control, EV: endoplasmic reticulum of Example 2.

[0073] FIG. 2c is a graph showing the change in nitric oxide production by endoplasmic reticulum derived from the Lacticase-Bacillus rhamnosus BBH112 strain after stimulation with LPS; N: negative control, P: untreated control, EV: endoplasmic reticulum of Example 2.

[0074] Figure 3a is a graph showing the protein expression levels of pro-inflammatory cytokines (TNF and IL-6) and anti-inflammatory cytokines (IL-10) by endoplasmic reticulum derived from the Bifidobacterium longum BBH110 strain after stimulation with LPS; N: negative control, P: untreated control, EV: endoplasmic reticulum of Example 2.

[0075] Figure 3b is a graph showing the protein expression levels of pro-inflammatory cytokines (TNF and IL-6) and anti-inflammatory cytokines (IL-10) by endoplasmic reticulum derived from the Lacticase-Bacillus rhamnosus BBH111 strain after stimulation with LPS; N: negative control, P: untreated control, EV: endoplasmic reticulum of Example 2.

[0076] FIG. 3c is a graph showing the protein expression levels of pro-inflammatory cytokines (TNF and IL-6) and anti-inflammatory cytokines (IL-10) by endoplasmic reticulum derived from the Lacticase-Bacillus rhamnosus BBH112 strain after stimulation with LPS; N: negative control, P: untreated control, EV: endoplasmic reticulum of Example 2.

[0077] Figure 4a is a graph showing the change in cell viability when treated with endoplasmic reticulum derived from Bifidobacterium longum BBH110 strain or endoplasmic reticulum derived from Bifidobacterium longum standard strain (T) in a TNF-α inflammatory response induced in colorectal cancer cell lines.

[0078] Figure 4b is a graph showing the change in cell viability when treated with endoplasmic reticulum derived from the Lacticase-Bacillus rhamnosus BBH111 strain or endoplasmic reticulum derived from the standard strain (T) of Lacticase-Bacillus rhamnosus in a TNF-α inflammatory response induced in colorectal cancer cell lines.

[0079] Figure 4c is a graph showing the change in cell viability when treated with endoplasmic reticulum derived from the Lacticase-Bacillus rhamnosus BBH112 strain or endoplasmic reticulum derived from the standard strain (T) of Lacticase-Bacillus rhamnosus in a TNF-α inflammatory response induced in colorectal cancer cell lines.

[0080] Figure 5a is a graph showing the protein expression levels of pro-inflammatory cytokines (TNF and IL-6) and anti-inflammatory cytokines (IL-10) when treated with endoplasmic reticulum derived from Bifidobacterium longum BBH110 strain in a TNF-α inflammatory response induced in colorectal cancer cell lines.

[0081] Figure 5b is a graph showing the protein expression levels of pro-inflammatory cytokines (TNF and IL-6) and anti-inflammatory cytokines (IL-10) when treated with endoplasmic reticulum derived from the Lacticase Bacillus rhamnosus BBH111 strain in a TNF-α inflammatory response induced in colorectal cancer cell lines.

[0082] Figure 5c is a graph showing the protein expression levels of pro-inflammatory cytokines (TNF and IL-6) and anti-inflammatory cytokines (IL-10) when treated with endoplasmic reticulum derived from the Lacticase Bacillus rhamnosus BBH112 strain in a TNF-α inflammatory response induced in colorectal cancer cell lines.

[0083] The following examples will be explained in more detail. However, these examples are intended to illustrate one or more specific examples, and the scope of the present invention is not limited to these examples.

[0084]

[0085] Example 1. Isolation and Identification of Strains

[0086] To isolate and identify strains from the feces of healthy individuals, the following procedures were performed.

[0087] First, 1 g of fecal matter collected from a healthy individual was mixed with 10 ml of 1 x PBS (Phosphate buffer saline) and vortexed to suspend the feces. Subsequently, the mixture was filtered using a cell strainer to remove undigested food and small particulate matter. The filtered fecal suspension was serially diluted and plated on EG plates 10 -6 to 10 -8 Spreading was performed, and bacteria were selected after incubation at 37°C for at least 3 days. PCR amplification was performed on the colonies after incubation was completed, and the nucleotide sequences of the determined 16S rRNA regions among the isolated cultured microbial colonies were compared and analyzed with other strains registered using the BLAST program provided on the website of the National Center for Biotechnology Information (NCBI).

[0088]

[0089] (1) Selection of BBH 110 strain

[0090] As a result of comparative analysis, strain 23-51-06 of Bifidobacterium longum with 99.93% homology was isolated. The selected strain 23-51-06 was named Bifidobacterium longum BBH110 and deposited with the Korea National Institute of Biological Resources on September 8, 2025, receiving accession number KCTC 16468BP. Bifidobacterium longum BBH110 possesses the 16S rRNA sequence of SEQ ID No. 1 (complementary DNA).

[0091]

[0092] (2) Selection of BBH 111 strain

[0093] As a result of comparative analysis, the Lacticaseibacillus rhamnosus strain 24-18-05 with 100% homology was isolated. The selected 24-18-05 strain was named Lacticaseibacillus rhamnosus BBH111 and deposited with the Korea National Center for Biological Resources on September 8, 2025, receiving accession number KCTC 16469BP. The Lacticaseibacillus rhamnosus BBH111 strain has the 16S rRNA sequence of SEQ ID No. 2 (complementary DNA).

[0094]

[0095] (3) Selection of BBH 112 strain

[0096] As a result of comparative analysis, the Lacticaseibacillus rhamnosus 24-24-05 strain with 100% homology was isolated. The selected 24-24-05 strain was named Lacticaseibacillus rhamnosus BBH112 and deposited with the Korea National Center for Biological Resources on September 8, 2025, receiving accession number KCTC 16470BP. The Lacticaseibacillus rhamnosus BBH112 strain possesses the 16S rRNA sequence of SEQ ID No. 3 (complementary DNA).

[0097]

[0098] Example 2. Separation of endoplasmic reticulum

[0099] In the above example, the vesicles of the isolated strain were isolated.

[0100] Specifically, to prepare the endoplasmic reticulum, the isolated strain was cultured for 3 days at 37°C under anaerobic conditions in YBHI broth supplemented with 0.1% cellobiose and 0.1% maltose. Subsequently, the culture medium was centrifuged at 5000 xg for 20 minutes to remove bacterial debris. Afterward, the mixture was filtered through a 0.45 μm filter and then filtered again through a 0.22 μm filter. Subsequently, a substance of 100 kD or more was concentrated using a centrifugation tube (Amicon® Ultra-15 Centrifugal Filter Unit), and the endoplasmic reticulum of the isolated strain was isolated.

[0101]

[0102] Experimental Example 1. Analysis of anti-inflammatory activity after LPS stimulation

[0103] 1.1 Confirmation of Cytotoxicity

[0104] First, changes in cytotoxicity induced by the endoplasmic reticulum were confirmed after stimulation with LPS. Specifically, Raw264.7 cells were placed in a 48-well plate at a rate of 6 × 10⁶ 4 300 μL aliquots were dispensed at a cell / well concentration and incubated in a CO2 incubator at 37°C for 24 hours. The well supernatant was discarded, and medium supplemented with 10 μg / ml lipopolysaccharide (LPS) was dispensed to induce inflammation, followed by an additional 4 hours of incubation. The supernatant containing LPS was discarded, and 1 x 10⁶ endoplasmic reticulum from Example 2 were added. -1 Up to 1 X 10 2The solution was added to the medium at a concentration of μg / ml and incubated at 37°C for 16 hours. Afterward, the supernatant was removed, and 300 μl of medium containing 10% WST-8 solution was added to each well and reacted for 4 hours. After 4 hours, cell viability was measured by determining the absorbance at 450 nm of the concentration of water-soluble formazan produced by succinate dehydrogenase present in the mitochondria of living cells, and the results are shown in Figures 1a, 1b, and 1c.

[0105] Figure 1a is a graph showing the change in cell viability by endoplasmic reticulum derived from the Bifidobacterium longum BBH110 strain after stimulation with LPS; N: negative control, P: untreated control, EV: endoplasmic reticulum of Example 2.

[0106] Figure 1b is a graph showing the change in cell viability by endoplasmic reticulum derived from the Lacticase-Bacillus rhamnosus BBH111 strain after stimulation with LPS; N: negative control, P: untreated control, EV: endoplasmic reticulum of Example 2.

[0107] Figure 1c is a graph showing the change in cell viability of endoplasmic reticulum derived from the Lacticase Bacillus rhamnosus BBH112 strain after stimulation with LPS; N: negative control, P: untreated control, EV: endoplasmic reticulum of Example 2. As shown in Figures 1a, 1b, and 1c, the endoplasmic reticulum of the strain according to one embodiment showed a cell viability of 90% or more, confirming that no cytotoxicity was observed.

[0108]

[0109] 1.2 Verification of Nitric Oxide (NO) Production Amount

[0110] Additionally, nitric oxide production was measured as follows. Mouse macrophage Raw264.7 cells were cultured at 37°C in the presence of 5% CO2 in DMEM (Dulbecco Modified Eagle Medium) containing 10% fetal bovine serum (FBS) and 1% antibiotics (100 U / mL penicillin and 100 μg / mL streptomycin). Subsequently, the Raw 264.7 cells were placed in a 48-well plate at a density of 6 × 10⁶ 4 300 μL aliquots were dispensed at a cell / well concentration and incubated in a CO2 incubator at 37°C for 24 hours. The well supernatant was discarded, and medium supplemented with 10 μg / ml lipopolysaccharide (LPS) was dispensed to induce inflammation, followed by an additional 4 hours of incubation. The supernatant containing LPS was discarded, and 1 x 10⁶ endoplasmic reticulum from Example 2 were added. -1 Up to 1 X 10 2 The substance was added to the medium at a concentration of μg / ml and treated, then incubated at 37°C for 16 hours. Afterward, 50 μL of the well supernatant was mixed with 50 μL of Griess reagent and reacted at room temperature for 10 minutes, after which the amount of nitric oxide produced was measured by measuring the absorbance at 540 nm using a plate reader.

[0111] Figure 2a is a graph showing the change in nitric oxide production by endoplasmic reticulum derived from the Bifidobacterium longum BBH110 strain after stimulation with LPS; N: negative control, P: untreated control, EV: endoplasmic reticulum of Example 2.

[0112] Figure 2b is a graph showing the change in nitric oxide production by endoplasmic reticulum derived from the Lacticase-Bacillus rhamnosus BBH111 strain after stimulation with LPS; N: negative control, P: untreated control, EV: endoplasmic reticulum of Example 2.

[0113] FIG. 2c is a graph showing the change in nitric oxide production by endoplasmic reticulum derived from the Lacticase-Bacillus rhamnosus BBH112 strain after stimulation with LPS; N: negative control, P: untreated control, EV: endoplasmic reticulum of Example 2.

[0114] As shown in Figures 2a, 2b, and 2c, the amount of NO produced in inflammation-induced cells treated with the endoplasmic reticulum of the strain according to one embodiment did not show a significant difference from the untreated control group.

[0115]

[0116] 1.3 Confirmation of Inflammatory Cytokine Production Levels

[0117] Next, the pro-inflammatory cytokine inhibitory activity and anti-inflammatory cytokine promoting activity of the endoplasmic reticulum of Example 2 were measured. Specifically, 10 endoplasmic reticulum of Example 2 were applied to Raw264.7 cells treated with LPS in the same manner as above. -1 Up to 1 X 10 2 After treatment with a concentration of μg / ml, the cells were cultured at 37°C for 16 hours. Subsequently, the protein expression of the pro-inflammatory cytokines TNF and IL-6 and the protein expression of the anti-inflammatory cytokine IL-10 in the cells were measured at 450 nm using an ELISA kit (BD Bioscience, USA) according to the manufacturer's instructions, and the results are shown in Figure 3.

[0118] Figure 3a is a graph showing the protein expression levels of pro-inflammatory cytokines (TNF and IL-6) and anti-inflammatory cytokines (IL-10) by endoplasmic reticulum derived from the Bifidobacterium longum BBH110 strain after stimulation with LPS; N: negative control, P: untreated control, EV: endoplasmic reticulum of Example 2.

[0119] Figure 3b is a graph showing the protein expression levels of pro-inflammatory cytokines (TNF and IL-6) and anti-inflammatory cytokines (IL-10) by endoplasmic reticulum derived from the Lacticase-Bacillus rhamnosus BBH111 strain after stimulation with LPS; N: negative control, P: untreated control, EV: endoplasmic reticulum of Example 2.

[0120] FIG. 3c is a graph showing the protein expression levels of pro-inflammatory cytokines (TNF and IL-6) and anti-inflammatory cytokines (IL-10) by endoplasmic reticulum derived from the Lacticase-Bacillus rhamnosus BBH112 strain after stimulation with LPS; N: negative control, P: untreated control, EV: endoplasmic reticulum of Example 2.

[0121] As shown in Figures 3a, 3b, and 3c, it was found that the endoplasmic reticulum of the strain according to one embodiment significantly reduced pro-inflammatory cytokines compared to the untreated control group and significantly increased anti-inflammatory cytokines compared to the untreated control group.

[0122]

[0123] The above results mean that the strain according to one embodiment can be usefully used for the prevention, improvement, or treatment of inflammatory diseases, particularly inflammatory bowel disease or irritable bowel syndrome.

[0124]

[0125] Experimental Example 2. Confirmation of anti-inflammatory activity in colorectal cancer cell lines

[0126] 2.1 Confirmation of Cytotoxicity

[0127] Cytotoxicity was confirmed using a cell viability assay kit (WST-8, Biomax, Korea). Specifically, 1 × 10⁶ Caco-2 cells were placed in a 48-well plate. 6300 μL aliquots were dispensed at a cell / well concentration and incubated in a CO2 incubator at 37°C for 24 hours. The well supernatant was discarded, and medium supplemented with 50 ng / ml of Tumor Necrosis Factor-α human (TNF-α) was dispensed to induce inflammation, followed by an additional 4 hours of incubation. The supernatant containing TNF-α was discarded, and 1 x 10⁶ endoplasmic reticulum from Example 2 were collected. -2 Up to 1 X 10 2 The solution was added to the medium at a concentration of μg / ml and incubated at 37°C for 16 hours. Afterward, the supernatant was removed, and 300 μl of medium containing 10% WST-8 solution was added to each well and reacted for 30 minutes. After 30 minutes, cell viability was measured by determining the absorbance at 450 nm of water-soluble formazan produced by succinate dehydrogenase present in the mitochondria of living cells, and the results are shown in Figure 4.

[0128] Figure 4a is a graph showing the change in cell viability when treated with endoplasmic reticulum derived from Bifidobacterium longum BBH110 strain or endoplasmic reticulum derived from Bifidobacterium longum standard strain (T) in a TNF-α inflammatory response induced in colorectal cancer cell lines.

[0129] Figure 4b is a graph showing the change in cell viability when treated with endoplasmic reticulum derived from the Lacticase-Bacillus rhamnosus BBH111 strain or endoplasmic reticulum derived from the standard strain (T) of Lacticase-Bacillus rhamnosus in a TNF-α inflammatory response induced in colorectal cancer cell lines.

[0130] Figure 4c is a graph showing the change in cell viability when treated with endoplasmic reticulum derived from the Lacticase-Bacillus rhamnosus BBH112 strain or endoplasmic reticulum derived from the standard strain (T) of Lacticase-Bacillus rhamnosus in a TNF-α inflammatory response induced in colorectal cancer cell lines.

[0131] As shown in FIGS. 4a, 4b, and 4c, the endoplasmic reticulum of the strain according to one embodiment is 1 x 10 -2 Up to 1 X 10 2 It was found that no cytotoxicity was observed at the μg / ml concentration.

[0132]

[0133] 2.2 Confirmation of Inflammatory Cytokine Production Levels

[0134] In the same manner as in Experimental Example 1.3, 1 x 10⁶ endoplasmic reticulum of Example 2 were injected into TNF-α-treated Caco-2 cells. -2 Up to 1 X 10 2 After treatment with a concentration of μg / ml, the cells were incubated at 37°C for 16 hours. Subsequently, the protein expression of the pro-inflammatory cytokines TNF and IL-8 and the protein expression of the anti-inflammatory cytokine IL-10 in the cells were measured at 450 nm using an ELISA set (BD Bioscience, USA) according to the manufacturer's instructions.

[0135] Figure 5a is a graph showing the protein expression levels of pro-inflammatory cytokines (TNF and IL-6) and anti-inflammatory cytokines (IL-10) when treated with endoplasmic reticulum derived from Bifidobacterium longum BBH110 strain in a TNF-α inflammatory response induced in colorectal cancer cell lines.

[0136] Figure 5b is a graph showing the protein expression levels of pro-inflammatory cytokines (TNF and IL-6) and anti-inflammatory cytokines (IL-10) when treated with endoplasmic reticulum derived from the Lacticase Bacillus rhamnosus BBH111 strain in a TNF-α inflammatory response induced in colorectal cancer cell lines.

[0137] Figure 5c is a graph showing the protein expression levels of pro-inflammatory cytokines (TNF and IL-6) and anti-inflammatory cytokines (IL-10) when treated with endoplasmic reticulum derived from the Lacticase Bacillus rhamnosus BBH112 strain in a TNF-α inflammatory response induced in colorectal cancer cell lines.

[0138] As shown in Figures 5a, 5b, and 5c, regarding protein expression in colorectal cancer cell lines, it was found that the endoplasmic reticulum according to one embodiment significantly reduced pro-inflammatory cytokines compared to the untreated control group and significantly increased anti-inflammatory cytokines compared to the untreated control group.

[0139]

[0140] The above results mean that the strain according to one embodiment and / or the endoplasmic reticulum derived therefrom can strengthen intestinal barrier function and alleviate intestinal inflammation, and can be usefully used for the prevention, improvement, or treatment of inflammatory diseases, particularly inflammatory bowel disease or irritable bowel syndrome.

[0141]

[0142] The foregoing description of the present invention is for illustrative purposes only, and those skilled in the art will understand that other specific forms can be easily modified without altering the technical spirit or essential features of the present invention. Therefore, the embodiments described above should be understood as illustrative in all respects and not restrictive.

[0143]

[0144] [Consignment Number]

[0145] 1. Bifidobacterium longum BBH110

[0146] Depository Name: Korea Research Institute of Biotechnology and Bioengineering Biological Resource Center (KCTC)

[0147] Trustee Number: KCTC16468BP

[0148] Date of Trust: 20250908

[0149]

[0150]

[0151] 2. Lacticaseibacillus rhamnosus BBH111

[0152] Depository Name: Korea Research Institute of Biotechnology and Bioengineering Biological Resource Center (KCTC)

[0153] Trustee Number: KCTC16469BP

[0154] Date of Trust: 20250908

[0155]

[0156]

[0157] 3. Lacticaseibacillus rhamnosus BBH112

[0158] Depository Name: Korea Research Institute of Biotechnology and Bioengineering Biological Resource Center (KCTC)

[0159] Trustee Number: KCTC16470BP

[0160] Date of Trust: 20250908

[0161]

Claims

1. Bifidobacterium longum strain BBH110 deposited under accession number KCTC 16468BP, Lacticaseibacillus rhamnosus strain BBH111 deposited under accession number KCTC 16469BP, or Lacticaseibacillus rhamnosus strain BBH112 deposited under accession number KCTC 16470BP.

2. An endoplasmic reticulum derived from the strain of Claim 1.

3. A lysate of the strain of Claim 1.

4. Culture solution of the strain of Claim 1.

5. A pharmaceutical composition for the prevention or treatment of inflammatory diseases comprising, as an active ingredient, a Bifidobacterium longum strain or a Lacticase Bacillus rhamnosus strain, an endoplasmic reticulum derived from said strain, a lysate of said strain, a culture medium of said strain, or a mixture thereof.

6. A pharmaceutical composition according to claim 5, wherein the inflammatory disease is an intestinal inflammatory disease.

7. A pharmaceutical composition according to claim 5, wherein the inflammatory disease comprises one or more selected from the group consisting of irritable bowel syndrome, inflammatory bowel diseases (IBD), enteritis, Crohn's disease, and ulcerative colitis.

8. A health functional food for improving intestinal health comprising, as an active ingredient, a Bifidobacterium longum strain, a Lacticase Bacillus rhamnosus strain, an endoplasmic reticulum derived from said strain, a lysate of said strain, a culture medium of said strain, or a mixture thereof.

9. A health functional food according to claim 8, wherein the improvement of intestinal health includes assistance in the proliferation of beneficial bacteria and inhibition of harmful bacteria in the intestines, assistance in intestinal health by regulating immunity, or assistance in facilitating bowel movements.

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